Biokar Catalogus

BIOKAR
DIAGNOSTICS
Products for Microbiology
3
contents
Index 5
Alphabetical index 6
Numerical index 13
The Company 23
Biokar Diagnostics Quality 25
Quality Assurance 26
Quality Control 27
Culture Media 31
Generalities
Product line 39
By methodology 40
By testing context 48
Product review 63
Culture media 65
Supplements, Reagents, Ringer 269
Microplates 287
Agars, Peptones, Extracts 299
4
Index
Alphabetical index
Culture media
Code Page
A
Azide Dextrose Broth BK125 66
Azide Dextrose Broth (acc. to Rothe) BK060 67
B
Bacillus cereus Agar (acc. to Mossel) BK116 / BM038 69
Baird-Parker Agar with Egg Yolk Tellurite BK055 / BM018 71
Baird-Parker RPF Agar BK055 / BM029 / BT005 73
BiGGY Agar (Nickerson) BK074 75
Bile Esculin Azide (BEA) Agar BK158 77
Bismuth Sulfite Agar BK004 78
Brain-Heart Agar BK029 80
Brain-Heart Broth BK015 81
Brilliant Green Agar (Edel and Kampelmacher) BK091 82
Brilliant Green Agar (Kristensen) BK071 84
Brilliant Green Bile Broth (BGBB) BK002 / BM011 85
Bromcresol Purple Lactose Agar BK023 87
Bromcresol Purple Lactose Broth BK119 89
Brucella Agar BK077 90
Brucella Broth BK122 91
Bryant and Burkey Broth with Resazurin (modified) BK141 92
Buffered Peptone Broth with sodium chloride pH 7.0 BK128 94
Buffered Peptone Water BK018 / BK131 / BM010 95
Buttiaux-Brogniart Hypersalted Broth (double strength) BK081 96
C
Cetrimide Agar BK049 98
CFC Agar BK118 99
Chloramphenicol Glucose Agar BK007 / BM021 101
CLED Agar BK020 102
Columbia Agar BK019 104
Columbia CNA Agar BK075 106
Cooked Meat Medium BK076 107
Count-Tact Agar BK130 108
D
DCLS Agar BK085 110
Desoxycholate Citrate Agar (DCA) BK044 111
Desoxycholate Lactose Agar BK065 113
Desoxycholate (0.1%) Lactose Agar BK062 114
Dextrose Tryptone Agar BK042 116
Drigalski Lactose Agar BK036 117
6
Code Page
E
EE Broth (acc. to Mossel) BK127 118
Elliker Broth BK054 119
EMB Agar (Levine) BK056 120
Endo Agar BK057 122
Ethyl Violet Azide Broth BK061 123
Eugon Agar BK096 / BM047 125
Eugon Broth BK068 126
F
Fraser Broth BK115 / BK133 / BM013 128
G
GC Agar BK051 130
Giolitti and Cantoni Broth BK080 131
H
Half-Fraser Broth BK133 / BM016 133
Hektoen Enteric Agar BK067 135
K
Karmali Agar BK117 137
KF Streptococcus Agar BK132 139
Kligler Iron Agar BK034 140
L
Lactose Broth BK082 143
Lactose-Sulfite Broth (LS) BK140 144
Laurylsulfate-Tryptose Broth BK010 146
Lecithin-Polysorbate-Triton X Agar BK138 / BM023 / BM044 / BM045 / BM046 147
Lecithin-Polysorbate-Triton X Broth BK137 / BM006 / BM042 / BM043 149
Listeria Enrichment Broth (LEB acc. to FDA) BK112 150
Listeria Enrichment Broth (LEB acc. to IDF) BK124 151
Listeria Enrichment Broth (UVM I modified) BK113 153
Listeria Enrichment Broth (UVM II modified) BK114 153
M
M17 Agar BK088 154
M17 Broth BK012 156
MacConkey Agar BK050 157
MacConkey Broth Purple BK107 159
MacConkey Sorbitol Agar (CT-SMAC) BK147 160
Malt Agar BK045 162
7
Code Page
Mannitol Salt Agar BK030 163
Meat-Liver Glucose Agar BK092 / BK157 164
Meat-Liver Glucose - 0.6% Agar BK024 166
Meat-Yeast Agar BK006 167
Meat-Yeast Glucose - 0.6 % Agar BK052 168
mEndo LES Agar BK155 169
mFC Agar BK154 171
Minerals Glutamate Medium BK005 172
Modified EC Broth (mEC) BK149 174
Modified Tryptone-Soy Broth (mTSB) BK150 175
MRS Agar BK089 177
MRS Broth BK070 178
MSE Agar BK087 180
MSRV Medium BK134 181
Mueller Hinton Agar BK048 182
Mueller Hinton Broth BK108 184
Müller-Kauffmann Broth BK135 186
N
Nutrient Agar BK021 187
Nutrient Broth BK003 188
O
Önöz Agar BK032 189
Orange Serum Agar BK103 191
Oxford Agar BK110 / BM019 192
Oxytetracycline Glucose Agar (OGA) BK053 / BM022 194
P
PALCAM Agar BK145 / BM020 195
Peptone Water BK084 198
Plate Count Agar (PCA) BK144 / BM015 / BM033 199
Plate Count Agar with skimmed milk BK161 200
Potato Dextrose Agar BK095 201
R
Rappaport-Vassiliadis Broth BK136 / BM012 202
Rappaport-Vassiliadis Soja Broth (RVS) BK148 204
Reinforced Clostridial Agar BK090 206
Reinforced Clostridial Medium BK094 207
Rogosa Agar BK033 208
Rose Bengal Chloramphenicol Agar BK151 210
Rose-Gal BCIG Agar BK142 211
8
Code Page
S
Sabouraud Chloramphenicol Agar BK027 214
Sabouraud Dextrose Agar BK025 / BM052 / BM053 215
Sabouraud Dextrose Broth BK026 216
Schaedler Agar BK063 217
Schaedler Broth BK064 219
Schubert Broth (modified) BK120 221
Selenite Broth (Leifson) BK079 222
Selenite-Cystine Broth BK009 223
Slanetz and Bartley Agar BK037 / BK129 / BM026 225
SS Agar BK022 226
Sugar-Free Agar (SFA) BK126 228
T
TBX Agar BK146 229
TCBS Agar BK040 231
Tergitol 7 Agar BK038 233
Tergitol 7 Agar (acc. to NF EN ISO) BK123 / BM024 234
Tergitol BCIG Agar BK139 / BM025 236
Tetrathionate Broth (USP) BK083 238
Thioglycollate Medium with Resazurin BK017 / BM007 / BM031 239
Todd-Hewitt Broth BK100 241
Tryptone-Salt Broth BK014 / BM008 242
Tryptone-Soy Agar BK047 / BM017 / BM049 / BM050 244
Tryptone-Soy Agar (Blood Agar) BK028 245
Tryptone-Soy Broth BK046 / BM009 / BM030 247
TSC Agar BK031 248
TSI Agar BK059 250
TSN Agar BK001 252
V
Violet Red Bile Agar (VRBL) BK152 / BM034 / BM035 253
Violet Red Bile Glucose Agar (VRBG) BK011 255
W
Wilkins-Chalgren Agar BK101 256
Wilkins-Chalgren Broth BK102 257
WL Differential Agar BK066 259
Wort Agar BK013 260
X
XLD Agar BK058 261
XLT4 Agar BK156 / BM036 263
9
Code Page
Y
Yeast Extract Agar BK153 / BM068 265
Yersinia Agar (CIN) BK039 266
Supplements, Reagents, Ringer
C
Cefixime-Tellurite Selective Supplement BS037 270
CFC Selective Supplement BS022 270
Chloramphenicol Selective Supplement BS021 271
CIN Selective Supplement BS023 271
Coagulase Rabbit Plasma BR002 272
D
D-Cycloserine Selective Supplement BS006 273
E
Egg Yolk Enrichment BS002 273
Egg Yolk Tellurite Enrichment BS001 274
F
Fraser Selective Supplement BS031 274
G
Gentamicin Selective Supplement BS009 275
H
Half-Fraser Selective Supplement BS030 / BS032 275
K
Karmali Selective Supplement BS017 276
M
MUG Supplement BS024 277
N
Novobiocin Selective Supplement BS033 278
10
Code Page
O
Oxford Selective Supplement (CCCFA) BS003 278
Oxytetracycline Selective Supplement BS008 279
P
PALCAM Selective Supplement BS004 / BS049 280
Polymyxin B Selective Supplement BS007 280
R
Rabbit Plasma Fibrinogen Supplement BS034 / BS038 281
Ringer's Solution (1/4 strength) BR001 282
Rosolic Acid Selective Supplement BS040 283
S
Sulfamethazine Selective Supplement BS028 283
Synthetic Sea Salt BR003 288 / 290
T
Tergitol 4 Selective Supplement BS039 284
Thayer-Martin Growth Supplement BS010 284
TTC Supplement 12.5 mg BS026 285
TTC Supplement 50 mg BS027 286
Kits
B
Baird-Parker RPF Agar BT005 73
M
Microplate MUD/SF BT003 / BT004 288
Microplate MUG/EC BT001 / BT002 290
11
Agars, Peptones, Extracts
Code Page
A
Acid Hydrolysate of Casein A1404 300
B
Bacteriological Agar Type A A1010 301
Bacteriological Agar Type E A1012 302
Bacteriological Malt Extract A1101 302
Bacteriological Meat Extract A1710 303
C
Casein-Meat Peptone A1408 304
M
Mycopeptone A1801 304
P
Pancreatic Digest of Casein Codex A1402 305
Pancreatic Digest of Gelatin A1001 306
Pancreatic Digest of Meat Type 2 A1702 307
Papaic Digest of Soybean Meal USP A1601 308
Peptic Digest of Meat USP A1708 309
T
Tryptone USP A1401 310
Y
Yeast Extract A1202 311
12
Numerical index
Code Page
A1001 Pancreatic Digest of Gelatin 306
A1010 Bacteriological Agar Type A 301
A1012 Bacteriological Agar Type E 302
A1101 Bacteriological Malt Extract 302
A1202 Yeast Extract 311
A1401 Tryptone USP 310
A1402 Pancreatic Digest of Casein Codex 305
A1404 Acid Hydrolysate of Casein 300
A1408 Casein-Meat Peptone 304
A1601 Papaic Digest of Soybean Meal USP 308
A1702 Pancreatic Digest of Meat Type 2 307
A1708 Peptic Digest of Meat USP 309
A1710 Bacteriological Meat Extract 303
A1801 Mycopeptone 304
BK001 TSN Agar 252
BK002 Brilliant Green Bile Broth (BGBB) 85
BK003 Nutrient Broth 188
BK004 Bismuth Sulfite Agar 78
BK005 Minerals Glutamate Medium (base) 172
BK006 Meat-Yeast Agar 167
BK007 Chloramphenicol Glucose Agar 101
BK009 Selenite-Cystine Broth 223
BK010 Laurylsulfate-Tryptose Broth 146
BK011 Violet Red Bile Glucose Agar (VRBG) 255
BK012 M17 Broth 156
13
Code Page
BK013 Wort Agar (base) 260
BK014 Tryptone-Salt Broth 242
BK015 Brain-Heart Broth 81
BK017 Thioglycollate Medium with Resazurin 239
BK018 Buffered Peptone Water (25.5 g/L) 95
BK019 Columbia Agar (base) 104
BK020 CLED Agar 102
BK021 Nutrient Agar 187
BK022 SS Agar 226
BK023 Bromcresol Purple Lactose Agar 87
BK024 Meat-Liver Glucose - 0.6% Agar 166
BK025 Sabouraud Dextrose Agar 215
BK026 Sabouraud Dextrose Broth 216
BK027 Sabouraud Chloramphenicol Agar 214
BK028 Tryptone-Soy Agar (Blood Agar base) 245
BK029 Brain-Heart Agar 80
BK030 Mannitol Salt Agar 163
BK031 TSC Agar (base) 248
BK032 Önöz Agar 189
BK033 Rogosa Agar 208
BK034 Kligler Iron Agar 140
BK036 Drigalski Lactose Agar 117
BK037 Slanetz and Bartley Agar 225
BK038 Tergitol 7 Agar (base) 233
14
Code Page
BK039 Yersinia Agar (CIN base) 266
BK040 TCBS Agar 231
BK042 Dextrose Tryptone Agar 116
BK044 Desoxycholate Citrate Agar (DCA) 111
BK045 Malt Agar 162
BK046 Tryptone-Soy Broth 247
BK047 Tryptone-Soy Agar 244
BK048 Mueller Hinton Agar 182
BK049 Cetrimide Agar (base) 98
BK050 MacConkey Agar 157
BK051 GC Agar (base) 130
BK052 Meat-Yeast Glucose - 0.6% Agar 168
BK053 Oxytetracycline Glucose Agar (OGA base) 194
BK054 Elliker Broth 119
BK055 Baird-Parker Agar with Egg Yolk Tellurite / Baird Parker RPF Agar (base / RPF base) 71 / 73
BK056 EMB Agar (Levine) 120
BK057 Endo Agar 122
BK058 XLD Agar 261
BK059 TSI Agar 250
BK060 Azide Dextrose Broth (acc. to Rothe) 67
BK061 Ethyl Violet Azide Broth 123
BK062 Desoxycholate (0.1%) Lactose Agar 113
BK063 Schaedler Agar 217
BK064 Schaedler Broth 219
15
Code Page
BK065 Desoxycholate Lactose Agar 113
BK066 WL Differential Agar 259
BK067 Hektoen Enteric Agar 137
BK068 Eugon Broth 126
BK070 MRS Broth 178
BK071 Brilliant Green Agar (Kristensen) 84
BK074 BiGGY Agar (Nickerson) 75
BK075 Columbia CNA Agar 106
BK076 Cooked Meat Medium 107
BK077 Brucella Agar 90
BK079 Selenite Broth (Leifson) 222
BK080 Giolitti and Cantoni Broth (base) 131
BK081 Buttiaux-Brogniart Hypersalted Broth (double strength) 96
BK082 Lactose Broth 143
BK083 Tetrathionate Broth (USP base) 238
BK084 Peptone Water 198
BK085 DCLS Agar 110
BK087 MSE Agar 180
BK088 M17 Agar 154
BK089 MRS Agar 177
BK090 Reinforced Clostridial Agar 206
BK091 Brilliant Green Agar (Edel and Kampelmacher) 82
BK092 Meat-Liver Glucose Agar (Base) 164
BK094 Reinforced Clostridial Medium 207
16
Code Page
BK095 Potato Dextrose Agar 201
BK096 Eugon Agar 125
BK100 Todd-Hewitt Broth 241
BK101 Wilkins-Chalgren Agar 256
BK102 Wilkins-Chalgren Broth 257
BK103 Orange Serum Agar 191
BK107 MacConkey Broth Purple 159
BK108 Mueller Hinton Broth 184
BK110 Oxford Agar (Base) 192
BK112 Listeria Enrichment Broth (LEB acc. to FDA) 150
BK113 Listeria Enrichment Broth (UVM I modified) 153
BK114 Listeria Enrichment Broth (UVM II modified) 153
BK115 Fraser Broth (Base) 128
BK116 Bacillus cereus Agar (Base acc. to Mossel) 69
BK117 Karmali Agar (Base) 137
BK118 CFC Agar (Base) 99
BK119 Bromcresol Purple Lactose Broth 89
BK120 Schubert Broth (modified) 221
BK122 Brucella Broth 91
BK123 Tergitol 7 Agar (Base acc. to NF EN ISO) 234
BK124 Listeria Enrichment Broth (LEB acc. to IDF) 151
BK125 Azide Dextrose Broth 66
BK126 Sugar-Free Agar (SFA) 228
BK127 EE Broth (acc. to Mossel) 118
17
Code Page
BK128 Buffered Peptone Broth with Sodium Chloride pH 7.0 94
BK129 Slanetz and Bartley Agar (Base) 225
BK130 Count-Tact Agar 108
BK131 Buffered Peptone Water (20 g/L) 95
BK132 KF Streptococcus Agar (Base) 139
BK133 Fraser Broth / Half-Fraser Broth (Bases) 128 / 133
BK134 MSRV Medium (Base) 181
BK135 Müller-Kauffmann Broth (Base) 186
BK136 Rappaport-Vassiliadis Broth 202
BK137 Lecithin-Polysorbate-Triton X Broth 149
BK138 Lecithin-Polysorbate-Triton X Agar 147
BK139 Tergitol BCIG Agar 236
BK140 Lactose Sulfite Broth (LS) 144
BK141 Bryant and Burkey Broth with Resazurin (modified) 92
BK142 Rose-Gal BCIG Agar 211
BK144 Plate Count Agar (PCA) 199
BK145 PALCAM Agar 195
BK146 TBX Agar 229
BK147 MacConkey Sorbitol Agar (CT-SMAC base) 160
BK148 Rappaport-Vassiliadis Soja Broth (RVS) 204
BK149 Modified EC Broth (mEC base) 174
BK150 Modified Tryptone-Soy Broth (mTSB base) 175
BK151 Rose Bengal Chloramphenicol Agar 210
BK152 Violet Red Bile Agar (VRBL) 253
18
Code Page
BK153 Yeast Extract Agar 265
BK154 mFC Agar (Base) 171
BK155 mEndo LES Agar 169
BK156 XLT4 Agar (Base) 263
BK157 Meat-Liver Glucose Agar 164
BK158 Bile Esculin Azide Agar 77
BK161 Plate Count Agar with skimmed milk 200
BM006 Lecithin-Polysorbate-Triton X Broth 149
BM007 Thioglycollate Medium with Resazurin 239
BM008 Tryptone-Salt Broth 242
BM009 Tryptone-Soy Broth 247
BM010 Buffered Peptone Water 95
BM011 Brilliant Green Bile Broth (BGBB) 85
BM012 Rappaport-Vassiliadis Broth 202
BM013 Fraser Broth 128
BM015 Plate Count Agar (PCA) 199
BM016 Half-Fraser Broth 133
BM017 Tryptone-Soy Agar 244
BM018 Baird-Parker Agar with Egg Yolk Tellurite 71
BM019 Oxford Agar 192
BM020 PALCAM Agar 195
BM021 Chloramphenicol Glucose Agar 101
BM022 Oxytetracycline Glucose Agar (OGA base) 194
19
Code Page
BM023 Lecithin-Polysorbate-Triton X Agar 147
BM024 Tergitol 7 Agar (acc. to NF EN ISO) 234
BM025 Tergitol BCIG Agar 236
BM026 Slanetz and Bartley Agar 225
BM029 Baird-Parker RPF Agar (Base) 73
BM030 Tryptone-Soy Broth 247
BM031 Thioglycollate Medium with Resazurin 239
BM033 Plate Count Agar (PCA) 199
BM034 Violet Red Bile Agar (VRBL) 253
BM035 Violet Red Bile Agar (VRBL) 253
BM036 XLT4 Agar 263
BM038 Bacillus cereus Agar (acc. to Mossel) 69
BM047 Eugon Agar 125
BM053 Sabouraud Dextrose Agar 215
BM068 Yeast Extract Agar 265
BR001 Ringer's Solution (1/4 strength) 282
BR002 Coagulase Rabbit Plasma 272
20
Code Page
BR003 Synthetic Sea Salt 288 / 290
BS001 Egg Yolk Tellurite Enrichment 274
BS002 Egg Yolk Enrichment 273
BS003 Oxford Selective Supplement (CCCFA) 278
BS004 PALCAM Selective Supplement (for 500 mL) 280
BS006 D-Cycloserine Selective Supplement 273
BS007 Polymyxin B Selective Supplement 280
BS008 Oxytetracycline Selective Supplement 279
BS009 Gentamicin Selective Supplement 275
BS010 Thayer-Martin Growth Supplement 284
BS017 Karmali Selective Supplement 276
BS021 Chloramphenicol Selective Supplement 271
BS022 CFC Selective Supplement 270
BS023 CIN Selective Supplement 271
BS024 MUG Supplement 277
BS026 TTC Supplement 12.5 mg 285
BS027 TTC Supplement 50 mg 286
BS028 Sulfamethazine Selective Supplement 283
BS030 Half-Fraser Selective Supplement (for 500 mL) 275
BS031 Fraser Selective Supplement 274
BS032 Half-Fraser Selective Supplement (for 2.25 liters) 275
BS033 Novobiocin Selective Supplement 278
BS034 Rabbit Plasma Fibrinogen Supplement (for 100 mL) 281
21
Numerical index
Page
BS037 Cefixime-Tellurite Selective Supplement 270
BS038 Rabbit Plasma Fibrinogen Supplement (for 500 mL) 281
BS039 Tergitol 4 Selective Supplement 284
BS040 Rosolic Acid Selective Supplement 283
BS049 PALCAM Selective Supplement (for 2.5 liters) 280
BT001 Microplate MUG/EC 290
BT002 Microplate MUG/EC 290
BT003 Microplate MUD/SF 288
BT004 Microplate MUD/SF 288
BT005 Baird-Parker RPF agar 73
22
The Company
Presentation of the Solabia Group
Founded in 1972, the Solabia Group is actively involved in the food, pharmaceutical, cosmetic and
biotechnology sectors in which it commercializes protein hydrolysates, molecules and active agents
obtained by chemical or biotechnological extraction.
The Solabia Group earns over 60% of its total sales outside France and devotes over 8% annually of these
sales to Research & Development, a demonstration of its determination to innovation.
The ISO 9002 certification of our production facilities is the guarantee of a complete mastery in the
reliability and reproducibility of our products. Dedicated to the manufacturing of high quality product
for numerous years, the Solabia Group answers in the most efficient way possible to the needs of its
corresponding markets.
Biokar Diagnostics
The Biokar Diagnostics division of the Solabia Group is entirely dedicated to the development, production
and the commercialization of diagnostic reagents for microbiology laboratories including dehydrated
and ready-to-use culture media, supplements and detection kits.
Strengthened by know-how developed over the past 30 years, Biokar Diagnostics serves markets as varied
as industry (agro-food, cosmetics, pharmaceuticals), the environment, (most notably in water analysis),
research, veterinary and human health.
Biokar Diagnostics owes its important strides in France, Europe, North Africa and Latin America to the high
standard of quality of its products, its capacity to innovate and a service that meets even the most
demanding customer.
Recently constructed, the industrial units are automatic and benefit from technically advanced equipment
and materials constantly updated and modernized, thereby reinforcing the vitality and longevity
of Biokar Diagnostics.
24
Biokar Diagnostics
Quality
Quality Assurance
Biokar Diagnostics :
Answers to your
microbiological needs
The certification ISO 9002 for the production of culture media

and associated supplements is only one tangible demonstration
of the objective Biokar Diagnostics has fixed in terms
of the commercialization of consistent and optimal quality products.
A division of the Solabia group, Biokar Diagnostics benefits

from the experience gained as a leading producer of peptones.
The elaboration of raw materials of this type, in the context
of a perfectly controlled process, largely contributes to the finality
of end products with very high quality. This competence, associated
with extensive quality control, imparts upon Biokar Diagnostic
products reliability and security to meet the highest standards
of our customers.
26
Quality Control
DEHYDRATED CULTURE MEDIA
Biokar Diagnostics performs controls targeting the raw materials, in-process intermediate products and the
final end product. All production batches are released for sale only when they meet or exceed pre-defined
acceptation criteria.
1. CHOICE AND CONTROL OF RAW MATERIALS

This is done in order to insure the :
- conformity to established specifications
- adaptation to the specified use
- compatibility of the ingredients between themselves
The raw materials are specially selected and analyzed as a function of their nature.
They are classified in several groups :
1. nitrogen source
2. carbon source
3. minerals
4. selective agents
5. indicators (pH, redox, flurogenes, chromogens)
6. solidification agents
1.1. NITROGEN SOURCES (PEPTONES, EXTRACTS….)
Nitrogen in bacterial growth is needed for the synthesis of amino acids, purines, pyrimidines, carbohydrates
and lipids, and enzyme cofactors, among others. While microorganisms are versatile in their ability to
utilize different nitrogen sources, in culture media nitrogen is provided essentially by peptones and extracts
for bacteriological use.
Peptones are protein hydrolysates prepared by proteolytic digestion of various protein sources, for example
meat, casein, soy flour, malt or other plant sources. By virtue of the biological nature of the hydrolyzed raw
materials, peptones enrich the media not only in nitrogen, but in carbohydrates, vitamins and oligo-
elements that have a direct contribution to the bacterial development.
Beyond the nutritional contribution in culture media, peptones also possess the faculty to influence osmotic
pressure, thereby influencing cell lysis or facilitating freezing or cell dehydration. They can also stimulate
the metabolism of carbon sources and can furnish elements that either favor or inhibit the induction of
metabolites and recombination products.
Peptones are differentiated by origin, by the nature of the raw material and by the process of digestion.
An acid hydrolysis, for example, gives rise to a non-specific, nearly total digestion of the raw material into
the individual amino acids, while an enzymatic hydrolysis cuts peptide bonds and specific sites, giving rise
to peptides of varying sizes. Finally the hydrolysis conditions themselves (pH, temperature, duration), the
neutralization steps, filtration and spray-drying all impact on the determination of the final product.
Given the critical nature of the peptones and extracts in culture media, these raw materials are analyzed
individually at Biokar Diagnostics and at great length, with strict criteria which must be met in terms of
physical, chemical and bacteriological characteristics.
1.1.1. Peptone physical characteristics
This control targets particle size, color, pH, odor, solubility in water and stability after autoclaving. Peptones
must be compatible between themselves and with the other ingredients. Complementary tests are made
concerning limpidity and the stability of the media in general. Peptones destined for broth formulas,
for example, should produce neither turbidity, precipitate nor marked opalescence after autoclaving
and storage.
1.1.2. Peptone chemical characteristics
Chemical characteristics of peptones are determined, such as total nitrogen count, -amino nitrogen
content, phosphates, calcium, chlorides and water. All these criteria can have differing impacts in a culture
media formulation. These analyses can, in certain cases, be completed according to criteria required for the
individual media formula.
27
1.1.3. Peptone bacteriological characteristics
The microbial load of a given peptone is systematically evaluated. The microbial performances are
controlled with test microorganisms that have been specially selected, and various methods are used
(peptone agar test, peptone broth cultures, etc.) to determine satisfactory growth of the test strains. The
strains used in these analyses originate principally from internationally recognized collections.
1.2. CARBON SOURCES (SUGARS)
The majority of microorganisms utilize carbon in its organic form expressly for the biosynthesis of cell
material. There is virtually no naturally occurring organic molecule that can not be used by some
microorganism. Depending on the species, this carbon requirement may be satisfied by simple two-carbon
compounds such as acetate, or by more complex molecules such as cellulose, which contains several
thousand carbon atoms.
Microbiological culture media capitalize on the ability of most bacteria to easily oxidize mono or
polysaccharides such as glucose or other carbohydrates including xylose or galactose. These sugars enable
bacterial growth and development, and are important ingredients in culture media. Tests involving purity
and non-toxicity are performed on every batch that goes into media formulations. Additional controls are
carried out that target color, particle size, water content and the compatibility of the sugars with other
ingredients.
1.3. MINERALS AND SALTS
The mineral requirements of bacteria vary considerably, from essential minerals like calcium or iron, to
trace amounts of zinc, cobalt or manganese. As it relates to micro or trace amounts of minerals, in most
instances these factors are usually normal “contaminants” of distilled water and there is consequently no
need to add them to culture media. Salts often serve to stabilize the osmotic balance in a medium,
although in some cases they can be used as a selective agent.
Specific tests concerning the physical and chemical characteristics of each mineral or salt are performed in
order in insure purity and their compatibility with other ingredients.
1.4. SELECTIVE AGENTS
Culture media are often called upon to selectively grow a certain bacterial strain or family of bacteria and
to inhibit the growth of other microorganisms that may be present in the same sample or inoculum. This
can be accomplished by a combination of nutrients, peptones or minerals that alone favor the growth of
only the species in question, or by this optimization of ingredients plus the addition of selective agents so
that unwanted bacterial growth is controlled without affecting the growth of the target organism.
Common selective agents include bile salts, desoxycholate, antiseptic dyes and antibiotics. Selection of a
particular agent depends on the bacteria that is targeted for inhibition at a concentration that will not be
counter-productive for growth of the bacteria needed. Gram positive bacteria are inhibited by the
judicious use of bile salts and desoxycholate, for example. Gram negative bacteria can be inhibited by the
addition of antibiotics such as cycloserine or polymyxin B. Media selectivity may also be controlled by the
osmotic pressure in the medium itself, for example through a high concentration of salt. Many selective
agents are found in the dehydrated media formulation, while some are heat sensitive (many antibiotics)
and need an extemporaneous addition. The latter selective agents are usually found as freeze-dried
supplements added to the media after sterilization.
1.5. INDICATORS
While selective agents are used with the primary objective of the selective growth of wanted bacteria
against that of unwanted bacteria, the use of indicators, in the form of dyes, pH indicators, reduction-
oxidation reactions (redox), chromogens or flurogens can render the selective media both selective and
differential. On the other hand, a purely general purpose media can be made differential by the addition
of these same indicators which while not giving total identity to the bacteria they affect, can serve as a
presumptive indication of the type of organism growing on a non-selective media.
The most common use of indicators is in the revelation of a change in the pH of the media, resulting from
the acid fermentation of one or several sugars. A number of pH indicators are frequently incorporated into
culture media to demonstrate pH changes during bacterial growth. They typically display somewhat poor
solubility in water so are used at low concentrations. The most common of these indicators are phenol red,
bromthymol blue or bromocresol purple.
28
A new type of indicator has recently been incorporated into culture media in order to specifically identify
certain biochemical characteristics. This indicator, a chromogenic substrate, is hydrolyzed by bacterial
enzymes that cut the chromogen at specific bonds, resulting in a dimerization that produces a visible
precipitate in the center of the target colony. Chromogenic media exploiting these new chromogenic
substrates have been developed that specifically identify, for example, -glucuronidase in Escherichia coli
or phospholipase C in Listeria monocytogenes.
All indicators are rigorously controlled as a function of their objective in a given media formula. They must
not contain any toxic substances that would provoke inhibition in sensitive strains. As with all other raw
materials, indicators must also be compatible with the other ingredients in the formula in order to fully
exploit their stated task.
1.6. SOLIDIFICATION AGENTS
In solid culture media, (plates, slants or deeps), agar is used as the solidification agent due to its unique
properties and neutral qualities that does not inhibit or encourage bacterial growth.
Agar is a phycocolloid extracted primarily for the marine algae Gelidium. It possesses the unique property
(known as “hysteresis”) of having different melting and solidification temperatures, making it an ideal
substrate for bacterial growth. It can be selected for different gel strengths, enabling either a strong
resistance to streaking for isolation, or a weaker resistance, more suitable for pour plate inoculations.
Agars are strictly controlled, as the slightest contaminant can be detrimental to bacterial growth.
They must contain neither inhibiting substances nor metabolically useful products such as peptides,
proteins or fermentable hydrocarbons. Agar is also controlled for absence of microbiological spores,
color (agars must be nearly transparent in order to correctly visualize colony pigmentation and type),
particle size and gel strength. Final controls assure the compatibility of the agar with the other raw
materials in the media formulation.
2. CONTROLS DURING MEDIA MANUFACTURING

2.1. ELABORATION OF THE FORMULA
Once all the pre-selected ingredients are controlled individually, a laboratory scale type-batch is made with
the representative samples of each raw material batch. This type-batch is generally examined in
comparison with a reference batch, in order to determine initial conformity with existing criteria for
quality. The criteria for each reference are unique and corresponds to the properties and specific
applications for each product.
2.2. INDUSTRIAL MANUFACTURING
When the results obtained from the type-batch are recognized as conform, the constituent ingredients of
the media batch can be submitted to industrial manufacturing on a much larger scale. This production
event differs from the type-batch elaboration in terms of volume, and includes mixing and grinding stages
that follow precise, established standard procedures.
3. FINAL CONTROL
Final control for each production batch is performed prior to its release for commercialization and entry
into stock. A sample is kept for reference in a sample collection for each batch for the duration of its
validity in case of need. Final quality control includes some of the following points :
Homogeneity
The resulting dehydrated powder must be fine, fluid and homogeneous.
Color
The powder must fall within defined limits for color or pigmentation, particularly when the formula
contains colorants, dyes or indicators.
pH
The measurement of pH is always taken on the complete media ready to use, after sterilization or heating,
at a standard temperature of 25°C, according to the standard ISO/TS 11133-1 : 2000. The results must be
conform to the specified values for each media formula.
29
Stability
Verification of the stability and the limpidity of liquid (broth) media is systematically performed
after autoclaving and storage at recommended temperatures for several days. No cloudiness, precipitate
or marked opalescence should be observed unless otherwise indicated. The aspect of the solid media
after preparation, sterilization and storage are equally verified.
Gel strength
For solid media, it is necessary to insure that the consistence and firmness of the gel (agar) corresponds to
the most recognized use of the formula by the customer. Not all uses can be anticipated, but if the
application of the agar involves a specific type of inoculation, the gel strength of the solid media must
reflect this constraint.
Additional parameters
For certain applications, additional analyses may be performed beyond that of standard controls. These
may include the maintaining of the molten media at high temperatures for extended periods, solidification
tests under more extreme conditions or the microbial performances of particularly difficult or fastidious
strains, sometimes involving wild-type strains found in the Biokar Diagnostics culture collection.
Microbiological performance
These tests serve to verify that the media are capable of favoring the growth of the target microorganisms
and that the media contains the necessary selective agents to totally or partially inhibit the multiplication
of unwanted or secondary contaminants. Performance tests call on specific analytical methods and
reference strains. The strains themselves are cultivated and maintained following rigorous methods,
following a defined quality procedure. In most cases, the tested media are compared to reference media
that have been recognized internally as presenting a consistent optimal quality. In other cases, the control
methods used are those foreseen in the project European standard ENV ISO/TR 11133-2 for food
microbiology or in the French project standard NF T90-461 for water microbiology.
READY-TO-USE CULTURE MEDIA,

DIAGNOSTIC KITS,
MICROPLATES,
SUPPLEMENTS AND REAGENTS FOR MICROBIOLOGY
Ready-to-use culture media in pre-packaged units or in kit format are manufactured from the dehydrated
media bases, and contain (where appropriate) the appropriate supplements. They are controlled in the
same fashion as their dehydrated counterparts, following similar procedures for microbiological
performance, stability and pH, among others.
Microplates for water analyses are controlled according to specified water control methods outlined in NF
EN ISO 7899-1 (intestinal enterococci) and NF EN ISO 9308-3 (Escherichia coli and coliform bacteria),
annexes E and F.
Supplements in liquid or freeze-dried form, as well as reagents, are controlled according to a test protocol
that directly involves both supplement, the corresponding media and their context for use. The methods
used for determination of supplement and reagent quality are similar to those used for the dehydrated
culture media formulations.
30
Culture Media
Generalities
General recommendations on the use
of culture media
COMPOSITION
The majority of the base constituents, including peptones and agar, are obtained from biological products
which can vary slightly from batch to batch. In order to obtain consistent and reproducible results for
microbiological performance, it is occasionally necessary to qualitatively optimize the ingredients used. For
this reason, the composition indicated for each reference is a type-formula which can be adjusted so that
the pre-defined specifications may be respected.
DEHYDRATED MEDIA
Dehydrated culture media from Biokar Diagnostics are produced in the form of a free-flowing powder. For
each batch, the constituent ingredients are ground and mixed to obtain a product that is fine, fluid and of
consistent color. In this fashion, a tremendous homogeneity can be achieved with a corresponding stability
that lasts for several years. The powders manufactured in this fashion have the advantage of presenting :
- a perfectly homogeneous distribution of their constituent ingredients

- a low residual moisture level
- a very low contamination rate or potential
- the absence of thermoresistant microorganisms
Culture media are packaged in water-tight containers, which is essential to assuring a long shelf life. In its
dehydrated form, the media occupies modest storage space and can be rapidly used when needed.
Occasionally, depending on the formula, it can be observed that some media are slightly clumped but easily
broken apart when manipulated for measurement and use. This phenomenon is normal and dependant on
the relative viscous nature of certain ingredients such as Tween or Tergitol.
Storage Conditions
Dehydrated culture media are, by definition, tremendously hygroscopic (readily taking up and retaining
moisture) and may contain some photosensitive substances. They should be stored in a dry environment,
shielded from excessive light, at a temperature normally comprised between 2 and 30°C. The vials and
plastic bags in the interior of drums should be resealed after each use, so moisture is not re-introduced
during storage. Water absorption generally is observed by the appearance of agglomerates or a
solidification of the normally “liquid” powder. Parallel to this may be the development of bacteria in the
powder itself, which can result in the consummation of nutrient elements, lowering of the pH or a
modification of the media tint, all which contribute to an overall reduction in media quality. Media that
has been subjected to any of these circumstances should be not used and discarded.
Alterations of pH may be observed after long periods of storage. The media should be readjusted to its
target pH by the addition of either a sterile base (NaOH) or acid (HCl). Under ideal storage conditions,
dehydrated culture media maintained in unopened, original containers have a shelf life from 3 to 5 years.
For each media formula, the expiration date is indicated on the package label. This date has been
established as a function of the nature and stability of the constituent ingredients. Once the bottles or
drums have been opened, responsibility for storage and shelf life transfers to the user and no guarantee as
to the shelf life can be made. This will largely depend on the conditions of storage and the quantity of
product remaining in the containers.
Preparation
Preparation steps specific to each media reference must be respected in order to insure that the initial
quality of the dehydrated media remains intact when needed. To obtain optimal quality, the media should
be totally dissolved and be subjected to strict minimum in terms of heating.
Dissolution
Media preparation should be performed with either freshly distilled or completely demineralized
water that is low in bacterial contamination and where inhibiting substances are totally absent.
32
Recipients used for media preparation should be cleaned and rinsed with distilled water to remove any
residues of detergents or disinfectants that could be detrimental to bacterial cultures. Media should be
prepared in the following manner :
- Weigh out the appropriate quantity of media taking care to avoid inhalation of the powder, particularly
with media containing irritating substances. Media manipulation can take place under a laminar flow
hood for added security.
- Progressively add the volume of water required for reconstitution. If a rapid dissolution of the powder is
desired, heating the water to approximately 50°C will speed up the process.
- Mix slowly and consistently in order to achieve complete dissolution of all the ingredients and to disperse
the agar homogeneously.
- Bring media containing agar, cystine or gelatin to a boil (without overheating) before dispensing in tubes
or vials. A complete dissolution of the agar is obtained when the viscous solution no longer contains any
particles of agar that attach to the walls of the preparation recipient. In the case of media that contain
natural precipitates, mix the resulting solution thoroughly before repartition. For liquid media (broths),
limpid solutions are obtained by a thorough mixing but without the need to heat the product before
sterilization.
pH adjustment
Biokar Diagnostics media are pre-adjusted to the pH of normal use of the product. Only pH readings of
complete media (with supplements), prior to inoculation and measured after autoclaving and cooling to
25°C are taken into consideration, conform to the standard ISO/TS 11133-1 : 2000. pH values depend on the
temperature of the media at the time of the analysis, as well as the heat treatment applied during the
course of the reconstitution and autoclaving steps.
Sterilization
Not all media are autoclaved. These media are identified in their corresponding catalogue sheets. For those
media that require autoclave sterilization, duration and temperature should be strictly applied. The
recommended values represent those intended for smaller containers (i.e. 500 to 1000 mL). For volumes
other than those represented, adjustments will be needed for the increase in interior temperature and the
subsequent cooling period. These characteristics depend on several factors, including the initial
temperature of the solution after repartition, the volume of the recipients used, the type of autoclave and
its volume coefficient. In order to sterilize large volumes, it is necessary to increase sterilization times,
without exceeding the recommended temperature for the media in question. At the end of the heat
treatment, a rapid cooling is required so that an excessive heating of critical components does not occur
which could be prejudicial to the nutritive or selective qualities of the media.
Repartition in Petri dishes

Agar media should be poured in Petri dishes at temperatures between 44 and 47°C in order to reduce
condensation in the covers. Plates should be poured so that at least a 2 mm thickness is obtained. Let the
plates cool and solidify by placing the plates with their covers on a flat, cool surface. The plates should be
dried, inverted and resting on their covers, in a 25-50°C incubator or sterile laminar flow hood, until the
evaporation of the fine droplets found on the surface of the media (usually around 30-60 minutes).
Preparation of freeze-dried supplements

Freeze-dried supplements should be reconstituted by scrupulously respecting the instructions provided in
the catalogue sheets for each product. Those supplements that contain toxic or irritating agents should be
handled with care, avoiding dissemination or spillage. It is recommended to consult the Material Safety
Data Sheets available for each product. Once the supplement reconstituted, the storage conditions must be
respected.
33
READY-TO-USE-MEDIA
Melting of solid media vials
It is recommended to melt media containing agar (tubes or vials) by placing them in a boiling water bath or
similar heating device. Media should be heated for the minimum amount of time necessary to achieve a total
liquefaction, so that the heating process itself does not affect the intrinsic qualities of the media. Avoid
excessive heating and remove the containers as soon as the agar has remelted. Cool and maintain in a molten
state in a heated water bath regulated at 44 to 47°C. The time necessary to reach this recommended
temperature range depends on the type of media, the volume and quantity of vials in the water bath. Media
remelted and cooled in this fashion should be used as quickly as possible, if possible within 4 hours. Never
remelt media having already been subjected to an initial liquefaction.
Supplement addition
In the case of solid media, liquid or freeze-dried supplements (once reconstituted) are to be added to the
melted media that has been maintained at 44-47°C. It is important to bring the reconstituted supplements
to room temperature before adding to the molten media, so as to avoid heat shock that could impact on
the supplement efficiency or quality. In addition, supplements held at low temperatures then added to
molten media can result in the formation of transparent plaques in the solution. These insoluble
agglomerations prevent a homogeneous mixture of the final product, and must be discarded. Mix rapidly
and progressively the reconstituted supplements into the molten media before repartition into plates or
other containers.
Storage
The stability and validity of prepared media, supplemented or not, is limited over time. Conservation times
range from a few days to several months under ideal conditions for each reference. Each catalogue entry
contains benchmark values, given on an informational basis only, for culture media prepared in the
laboratory from the dehydrated base. These recommendations can not be guaranteed, as they are
dependant on the preparation steps inherent to each laboratory.
Once poured into plates, the media should be cooled on a flat, cool surface until solidification. If possible,
they should be used immediately after drying. If the plates are to be used at a later date, they can
nevertheless be stored shielded from light, in a cold room or refrigerator at 2-8°C, and in airtight plastic
bags that have been carefully sealed. Solid media should never be stored at temperatures lower than 0°C,
as the crystallizations that occur would destroy the gel structure. Prepoured plates that are stored too long
or under unfavorable conditions often suffer from marked desiccation. The loss of moisture favors the
formation of precipitates, crystallizations or changes in the composition that render the plates unusable. As
it concerns commercially available prepoured plates, the instructions governing their use and storage
conditions, found either in the catalogue entries or on the product labels must be followed to insure
quality results. They should be stored under the conditions specific to each reference immediately after
reception.
PRODUCTS PRESENTING DANGER OR RISK

The vast majority of the products commercialized by Biokar Diagnostics present no particular risk other
than that linked to the use of finely ground powder. There exists, however, certain specific products which
contain toxic or irritating substances including sodium azide, sodium biselenite or cycloheximide. These
products are clearly indicated and should be handled with care. To avoid the inhalation of the powder,
which can lead to respiratory irritations, it is recommended to wear the appropriate respiratory mask. In all
cases, it is absolutely essential to familiarize oneself with the measures described in each Material Safety
Data Sheet, available for each product.
34
DECONTAMINATION AND DESTRUCTION OF MEDIA AFTER USE
Once the culture media have completed their objective, there is a high probability that they may contain a
large number of potentially pathogenic microorganisms. Consequently, the elimination of contaminated
media must be done by rigorous and reliable means. In general, it is necessary to neutralize the media by
means of an appropriate heat treatment before re-using glassware or prior to the disposal of the plastic
waste used for microbial growth. Effective destruction of microbial cultures in Petri dishes, tubes or vials
can be achieved by autoclaving a minimum of one hour at 121°C. For waste elimination, consult the
appropriate legislative texts in vigor in each country.
Chemical destruction is active against vegetative forms of bacteria but should not be used against bacterial
spores or spore-forming bacteria, as the resistance to these decontamination agents might not allow the
required level of sanitation. For more information, it is recommended to consult the Material Safety Data
Sheets in addition to the standard NF EN 12740 which applies to the manipulation, inactivation and control
of microbial wastes in the context of environmental protection.
35
Table of the main unfavorable conditions
which may be encountered in the preparation
and storage of culture media
Dehydrated medium solidified Relative humidity too high

in clumps or in a solid block
Recipient left open too long
Recipient not tightly closed after use
Shelf life exceeded (expiration)
pH incorrect Recipient stored in unfavorable conditions
Shelf life exceeded
Weighing error
Water for reconstitution poorly tested
Glassware (flasks or tubes) poorly washed or rinsed
Medium overheated or remelted several times
Autoclave incorrectly adjusted
Turbidity Shelf life exceeded
Opalescence Weighing error
Precipitation Glassware (flasks or tubes) poorly washed or rinsed
Water improperly deionized
Wrong pH
Incomplete homogenization
Overheating when preparing or autoclaving

Excessive evaporation of water during
preparation or storage
Color changes Dehydrated medium deteriorated
Shelf life exceeded
Weighing error
Wrong pH
Overheating when preparing or autoclaving
Excessive evaporation of water during preparation

or storage
36
Abnormal gel strength Dehydrated medium deteriorated
Shelf life exceeded
Weighing error
Wrong pH
Medium overheated or remelted several times (especially in the

case of acid media)
Prepared medium is contaminated Dehydrated medium deteriorated
Shelf life exceeded
Sterilization fault
Recontamination of tubes or flasks after autoclaving
Petri dishes contaminated
Atypical cultures Dehydrated medium deteriorated
Shelf life exceeded

Glassware poorly rinsed, may contain toxic residues
(detergents, antiseptics, other inhibitors)
Water for reconstitution improperly deionized
Wrong pH
Quantities of additives incorrect
Medium overheated or remelted several times
Pouring temperature too high
Medium incubated with an insufficient quantity of sample
Petri dishes insufficiently dried
Poor incubation conditions
37
38
Product line
By methodology
Suspensions, Dilutions, Resuscitation
Buffered Peptone Broth with sodium chloride pH 7.0 BK128
Buffered Peptone Water BK018 / BK131 / BM010
Lactose Broth BK082
Ringer's Solution (1/4 strength) BR001
Tryptone-Salt Broth BK014 / BM008
Antibiotic susceptibility testing
Mueller Hinton Agar BK048
Mueller Hinton Broth BK108
Wilkins-Chalgren Agar BK101
Wilkins-Chalgren Broth BK102
Sterility testing
Eugon Broth BK068
Lecithin-Polysorbate-Triton X Agar BK138 / BM023 / BM044 / BM045 / BM046
Lecithin-Polysorbate-Triton X Broth BK137 / BM006 / BM042 / BM043
Sabouraud Dextrose Broth BK026
Schaedler Broth BK064
Thioglycollate Medium with Resazurin BK017 / BM007 / BM031
Tryptone-Soy Agar BK047 / BM017 / BM049 / BM050
Tryptone-Soy Broth BK046 / BM009 / BM030
Blood culture
Brain-Heart Agar BK029
Brucella Agar BK077
Columbia Agar BK019
Columbia CNA Agar BK075
GC Agar BK051
Schaedler Agar BK063
Tryptone-Soy Agar (Blood Agar) BK028
Aerobic microorganisms
Brain-Heart Broth BK015
40
Count-Tact Agar BK130
Eugon Agar BK096 / BM047
Eugon Broth BK068
Lecithin-Polysorbate-Triton X Agar BK138 / BM023 / BM044 / BM045 / BM046
Lecithin-Polysorbate-Triton X Broth BK137 / BM006 / BM042 / BM043
Nutrient Agar BK021
Nutrient Broth BK003
Plate Count Agar (PCA) BK144 / BM015 / BM033
Plate Count Agar with skimmed milk BK161
Sugar-Free Agar (SFA) BK126
Tryptone-Soy Agar BK047 / BM017 / BM049 / BM050
WL Differential Agar BK066
Yeast Extract Agar BK153 / BM067 / BM068
Anaerobic microorganisms
Cooked Meat Medium BK076
Meat-Liver Glucose - 0.6% Agar BK024
Meat-Yeast Glucose - 0.6% Agar BK052
Reinforced Clostridial Agar BK090
Reinforced Clostridial Medium BK094
Schaedler Agar BK063
Schaedler Broth BK064
Wilkins-Chalgren Agar BK101
Wilkins-Chalgren Broth BK102
WL Differential Agar BK066
Bacillus
Bacillus cereus Agar (acc. to Mossel) BK116 / BM038
Dextrose Tryptone Agar BK042
Brucella
Brucella Agar BK077
Brucella Broth BK122
41
Candida
BiGGY Agar BK074
Campylobacter
Brucella Agar BK077
Brucella Broth BK122
Columbia Agar BK019
Karmali Agar BK117
Clostridia
Bryant and Burkey Broth with Resazurin (modified) BK141
Lactose Sulfite Broth (LS) BK140
Meat-Liver Glucose - 0.6% Agar BK024
Meat-Liver Glucose Agar BK092 / BK157
Meat-Yeast Glucose - 0.6% Agar BK052
Meat-Yeast Agar BK006
Reinforced Clostridial Agar BK090
Reinforced Clostridial Medium BK094
TSC Agar BK031
TSN Agar BK001
Coliforms, Heat-tolerant coliforms, Escherichia coli
Brilliant Green Bile Broth (BGBB) BK002 / BM011
Bromcresol Purple Lactose Agar BK023
Bromcresol Purple Lactose Broth BK119
CLED Agar BK020
Desoxycholate Lactose Agar BK065
Desoxycholate (0.1%) Lactose Agar BK062
Drigalski Lactose Agar BK036
EE Broth (acc.to Mossel) BK127
EMB Agar (Levine) BK056
Endo Agar BK057
Kligler Iron Agar BK034
Lactose Broth BK082
42
Laurylsulfate-Tryptose Broth BK010
MacConkey Agar BK050
MacConkey Broth Purple BK107
mEndo LES Agar BK155
mFC Agar BK154
Microplate MUG/EC BT001 / BT002
Minerals Glutamate Medium BK005
Peptone Water BK084
Rose-Gal BCIG Agar BK142
Schubert Broth (modified) BK120
TBX Agar BK146
Tergitol 7 Agar BK038
Tergitol 7 Agar (acc. to NF EN ISO) BK123 / BM024
Tergitol BCIG Agar BK139 / BM025
TSI Agar BK059
Violet Red Bile Agar (VRBL) BK152 / BM034 / BM035
Violet Red Bile Glucose Agar (VRBG) BK011
Escherichia coli O157 : H7
MacConkey Sorbitol Agar (CT-SMAC) BK147
Modified EC Broth (mEC) BK149
Modified Tryptone-Soy Broth (mTSB) BK150
Fecal Streptococci (Enterococci)
Azide Dextrose Broth BK125
Azide Dextrose Broth (acc. to Rothe) BK060
Bile Esculin Azide Agar BK158
Ethyl Violet Azide Broth BK061
Slanetz and Bartley Agar BK037 / BK129 / BM026
Microplate MUD/SF BT003 / BT004
Haemophilus
GC Agar BK051
43
Hemolytic Streptococci
Columbia Agar BK019
Columbia CNA Agar BK075
Todd Hewitt Broth BK100
Lactic Streptococci
Elliker Broth BK054
M17 Agar BK088
M17 Broth BK012
Lactobacilli
Eugon Agar BK096 / BM047
MRS Agar BK089
MRS Broth BK070
Rogosa Agar BK033
Leuconostoc
MSE Agar BK087
Listeria
Fraser Broth BK115 / BK133 / BM013
Half-Fraser Broth BK133 / BM016
Listeria Enrichment Broth (LEB acc. to FDA) BK112
Listeria Enrichment Broth (LEB acc. to IDF) BK124
Listeria Enrichment Broth (UVM I and UVM II modified) BK113 / BK114
Oxford Agar BK110 / BM019
PALCAM Agar BK145 / BM020
Neisseria
GC Agar BK051
Mueller Hinton Broth BK108
44
Pseudomonas
Cetrimide Agar BK049
CFC Agar BK118
Salmonella and Shigella
Bismuth Sulfite Agar BK004
Brilliant Green Agar (Edel and Kampelmacher) BK091
Brilliant Green Agar (Kristensen) BK071
Bromcresol Purple Lactose Agar BK023
Buffered Peptone Water BK018 / BK131 / BM010
DCLS Agar BK085
Desoxycholate Citrate Agar (DCA) BK044
Drigalski Lactose Agar BK036
EMB Agar (Levine) BK056
Endo Agar BK057
Hektoen Enteric Agar BK067
Kligler Iron Agar BK034
Lactose Broth BK082
MacConkey Agar BK050
MSRV Medium BK134
Müller-Kauffmann Broth BK135
Nutrient Broth BK003
Önöz Agar BK032
Rappaport-Vassiliadis Soja Broth BK148
SS Agar BK022
Selenite Broth (Leifson) BK079
Selenite-Cystine Broth BK009
Tetrathionate Broth BK083
TSI Agar BK059
XLD Agar BK058
XLT4 Agar BK156 / BM036
45
Staphylococci
Baird-Parker Agar with Egg Yolk Tellurite BK055 / BM018
Baird-Parker RPF Agar BK055 / BM029 / BT005
Brain-Heart Broth BK015
Buttiaux-Brogniart Hypersalted Broth (double strength) BK081
Giolitti and Cantoni Broth BK080
Mannitol Salt Agar BK030
Vibrio
TCBS Agar BK040
Yeasts and Molds
BiGGY Agar BK074
Chloramphenicol Glucose Agar BK007 / BM021
Malt Agar BK045
Orange Serum Agar BK103
Oxytetracycline Glucose Agar (OGA) BK053 / BM022
Potato Dextrose Agar BK095
Rose Bengal Chloramphenicol Agar BK151
Sabouraud Chloramphenicol Agar BK027
Sabouraud Dextrose Agar BK025 / BM052 / BM053
Sabouraud Dextrose Broth BK026
Wort Agar BK013
Yersinia
Yersinia Agar (CIN) BK039
46
By testing context
EXAMINATION OF FOOD PRODUCTS
Examination of beverages
Target Culture media Use
Total count Plate Count Agar (PCA) Enumeration
Spoilage microorganisms Orange Serum Agar Isolation, culture and enumeration of acid-tolerant
microorganisms in fruit juices
WL Differential Agar Isolation, culture, enumeration of microorganisms

in beer
Anaerobic microorganisms Reinforced Clostridial Agar Enumeration
Reinforced Clostridial Medium Enumeration
Bacillus Dextrose Tryptone Agar Enumeration of spore forming aerobies

lowering or not pH level
Coliforms Laurylsulftate-Tryptose Agar Selective enumeration
Violet Red Bile Agar (VRBL) Selective enumeration
Total enterobacteriaceae Violet Red Bile Glucose Agar (VRBG) Selective enumeration
Lactobacilli MRS Agar Selective isolation
MRS Broth Culture and enumeration
Rogosa Agar Selective isolation
Yeasts and molds Chloramphenicol Glucose Agar Enumeration
Orange Serum Agar Enumeration
Oxytetracycline Glucose Agar (OGA) Enumeration
Rose Bengal Chloramphenicol Agar Enumeration
Wort Agar Non selective enumeration
48
Examination of milk and dairy products
Suspensions, Dilutions Ringer's Solution (1/4 strength) Diluent
Tryptone-Salt Broth Diluent
Buffered Peptone Water Diluent
Total aerobic mesophilic count Plate Count Agar (PCA) Enumeration

Total aerobic psychrophilic count
Plate Count Agar with Skimmed Milk Enumeration
Spoilage flora Sugar-Free Agar Enumeration
Clostridium perfringens TSC Agar Enumeration
TSN Agar Enumeration
Thioglycollate Medium with Resazurin Culture
Lactose-Sulfite Broth (LS) Confirmation
Clostridium tyrobutyricum Bryant and Burkey Broth with Enumeration by the most probable number method
Resazurin (modified)
Coliforms Brilliant Green Bile Broth (BGBB) Selective enumeration
Laurylsulfate-Tryptose Broth Selective enumeration
MacConkey Broth Purple Presumptive test
Desoxycholate Lactose Agar Selective enumeration
Desoxycholate (0.1%) Lactose Agar Selective enumeration
MacConkey Agar Selective enumeration
Rose-Gal BCIG Agar Selective enumeration
Total enterobacteriaceae EE Broth (acc. to Mossel) Selective enrichment
Violet Red Bile Glucose Agar (VRBG) Selective isolation
Enterococci Bile Esculin Azide Agar Partially selective isolation
KF Streptococcus Agar Selective isolation
Escherichia coli Brilliant Green Bile Broth (BGBB) Mackenzie test
Peptone Water Mackenzie test
Rose-Gal BCIG Agar Selective isolation
TBX Agar Selective isolation
Tergitol BCIG Agar Selective isolation
Violet Red Bile Agar (VRBL) Selective isolation, fluorogenic procedure

+ MUG Supplement
49
Examination of milk and dairy products
Lactococci Elliker Broth Enumeration
M17 Broth Culture
M17 Agar Enumeration
Leuconostoc MSE Agar Selective enumeration
Listeria monocytogenes Listeria Enrichment Broth (LEB acc. to FDA) Enrichment
Listeria Enrichment Broth (LEB acc. to IDF) Enrichment
Listeria Enrichment Broth (UVM I modified) Primary enrichment
Listeria Enrichment Broth (UVM II modified) Secondary enrichment
Half-Fraser Broth Primary enrichment
Fraser Broth Secondary enrichment
Oxford Agar Selective isolation
PALCAM Agar Selective isolation
Salmonella Buffered Peptone Water Preenrichment
Rappaport-Vassiliadis Broth Selective enrichment
Rappaport-Vassiliadis Soja Broth (RVS) Selective enrichment
Selenite-Cystine Broth Selective enrichment
Bismuth Sulfite Agar Selective isolation
Brilliant Green Agar Selective isolation

(Edel and Kampelmacher)
DCLS Agar Selective isolation
Hektoen Enteric Agar Selective isolation
MSRV Medium Selective isolation
XLD Agar Selective isolation
XLT4 Agar Selective isolation
Kligler Iron Agar Differentiation
Triple Sugar Iron Agar Differentiation
Staphylococci Buttiaux-Brogniart Hypersalted Broth Selective enrichment
Giolitti and Cantoni Broth Selective enrichment
Baird-Parker Agar with Egg Yolk Tellurite Selective isolation
Baird-Parker RPF Agar Selective isolation
Malt Agar Enumeration
Potato Glucose Agar Enumeration
50
Examination of egg products
Campylobacter Karmali Agar Selective isolation
Laurylsulfate-Tryptose Agar Selective enumeration
Total enterobacteriaceae EE Broth (acc. to Mossel) Selective enrichment
Yeast and molds Chloramphenicol Glucose Agar Enumeration
Malt Agar Enumeration
Potato Dextrose Agar Enumeration

Staphylococci Baird-Parker Agar with Egg Yolk Tellurite Selective isolation
51
Examination of meat, meat based products and prepared dishes
Suspensions, Dilutions Tryptone-Salt Broth Diluent
Total count Plate Count Agar (PCA) Enumeration
Sulfite-reducing anaerobic Meat-Liver Glucose Agar Enumeration

microorganisms
Laurylsulfate-Tryptose Agar Selective enumeration
Clostridium perfringens TSC Agar Selective enumeration
TSN Agar Selective enumeration
Enterobacteriaceae EE Broth (acc. to Mossel) Selective enrichment
Escherichia coli Bromcresol Purple Lactose Agar Selective isolation
EMB Agar (Levine) Selective isolation
TBX Agar Selective enumeration
Tergitol BCIG Agar Selective enumeration
Escherichia coli O157 : H7 Modified EC Broth (mEC) Selective enrichment
Modified Tryptone Soy Broth (mTSB) Selective enrichment
MacConkey Sorbitol Agar (CT-SMAC) Selective isolation
Lactobacillus MRS Broth Culture and enumeration
MRS Agar Selective isolation
52
Examination of meat, meat based products and prepared dishes
Listeria monocytogenes Listeria Enrichment Broth (UVM I modified) Primary enrichment
Listeria Enrichment Broth ( UVM II modified) Secondary enrichment
Half-Fraser Broth Primary enrichment
Fraser Broth Secondary enrichment
Pseudomonas CFC Agar Selective isolation
Önöz Agar Selective isolation, differentiation of Proteus
Staphylococci Baird-Parker Agar with Egg Yolk Tellurite Selective isolation
Baird-Parker RPF Agar Selective isolation
Potato Dextrose Agar Enumeration
53
Examination of seafood
Suspensions, Dilutions Tryptone-Salt Broth Diluent
Coliforms Brilliant Green Bile Broth (BGGB) Selective enumeration
Laurylsulfate-Tryptose Broth Selective enumeration
Clostridium perfringens TSC Agar Enumeration
TSN Agar Enumeration
KF Steptococcus Agar Selective isolation
Lactobacillus MRS Broth Culture and enumeration
MRS Agar Selective isolation
Pseudomonas CFC Agar Selective enumeration
Salmonella and Shigella Buffered Peptone Water Preenrichment

MSRV Agar Selective isolation
Salmonella-Shigella Agar Selective isolation
Vibrio TCBS Agar Selective isolation
54
EXAMINATION OF DRINKING, SWIMMING, INDUSTRIAL AND WASTE WATER
Stock solution Ringer's Solution (1/4 strength) Diluent
Synthetic Sea Salt Diluent
Total aerobic mesophilic count, Yeast Extract Agar Enumeration

Total aerobic psychrophilic count
Sulfite-reducing anaerobic Meat-Liver Glucose Agar Enumeration

microorganisms
Coliforms Lactose Broth Presumptive test
Laurylsulfate-Tryptose Broth Presumptive test
mENDO LES Agar Presence / Absence test, enumeration
mFC Agar Presence / Absence test, enumeration
Tergitol 7 Agar (acc. to EN NF ISO) Presence / Absence test, enumeration
Brilliant Green Bile Broth (BGBB) Confirmation
Schubert Broth (modified) Confirmation
Escherichia coli Laurylsulfate-Tryptose Broth + MUG Fluorogenic detection
Bromcresol Purple Lactose Agar Isolation
EMB Agar (Levine) Isolation
Kligler Iron Agar Identification
Triple Sugar Iron Agar Identification
Microplate MUG/EC Fluorogenic enumeration
Enterococci Azide Dextrose Broth Presumptive test
Ethyl Violet Azide Broth Confirmation
Slanetz and Bartley Agar Selective enumeration
Microplate MUD/SF Fluorogenic enumeration
55
EXAMINATION OF DRINKING, SWIMMING, INDUSTRIAL AND WASTE WATER
Salmonella and Shigella Buffered Peptone Water Preenrichment
Selenite Broth (Leifson) Selective enrichment

Staphylococcus Mannitol Salt Agar Partially selective isolation
Sulfite-reducing clostridia TSC Agar Isolation and enumeration
Vibrio TCBS Agar Selective isolation
Yersinia enterocolitica Yersinia Agar Selective isolation
56
EXAMINATION OF PHARMACEUTICAL PRODUCTS , COSMETICS
AND THEIR RAW MATERIALS
Stock suspension Buffered Peptone Broth with Sodium Diluent

Chloride pH 7.0
Spoilage flora Tryptone-Soy Broth Sterility testing
Eugon Broth Sterility testing
Thioglycollate Medium with Resazurin Sterility testing - Culture of aerobic and anaerobic
microorganisms
Lecithin-Polysorbate-Triton X Broth Sterility testing - Microbiological count

of microorganisms in products with preservatives
Schaedler Broth Sterility testing - Culture of anaerobic

microorganisms
Sabouraud Dextrose Broth Sterility testing - Culture of yeasts and molds
Tryptone-Soy Agar Enumeration of spoilage microorganisms
Eugon Agar Enumeration of spoilage microorganisms
Lecithin-Polysorbate-Triton X Agar Enumeration of microbial contamination

of products with preservatives
Sabouraud Dextrose Agar Non selective enumeration of yeasts and molds
Count-Tact Agar Detection of the residual contamination

of equipment and surfaces after cleaning
and disinfection
Enterobacteriaceae Lactose Broth Resuscitation
EE Broth (acc. to Mossel) Selective enrichment
Violet Red Bile Glucose Agar (VRBG) Selective isolation, enumeration
Escherichia coli Lactose Broth Non selective enrichment
MacConkey Broth Purple Presumptive test
MacConkey Agar Selective isolation
EMB Agar (Levine) Partially selective isolation
Peptone Water Confirmation
57
EXAMINATION OF PHARMACEUTICAL PRODUCTS , COSMETICS
AND THEIR RAW MATERIALS
Pseudomonas aeruginosa Tryptone-Soy Agar Non selective enrichment
Cetrimide Agar Isolation
Salmonella Lactose Broth Non selective enrichment
Tetrathionate Broth Selective enrichment
Brilliant Green Agar (Kristensen) Selective isolation
Desoxycholate Citrate Agar (DCA) Selective isolation
Staphylococci Tryptone-Soy Agar Non selective enrichment
Baird-Parker Agar with Egg Yolk Tellurite Selective isolation, enumeration
Mannitol Salt Agar Isolation, enumeration
Yeasts and molds Chloramphenicol Glucose Agar Selective isolation
Sabouraud Dextrose Agar Non selective isolation, enumeration
Potato Dextrose Agar Non selective isolation, culture
58
EXAMINATION OF MEDICAL SAMPLES
Throat, nose, pharynx, expectorations

General use Tryptone-Soy Agar + sheep blood Culture and non selective isolation
Anaerobic microorganisms Schaedler Agar Isolation, culture
-hemolytic streptococci Columbia CNA Agar + sheep blood Selective isolation, hemolysis identification
Streptococcus pneumoniae
Haemophilus influenzae GC Agar Isolation

+ Thayer-Martin Growth Supplement
Lactose-positive enterobacteria Bromcresol Purple Lactose Agar Non selective isolation

(Klebsiella pneumoniae)
MacConkey Agar Selective isolation, differentiation
Neisseria GC Agar Selective isolation

Pseudomonas aeruginosa Cetrimide Agar Selective isolation
Staphylococcus aureus Mannitol Salt Agar Selective isolation
Yeasts BiGGY Agar Selective isolation
Sabouraud Chloramphenicol Agar Selective isolation
Sabouraud Chloramphenicol Agar Isolation and identification of Candida

+ Gentamicin (Candida albicans)
Eye (conjunctivitis)
Anaerobic bacteria Cooked Meat Medium Non selective enrichment
Schaedler Agar Isolation, culture
Wilkins-Chalgren Agar Isolation, culture

Molds Sabouraud Dextrose Agar Isolation (partially selective), hyphae formation
Neisseria GC Agar Selective isolation

Pseudomonas Cetrimide Agar Selective isolation of Pseudomonas aeruginosa
Staphylococcus Mannitol salt Agar Selective isolation of Staphylococcus aureus
Streptococcus Columbia CNA Agar + sheep blood Selective isolation, hemolysis identification
Yeasts (Candida) BiGGY Agar Isolation and identification of Candida

(Candida albicans)

+ Gentamicin
59
Ear
Enterobacteriaceae Bromcresol Purple Lactose Agar Non selective isolation
EMB Agar (Levine) Partially selective isolation, differentiation
XLD Agar Partially selective isolation (Salmonella, Shigella)

Staphylococci Mannitol Salt Agar Partially selective isolation of Staphylococcus aureus
Streptococci Columbia CNA Agar + sheep blood Selective isolation, hemolysis identification
Urine
CLED Agar Enumeration, isolation and differentiation
Drigalski Lactose Agar Selective isolation
MacConkey Agar Selective isolation
Escherichia coli Bromcresol Purple Lactose Agar Fluorogenic procedure

+ MUG Supplement
Staphylococci Mannitol Salt Agar Partially selective isolation of Staphylococcus aureus
Urogenital tract
-hemolytic streptococci Columbia CNA Agar + sheep blood Selective isolation, hemolysis identification
Candida BiGGY Agar Isolation and identification of Candida

(Candida albicans)

+ Gentamicin
Enterococci Bile Esculin Azide Agar Selective isolation

Neisseria gonorrhoeae GC agar Selective isolation

Staphylococcus aureus Mannitol Salt Agar Partially selective isolation
60
Feces
Candida BiGGY Agar Isolation and identification of Candida

(Candida albicans)

+ Gentamicin
Lactose-positive Enterobacteriaceae Bromcresol Purple Lactose Agar Non selective isolation
Bromcresol Purple Lactose Agar Fluorescent procedure for the detection

+ MUG Supplement of Escherichia coli
Desoxycholate Lactose Agar Selective isolation, differentiation
Drigalski Lactose Agar Selective isolation
EMB Agar (Levine) Isolation (partially selective), differentiation
Lactose-negative Enterobacteriaceae Rappaport-Vassiliadis Broth Selective enrichment of Salmonella other

(Salmonella, Shigella) than Salmonella Typhi
Selenite Broth (Leifson) Selective enrichment
Bismuth Sulfite Agar Selective isolation (Salmonella Typhi)
Brilliant Green Agar (Kristensen) Selective isolation of Salmonella other

than Salmonella Typhi
Bromcresol Purple Lactose Agar Non selective isolation
DCLS Agar Selective isolation, differentiation
Desoxycholate Citrate Agar (DCA) Selective isolation
Drigalski Lactose Agar Selective isolation, differentiation
EMB Agar Partially selective isolation, differentiation
Hektoen Enteric Agar Selective isolation, differentiation,

culture of Shigella
Önöz Agar Selective isolation, differentiation of Proteus
Salmonella-Shigella Agar Selective isolation
Listeria monocytogenes Enrichment Broths for Listeria Selective enrichment
Staphylococcus aureus Mannitol Salt Agar Partially selective isolation
Yersinia enterocolitica Yersinia Agar Selective isolation
61
Pus, abscesses, wounds
General use Tryptone-Soy Agar Culture and non selective isolation
Anaerobic bacteria Cooked Meat Medium Non selective enrichment, cullture
Schaedler Agar Isolation, culture
Wilkins-Chalgren Agar Isolation, culture
EMB Agar (Levine) Partially selective isolation, differentiation
XLD Agar Partially selective isolation
Staphylococci Mannitol salt Agar Selective isolation
62
Product review
63
General indications
● The autonomy indicated in the "Product Profile" boxes is based on the following calculations :
15 mL per Petri dish, 10 mL for tubes and 225 mL for vials (unless otherwise specified).
● The information contained on the product labels or on the product instruction sheets take precedence
over the information or formulas described in this catalogue.
● The information and specifications contained in the following pages have been established at the time
of printing of this catalogue. They are susceptible to change at any time, without warning.
● The indications relative to the strains used in Quality Control can be modified as a function of an
improvement in available performance tests and the evolution of appropriate standards.
● The following table lists the equivalent terms between the designations of certain culture media
components according the standard ISO/TS 11133-1 (2000) and those found in the individual
product sheets :
Designation according to ISO/TS 11133-1 Product sheet designations
Enzymatic digest of casein Tryptone

Enzymatic digest of soybean meal Papaic Digest of Soybean Meal
Enzymatic digest of animal tissues Peptic Digest of Meat
Pancreatic Digest of Meat
Meat-Liver Peptone
Enzymatic digest of heart Pancreatic Digest of Heart
Enzymatic digest of gelatin Pancreatic Digest of Gelatin
Enzymatic digest of animal and plant tissues Polypeptone
64
Culture media
65
Azide Dextrose Broth
Intended Use Product Profile

Target : Intestinal enterococci.
Azide Dextrose Broth is used as a presumptive test for the detection
and enumeration of fecal streptococci in drinking water, in Reconstitution : 34.7 g / L.
swimming water, in surface water, as well as in other water samples, Autonomy (500 g) : 1440 tubes.
by the most probable number method. This procedure involves two
pH (25°C) : 7.2 ± 0.2.
steps :
- presumptive test in Azide Dextrose Broth, Supplement required : None.
- confirmation test in Ethyl Violet Azide Broth. Sterilization : 121°C / 15 minutes.
History
Preparation
Sodium azide glucose broth is inspired by the formula of Rothe.
It was recommended by Malmann and Seligman for the Single strength medium
enumeration of fecal streptococci in water, waste water and foods. - Dissolve 34.7 g of dehydrated medium
Malmann, Botwright and Churchill showed the bacteriostatic action (BK125) in 1 liter of distilled or deionized
of sodium azide on Gram-negative flora, thereby favoring the water.
growth of streptococci (Gram-positive). - Stir slowly until complete dissolution.
- Dispense in 16 x 160 mm tubes at 10 mL
Principles per tube.
- Sterilize in an autoclave at 121°C for
- The nutritive capacity of Azide Dextrose Broth is due to the 15 minutes.
presence of Tryptone and meat extract, as well as glucose.
- Sodium chloride maintains the osmotic balance. Double strength medium
- Sodium azide inhibits the growth of Gram-negative bacteria by its - Dissolve 69.4 g of dehydrated medium
bacteriostatic action and favors that of enterococci. (BK125) in 1 liter of distilled or deionized
- After observing cultures in tubes (presumptive test), it is necessary water.
to carry out a confirmation in Ethyl Violet Azide Broth. The latter - Stir slowly until complete dissolution.
method combines the effect of sodium azide and that of a dye, - Dispense in 20 x 200 mm tubes at 10 mL
ethyl violet. per tube.
Typical Composition 15 minutes.
(can be adjusted to obtain optimal performance)
Instructions for Use
For 1 liter of medium :
- Tryptone 15.0 g Single strength medium
- Meat extract 4.5 g - Inoculate tubes with 1 mL of inoculum and
- Glucose 7.5 g its serial tenfold dilutions.
- Sodium chloride 7.5 g
- Sodium azide 0.2 g Double strength medium
- Inoculate tubes with 10 mL of inoculum.
pH of the ready-to-use medium at 25°C : 7.2 ± 0.2.
Incubation
- Incubate all the tubes at 37°C for 24 and
Quality Control 48 hours.
- Dehydrated medium : cream-white powder, free-flowing and Results

homogeneous.
- Prepared medium : amber solution, limpid. Positive tubes are cloudy, resulting from
- Typical culture response after 24 hours of incubation at 37°C, bacterial growth.
followed by subculture in Ethyl Violet Azide Broth : Positive tubes must be subjected to the
confirmation test in Ethyl Violet Azide
Microorganisms Growth Broth (BK061).
Enterococcus faecalis ATCC ®
29212 good
Enterococcus faecalis ATCC 33186 good
Escherichia coli ATCC 25922 inhibited
Staphylococcus aureus ATCC 25923 inhibited
66
Storage / Shelf Life
Dehydrated medium : 2-30°C.

- The expiration date is indicated on the label.
Prepared medium (benchmark value) :
- Media in tubes : 6 months at 2-8°C.
Packaging
Dehydrated medium :
- 500 g bottle : BK125HA
Bibliography
(1) Rothe. Illinois State Health Department. (3) NF T 90-411 : October 1989. Testing water. Detection and
(2) Mallmann, W.L., and Seligmann, E.B. 1950. A comparative enumeration of group D Streptococci. General method by
study of media for the detection of streptococci in water and culture in liquid media (MPN).
sewage. Am. J. Pub. Health, 40 : 286.
Azide Dextrose Broth (acc. to Rothe)

Target : Enterococci.
Azide Dextrose Broth is used for the presumptive test of detection
and enumeration of enterococci in drinking water, frozen foods Reconstitution : 35.6 g / L.
and other food products by the most probable number method. Autonomy (500 g) : 1405 tubes.
The tests involves two steps :
pH (25°C) : 6.8 ± 0.2.
- presumptive test in Azide Dextrose Broth,
- confirmation test in Ethyl Violet Azide Broth. Supplement required : None.
Sterilization : 121°C / 15 minutes.
History
Preparation
This sodium azide glucose broth is prepared according to the
formula of Rothe. It was recommended by Malmann and Seligman Single strength medium
for the enumeration of fecal streptococci in waste water and foods. - Dissolve 35.6 g of dehydrated medium
Malmann, Botwright and Churchill showed the bacteriostatic action (BK060) in 1 liter of distilled or deionized
of sodium azide on Gram-negative flora, thereby favoring the water.
growth of enterococci. - Stir slowly until complete dissolution.
- Dispense in tubes at 10 mL per
Principles 16 x 160 mm tube.
- The high nutritive capacity of Azide Dextrose Broth is due to the 15 minutes.
presence of a high concentration of polypeptone and glucose.
- Sodium chloride maintains the osmotic equilibrium. Double strength medium
- Potassium phosphates buffer the medium. - Dissolve 71.2 g of dehydrated medium
- Sodium azide inhibits the growth of Gram-negative bacteria by its (BK060) in 1 liter of distilled or deionized
bacteriostatic action and favors that of fecal streptococci. water.
- After observing cultures in tubes (presumptive test), it is necessary - Stir slowly until complete dissolution.
to carry out a confirmation in Ethyl Violet Azide Broth. The latter - Dispense in tubes at 10 mL per
method combines the effect of sodium azide and that of a dye, 20 x 200 mm tube.
ethyl violet. - Sterilize in an autoclave at 121°C for
15 minutes.
67
Typical Composition Instructions for Use
Single strength medium
For 1 liter of medium : - Inoculate tubes with 1 mL of inoculum and
- Polypeptone 20.0 g its serial tenfold dilutions.
- Glucose 5.0 g
- Sodium chloride 5.0 g Double strength medium
- Potassium dihydrogen phosphate 2.7 g - Inoculate tubes with 10 mL of inoculum.
- Dipotassium phosphate 2.7 g
- Sodium azide 0.2 g Incubation
- Incubate all the tubes at 37°C for 24 and
pH of the ready-to-use medium at 25°C : 6.8 ± 0.2. 48 hours.
Quality Control Results
- Dehydrated medium : cream-white powder, free-flowing and Positive tubes are cloudy and must be
homogeneous. subjected to the confirmation test in
- Prepared medium : amber solution, limpid. Ethyl Violet Azide Broth (BK061).
- Typical culture response after 24 hours of incubation at 37°C,
followed by subculture on Ethyl Violet Azide Broth :
Microorganisms Growth
Enterococcus faecalis ATCC® 33186 good
Enterococcus faecalis ATCC 29212 good

Packaging
Dehydrated medium :
Bibliography
(1) Rothe. Illinois State Health Department. (3) Rodier, J. 1984. L’analyse de l’eau. Dénombrement des
(2) Mallmann, W.L., and Seligmann, E.B. 1950. A comparative streptocoques fécaux présumés (Méthode par ensemencement
study of media for the detection of streptococci in water and en milieux liquides). Dunod 7ème Ed., 825-828.
sewage. Ann. J. Pub. Health, 40 : 286.
68
Bacillus cereus Agar (acc. to Mossel)
Intended Use
Bacillus cereus Agar is used for the detection and enumeration of

spores and vegetative cells of Bacillus cereus in food products.
History
In 1967, Mossel et al. recommended the use of a mannitol-phenol

red-egg yolk medium, whose principles were based on two factors :
- The lack of mannitol fermentation by Bacillus cereus.
- The presence of a lecithinase in the majority of tested strains.
The authors showed that satisfactory selectivity was obtained with
polymyxin B at 10 mg / liter.
Principles Bacillus cereus
- Tryptone and meat extract favor the growth of Bacillus cereus.

- Sterile egg yolk emulsion is used to detect the presence of Product Profile
lecithinase present in most Bacillus cereus strains. Insoluble Target : Bacillus cereus.
breakdown products of egg yolk lecithin accumulate around the Reconstitution : 44.5 g / 900 mL.
colonies, forming a whitish precipitate.
- Mannitol is used to differentiate contaminating microorganisms Autonomy (500 g) : 674 plates.
which ferment it, causing phenol red to turn yellow. pH (25°C) : 7.2 ± 0.2.
- Polymyxin B is used to inhibit accompanying microflora when the Supplement required : Egg Yolk Enrichment.
tested sample is heavily contaminated. Polymyxin B Selective Supplement.
Typical Composition
Preparation
For 0.9 liters of base medium :
- Tryptone 10.0 g - Suspend 44.5 g of dehydrated medium
- Meat extract 1.0 g (BK116) in 0.9 L of distilled or deionized
- D-mannitol 10.0 g water.
- Sodium chloride 10.0 g - Slowly bring to boiling, stirring until
- Phenol red 25 mg complete dissolution.
- Sterile egg yolk emulsion 100 mL - Dispense 90 mL in flasks.
- Polymyxin B 1 x 105 IU - Sterilize in an autoclave at 121°C for
- Bacteriological agar 13.5 g 15 minutes.
pH of the ready-to-use medium at 25°C : 7.2 ± 0.2. Instructions for Use
Quality Control - Melt the medium (if it was prepared in

advance).
- Dehydrated medium : pinkish powder, free-flowing and - Cool and maintain at 47°C.
homogeneous. - Per 90 mL of base, aseptically add 10 mL
- Prepared medium (complete) : pinkish to orange opaque agar. of Egg Yolk Enrichment (BS002) and 1 mL
- Typical culture response after 24 hours of incubation at 30°C : of reconstituted Polymyxin B Selective
Supplement (BS007).
Characteristics - Mix rapidly and thoroughly.
Microorganisms Growth Color of Lecithinase - Pour into sterile Petri dishes.
colonies - Let solidify on a cold surface.
Bacillus cereus ATCC® 11778 good pinkish positive - Dry in an incubator with the covers
Bacillus cereus ATCC 14579 good pinkish positive partially removed.
Pseudomonas aeruginosa ATCC 9027 inhibited
69
Storage / Shelf Life - Transfer 0.1 mL of the sample to analyze
and its serial tenfold dilutions to the plates
Dehydrated medium (without Egg Yolk nor Polymyxin B) : 2-30°C. prepared as above or onto complete
- The expiration date is indicated on the label. ready-to-use plates (BM038).
Prepared medium from dehydrated base (benchmark value) : - Spread the inoculum on the surface of the
- Base media in flasks : 6 months at 2-8°C. agar with a sterile triangle.
- Complete media in plates : 5 days at 2-8°C. - Incubate at 30°C for 24 and 48 hours.
Ready-to-use media in Petri plates,
Sterile Egg Yolk Emulsion, Results
Polymyxin B Selective Supplement :
- Store between 2-8°C, shielded from light. Presumed colonies of Bacillus cereus are
- The expiration dates are indicated on the labels. pink (mannitol-negative) and almost always
surrounded by a halo of precipitate,
Packaging indicating the production of lecithinase.
Proceed with the selection and
Complete ready-to-use media : identification of doubtful colonies for
- 20 plates : BM03808 confirmation by the appropriate
Dehydrated base medium biochemical tests.
(without Egg Yolk Emulsion nor Polymyxin B) :
Egg Yolk Emulsion :
- 50 mL vial : BS00208
Polymyxin B Selective Supplement :
- 10 vial pack : BS00708
Bibliography
(1) Mossel, D.A.A., Koopman, M.J., and Jongerius, E. 1967. (3) NF EN ISO 7932 : March 1998. Microbiology. General guidance for
Enumeration of Bacillus cereus in Foods. App. Microb., 15, (3) : enumeration of Bacillus cereus. Colony-count technique at 30°C.
650-653. (4) IDF provisional 181: 1998. Dried milk products. Enumeration of
(2) XP V08-058. Nov. 1995. Food and animal feeding stuffs. Bacillus cereus. Most probable technique.
Microbiology. Enumeration of Bacillus cereus by colony-count
technique at 30°C. Routine method.
70
Baird-Parker Agar with Egg Yolk Tellurite
Intended Use
Baird-Parker Agar with Egg Yolk Tellurite is a selective medium for

the detection and enumeration of Staphylococcus aureus in
biological samples, pharmaceutical products, cosmetics and foods.
History
The formula, developed by Baird-Parker in 1962, was found to be

particularly appropriate for the enumeration of coagulase positive
staphylococci. In 1964, Smith and Baird-Parker showed that adding
sulfamethazine to the medium inhibited the growth of Proteus and
in 1971, Tardio and Baer observed, that among 18 selective isolation
media tested, the Baird-Parker formulation was less inhibitory than
Vogel-Johnson medium, used previously with some frequency.
Principles Staphylococcus aureus
- The growth of staphylococci is favored by sodium pyruvate and

glycine.
- Accompanying microflora are inhibited by lithium chloride and Product Profile
potassium tellurite (added extemporaneously), as well as a high
Target : Coagulase positive staphylococci.
concentration of glycine.
- The addition of sulfamethazine after autoclaving inhibits most Reconstitution : 58.0 g / 950 mL .
Proteus and thus limits the invasion of the medium by this species. Autonomy (500 g) : 575 plates.
- Enrichment with egg yolk aids in identification by demostrating
pH (25°C) : 7.2 ± 0.2.
lecithinase activity.
- The characterization of Staphylococcus aureus, black colonies due Supplement required : Egg Yolk Tellurite
to the reduction of tellurite to telluride and surrounded by clear Enrichment.
halos, may be complemented by the coagulase test and optionally Sulfamethazine Selective
by the desoxyribonuclease and phosphatase tests. Supplement (optional).
Typical Composition of the complete medium
(can be adjusted to obtained optimal performance)
Preparation
- Tryptone 10 g - Suspend 58.0 g of dehydrated medium
- Meat extract 5g (BK055) in 950 mL of distilled or deionized
- Yeast extract 1g water.
- Sodium pyruvate 10 g - Slowly bring to boiling, stirring until
- Glycine 12 g complete dissolution.
- Lithium chloride 5g - Dispense in flasks by adding 95 mL per
- Bacteriological agar 15 g flask.
- Egg Yolk Emulsion 47 mL - Sterilize in an autoclave at 121°C for
- Potassium tellurite (3.5%) 3 mL 15 minutes.
Quality Control - Melt the medium (if prepared in advance).
- Cool and maintain at 47°C.
- Dehydrated (base) medium : cream-white powder, free-flowing - Aseptically add 5 mL of Egg Yolk Tellurite
and homogeneous. Enrichment (BS001) to 95 mL of base and,
- Prepared medium (complete) : yellowish opaque agar. if necessary, 1 mL of reconstituted
- Typical culture response after 48 hours of incubation at 37°C : Sulfamethazine Selective Supplement
Characteristics (BS028) when Proteus is suspected.
Microorganisms Growth Colony Clear - Mix rapidly and thoroughly.
aspect zones - Pour into sterile Petri dishes.
Staphylococcus aureus ATCC® 6538 good to excellent black present - Let solidify on a cool surface.
Staphylococcus aureus ATCC 25923 good to excellent black present - Dry the plates in an incubator with the
covers partially removed.
Staphylococcus epidermidis ATCC 12228 slow black absent
71
Storage / Shelf Life Results
Dehydrated base medium (without Egg Yolk Tellurite) : 2-30°C. Staphylococcus aureus is characterized by
- The expiration date is indicated on the label. the formation of black colonies (reduction
Prepared medium from dehydrated base (benchmark value) : of tellurite to telluride), which are shiny,
- Base in flasks : 6 months at 2-8°C. convex and surrounded by clear zones
- Complete media in plates : 5 days at 2-8°C. of 2 to 5 mm in diameter, resulting from
Ready-to-use media in Petri plates, proteolysis (egg yolk reaction). An opaque
Sterile Egg Yolk Tellurite, zone due to the action of lecithinase may
Sulfamethazine Selective Supplement, appear inside the halo. Other bacteria are,
Coagulase Rabbit Plasma : in principle, inhibited. Nevertheless, it is
- Store between 2-8°C, shielded from light. possible to observe brown colonies of
- The expiration dates are indicated on the labels. micrococci, white colonies of yeast and
matte brown colonies of Bacillus or Proteus.
Packaging Both characteristic and/or non-characteristic
colonies must be verified with the
Ready-to-use media : coagulase tube test (refer to the
- 20 plates : BM01808 monograph on the Coagulase Rabbit
Dehydrated base medium (without Egg Yolk Tellurite) : Plasma, BR002) in order to confirm their
- 500 g bottle : BK055HA pathogenicity. Coagulase negative
- 5 kg drum : BK055GC staphylococci are usually inhibited. If a
Egg Yolk Tellurite enrichment : culture appears, however, typical clear
- 50 mL vial : BS00108 zones are absent.
Sulfamethazine Selective Supplement :
Coagulase Rabbit Plasma :
- 10 vial pack (20 tests per vial) : BR00208
Bibliography
(1) Duthie, E.S. 1954. Evidence of two forms of staphylococcal (8) NF V 04-415. Février 1984. Laits de conserve. Microbiologie.
coagulase. J. Gen. Microbiol., 10: 427-436. (9) NF V04-502. Dec. 1992. Viandes et produits à base de viande.
(2) Klempereur, R., and Haughton, G. 1957. A medium for the Examen microbiologique. Partie 2 : adaptation des directives
rapid recognition of penicillin-resistant coagulase-positive générales.
staphylococci. Jour. of Appl. Bact., 10: 96-99. (10) V08-057-1. Nov. 1994. Food microbiology. Routine method for
(3) Baird-Parker, A.C. 1962. An improved diagnostic and selective enumeration of coagulase positive Staphylococci by colony-
medium for isolating coagulase positive staphylococci. Jour. of count technique at 37°C. Part 1: Technique with confirmation
Appl. Bact., 25: 12-19. of the colonies.
(4) Smith, B.A., and Baird-Parker, A.C. 1964. The use of (11) FIL-IDF provisional 60C: 1997. Dried milk products.
sulphamezathine for inhibiting Proteus spp. on Baird-Parker’s Enumeration of Staphylococcus aureus. MPN procedure.
isolation medium for Staphylococcus aureus. J. of Appl. Bact., (12) FIL-IDF provisional 145A: 1997. Milk and milk-based
27: 78. products. Enumeration of Staphylococcus aureus. Colony count
(5) Thieulin, G., Basille, D., Pantaléon, J., Rosset, R., Gandon, Y., et technique.
Petit A. 1966. Recherche des staphylocoques pathogènes dans le (13) NF EN ISO 6888-1: Oct. 1999. Microbiology of food and
lait et les produits laitiers. Le lait, 46: 131. animal feeding stuffs - Horizontal method for the
(6) Holbrook, R., Anderson, J.M., and Baird-Parker, A.C. 1969. The enumeration of coagulase-positive staphylococci
performance of a stable version of Baird-Parker’s medium for (Staphylococcus aureus and other species) - Part 1: Technique
isolating Staphylococcus aureus. J. App. Bact., 32, (2): 187. using Baird-Parker medium.
(7) Sperber, W.H., and Tatini, S.R. 1975. Interpretation of the tube (14) Pharmacopée européenne. Addendum 2000. Contrôle
coagulase test for the identification of Staphylococcus aureus. microbiologique des produits non stériles (recherche de
App. microb., 29: 502-505. microorganismes spécifiés). Solution et milieux de culture
recommandés, p. 56-60.
(15) United States Pharmacopoeia 24. 2000. Microbial Limit Tests,
1814-1823.
72
Baird-Parker RPF Agar
Intended Use
Baird Parker RPF (RPF = Rabbit Plasma Fibrinogen) Agar is used for
the direct detection and enumeration of coagulase positive
staphylococci. The medium has the advantage of considerably
reducing the number of confirmation tests for the presence of
coagulase positive staphylococci particularly when atypical colonies
are observed on other selective media. The medium allows the
simultaneous enumeration and confirmation to be performed
in a single operation.
History
The production of free coagulase, considered to be the principle

characteristic for recognizing the pathogenicity of staphylococci, in
particular Staphylococcus aureus, was at the origin of the
development of Baird Parker with Rabbit Plasma Fibrinogen. Initial Staphylococcus aureus
formulations of the incorporation of plasma in solid culture media
revealed inconsistencies between the results obtained by the
coagulase tube test and the formation of a characteristic halo of Product Profile
fibrin around colonies. The differences arose from the fact that Target : Coagulase positive staphylococci.
certain strains possessed a coagulase that also activated the
Reconstitution : 54.9 g / 900 mL.
plasminogen-plasmin system as well, resulting in fibrinolysis and the
disappearance of the fibrin halo. In order to overcome this Autonomy (500 g) : 607 plates.
difficulty, it was recommended to add soybean trypsin inhibitor. pH (25°C) : 7.2 ± 0.2.
Supplement required : Rabbit Plasma
In order to favor the detection of coagulase produced by
Fibrinogen Supplement.
Staphylococcus aureus, Devoyod et al. studied in 1976, the
incorporation of pork plasma into Baird-Parker medium. Hauschild Sterilization : 121°C / 15 minutes.
subsequently improved the performance of Devoyod’s medium
through the inclusion of bovine fibrinogen and a trypsin inhibitor, Preparation
with a correlative decrease in the amount of plasma. In 1983,
Beckers et al. modified Hauschild's medium, replacing the pork - Suspend 54.9 g of dehydrated base
plasma with rabbit plasma, classically used in the coagulase tube medium (BK055) in 900 mL of distilled or
test. These authors also inoculated the medium in depth, rather than deionized water.
the previously used double layer technique of Devoyod. Finally, the - Slowly bring to boiling, stirring until
formulation was improved in 1986 by Sawhney, after completing complete dissolution.
studies relative to the toxicity of potassium tellurite towards - Dispense in flasks by adding 90 mL per
Staphylococcus aureus in a rabbit plasma fibrinogen medium. flask, if to be used with Rabbit Plasma
Fibrinogen Supplement (BS034 or BS038).
Principles - For large volume preparation, it is also
possible to dispense into 1000 mL capacity
- The growth of staphylococci is favored by sodium pyruvate flasks by adding 450 mL of medium per
and glycine. flask, if the RPF Supplement for 500 mL is
- Accompanying microflora are inhibited by lithium chloride and chosen (BS038).
potassium tellurite (added extemporaneously), as well as a high - Sterilize in an autoclave at 121°C for
15 minutes.
concentration of glycine.
- Rabbit plasma was chosen for its excellent specificity towards
staphylococcal coagulase and by its aptitude to rapidly produce
clot formation by forming staphylothrombin from prothrombin. - Melt the medium for the minimum
The rabbit plasma is reinforced with bovine fibrinogen. amount of time necessary in order to
Staphylothrombin acts by cutting the A and B fibrinopeptides of achieve total liquefaction (if prepared in
fibrinogen, thereby initiating the polymerization process that advance or if vials of ready-to-use base
results in the appearance of fibrin halos surrounding the colonies. media are used, i.e. BM029 or RPF kit
- Soybean trypsin inhibitor prevents fibrinolysis. BT005, reagent R1).
- The black coloration of staphylococcal colonies is due to the - Cool and maintain at 47°C.
reduction of potassium tellurite to telluride. In addition, the - For 90 mL of base media, add 10 mL of
presence of tellurite favors the inhibition of contaminating Gram- reconstituted Rabbit Plasma Fibrinogen
positive microflora. Supplement (BS034 or reagent R2 from
RPF kit BT005).
73
Typical Compositions - For 450 mL of base media, add 50 mL of
(can be adjusted to obtained optimal performance) the reconstituted Rabbit Plasma
Fibrinogen Supplement BS038.
For 950 mL of base medium - Mix well to insure a proper and complete
homogenization. Use immediately.
- Tryptone 10 g - Transfer 1 mL of the sample to analyze
- Meat extract 5 g and its tenfold dilution series into sterile
- Yeast extract 1 g Petri dishes.
- Sodium pyruvate 10 g - Pour 10 to 15 mL of complete medium.
- Glycine 12 g - Homogenize by swirling.
- Lithium chloride 5 g - Let solidify or a cool surface.
- Bacteriological agar 15 g - Incubate at 37°C for 24 and 48 hours.
For a supplement vial to be added to 90 mL of base media Results
- Rabbit plasma, EDTA 2.5 mL Coagulase positive staphylococci are

- Bovine fibrinogen 0.5 g characterized by the formation of gray or
- Trypsin inhibitor 2.5 mg black colonies surrounded by an opaque
- Potassium tellurite 2.5 mg halo of fibrin that is clearcut, stable and
well visible. Count only those plates
pH of the complete medium at 25°C : 7.2 ± 0.2. containing less than 100 characteristic
colonies. Use of the RPF technique
Quality Control eliminates the need for confirming the
results by the coagulase tube test.
- Dehydrated base medium : cream-white powder, free-flowing
and homogeneous.
- Complete prepared medium : amber agar.
- Typical culture response after 48 hours of incubation at 37°C :
Characteristics
Microorganisms Growth Colony Fibrin
aspect halo
Staphylococcus aureus ATCC® 6538 good to excellent black present
Staphylococcus aureus ATCC 25923 good to excellent black present
Staphylococcus epidermidis ATCC 12228 partially inhibited black absent
Dehydrated base medium : 2-30°C.

Prepared medium from dehydrated base (benchmark value) :
- Base in flasks : 6 months at 2-8°C.
- Complete media in plates : use immediately after preparation.
Ready-to-use base media in vials,
Rabbit Plasma Fibrinogen Supplement,
RPF kit :
- Store between 2-8°C, shielded from light.
- The expiration dates are indicated on the labels.
Packaging
RPF kit :
- Composed of 6 x 90 mL vials of base media (R1) and
6 freeze-dried RPF supplement vials (R2) : BT00508
Ready-to-use base media :
- 10 x 90 mL vials : BM02908
Dehydrated base medium :
- 5 kg drum : BK055GC
RPF Supplement :
- 8 vial pack for 100 mL complete media / vial : BS03408
- 1 vial pack for 500 mL complete media / vial : BS03808
74
Bibliography
(1) Loeb, L. 1903. The influence of certain bacteria on the (9) V08-057-2. Nov. 1994. Food microbiology. Routine method for
coagulation of blood. Journ. Med. Res., 10: 407. enumeration of coagulase positive Staphylococci by colony-
(2) Duthie, E.S. 1954. Evidence of two forms of staphylococcal count technique at 37°C. Part 2: Technique without colonies
coagulase. J. gen. Microbiol., 10: 427-436. confirmation.
(3) Baird-Parker, A.C. 1962. An improved diagnostic and selective (10) FIL-IDF provisional 145A: 1997. Milk and milk-based
medium for isolating coagulase positive staphylococci. Jour. of products. Enumeration of Staphylococcus aureus. Colony count
Appl. Bact., 25: 12-19. technique.
(4) Devoyod, J.J., Millet, L., et Mocquot G. 1976. Un milieu gélosé (11) De Buyser, M.L., Audinet, N., Delbart, M. O., Maire, M., and
pour le dénombrement direct de Staphylococcus aureus: milieu Françoise, F. 1998. Comparison of selective culture media to
au plasma de porc pour Staphylococcus aureus (PPSA). Can. enumerate coagulase-positive staphylococci in cheeses made
Jour. of Microb., 22 (11): 1603-1611. from raw milk. Food microbiol. 15: 339-346.
(5) Stadhouders, J., Hassing, F., and van Aalst-van Maren, N.O. (12) prEN ISO/CD 6888-3. September 1999. Microbiology of food
1976. A pour-plate method for the detection and enumeration and animal feeding stuffs - Horizontal method for the
of coagulase-positive Staphylococcus aureus in the Baird-Parker enumeration of coagulase-positive staphylococci
medium without egg yolk. Neth. Milk Dairy J., 30: 222-229. (Staphylococcus aureus and other species) - Part 3 : MPN
(6) Hauschild, A.H.W., Park, C.E., and Hilsheimer, R. 1979. A Technique for low numbers.
modified pork plasma agar for the enumeration of (13) NF EN ISO 6888-1: Oct. 1999. Microbiology of food and
Staphylococcus aureus in foods. Can. J. of Microbiol., 25: animal feeding stuffs - Horizontal method for the
1052-1057. enumeration of coagulase-positive staphylococci
(7) Beckers, H.J., van Leusden, F.M., Bindschedler, O., and Guerraz, (Staphylococcus aureus and other species) - Part 1: Technique
D. 1984. Evaluation of a pour-plate system with a rabbit plasma- using Baird-Parker agar medium.
bovine fibrinogen agar for the enumeration of Staphylococcus (14) NF EN ISO 6888-2: Oct. 1999. Microbiology of food and
aureus in food. Can. Jour. of Microb., 30: 470-474. animal feeding stuffs - Horizontal method for the
(8) Sawhney, D. 1986. The toxicity of potassium tellurite to enumeration of coagulase-positive staphylococci
Staphylococcus aureus in rabbit plasma fibrinogen agar. Journ. (Staphylococcus aureus and other species) - Part 2: Technique
of Appl. Bact. 61: 149-155. using rabbit plasma fibrinogen agar.
BiGGY Agar (Nickerson)

Intended Use
Product Profile
BiGGY (Bismuth Sulfite Glucose Glycine Yeast) Agar, based on the Target : Candida.
formula of Nickerson, is a differentiation medium recommended for Reconstitution : 45.0 g / L.
the detection, isolation and presumptive identification of Candida. Autonomy (500 g) : 740 plates.
History pH (25°C) : 6.8 ± 0.2.

Supplement required : None.
Nickerson developed the use of this medium in 1953, following a Sterilization : Heat to boiling.
study on the reduction of sulfites by various strains of Candida. Do not autoclave.
Principles Preparation
- Yeast extract, glycine and glucose are nutrient substrates required - Suspend 45.0 g of dehydrated medium
for the growth of Candida. (BK074) in 1 liter of distilled or deionized
- Bismuth sulfite, which inhibits bacterial growth, is reduced by water.
Candida to bismuth sulfide which colors the colonies brown and - Slowly bring to boiling, stirring the
sometimes also the surrounding medium in the case of several precipitate that can be formed.
strains. - Continue boiling for a maximum of
- Barr and Collins recommended adding neomycin sulfate at 2 minutes.
2 mg / liter to better inhibit accompanying bacterial flora. - Do not autoclave.
75
- Cool and maintain the medium at 47°C.
For 1 liter of medium : - Disperse the precipitate.
- Yeast extract 1 g - Pour into sterile Petri dishes.
- Glycine 10 g - Let solidify on a cold surface.
- Glucose 10 g - Dry in an incubator with the covers
- Bismuth ammonium citrate 5 g partially removed.
- Sodium sulfite 3 g - Inoculate by streaking with a platinum
- Bacteriological agar 16 g loop or spread with a sampling swab.
- Incubate at 22°C or, if necessary, at 37°C
pH of the ready-to-use medium at 25°C : 6.8 ± 0.2. for 3 to 5 days.
- Examine the plates daily.
Quality Control
Results
- Dehydrated medium : cream-white powder, free-flowing and
The colonies have the following
homogeneous.
appearance :
- Prepared medium : amber agar, with a slight flocculent
precipitate.
- Dark brown to black colonies without a
metallic reflection or diffusion of pigments :
Candida albicans
Microorganisms Growth Characteristics
- Dark brown colonies with a black center,
Candida albicans ATCC 10231
®
good to excellent brown colonies with metallic reflection and black pigment
Candida tropicalis ATCC 750 good to excellent brownish colonies which diffuses around the colonies within
Escherichia coli ATCC 25922 inhibited 72 hours of incubation :
Candida tropicalis
- Dark red-brown colonies, shiny, with no
pigment diffusion :
Storage / Shelf Life Candida kefyr
- Rough silvery black-brown colonies with a
Dehydrated medium : 2-30°C. yellow diffusion halo in the medium :
- The expiration date is indicated on the label. Candida krusei
Prepared medium (benchmark value) : - Rough colonies, dark red-brown with
- Media in plates : use on the day of preparation. yellow mycelial fringes :
Candida parakrusei
Packaging - Dark brown colonies with slight
mycelial fringe :
Dehydrated medium Candida stellatoidea
Bibliography
(1) Nickerson, W.J. 1947. Biology of pathogenic fungi. Waltham, (3) Barr, F.S., and Collins, G.F. 1966. A rapid method for the
Mass. The Chronica Botanica. isolation and identification of Candida. South. Med. J.,
(2) Nickerson, W.J. 1953. Reduction of inorganic substances by 59: 694.
yeasts. Extracellular reduction of sulfite by species of Candida.
J. Infect. Dis., 93: 43.
76
Bile Esculin Azide (BEA) Agar
Intended Use
Bile Esculin Azide Agar is a selective medium used to isolate and

enumerate fecal streptococci (Lancefield group D) in food and
pharmaceutical products. It is also used for the enumeration of
intestinal enterococci in water.
History
Rochaix first showed the interest of esculin hydrolysis for the

identification of enterococci. Meyer and Schoefeld then showed that
this hydrolysis in medium containing bile was an excellent test for
the detection of group D streptococci. The first formulations
described by Swan were subsequently modified by Isenberg, who
obtained a medium which was both more selective and at the same
time favored the rapid growth and good recovery of the bacteria Enterococcus faecalis
sought. The current formula is described in the project standard
NF EN 7899-2 as confirmation media for intestinal enterococci in
water, when the enumeration is carried out by membrane filtration.
Product Profile
Principles Target : Intestinal enterococci.
Reconstitution : 54.6 g / L.
- Sodium azide inhibits contaminating Gram-negative bacteria.
- Bacteriological bile inhibits the growth of Gram-positive Autonomy (500 g) : 610 plates.
microorganisms. pH (25°C) : 7.1 ± 0.2.
- Fecal streptococci hydrolyze esculin to glucose and esculetin. The Supplement required : None.
esculetin produced forms a black complex in the presence of ferric
ions arising from ferric citrate in the medium. Sterilization : 121°C / 15 minutes.
Typical Composition
Preparation
- Suspend 54.6 g of dehydrated medium
(BK158) in 1 liter of distilled or deionized
- Tryptone 17.00 g
water.
- Peptic digest of meat 3.00 g
- Slowly bring to boiling, stirring until
- Yeast extract 5.00 g
complete dissolution.
- Bacteriological ox bile 10.00 g
- Dispense in tubes or flasks.
- Esculin 1.00 g
15 minutes.
- Ferric ammonium citrate 0.50 g
- Sodium azide 0.15 g
- Bacteriological agar 13.00 g
- Pour into sterile Petri dishes.
- Let solidify on a cold surface.
Quality Control
- Dry in an incubator with the covers
partially removed.
- Dehydrated medium : cream powder, free-flowing and
- Inoculate on the surface of the medium.
homogeneous.
- Incubate at 37°C for 24 to 72 hours.
- Prepared medium : amber agar with a bluish glint.
NOTE
For the confirmation of enterococci in water, and
Microorganisms Growth Esculin hydrolysis
if there are typical colonies on the enumeration
Enterococcus faecalis ATCC® 29212 good positive media (Slanetz & Bartley), transfer the membrane
Streptococcus pyogenes ATCC 19615 poor to inhibited negative and the colonies onto an Petri dish of Bile Esculin
Escherichia coli ATCC 25922 inhibited Azide Agar preheated to 44°C. Incubate at 44°C
for 2 hours. Consider as positives all colonies that
give rise to a brown to black coloration in the
media, recording these as intestinal enterococci.
77
Dehydrated medium : 2-30°C. Group D streptococci appear as small
- The expiration date is indicated on the label. translucent colonies surrounded by
Prepared medium (benchmark value) : a black halo.
- Media in vials : 6 months at 2-8°C. Staphylococci and yeasts may yield opaque
- Media in plates : 5 days at 2-8°C. colonies without a black halo. It is
indispensable to identify suspected bacteria,
Packaging especially to eliminate confusion with
Listeria which may give rise to colonies
Dehydrated medium
similar to those of group D streptococci.
Bibliography
(1) Isenberg, H.D., Goldberg, D., and Sampson, J. 1970. (3) NF T 90 – 411. Octobre 1989. Essais des eaux. Recherche et
Laboratory studies with a selective enterococcus medium. dénombrement des streptocoques du groupe D. Méthode
Appl. Microbiol., 20 (3): 433-436. générale par ensemencement en milieu liquide (MPN).
(2) Rodier, J. 1984. L’analyse de l’eau. Dénombrement des (4) NF EN ISO 7899-2. Aug. 2000. Water Quality. Detection and
streptocoques fécaux présumés. Dunod 7ème Ed: 827. enumeration of intestinal enterococci - Part 2: Membrane
filtration method.
Bismuth Sulfite Agar

Intended Use
Bismuth Sulfite Agar is a selective medium used to isolate

Salmonella Typhi and other salmonellaes in pathological products,
water, dairy and other food products.
History
In 1926, Wilson and Blair combined bismuth and sodium sulfite in a

medium destined to isolate Salmonella of the typhi and paratyphi
groups. In 1956, Hajna and Damon described a modified formula
which was recommended by the United States Pharmacopoeia.
Principles
- The concentrations of brilliant green and bismuth sulfite inhibit

accompanying Gram-positive flora and most enterobacteria, except Salmonella Typhi
for Salmonella and several Shigella.
- Using the sulfur compounds in the medium, Salmonella releases
hydrogen sulfide which produces a metallic precipitate in the Product Profile
presence of ferrous sulfate, giving the colonies a black or Target : Salmonella, Salmonella Typhi.
sometimes green color. Reconstitution : 47.0 g / L.
- It is particularly recommended to first enrich using Tetrathionate,
Selenite or Rappaport-Vassiliadis broths and to simultaneously Autonomy (500 g) : 532 plates.
(20 mL per plate).
inoculate onto other less selective media : MacConkey, XLD or
Hektoen Enteric agars, for example. pH (25°C) : 7.6 ± 0.2.
- Because of its elevated inhibitory power, this medium enables a Supplement required : None.
highly contaminated inoculum to be used.
Sterilization : Heat to boiling.
Do not autoclave.
78
Typical Composition Preparation
For 1 liter of medium : (BK004) in 1 liter of distilled or deionized
- Tryptone 5.00 g water.
- Peptic digest of meat 5.00 g - Slowly bring to boiling, stirring until
- Meat extract 5.00 g complete dissolution.
- Glucose 5.00 g - Do not autoclave.
- Disodium phosphate 4.00 g
- Ferrous sulfate 0.30 g Instructions for Use
- Bismuth ammoniacal citrate 1.85 g
- Sodium sulfite 6.15 g - Cool and maintain the medium at 47°C.
- Brilliant green 25 mg - Homogenize the medium in order to
- Bacteriological agar 14.70 g disperse the precipitate.
- Pour into sterile Petri dishes at 20 mL per dish.
pH of the ready-to-use medium at 25°C : 7.6 ± 0.2. - Let solidify on a cold surface.
Quality Control partially removed.
- Inoculate by streaking the medium with
- Dehydrated medium : greenish-beige powder, free-flowing and the enrichment media used.
homogeneous. - In parallel, transfer the inoculum to
- Prepared medium : green-beige agar. another selective medium.
- Typical culture response after 48 hours of incubation at 37°C : - Incubate at 37°C for 24 and 48 hours.
Microorganisms Growth Characteristics NOTE

Salmonella Typhi sp. good black colonies surrounded Immediately after its preparation, the medium
by a black zone, with has optimal selectivity which gradually decreases
with time. This is why it is not recommended to
metallic sheen
store the ready-to-use medium more than 4 days
Salmonella Typhimurium ATCC ®14028 good brownish colonies with at 2-8°C. Once the medium has set, do not melt it
metallic sheen a second time.
Shigella flexneri ATCC 29903 partially inhibited
Escherichia coli ATCC 25922 inhibited Results
Enterococcus faecalis ATCC 29212 inhibited
Staphylococcus aureus ATCC 25923 inhibited Colonies have the following appearance :
- black, flat, dry colonies surrounded by a
Storage / Shelf Life black-brown zone with metallic reflections
usually present : Salmonella Typhi.
Dehydrated medium : 2-30°C. - black colonies without zones : Salmonella
- The expiration date is indicated on the label. Enteritidis.
Prepared medium (benchmark value) : - green or brownish colonies without
- Media in plates : 4 days at 2-8°C. zones : Salmonella Gallinarium, Salmonella
Choleraesuis, Salmonella Paratyphi,
Packaging Shigella.
Coliform bacteria, Proteus and Shigella are
Dehydrated medium strongly inhibited but may sometimes yield
- 500 g bottle : BK004HA small greenish or brownish colonies.
Bibliography
(1) Wilson, J.W., and Blair, E.M. 1931. Further experience of the (4) NF V 08-052. May 1997. Microbiology of food and animal
Bismuth Sulphite Media in the isolation of Bacillus typhosus feeding stuffs. Detection of Salmonella. Routine method.
and Bacillus paratyphosus B from faeces, sewage and water. J. (5) NF EN 12824. February 1998. Microbiology of food and animal
Hyg., 31: 138. feeding stuffs. Horizontal method for the detection of
(2) Wilson, J.W. 1938. Isolation of Bact. typhosum by means Salmonella.
Bismuth Sulphite Medium in water and milk born epidemies. J. (6) United States Pharmacopoeia 24. 2000. Microbial Limit Tests.
Hyg., 38: 507-519. 1814-1818.
(3) NF V 04-015: February 1984. Dried milk and sweetened
condensed milk. Microbiology.
79
Brain-Heart Agar

Brain-Heart Agar is suitable for the culture of a wide variety of Target : streptococci, pneumococci,
aerobic and anaerobic microorganisms including yeasts and molds. meningococci, pathogenic fungi.
After adding sheep blood, the medium is used to culture fastidious Reconstitution : 52.0 g / L.
bacteria : streptococci, pneumococci or meningococci. After adding Autonomy (500 g) : 641 plates.
gentamicin and chloramphenicol, the resulting selective medium is
pH (25°C) : 7.4 ± 0.2.
suitable for the growth of pathogenic fungi from samples highly
contaminated by bacteria and saprophytic molds. The medium is Supplement required : Defibrinated sheep
unsuitable for characterizing hemolysis types, because of its glucose blood, Gentamicin Selective Supplement
content. (depending on use).
Principles
- Brain and heart extracts and gelatin digest are the essential Preparation
elements for growing fastidious organisms.
- Glucose is utilized through the fermentation process. - Suspend 52.0 g of dehydrated medium
- The medium is buffered by the use of disodium phosphate. (BK029) in 1 liter of distilled or deionized
water.
Typical Composition - Slowly bring to boiling, stirring until
(can be adjusted to obtain optimal performance) complete dissolution.
For 1 liter of medium : - Sterilize in an autoclave at 121°C for
- Brain heart infusion 17.5 g 15 minutes.
- Pancreatic digest of gelatin 10.0 g
- Sodium chloride 5.0 g Instructions for Use
- Glucose 2.0 g - Melt the medium (if prepared in advance).
- Bacteriological agar 15.0 g - Cool and maintain at 47°C.
- Add the supplements required for the
pH of the ready-to-use medium at 25°C : 7.4 ± 0.2. growth of the bacteria to be detected
(defibrinated sheep blood, Gentamicin
Quality Control [BS009], Chloramphenicol [BS021]).
- Dehydrated medium : cream powder, free-flowing and - Let solidify on a cold surface.
homogeneous. - Dry in an incubator with the covers
- Prepared medium with 5% defibrinated sheep blood : red partially removed.
opaque agar - Inoculate.
- Typical culture response in medium prepared with 5% sheep - Incubate at 37°C for 24 to 72 hours.
blood, after 24-48 hours of incubation at 37°C :
Clostridium difficile ATCC® 9689 good
Neisseria meningitidis ATCC 13090 good
Streptococcus pneumoniae ATCC 6303 good to excellent
Streptococcus pyogenes ATCC 19615 good to excellent
Candida albicans ATCC 10231 good

- Base media in vials : 6 months at 2-8°C.
- Complete media in plates (with sheep blood) : 1 month at 2-8°C.
Gentamicin Selective Supplement,
Chloramphenicol Selective Supplement :
80
Packaging

Gentamicin Selective Supplement :
Bibliography
(1) Rosenow, E.C. 1919. Studies on elective localization, focal (3) Forney, J.E., and Miller, J.M. 1985. In Lennette, Balows,
infection with special reference to oral sepsis. Jour. Dent. Hausler and Shadomy (Ed). Manual of Clinical Microbiology,
Res., 1: 205-249. 4th Ed., A.S.M. Washington, D.C.
(2) Ajello, L. Georg, Kaplan, and Kaufman. 1963. Laboratory
manual for medical mycology. PHS Publication n° 994, U.S.
Gov. Printing Office, Washington, D.C.
Brain-Heart Broth
Intended Use
Product Profile
Brain-Heart Broth is a buffered nutrient medium used for the Target : Aerobic microorganisms.
culture of a wide variety of aerobic and anaerobic microorganisms Reconstitution : 37.0 g / L.
including yeasts and molds. It is particularly suited for the growth of Autonomy (500 g) : 1351 tubes
such fastidious bacteria as streptococci, pneumococci or (10 mL per tube).
meningococci in samples of varied origins. It is suitable for the
detection of staphylocoagulase. pH (25°C) : 7.4 ± 0.2.
History Sterilization : 121°C / 15 minutes.
In 1919, Rosenow formulated an excellent medium for the growth

Preparation
of streptococci by adding fragments of brain tissue to glucose broth.
Brain-Heart Broth has derived from this.
- Dissolve 37.0 g of dehydrated medium
Principles
water.
- Stir slowly until complete dissolution.
- Brain-Heart Broth uses the original principle of the Rosenow
formula.
- Its elevated content in nutritive compounds enables fastidious
15 minutes.
bacteria to rapidly reach the stationary phase of growth.
Typical Composition
- Inoculated media are incubated at 30 or
37°C for 3 to 15 days.
- If Brain-Heart Broth is used for the
- Brain heart infusion 17.5 g
confirmation of staphylocoagulase,
inoculate 10 mL tubes of media with
suspect colonies and incubate 24 hours at
37°C. Proceed with the coagulase tube test
- Glucose 2.0 g
using Coagulase Rabbit Plasma (BR002)
and refer to the catalogue entry for more
information.
Quality Control
Results
Growth is detected by turbidity in the
homogeneous.
medium. Subcultures must be prepared in
- Prepared medium : light amber solution, limpid.
order to purify the strains to be identified.
81
Neisseria meningitidis ATCC® 13090 good to excellent
Staphylococcus aureus ATCC 25923 good to excellent
Clostridium difficile ATCC 9689 good to excellent
Candida albicans ATCC 10231 good to excellent

- Media in vials or tubes : 6 months at 2-8°C.
Coagulase Rabbit Plasma :
- The expiration date.
Packaging
Dehydrated medium :
Coagulase rabit plasma :
- 10 vials pack, 20 tests per vial : BK00208
Bibliography
(1) Rosenow, E.C. 1919. Studies on elective localization. Focal (7) XP V08-057-1. Nov. 1994. Food microbiology. Routine method
infection with special reference to oral sepsis. Jour. Dent. for enumeration of coagulase positive Staphylococcus by
Res., 1: 205-249. colony-count technique at 37°C. Part 1: Technique with
(2) NF V04-502. December 1981. Meat and meat products. confirmation of the colonies.
Microbiology analysis. Part 2: General guidance adaptation. (8) FIL-IDF 60C: 1997. Dried milk. Staphylococcus aureus. MPN
(3) NF V59-105. October 1982. Edible gelatin. Detection of procedure.
Staphylococcus aureus. (9) FIL-IDF provisional 145A: 1997. Milk and milk based
(4) NF V 04-015: February 1984. Dry milk and sweetened products. Enumeration of Staphylococcus aureus in products
condensed milk. Microbiology. other than dried milk. Colony count technique.
(5) Jones, R.N., Barry, A.L., Gavan, T.L., and Washington, J.A. (10) NF EN ISO 6888-1. Oct. 1999. Microbiology of food and
1985. In Lennette, Balows, Hausler and Shadomy (Ed). Manual animal feeding stuffs - Horizontal method for the
of Clinical Microbiology. 4th ed. A.S.M. Washington. D.C. enumeration of coagulase-positive staphylococci
(6) NF T 90-421: October 1989. Testing water. Bacteriological (Staphylococcus aureus and other species) - Part 1: Technique
examinations of swimming pool water. using Baird-Parker agar medium.
Brilliant Green Agar (Edel and Kampelmacher)

Intended Use
Brilliant Green Agar of Edel and Kampelmacher is a selective

medium used to isolate Salmonella in food products, especially
when the bacteria are present in small numbers.
History
In 1925, Kristensen, Lester and Jurgens described this medium, later

modified by Kauffmann and then at the Rijks Instituut voor de
Volksgezondheid in Utrecht, under the responsibility of Edel and
Kampelmacher.
Salmonella Typhimurium
82
Principles Product Profile
Target : Salmonella.
- Brilliant green inhibits Gram-positive bacteria and most Gram-
negative bacteria. Reconstitution : 53.7 g / L.
- In comparison to the Kristensen medium, this medium was enriched Autonomy (500 g) : 620 plates.
with nutritive factors and the inhibiting power was reduced.
pH (25°C) : 6.9 ± 0.2.
- The medium contains two carbohydrates, lactose and sucrose for
which the fermentation of either results in a decrease in pH. This Supplement required : None.
results in the appearance of yellow-green colonies in the presence Sterilization : Heat to boiling.
of the pH indicator, phenol red. Do not autoclave.
- Salmonellae produce colorless to pinkish colonies.
- It is particularly recommended to carry out a prior enrichment on Preparation
Tetrathionate, Selenite-cystine or Rappaport-Vassiliadis broths and
to simultaneously inoculate other more selective media : DCLS - Suspend 53.7 g of dehydrated medium
Agar, Bismuth Sulfite Agar, XLT4 Agar, etc... (BK091) in 1 liter of distilled or deionized
water.
Typical Composition - Slowly bring to boiling, stirring until
(can be adjusted to obtain optimal performance) complete dissolution.
For 1 liter of medium : - Do not autoclave.
- Tryptone 10.0 g
- Meat extract 5.0 g Instructions for Use
- Lactose 10.0 g - Cool and maintain the medium at 47°C.
- Sucrose 10.0 g - Pour into sterile Petri dishes.
- Disodium phosphate 1.0 g - Let solidify on a cold surface.
- Monosodium phosphate 0.6 g - Dry in an incubator with the covers
- Phenol red 90 mg partially removed.
- Brilliant green 5 mg - Inoculate by streaking on plates with the
- Bacteriological agar 14.0 g enrichment media used for the detection
of Salmonella.
pH of the ready-to-use medium at 25°C : 6.9 ± 0.2. - In parallel, transfer the inoculum to
another selective medium.
Quality Control - Incubate at 37°C for 24 and 48 hours.
- Dehydrated medium : pinkish powder, free-flowing and NOTE:

homogeneous. The medium, normally red-brown, becomes
bright red after incubation and assumes its
- Prepared medium : orange-brown agar. original color after returning to room
- Typical culture response after 24-48 hours of incubation at 37°C : temperature.
Microorganisms Growth Characteristics Results

Salmonella Enteritidis ATCC® 13076 good pinkish colonies
Salmonella Typhimurium ATCC 14028 good pinkish colonies The colonies have the following appearance :
- Colorless to pink colonies surrounded by a
Staphylococcus aureus ATCC 25923 inhibited red zone in the medium :
Salmonella (lactose/sucrose negative).
Storage / Shelf Life - Pink colonies without proliferation :
Proteus
Dehydrated medium : 2-30°C. - Small pink colonies :
- The expiration date is indicated on the label. Pseudomonas
Prepared medium (benchmark value) : - Greenish-yellow colonies surrounded by a
- Media in plates : 1 month at 2-8°C. yellow zone in the medium :
Escherichia, Citrobacter, Klebsiella,
Packaging Enterobacter (lactose/sucrose positive).
- Totally or almost totally inhibited :
Dehydrated medium : Gram-positive bacteria.
83
Bibliography
(1) Kristensen, M., Lester, V., and Jurgens, A. 1925. On the use of (5) FIL-IDF 93B: 1995. Milk and milk products. Detection of
trypsinized casein, bromthymol blue, bromcresol purple, phenol Salmonella.
red and brilliant green for bacteriological nutrient media. Br. (6) ISO 6340: 1995. Water Quality. Detection of Salmonella.
J. Exp. Pathol., 6: 291. (7) NF V 08-052. May 1997. Microbiology of food and animal
(2) Edel, W., and Kampelmacher, E.H. 1968. Comparative studies of feeding stuffs. Detection of Salmonella. Routine method.
Salmonella isolation in eight European Laboratories. Bull. WHO, (8) NF EN 12824. February 1998. Microbiology of food and animal
39 (3): 487. feeding stuffs. Horizontal method for the detection of
(3) Edel, W., and Kampelmacher, E.H. 1969. Salmonella- isolation Salmonella.
in nine European Laboratories using a standardized technique.
Bull. Wld. Health. Org., 41: 297-306.
(4) NF V 04-015: February 1984. Dried milk and sweetened
Brilliant Green Agar (Kristensen)
Intended Use
Product Profile
Brilliant Green Agar of Kristensen is a highly selective medium used Target : Salmonella.
to isolate salmonella, except for Salmonella Typhi, in biological Reconstitution : 51.6 g / L.
samples and food products. Autonomy (500 g) : 646 plates.
History pH (25°C) : 6.9 ± 0.2.

In 1925, Kristensen, Lester and Jurgens described this medium, later Sterilization : 121°C / 15 minutes.
modified by Kauffmann and then used by Broh-Kahn and Edwards
with satisfaction. Preparation
Principles - Suspend 51.6 g of dehydrated medium
- Brilliant green inhibits Gram-positive bacteria and most Gram- water.
negative bacteria. - Slowly bring to boiling, stirring until
- The medium contains two carbohydrates : lactose and sucrose for complete dissolution.
which the fermentation of either results in a decrease in pH, - Dispense in tubes or flasks.
causing the appearance of yellow-green colonies in the presence - Sterilize in an autoclave at 121°C for
of the pH indicator, phenol red. Salmonellae produce colorless to 15 minutes.
pinkish colonies. - Do not overheat.
- It is particularly recommended to carry out a prior enrichment in
Tetrathionate, Selenite or Rappaport-Vassiliadis broths and to Instructions for Use
simultaneously inoculate another less selective media : i.e.
MacConkey Agar, XLD Agar, or Hektoen Enteric Agar. - Cool and maintain the medium at 47°C.
- Because of its elevated inhibitory power, this medium enables a - Pour into sterile Petri dishes.
highly contaminated inoculum to be used. - Let solidify on a cold surface.
Typical Composition partially removed.
(can be adjusted to obtain optimal performance) - Inoculate by streaking on plates, using the
For 1 liter of medium : enrichment media, as inoculum.
- Tryptone 5.0 g - In parallel, transfer the inoculum to
- Peptic digest of meat 5.0 g another selective medium.
- Yeast extract 3.0 g - Incubate at 37°C for 24 and 48 hours.
- Lactose 10.0 g
NOTE:
- Sucrose 10.0 g
The medium, normally red-brown, becomes
- Sodium chloride 5.0 g bright red after incubation and assumes its
- Phenol red 80 mg original color after returning to room
- Brilliant green 12.5 mg temperature.
84
- Dehydrated medium : pinkish powder, free-flowing and The colonies have the following appearance :
homogeneous.
- Prepared medium : orange-brown agar. - Colorless to pink colonies, smooth and
- Typical culture response after 24-48 hours of incubation at 37°C : surrounded by red zones in the medium :
Salmonella (except for Salmonella Typhi
Microorganisms Growth Characteristics and Paratyphi lactose/sucrose negative)
Salmonella Enteritidis ATCC ®
13076 good pinkish colonies - No or very little growth : Shigella
- Yellow to greenish-yellow colonies
Salmonella Typhimurium ATCC 14028 good pinkish colonies
surrounded by greenish yellow zones in the
Enterococcus faecalis ATCC 29212 inhibited medium : Escherichia, Citrobacter,
Staphylococcus aureus ATCC 25923 inhibited Klebsiella, Enterobacter (lactose/sucrose
positive)
Storage / Shelf Life - Totally or almost totally inhibited :
Gram-positive bacteria
- Media in plates : 1 month at 2-8°C.
Packaging
Dehydrated medium :
Bibliography
(1) Kristensen, M., Lester, V., and Jurgens, A. 1925. On the use of (3) Pharmacopée européenne. Addendum 2000. Contrôle
trypsinized casein, bromthymol blue, bromcresol purple, phenol microbiologique des produits non stériles (Recherche de
red and brilliant green for bacteriological nutrient media. microorganismes spécifiés). Solution et milieux de culture
Br. J. Exp. Pathol., 6: 291. recommandés, 56-61.
(2) United States Pharmacopoeia 24. 2000. Microbial Limit Test.
1814-1818.
Brilliant Green Bile Broth (BGBB)

Brilliant Green Bile Broth is used for the detection and enumeration Target : Coliforms, Thermotolerant coliforms,
of coliform, heat-tolerant coliform bacteria and Escherichia coli Escherichia coli.
(Mackenzie test) in milk, milk-based products and other food Reconstitution : 40.0 g / L.
products, drinking water and waste water. Autonomy (500 g) : 1250 tubes.
History pH (25°C) : 7.2 ± 0.2.

Supplement required : MUG Supplement
Research and development of a culture medium inhibiting (optional).
microorganisms other than coliform bacteria has long interested Sterilization : 121°C / 15 minutes.
bacteriologists. In 1926, Dunham and Schoenlein studied the
proportions of bile and brilliant green which could give good
results. Jordan showed that this medium was better than Lactose
Broth for the detection of coliform bacteria in water. For the control
of milk pasteurization, MacCrady and Langevin satisfactorily used
Brilliant Green Bile Broth for the detection of coliform bacteria.
Mackenzie verified that the brilliant green concentration was
sufficient to effectively inhibit the culture of lactose-fermenting
anaerobes, in particular Clostridium perfringens.
85
- The simultaneous presence of ox bile and brilliant green inhibit Single strength medium
almost all Gram-positive organisms and Gram-negative bacteria - Dissolve 40.0 g of dehydrated medium
other than coliforms. (BK002) in 1 liter of distilled or deionized
- The brilliant green concentration was specially determined in water.
order to prevent the growth of lactose-fermenting anaerobes at - Stir slowly until complete dissolution.
44°C, which avoids false positives. - Dispense in 16 x 160 mm tubes containing
- Development of coliform bacteria is shown by turbidity and gas a Durham tube.
production in the Durham tubes, as a result of lactose - Sterilize in an autoclave at 121°C for
fermentation. 15 minutes.
- After cooling, the Durham tubes should
not contain trapped air.
Typical Composition of single strength medium
NOTE
MUG (4-methylumbelliferyl-ß-D-glucuronide) can
For 1 liter of medium : be added to the medium in order to detect
- Tryptone 10.0 g Escherichia coli (refer to the monograph on MUG
- Bacteriological ox bile 20.0 g Supplement, BS024).
- Lactose 10.0 g
- Brilliant green 13.3 mg Double strength medium
pH of the ready-to-use medium at 25°C : 7.2 ± 0.2. (BK002) in 1 liter of distilled or deionized
water.
Quality Control - Stir slowly until complete dissolution.
- Dispense 10 mL in 20 x 200 mm tubes
- Dehydrated medium : greenish powder, free-flowing and (without a Durham tube).
homogeneous. - Sterilize in an autoclave at 121°C for 15
- Prepared medium : green solution, limpid. minutes.
- Typical culture response after 24-48 hours of incubation at 30°C :
Microorganisms Growth Gas production
Escherichia coli ATCC® 25922 good to excellent positive
- Cool the medium to 25°C.
Enterobacter cloacae ATCC 13047 good to excellent positive - Inoculate the tubes with 1 mL of inoculum
Citrobacter freundii ATCC 43864 good to excellent positive and its serial tenfold dilutions.
Enterococcus faecalis ATCC 29212 partially inhibited negative
Staphylococcus aureus ATCC 25923 inhibited Double strength medium
- Inoculate the tubes with 10 mL of
inoculum.
- Cover with a plug of sterile agar
(Bacteriological Agar type E), previously
maintained at 47°C.
- Media in tubes : 3 months at 2-8°C, shielded from light. Incubation
Ready-to-use media in tubes containing Durham tubes, - For coliform bacteria, incubate for 24 and
MUG Supplement : 48 hours at 30 or 37°C, depending on the
- Store between 2-8°C, shielded from light. analysis protocol followed.
- The expiration dates are indicated on the labels. - For heat-tolerant coliform bacteria,
incubate for 24 and 48 hours at 44°C.
- Transfer one inoculating loop of double
strength culture to a single strength tube
and incubate for an additional 24 to 48
hours before reading.
86
Packaging Results
Ready-to-use media : Lactose fermentation, which results in the

- 50 x 10 mL tubes : BM01108 production of gas in the Durham tubes
Dehydrated medium : (volume at least equal to 1/10 of the tube
- 500 g bottle : BK002HA volume) in less than 48 hours, indicates the
MUG Supplement : presence of coliform bacteria. The bacteria
- 10 vial pack : BS02408 can be identified by subculturing from the
gas-producing tube on suitable agar media :
- BCP Lactose Agar (BK023)
- EMB Agar (BK056).
The presence of Escherichia coli can be
confirmed by carrying out the Mackenzie
test as follows :
- Inoculate a loop from the gas-producing
tubes into a tube of BGBB with a Durham
tube and also in a tube of peptone water
(BK084).
- Incubate at 44 ± 0.5°C in a water bath for
24 and 48 hours.
Under these conditions, Escherichia coli
simultaneously presents the following
characteristics :
- growth in BGBB : positive and gas-
producing.
- indole production (Ehrlich-Kovacs
reagent) : positive.
Bibliography
(1) Dunham, and Schoenlein. 1926. Stain technology, 1: 129. (7) Rodier, J. 1984. L’analyse de l’eau. Dénombrement des
(2) Jordan. 1927. J. American Water Works Association, 18: 337. coliformes, coliformes fécaux, et Escherichia coli présumés.
(3) McCrady, and Langevin. 1932. J. Dairy Science, 15: 321. Dunod 7ème Ed: 793-798.
(4) Mackenzie, E.F.W., Taylor, W.E., and Gilbert, W.E. 1948. Recent (8) NF V 04-015: February 1984. Dried milk and sweetened
experiments in the rapid identification of Bacterium coli type condensed milk. Microbiology.
I. J. Gen. Microb., 2: 197-204. (9) NF T 90-413. October 1985: Testing water. Detection and
(5) Journal Officiel (French) du 21 Septembre 1968. Méthode de enumeration of coliforms and thermotolerant coliforms.
prélèvement et d’analyse bactériologique des glaces et crèmes General method by culture in liquid media (MPN).
glacées (arrêté du 30 Août 1968). (10) NF ISO 4831. July 1991. Microbiology. General guidance for the
(6) NF V 45-110. June1981. Fish products. Enumeration of faecal enumeration of coliforms. Most probable number technique.
coliforms in conchological waters and in living shell fishes. (11) FIL-IDF provisional 73B: 1998. Milk and milk products.
Enumeration of coliforms. Part 1: Colony count technique at
30°C without resuscitation. Part 2: Most probable number
technique at 30°C without resuscitation.
Bromcresol Purple Lactose Agar

Target : Enterobacteria.
Bromcresol Purple (BCP) Lactose Agar is a non-selective medium for
the detection and isolation of enteric bacteria in water, food and Reconstitution : 31.0 g / L.
biological products. Autonomy (500 g) : 1075 plates.
pH (25°C) : 7.0 ± 0.2.
History
Initially described by Wurtz in 1892, litmus was replaced by Sterilization : 121°C / 15 minutes.
bromcresol purple which is more sensitive and more stable.
87
- Fermentation of lactose to acid is revealed in the presence of - Suspend 31.0 g of dehydrated medium
bromcresol purple by a color change from violet-blue to yellow. (BK023) in 1 liter of distilled or deionized
- Lactose-negative bacteria form blue colonies. water.
Typical Composition complete dissolution.
(can be adjusted to obtain optimal performance) - Dispense in tubes or flasks.
For 1 liter of medium : 15 minutes.
- Tryptone 5g
- Meat extract 3g Instructions for Use
- Lactose 10 g
- Bromcresol purple 25 mg - Cool and maintain the medium at 47°C.
- Bacteriological agar 13 g - Pour into sterile Petri dishes.
pH of the ready-to-use medium at 25°C : 7.0 ± 0.2. - Dry in an incubator with the covers
partially removed.
Quality Control - Inoculate by streaking.
- Dehydrated medium : beige-green powder, free-flowing and
homogeneous. NOTE
- Prepared medium : violet agar. This incubation must not be exceeded, or else
acidification of the medium may cause
interpretation errors.
Microorganisms Growth Acid production

Results
Enterobacter aerogenes ATCC 13048 good to excellent positive
Lactose-positive colonies are yellow and are
Salmonella Enteritidis ATCC 13076 good to excellent negative
differentiated as a function of the following
Staphylococcus aureus ATCC 25923 good to excellent positive
appearances :
- mucoid colonies : Klebsiella, Escherichia coli.
- colonies bluish at the edges : slow lactose-
fermenting Escherichia coli.
- smooth or rough colonies which are
transparent when examined with back
- Media in vials : 6 months at 2-8°C, shielded from light.
lighting : Escherichia coli, Citrobacter.
- Media in plates : 15 days at 2-8°C, shielded from light.
Lactose-negative colonies are blue.
Packaging
Dehydrated medium :
Bibliography
(1) NF V08-017: June 1980. General guidance for the enumeration (3) Rodier, J. 1984. L’analyse de l’eau. Identification des coliformes
of fecal coliforms and Escherichia coli. et en particulier d’Escherichia coli. Dunod 7ème Ed., 809.
(2) NF V08-301: June 1983. Microbiology of food and feeding (4) NF T90-425: February 1992. Testing water. Bacteriological
stuffs. Desiccated products. Microbiology analysis. examinations of the containers and sealing systems intended
for the conditioned waters.
88
Bromcresol Purple Lactose Broth

Target : Coliforms.
Bromcresol Purple (BCP) Lactose Broth is a presumptive medium for
the detection of coliform bacteria in water. Reconstitution : 13.0 g / L.
Autonomy (500 g) : 3846 tubes.
Principles
pH (25°C) : 6.7± 0.2.
- Tryptone and meat extract supply nitrogen nutrients required for Supplement required : None.
bacterial growth. Sterilization : 121°C / 15 minutes.
- The fermentation of lactose is shown by the acidification of the
medium that causes the pH indicator (bromcresol purple) to turn
yellow, as well as by gas production in Durham tubes. Preparation
- In order to confirm the presence of coliform bacteria, subcultures
must be prepared using the appropriate confirmation media. Single strength medium
- Dissolve 13.0 g of dehydrated medium (BK119)
Typical Composition in 1 liter of distilled or deionized water.
(can be adjusted to obtain optimal performance) - Stir slowly until complete dissolution.
For 1 liter of medium : containing a Durham tube.
- Tryptone 5g - Sterilize in an autoclave at 121°C for
- Meat extract 3g 15 minutes.
- Lactose 5g - After cooling, the Durham tubes should
- Bromcresol purple 25 mg not contain trapped air.
pH of the ready to use medium at 25°C : 6.7 ± 0.2. Double strength medium
Quality Control in 1 liter of distilled or deionized water.
- Dehydrated medium : cream-white powder. - Dispense 10 mL in 20 x 200 mm tubes
- Prepared medium : violet, limpid solution. containing a Durham tube.
- Typical culture response after 24-48 hours of incubation at 30°C : - Sterilize in an autoclave at 121°C for
15 minutes.
Enterobacter cloacae ATCC 13047 good to excellent positive Single strength medium
Enterococcus faecalis ATCC 29212 good negative - Inoculate the tubes with 1 mL of inoculum
Staphylococcus aureus ATCC 25923 good negative and its serial tenfold dilutions.
Storage / Shelf Life Double strength medium

- Inoculate the tubes with 10 mL of
Dehydrated medium : 2-30°C. inoculum.
Prepared medium (benchmark value) : Incubation
- Media in tubes or vials : 6 months at 2-8°C. - Incubate the inoculated tubes at 30°C for
24 and 48 hours.
Packaging
Results
Dehydrated medium :
- 500 g bottle : BK119HA The fermentation of lactose results in the
appearance of cloudiness due to bacterial
development, as well as the release of gas
in the Durham tubes. Using these positive
cultures in the presumptive medium,
subcultures must be prepared with the
appropriate confirmation media :
- Brilliant Green Bile Broth (BK002) for
coliform bacteria.
- Schubert Broth (BK120) for thermotolerant
coliform bacteria.
89
Bibliography
(1) NF T 90-413: October 1985. Testing water. Detection and (2) Rodier, J. 1984. L'analyse de l'eau. Dénombrement des
enumeration of coliforms and thermotolerant coliforms. General coliformes, coliformes fécaux et Escherichia coli présumés.
method by culture in liquid media (MPN). Méthode de dénombrement par inoculation en milieux liquides.
Dunod 7ème Ed., 793-798.
Brucella Agar
Brucella Agar is recommended for the culture and isolation of Target : Brucella, Campylobacter.
Brucella in pathologic samples and dairy products. It can be Reconstitution : 43.1 g / L.
supplemented with 5% horse blood for the culture of particularly Autonomy (500 g) : 773 plates.
fastidious aerobic and anaerobic bacteria (Streptococcus
pneumoniae, Streptococcus viridans, Listeria monocytogenes, pH (25°C) : 7.0 ± 0.2.
Neisseria meningitidis, Haemophilus influenzae). The medium can Supplement required : None.
also be used as a nutritive base for the isolation of Campylobacter. Sterilization : 121°C / 15 minutes.
Principles
Preparation
- The high nutritive value of this medium is due to its content in
peptones, yeast extract and glucose. - Suspend 43.1 g of dehydrated base
- Yeast extract is a source of the vitamin B complex. medium (BK077) in 1 liter of distilled or
- Glucose is an energy source. deionized water.
- The medium can be supplemented with vitamin K1 (10 mg / L) and - Slowly bring to boiling, stirring until
hemin (10 mg / L) to favor the growth of Bacteroides. complete dissolution.
- The addition of 5% horse blood supplies all factors X (heme) and - Dispense in tubes or flasks.
V (nicotinamide adenine dinucleotide) required for the growth of - Sterilize in an autoclave at 121°C for
Haemophilus influenzae. Sheep blood depleted of factor V is not 15 minutes.
suitable for this application.
- In order to obtain a selective medium for Brucella, it is useful to Instructions for Use
add an appropriate supplement.
- Melt and cool the medium (if prepared in
Typical Composition advance).
(can be adjusted to obtained optimal performance) - Cool and maintain at 47°C.
- Add (if necessary) 5% sterile defibrinated
For 1 liter of medium : sheep or horse blood, as well as the
- Tryptone 10.0 g selective supplements required.
- Peptic digest of meat 10.0 g - Homogenize well.
- Yeast extract 2.0 g - Pour into sterile Petri dishes.
- Sodium chloride 5.0 g - Let solidify on a cold surface.
- Glucose 1.0 g - Dry in an incubator with the covers
- Sodium bisulfite 0.1 g partially removed.
- Bacteriological agar 15.0 g - Inoculate.
- Incubate in a carbon dioxide-enriched
pH of the ready-to-use medium at 25°C : 7.0 ± 0.2. atmosphere for 24 to 72 hours at 37°C for
the culture of Brucella and at 42°C in a
Quality Control microaerophilic atmosphere for the culture
of Campylobacter.
homogeneous.
- Prepared medium : light amber agar.
Brucella abortus sp. good to excellent
Streptococccus pneumoniae ATCC 6303
®
good
Staphylococcus aureus ATCC 25923 good
90

- Media in plates (with added sheep blood) : 1 month at 2-8°C.
Packaging

Bibliography
(1) APHA. 1960. Standard methods for the examination of dairy (4) Diagnostic Microbiology. 1982. The C.V. Mosby Co. St. Louis,
products, 11 th Ed. 6 th Ed.
(2) APHA. 1963. Diagnostic procedures and reagents, 4 th Ed. (5) MacFaddin, J.F. 1985. Media for Isolation - Cultivation -
(3) Lennette, E.H., Balows, A., Hausler, Jr, W.J., and Shadomy, Identification - Maintenance of Medical Bacteria, vol 1 -
H.J. 1985. Manual of Clinical Microbiology, 4 th Ed. American Williams et Wilkins Baltimore: 110-114.
Society for Microbiology. Washington D.C.
Brucella Broth
Brucella Broth is a non-selective medium for the culture of Brucella, Target : Brucella, Campylobacter.
Campylobacter and other particularly fastidious aerobic and Reconstitution : 28.1 g / L.
anaerobic bacteria. Autonomy (500 g) : 1779 tubes.
Principles pH (25°C) : 7.0 ± 0.2.

- The high nutritive value of this medium is due to its richness in Sterilization : 121°C / 15 minutes.
peptones, yeast extract and glucose.
- Yeast extract is a source of the vitamin B complex.
- Glucose is an energy source. Preparation
- Sodium chloride maintains osmotic balance.
- Sodium bisulfite is a reducing agent. - Dissolve 28.1 g of dehydrated medium
Typical Composition water.
(can be adjusted to obtained optimal performance) - Stir slowly until complete dissolution.
- Tryptone 10.0 g 15 minutes.
- Yeast extract 2.0 g Instructions for Use
- Glucose 1.0 g
- Sodium chloride 5.0 g - Inoculate the medium with purified
cultures or with other types of mixed
- Sodium bisulfite 0.1 g
microflora inoculum.
- For the growth of Brucella, incubate up to
pH of the ready to use medium at 25°C : 7.0 ± 0.2.
7 days at 37°C in an anaerobiosis jar with
CO2.
Quality Control
- For other microorganisms, incubate at the
- Dehydrated medium : cream powder, free-flowing and required optimal temperature for the
homogeneous. period of time necessary for the
- Prepared medium : amber solution, limpid. development of cultures.
- Typical culture response after 24-48 hours of incubation at
37°C :
91
Microorganisms Growth Results
Brucella spp. (1) good to excellent
Growth is shown by turbidity, resulting from
Streptococcus pyogenes ATCC® 19615 good
microbial multiplication. The resulting
Staphylococcus aureus ATCC 25923 good cultures are subcultured on the appropriate
Campylobacter jejuni ATCC 33291 good media before being subjected to
(1)
Incubation under CO2 identification tests.

- Media in tubes or vials : 6 months at 2-8°C.
Packaging
Dehydrated medium :
Bibliography
(1) MacFaddin, J.F. 1985. Media for Isolation - Cultivation - (3) NF ISO 10272. January 1996. Microbiology of food and animal
Identification - Maintenance of medical bacteria. Williams and feeding stuffs. Horizontal method for detection of
Wilkins Baltimore/London. Volume 1, 125-127. thermotolerant Campylobacter.
(2) Hausler, W.J. Jr., Moyer, N. P., and Holcomb L.A. 1985.
Brucella. Manual of Clinical Microbiology. 4th Ed., 382-386.
Bryant and Burkey Broth with Resazurin

Intended Use
Product Profile
Bryant and Burkey Broth with Resazurin (modified by Bergère) is Target : Clostridium tyrobutyricum.
used to enumerate the spores of lactate fermenting Clostridium in Reconstitution : 38.0 g / L.
milk and dairy products. In particular, it is used to detect Clostridium
tyrobutyricum, responsible for the late blowing of cheeses such as
gruyère, emmental, gouda, edam, cheddar or parmesan. The pH (25°C) : 5.9 ± 0.1.
phenomenon arises from a high number of spores in the milk used Supplement required : None.
to prepare the cheese. This number depends above all on the type Sterilization : 121°C / 15 minutes.
of food given to the animals, since the main source of
contamination is silage. Preparation
History - Suspend 38.0 g of dehydrated medium

Originally described by Bryant and Burkey, the medium was water.
modified by Bergère, who in 1968, carried out a study on the role of - Stir slowly until complete dissolution.
silage in butyric contamination of milk and clearly differentiated - Dispense 10 mL in 16 x 160 mm tubes.
Clostridium butyricum from Clostridium tyrobutyricum, the latter - Sterilize in an autoclave at 121°C for
being the primary agent in cheese degradation. 15 minutes.
Principles Instructions for Use
- Tryptone, meat and yeast extracts, and cysteine are the nutritive - Cool tubes coming from the autoclave to
substrates required for the rapid multiplication of Clostridium 25°C in a cold water bath.
tyrobutyricum under anaerobic conditions. - If after storage the medium is pink (sign of
- Sodium acetate is the principle promoter of spore germination, a oxidation) for more than 1/3 of its height
process which is also activated by heating to 75°C, which under the surface, regenerate anaerobic
simultaneously destroys the vegetative forms. conditions by heating at 100°C for 20
minutes.
- Do not carry out this operation more than
once.
92
- Resazurin is a redox indicator and should remain colorless after - Inoculate with 1 mL of inoculum and each
heating the inoculum in the medium. Any increase in oxygen levels of its tenfold dilutions.
is reflected by the appearance of a pink color. Pink tubes are not - Pour 2 mL of melted (58-60°C) paraffin,
to be used when analyzing the results. previously sterilized at 121°C for 20
- The fermentation of lactate in the presence of acetate leads to the minutes.
gas (hydrogen + carbon dioxide) production. Gas release is - Heat the tubes at 75°C for 15 minutes to
visualized with a paraffin plug which is raised. destroy vegetative cells and activate
spores.
Typical Composition - Cool in an ice-water bath to solidify the
(can be adjusted to obtained optimal performance) paraffin.
- Incubate at 37°C for 7 days.
- Tryptone 15.0 g Results
- Meat extract 7.5 g
Tubes with growth and gas generation,
- Cysteine hydrochloride 0.5 g
raising the paraffin plug, are considered
- Sodium lactate 5.0 g
positive. Enumerate using the most
- Sodium acetate 5.0 g
probable number method.
- Resazurin 2.5 mg
NOTE
pH of the ready-to-use medium at 25°C : 5.9 ± 0.1. This medium is not selective and only an
identification of colonies isolated from positive
Quality Control tubes can be used to confirm the presence of
Clostridium tyrobutyricum. Experience has shown
- Dehydrated medium : cream-white powder, free-flowing and that the results obtained with this medium are
homogeneous. well correlated with damage occurring in cheese
- Prepared medium : amber solution, limpid, with a pink ring on manufacturing.
the surface.
- Typical culture response after 7 days of anaerobic incubation
at 37°C :

Clostridium tyrobutyricum CNRZ 500 good to excellent positive

- Regenerate 20 minutes at 100°C before inoculating.
Packaging
Dehydrated media :
Bibliography
(1) Rosenberger, K.F. 1951. The development of methods for the (6) Cerf, O., et Bergère, J.L. 1968. La numération des spores de
study of obligate anaerobics in silage. Proc. Soc. Appl. Clostridium et son application au lait et aux produits laitiers.
Bacteriol., 14: 161-164. Numération des différents groupes de Clostridium. Le lait, 48:
(2) Bryant, M.P,. Kroulik, S.T., Burkey, L.A., Wiseman, H.G. 1952. A 501-519.
study of anaerobic bacteria present in grass silage. Bact. proc., 21. (7) Touraille, C., et Bergère, J.L. 1974. La germination de la spore
(3) Bryant, M.P., and Burkey, L.A. 1953. Cultural methods and de Clostridium tyrobutyricum. Biochimie, 56 (3): 405-421.
some characteristics of some of the more numerous groups of (8) Bergère, J.L. Septembre 1979. Développement de l’ensilage.
bacteria with bovine rumen. J. Dairy. Science, 36: 205-217. Ses conséquences sur la qualité du lait et des produits
(4) Bryant, M.P., and Burkey, L.A. 1956. The characteristics of laitiers. Revue laitière française.
lactate-fermenting sporeforming anaerobes from silage. J. (9) Frankinet, J. et de Carheil, M. 1983. Les tests de contrôle des
Bact., 71: 43-46. germes butyriques. La technique laitière, 977: 15-18.
(5) Bergère, J.L., Grouet, P., Hermier, P., et Mocquot. 1968. Les (10) CNERNA. Avril 1986. Recommandations pour l’estimation de la
Clostridium du groupe butyrique dans les produits laitiers. Ann. contamination du lait en spores de Clostridia par la méthode
Institut Pasteur Lille, 19: 41-54. en milieu liquide. Revue laitière française, 451: 39-43.
93
Buffered Peptone Broth with sodium chloride
pH 7.0
Intended Use
Product Profile
Buffered Peptone Broth with sodium chloride pH 7.0 is a diluent Target : Dilutions, Suspensions.
used to prepare stock solutions or suspensions of water-soluble or Reconstitution : 16.1 g / L.
non-lipid water-insoluble pharmaceutical products. The medium is Autonomy (500 g) : 345 vials.
also used to rinse membranes when the filtration method is used to (90 mL by vial).
enumerate bacteria, yeasts and molds on the surface of appropriate
agar media. For lipid products, a surface active agent can be added pH (25°C) : 7.0 ± 0.2.
to the diluent, such as polysorbate 20 or 80, at concentrations Supplement required : None.
included between 0.1 and 1.0%. Sterilization : 121°C / 15 minutes.
Principles
Preparation
- Tryptone assures the resuscitation of microorganisms having
undergone sublethal treatments.
- Sodium chloride maintains the osmotic balance.
water.
- The medium is buffered with disodium and potassium dihydrogen
phosphates.
Typical Composition
15 minutes.
- Tryptone 1.00 g - Cool to 25°C.
- Sodium chloride 4.30 g - Aseptically add 10 g of product to analyze
- Disodium phosphate 7.23 g to a tared flask containing 90 mL of
- Potassium dihydrogen phosphate 3.56 g medium.
- Homogenize thoroughly.
Quality Control
- Dehydrated medium : white powder, free-flowing and

homogeneous.
- Prepared medium : very light amber solution, limpid.
Escherichia coli ATCC® 25922 good
Salmonella Typhimurium ATCC 14028 good

- Media in vials : 6 months at 2-8°C.
Packaging
Dehydrated medium
Bibliography
(1) Pharmacopée européenne. Addendum 2000. Contrôle

microbiologique des produits non stériles (Recherche de
microorganismes spécifiés). Solution et milieux de culture
recommandés, 56-61.
94
Buffered Peptone Water
Intended Use
Product Profile
Buffered Peptone Water is a diluent destined for the preparation of Target : Suspensions, Dilutions, Salmonella.
stock suspensions of powdered and concentrated milk, yogurts, Reconstitution : 25.5 g / L (BK018).
dairy products, animal products and other food products. The 20.0 g / L (BK131).
medium can also be used for pre-enrichment of Salmonella, prior to Autonomy (500 g) : 87 vials (BK018).
the selective enrichment and isolation phases. It allows in particular 111 vials (BK131).
to resuscitate microorganisms which have undergone sub-lethal
treatments. It is also used for serial dilutions during microbiological pH (25°C) : 7.0 ± 0.2.
analysis. Supplement required : None.
Principles
Preparation
- Pre-enrichment of Salmonella has for objective the resuscitation
of microorganisms having undergone damage during industrial - Dissolve 25.5 g of dehydrated medium
preservation treatments of food products, such as spray-drying, (BK018) or 20.0 g (BK131) in 1 liter of
pasteurizing, preservatives, high osmotic pressure, strong acidity, distilled or deionized water.
etc. - Stir slowly until complete dissolution.
- Sodium chloride maintains the osmotic balance. - Dispense in vials at 90 or 225 mL per vial.
- The medium is buffered with sodium and potassium phosphates. - Sterilize in an autoclave at 121°C for
15 minutes.
Typical Composition
(can be adjusted to obtain optimal performance) Instructions for Use
For 1 liter of medium (BK018, BM010) : - Cool the medium to 25°C.

- Peptone 10.0 g - Aseptically add 10 or 25 g of the product
- Sodium chloride 5.0 g to analyze to a tared flask containing 90
- Disodium phosphate, x 7H2O 9.0 g or 225 mL of media prepared as above, or
- Potassium dihydrogen phosphate 1.5 g to the ready-to-use media BM010.
- Homogenize thoroughly with an
For 1 liter of medium (BK131) : appropriate mixer in order to obtain a
- Peptone 10.0 g stock suspension or a preenrichment broth.
- Sodium chloride 5.0 g - Incubate respecting the analytical protocol
- Disodium phosphate, anhydrous 3.56 g applied.
- Potassium dihydrogen phosphate 1.5 g
pH of the ready-to-use media at 25°C : 7.0 ± 0.2.
Quality Control
- Dehydrated media : cream-white powder, free-flowing and

homogeneous.
- Prepared media : amber solution, limpid.
Salmonella Typhimurium ATCC® 14028 good to excellent
Salmonella Enteritidis ATCC 13076 good to excellent
Escherichia coli ATCC 25922 good to excellent
Dehydrated media : 2-30°C.

Ready-to-use media in vials :
95
Packaging
Ready-to-use media :
- 10 x 225 mL vials : BM01008
Dehydrated medium (25.5 g/L) :
Dehydrated medium (20.0 g/L) :
Bibliography
(1) Journal Officiel (French) du 19 janvier 1980. Critères (8) NF EN 12824. February 1998. Microbiology of food and animal
microbiologiques auxquels doivent satisfaire certaines denrées feeding stuffs. Horizontal method for the detection of
animales ou d’origine animale. Méthodes générales d'analyse Salmonella.
bactériologique. (arrêté du 21 décembre 1979). (9) NF EN ISO 11290-2. August 1998. Microbiology of food and
(2) NF V 04-501. June 1990. Meat and meat products. animal feeding stuffs. Horizontal method for the detection
Microbiological analysis. Part 1: test sample. Initial suspension and enumeration of Listeria monocytogenes. Part 2:
and dilutions. Enumeration method.
(3) ISO 6579: 1993. Microbiology. General guidance on methods (10) NF EN ISO 6887-1: September 1999. Microbiology of food and
for the detection of Salmonella. animal feeding stuffs. Preparation of test samples, initial
(4) FIL-IDF 93B: 1995. Milk and milk products. Detection of suspension and decimal dilutions for microbiological
Salmonella. examination. Part 1: General rules for preparation of the
(5) ISO 6340 :1995. Water quality. Detection of Salmonella. initial suspension and decimal dilutions.
(6) FIL-IDF 122C: 1996. Milk and milk products. Preparation of (11) XP V08-062. Octobre 2000. Microbiologie des aliments.
samples and dilutions for microbiological examination. Méthode de dénombrement de Listeria monocytogenes.
(7) NF V 08-052. May 1997. Microbiology of food and animal Méthode de routine.
feeding stuffs. Detection of Salmonella. Routine method.
Buttiaux-Brogniart Hypersalted Broth

(double strength)
Target : Staphylococcus aureus.
Buttiaux-Brogniart Hypersalted Broth is a double strength Reconstitution : 192 g / L.
enrichment medium for the detection of Staphylococcus aureus in
biological samples and food products.
pH (25°C) : 7.4 ± 0.2.
History Supplement required : None.
Following work by Koch, Chapman formulated a selective media
which enabled the rapid development of Staphylococcus aureus, to
the practically total exclusion of other microorganisms. The Preparation
formulation of the hypersalted broth is derived from that developed
by Buttiaux and Brogniart, who used it successfully for the detection - Suspend 192.0 g of dehydrated medium
of pathogenic staphylococci in suspected foods and in feces. (BK081) in 1 liter of distilled or deionized
water.
Principles - Slowly bring to boiling, stirring until
- Selectivity is based on the resistance of staphylococci to a sodium - Dispense 10 mL in 20 x 200 mm tubes.
chloride concentration of 75 g per liter. Other bacteria are strongly - Sterilize in an autoclave at 121°C for
inhibited in this hypertonic medium. 15 minutes.
- Nutrients which favor growth are supplied by the peptones
and by lactose.
96
- Rapidly cool to 25°C.
For 1 liter of medium : - Transfer 10 mL of inoculum to each tube.
- Tryptone 20 g - Homogenize the sample with the medium,
- Meat extract 6 g avoiding the formation of air bubbles.
- Lactose 15 g - Incubate the tubes 24 to 48 hours at 37°C.
- Sodium chloride 150 g
- Bacteriological agar 1 g NOTE
The medium can also be used single strength in
pH of the ready-to-use medium at 25°C : 7.4 ± 0.2. compliance with AFNOR standard NF V59-105.
Results
Quality Control
Using the cultures obtained, inoculate
Baird-Parker Agar (BM018) by streaking.
homogeneous.
- Prepared medium : semi-fluid medium, slightly amber.
Staphylococcus aureus ATCC 25923
®
good
Staphylococcus aureus ATCC 6538P good

- Media in tubes : 6 months at 2-8°C
- Regenerate the medium by heating for 15 minutes at 100°C at the
time of use.
Packaging
Dehydrated medium :
Bibliography
(1) Buttiaux, R., et Brogniart, R.S. 1947. Techniques d’isolement des (2) NF V 59-105: October 1982. Edible gelatin. Detection of
staphylocoques pathogènes. Identification des staphylocoques Staphylococcus aureus.
entérotoxiques. Ann. Inst. Pasteur Paris, 73: 830.
97
Cetrimide Agar
Intended Use
Product Profile
Cetrimide Agar is a selective medium for the isolation and Target : Pseudomonas aeruginosa.
enumeration of Pseudomonas aeruginosa in biological, Reconstitution : 46.7 g / L.
pharmaceutical and cosmetic products. Its composition is that of the Autonomy (500 g) : 713 plates.
United States Pharmacopoeia.
pH (25°C) : 7.2 ± 0.2.
History Supplement required : Glycerol.
The formula of this medium was derived from the King A medium,
favoring pyocyanin production by Pseudomonas aeruginosa. Preparation
In 1951, Lowbury recommended the use of cetrimide in a selective
medium for the isolation of Pseudomonas. The concentration of the - Suspend 46.7 g of dehydrated base
inhibitor was reduced by Lowbury and Collins (1955) as a result of medium (BK049) in 1 liter of distilled or
its improved purity. deionized water.
- Add 10 mL of glycerol.
- Cetrimide (cetyltrimethylammonium bromide) is a quaternary - Dispense in tubes or flasks.
ammonium compound which inhibits a large number of bacteria - Sterilize in an autoclave at 121°C for
including species of Pseudomonas other than Pseudomonas 15 minutes.
aeruginosa.
- The production of pyocyanin (a blue, non-fluorescent pigment Instructions for Use
soluble in water and chloroform) is stimulated by magnesium
chloride and potassium sulfate. - Cool and maintain the medium at 47°C.
- The medium also favors the production of fluorescent pigments - Pour into sterile Petri dishes.
(pyoverdins) by some strains of Pseudomonas aeruginosa. - Let solidify on a cold surface.
- Most Pseudomonas aeruginosa can be identified by the - Dry in an incubator with the covers
characteristic grapelike odor of aminoacetophenone. partially removed.
- Transfer 0.1 mL of the product and its
Typical Composition serial tenfold dilutions to the plates
(can be adjusted to obtain optimal performance) containing the medium.
- Spread the inoculum on the surface with a
For 1 liter of medium : sterile triangle.
- Pancreatic digest of gelatin 20.0 g - Incubate at 37°C for 48 hours.
- Cetrimide 0.3 g
- Magnesium chloride 1.4 g Results
- Potassium sulfate 10.0 g
- Bacteriological agar 15.0 g The following are suspected as positive :
- colonies with a characteristic blue or blue-
pH of the ready-to-use medium at 25°C : 7.2 ± 0.2. green pigmentation surrounding the
colonies and which fluoresces under
Quality Control ultraviolet light at 254 nm ;
- grayish mucous colonies, pigmented or not.
- Dehydrated medium : cream-white powder, free-flowing and The presence of pyocyanin can be
homogeneous. confirmed by extracting with chloroform.
- Complete prepared medium : whitish agar, may contain a slight Pseudomonas aeruginosa typically produces
precipitate after autoclaving. both pyocyanin and fluorescein.
- Typical culture response after 24-48 hours of incubation at 37°C : Occasionally, strains of Klebsiella,
Enterobacter, Citrobacter, Proteus,
Microorganisms Growth Providencia, Alcaligenes and Aeromonas
Pseudomonas aeruginosa ATCC
®
10145 good to excellent may also grow, causing a slight yellowing of
Escherichia coli ATCC 25922 inhibited the medium. This color is easily
Proteus vulgaris ATCC 13315 inhibited differentiated from fluorescein production,
since the former does not fluoresce. Isolated
colonies are subjected to the following
three confirmation tests :
- Detection of oxidase with the Kovacs
reagent (1% solution of tetramethyl-p-
phenylenediamine hydrochloride).
98
Storage / Shelf Life - Pyocyanin production on King A medium.
- Growth at 42°C in Tryptone-Soy Broth
Dehydrated base medium : 2-30°C. (BK046).
- The expiration date is indicated on the label. A Gram-negative bacillus growing on
Prepared medium (benchmark value) : Cetrimide Agar is considered to be
- Base media in vials : 6 months at 2-8°C. Pseudomonas aeruginosa when a typical
- Complete media in plates : 1 month at 2-8°C, shielded from light. colony can be characterized with the
following tests :
Packaging - Oxidase : positive
- Pyocyanin : positive
Dehydrated base medium : - Growth at 42°C : positive
Bibliography
(1) Lowbury, E.J.L., and Collins, A.G., 1955. The use of a new (4) United States Pharmacopeia 24. 2000. Microbial Limit Tests:
cetrimide product in a selective medium for Pseudomonas 1814 – 1818.
aeruginosa., J. Clin. Pathol., 8: 47. (5) Pharmacopée européenne. Addendum 2000. Contrôle
(2) Brown, V.I., and Lowbury, E.J.L. 1965. Use of an improved microbiologique des produits non stériles (Recherche de
Cetrimide Agar Medium and of culture methods for microorganismes spécifiés). Solution et milieux de culture
Pseudomonas aeruginosa. J. Clin. Pathol.,18: 752. recommandés, 56 - 61.
(3) Ministère de l’Agriculture (French). Commission XXX.
Cosmétologie. Recherche de Pseudomonas aeruginosa dans les
produits cosmétiques.
CFC Agar
Intended Use
CFC Agar is a selective medium for the isolation and enumeration of

pigmented and non-pigmented Pseudomonas that contaminate
meats and meat-based products during their cold storage.
History
The formula of the base medium is a modification of King A

medium, in which magnesium chloride and potassium sulfate favor
pyocyanin production. CFC Supplement was developed by Mead and
Adams in 1976 in order to enable the selective development of most
psychrophilic Pseudomonas that contaminate poultry products.
Principles
- Pancreatic digest of gelatin and Tryptone are the nutrient Pseudomonas aeruginosa
substrates required for the rapid multiplication of Pseudomonas.
- The production of pyocyanin (a blue, non-fluorescent pigment,
soluble in water and in chloroform) is stimulated by magnesium Product Profile
chloride and potassium sulfate. Target : Pseudomonas.
- The cephalosporin concentration inhibits most contaminating Reconstitution : 49.4 g / L.
bacteria, in particular enterobacteria, staphylococci and
streptococci. Autonomy (500 g) : 675 plates.
- Fucidin inhibits the development of Acinetobacter / Moraxella pH (25°C) : 7.2 ± 0.2.
without affecting the growth of Pseudomonas. Supplement required : CFC Selective
- Contaminating yeasts are inhibited by cetrimide. Supplement.
- The cephalosporin, fucidin and cetrimide are added just
before use.
99
Typical Composition of complete medium Preparation
- Suspend 49.4 g of dehydrated base
For 1 liter of medium : medium (BK118) in 1 liter of distilled or
- Pancreatic digest of gelatin 16.0 g deionized water.
- Tryptone 10.0 g - Slowly bring to boiling, stirring until
- Potassium sulfate 10.0 g complete dissolution.
- Magnesium chloride 1.4 g - Dispense 100 mL in flasks.
- Bacteriological agar 12.0 g - Sterilize in an autoclave at 121°C for
- Cetrimide 10 mg 15 minutes.
- Fucidin 10 mg
- Cephalosporin 50 mg Instructions for Use
pH of the ready to use medium at 25°C : 7.2 ± 0.2. - Melt the medium (if prepared in advance).
Quality Control - Aseptically add 1 mL of reconstituted CFC
Selective Supplement (BS022) to each flask.
- Dehydrated base medium : cream-white powder, free-flowing - Homogenize thoroughly.
and homogeneous. - Transfer 1 mL of the product to analyze
- Prepared medium (complete) : whitish, agar. and its serial tenfold dilutions to sterile
- Typical culture response on complete medium after 72 hours of Petri dishes.
incubation at 25°C : - Pour 15 mL of complete medium.
- Mix well.
Microorganisms Growth - Let solidify on a cold surface.
Pseudomonas aeruginosa ATCC® 9027 good to excellent - Incubate at 22°C for 48 and 72 hours.
Pseudomonas aeruginosa ATCC 27853 good to excellent
NOTE
Pseudomonas putida ATCC 12633 good It is equally possible to inoculate on the surface
Proteus vulgaris ATCC 13315 partially inhibited of prepared plates.
Results
Storage / Shelf Life Only count plates containing fewer than

150 colonies. Pigmented or fluorescent
Dehydrated base medium : 2-30°C. colonies can be considered as presumptively
- The expiration date is indicated on the label. belonging to Pseudomonas.
Prepared medium (benchmark value) : The results must be confirmed by additional
- Base media in flasks : 6 months at 2-8°C. appropriate tests such as : Gram-staining,
- Complete media in plates : 8 days at 2-8°C, shielded from light. mobility, pigment production, oxidase
CFC Selective Supplement : reaction.
Packaging
Dehydrated base medium

(without cetrimide, fucidin and cephalosporin) :
CFC Selective Supplement :
Bibliography
(1) Barnes, E.M., and Thornley, M.I. 1966. the spoilage flora of (3) ISO 13720. December 1995. Meat and meat products -
eviscerated chickens stored at different temperatures. J. Food. Enumeration of Pseudomonas spp.
Technol., 1: 113-119. (4) NF V 04-504: April 1998. Microbiology of food and animal
(2) Mead, G.C., and Adams, B.W. 1977. A selective medium for the feeding stuffs. Enumeration of Pseudomonas spp. in meat and
rapid isolation of pseudomonas associated with poultry meat meat products.
spoilage. Br. Poult. Sci., 18: 661-670. (5) ISO/WD 13720-2000. Microbiology of food and animal
feeding stuffs. Horizontal method for the emuneration
of Pseudomonas spp.
100
Chloramphenicol Glucose Agar
Chloramphenicol Glucose Agar is destined for the detection and Target : Yeasts, Molds.
enumeration of yeasts and molds in dairy and other food products. Reconstitution : 40.1 g / L.
Autonomy (500 g) : 831 plates.
Principles
pH (25°C) : 6.6 ± 0.2.
- Yeast extract and glucose favor the growth of yeasts and molds. Supplement required : Gentamicin
- The presence of chloramphenicol, a heat-stable antibiotic, inhibits Selective Supplement.
the growth of contaminating bacteria. Sterilization : 121°C / 15 minutes.
- Gentamicin, added extemporaneously, increases selectivity
particularly when analyzing meat and raw seafood products, often Preparation
contaminated with Gram-positive bacteria.
Typical Composition of media without Gentamicin (BK007) in 1 liter of distilled or deionized
(can be adjusted to obtain optimal performance) water.
For 1 liter of medium : complete dissolution.
- Yeast extract 5.0 g - Dispense at 100 mL per flask.
- Glucose 20.0 g - Sterilize in an autoclave at 121°C for
- Chloramphenicol 0.1 g 15 minutes.
- With the ready-to-use base media BM021
pH of the ready-to-use medium at 25°C : 6.6 ± 0.2. (or if the media is prepared in advance
from the dehydrated product), melt the
Quality Control agar for the minimum amount of time
necessary to achieve total liquefaction.
- Dehydrated medium : beige powder, free-flowing and
homogeneous. Instructions for Use
- Prepared medium : amber agar.
- Typical culture response after 72 hours of incubation at 25°C : - Cool and maintain at 47°C.
- If necessary, add the reconstituted
Microorganisms Growth Gentamicin Selective Supplement (BS009)
Saccharomyces cerevisiae ATCC® 9763 good to excellent according to the protocol being followed.
Aspergillus niger ATCC 16404 good to excellent Consult the monograph concerning this
Escherichia coli ATCC 25922 inhibited product for more information.
Storage / Shelf Life - Dry in an incubator with the covers
partially removed.
Dehydrated medium (without Gentamicin) : 2-30°C. - Transfer 0.1 mL of the sample to analyze
- The expiration date is indicated on the label. or its serial tenfold dilutions onto the
Prepared medium (benchmark value) : plates.
- Media in vials : 6 months at 2-8°C. - Spread the inoculum on the surface with a
- Media in plates :1 month at 2-8°C. sterile triangle.
Ready-to-use media in vials, - Incubate at 25°C for 3 to 5 days.
Gentamicin selective supplement :
- Store between 2-8°C, shielded from light. Results
Separately count yeasts and molds. Carry out
Packaging a confirmation test under the microscope on
each type of colony encountered.
Ready-to-use media (without Gentamicin) :
- 10 x 100 mL vials : BM02108
Dehydrated media (without Gentamicin) :
101
Bibliography
(1) NF V 04-015: February 1984. Dried milk and sweetened (5) ISO 6611. 1992. Milk and milk products. Enumeration of colony
condensed milk. Microbiology. forming units of yeasts and/or moulds. Colony-count
(2) NF ISO 7954: August 1988. Microbiology. General guidance for technique at 25°C.
enumeration of yeasts and moulds. Colony count technique at 25°C. (6) XP V08-059. November 1995. Microbiology of food and animal
(3) FIL-IDF 94 B: 1991. Milk and milk products. Yeasts and feeding stuffs. Enumeration of yeasts and moulds by colony-
moulds. Colony-count technique at 25°C. count technique at 25°C. Routine method.
(4) NF ISO 7698: August 1991. Cereals, pulses and derived
products. Enumeration of bacteria, yeasts and moulds.
CLED Agar
Escherichia coli
Intended Use
CLED Agar (Cystine Lactose Electrolyte Deficient) is used for the

isolation, enumeration and differentiation of urinary
microorganisms.
History
In 1960, Sandys investigated a means for preventing the invasion of

plates by Proteus by the use of an electrolyte-deficient medium, in
order to favor the observation of colonies formed by other
microorganisms. A subsequent modification by Mackey and Sandys
involved the addition of cystine to favor the growth of coliform
bacteria. This new formulation, applied to urinary bacteriology, was
successfully used in the manufacture of agar slides for immersion in
samples.
Principles Proteus vulgaris Staphylococcus aureus
- Lactose fermentation in acid medium is detected when the pH Product Profile

indicator (bromthymol blue) turns from green to yellow. Target : Urinary microorganisms.
- Cystine favors the growth of coliform bacteria, usually forming Reconstitution : 36.1 g / L.
small colonies on other media.
- The electrolyte deficiency reduces invasion by Proteus.
pH (25°C) : 7.3 ± 0.2.
Typical Composition Supplement required : None.
- Pancreatic digest of gelatin 4g Preparation
- Tryptone 4g
- Meat extract 3g - Suspend 36.1 g of dehydrated medium
- L-cystine 128 mg (BK020) in 1 liter of distilled or deionized
- Lactose 10 g water.
- Bromthymol blue 20 mg - Slowly bring to boiling, stirring until
- Bacteriological agar 15 g complete dissolution.
pH of the ready-to-use medium at 25°C : 7.3 ± 0.2. - Sterilize in an autoclave at 115°C for
20 minutes.
102
Quality Control Instructions for Use
- Dehydrated medium : beige powder, free-flowing and - Cool and maintain the medium at 47°C.
homogeneous. - Pour into sterile Petri dishes.
- Prepared medium : bluish-green agar. - Let solidify on a cold surface.
- Typical culture response after 24-48 hours of incubation at 37°C : - Dry in an incubator with the covers
partially removed.
Microorganisms Growth Characteristics - Inoculate.
Escherichia coli ATCC 11775
®
good to excellent yellow colonies with - Incubate at 37°C for 18 hours.
a dark center
Results
Enterobacter aerogenes ATCC 13048 good to excellent yellowish colonies
Proteus vulgaris ATCC 13315 good to excellent blue translucent colonies The appearance of various bacterial colonies
without invasion are as follows :
Enterococcus faecalis ATCC 29212 good opaque pale yellow
colonies - Large golden yellow colonies surrounded
by a yellow halo :
Staphylococcus aureus ATCC 25923 good small dark yellow colonies Escherichia coli, Citrobacter
- Large viscous golden yellow colonies
Storage / Shelf Life surrounded by a yellow halo :
Enterobacter, Klebsiella
Dehydrated medium : 2-30°C. - Large transparent colonies surrounded by
- The expiration date is indicated on the label. a blue halo :
Prepared medium (benchmark value) : Proteus, Serratia
- Media in vials : 6 months at 2-8°C. - Large green colonies with a brownish
- Media in plates : 1 month at 2-8°C. center, surrounded by a blue halo :
Pseudomonas
Packaging - Small, opaque, pale yellow colonies :
streptococci
Dehydrated medium : - Very small, opaque, yellow colonies :
- 500 g bottle : BK020HA staphylococci
- 5 kg drum : BK020GC - Small gray colonies :
corynebacteria
The presumptive diagnosis must be

confirmed by a complete identification in
an appropriate medium.
Bibliography
(1) Sandys, G.H. 1960. A new method of preventing swarming of (4) Bevis, T.D. 1968. A modified electrolyte deficient culture
Proteus spp. with a description of a new medium suitable for use medium. J. Med. Technol., 25: 38.
in routine laboratory practice. J. Med. Lab. Technol., 17: 224. (5) Dixon, J.M.S., and Clark, M.A. 1968. Experience with agar-
(2) Mackey, J.P., and Sandys, G.H. 1965. Laboratory diagnosis of filled spoons for the transport of specimens of urine. Can. Med.
infections of the urinary tract in general practice by means of Assoc. J., 99(15): 741.
a dip-inoculum transport medium. Br. Med. J., 2: 1286. (6) Benner, E.J. 1970. Simple disposable method for quantitative
(3) Mackey, J.P., and Sandys, G.H. 1966. Diagnosis of urinary tract cultures of urine. Appl. Microbiol., 19 (3): 409.
infections. Br. Med. J., 1: 1173.
103
Columbia Agar
Intended Use
Columbia Agar is a highly nutritive medium used for the growth

and isolation of a large variety of microorganisms, particularly very
fastidious bacteria : streptococci and pneumococci in samples. When
blood, selective agents or growth accelerators are added, it
becomes possible to prepare a wide variety of media adapted to
specific uses.
History
Developed by Ellner in 1966, Columbia Agar enables luxuriant

colonies, perfectly defined hemolytic zones and well characterized
colonies and pigmentation to be obtained.
Principles
Streptococcus pyogenes
- Peptones included in the composition of the medium favor the
excellent growth of colonies.
- Yeast extract is a source of vitamin B complex. Product Profile
- Starch is a detoxifying agent and also an energy source. Target : streptococci, pneumococci.
- Defibrinated sheep blood, which can be added to the medium, Reconstitution : 42.5 g / L.
favors the detection of hemolytic reactions and supplies X factor
(heme) required for the growth of a large number of bacteria, but
lacks V factor (nicotinamide adenine dinucleotide) due to the pH (25°C) : 7.3 ± 0.2.
presence of an NADase, which destroys any NAD present. Supplement required : Defibrinated sheep/
Haemophilus influenzae, which requires both X and V factors, does horse blood (depending on use).
not grow on agar containing ordinary blood.
- The following media can be prepared with Columbia base :
1 - Blood agar Preparation

By adding 5 or 10% sterile sheep blood after autoclaving and
cooling, the medium is suitable for the growth of Streptococcus, - Suspend 42.5 g of dehydrated medium
Pneumococcus, Staphylococcus, Listeria and Erysipelothrix. It can be (BK019) in 1 liter of distilled or deionized
made selective by adding colistin and nalidixic acid to preclude the water.
development of Gram-negative bacteria and Bacillus. - Slowly bring to boiling, stirring until
2 - Chocolate agar - Dispense in tubes or flasks.
By adding 10% blood to sterile Columbia Agar and heating at 80°C - Sterilize in an autoclave at 121°C for
until a chocolate color develops, an excellent medium is obtained for 15 minutes.
the growth of Haemophilus influenzae and Neisseria species. Sheep
blood does not favor the growth of most Haemophilus strains. Instructions for Use
3 - Base without enrichment - Melt the medium (if prepared in advance).

Columbia Agar can be used to grow Brucella abortus, Yersinia pestis - Cool and maintain at 47°C.
and Clostridium perfringens, as well as all enterobacteria. - Add the supplements required for the
growth of the bacteria to be detected
Typical Composition (sterile defibrinated horse or sheep blood,
(can be adjusted to obtain optimal performance) growth accelerators, selective agents).
- Homogenize well.
For 1 liter of medium : - Pour into sterile Petri dishes.
- Polypeptone 17.0 g - Let solidify on a cold surface.
- Pancreatic digest of heart 3.0 g - Dry the plates with the covers partially
- Yeast extract 3.0 g removed.
- Corn starch 1.0 g - Inoculate.
- Sodium chloride 5.0 g - Incubate at 37°C for 24 to 48 hours in the
- Bacteriological agar 13.5 g optimal culture conditions of the bacteria
inoculated.
104
Quality Control

homogeneous.
- Prepared medium (with 5% defibrinated sheep blood) : opaque
red agar.
- Typical culture response after 24-48 hours of incubation at 37°C
with 5% sheep blood :
Microorganisms Growth Type of hemolysis

Streptococcus pyogenes ATCC® 19615 good to excellent beta
Streptococcus pneumoniae ATCC 6303 good to excellent alpha
Staphylococcus aureus ATCC 25923 excellent beta / gamma
Escherichia coli ATCC 25922 excellent

- Media in plates (with sheep blood) : 1 month at 2-8°C.
Packaging
Dehydrated medium :
Bibliography
(1) Neter, E. 1947. The effect of yeast concentrate on the growth (4) NF V 08-405: December 1986. Microbiology of food products.
and survival of Haemophilus influenza in infusion broth. J. Preserves. Detection of Clostridium thermophilus.
Bact., 54: 70-71. (5) NF ISO 10272. January 1996. Microbiology of food and animal
(2) Thayer, J.D., and Martin, J.E. 1966. Improved medium feeding stuffs. Horizontal method for detection of
selective for cultivation of Neisseria gonorrhoeae and Neisseria thermotolerant Campylobacter.
meningitidis. Publ. Health Rep., 81: 559-562. (6) Pharmacopée européenne. Addendum 2000. Contrôle
(3) Ellner, P.D. , Stoessel, C.I., Drake Ford, E., and Vasi, F. 1966. A microbiologique des produits non stériles (Recherche de
new culture medium for medical bacteriology. Amer. J. Clin. microorganismes spécifiés). Solution et milieux de culture
Path., 45: 502-504. recommandés, 56-61.
105
Columbia CNA Agar

Columbia Agar containing nalidixic acid and colistin is a selective Target : Gram-positive cocci.
base medium. When 5% sheep blood is added, it is used to detect, Reconstitution : 43.0 g / L.
isolate and determine the hemolytic characteristics of Gram-positive Autonomy (500 g) : 775 plates.
cocci in biological samples.
pH (25°C) : 7.3 ± 0.2.
History Supplement required : Defibrinated
sheep blood.
Ellner et al. found that after adding colistin and nalidixic acid in Sterilization : 121°C / 15 minutes.
Columbia Agar enriched with 5% sheep blood, the resulting
medium was favorable for the growth of staphylococci, enterococci
Preparation
and hemolytic streptococci, while Proteus, Klebsiella and Bacillus
were inhibited.
Principles (BK075) in 1 liter of distilled or deionized
water.
- Peptones included in the composition favor the excellent growth - Slowly bring to boiling, stirring until
of colonies. complete dissolution.
- Yeast extract is a source of vitamin B complex. - Dispense in tubes or flasks.
- Starch is a detoxifying agent and also an energy source. - Sterilize in an autoclave at 121°C for
- Defibrinated sheep blood added to the medium favors the 15 minutes.
detection of hemolytic reactions and supplies X factor (heme) - Do not overheat the medium.
required for the growth of bacteria, but lacks V factor
(nicotinamide adenine dinucleotide) because of the presence of an Instructions for Use
NADase, which destroys any NAD present.
- The selectivity of the medium is due to the simultaneous presence - Cool and maintain at 47°C.
of colistin and nalidixic acid. Colistin ruptures the cell membranes - Add 5% sterile defibrinated sheep blood.
of Gram-negative bacteria. Nalidixic acid blocks DNA replication of - Pour into sterile Petri dishes.
bacteria sensitive to this antibacterial agent. - Let solidify on a cold surface.
- Dry the plates with the covers partially
Typical Composition removed.
(can be adjusted to obtain optimal performance) - Inoculate.
- Polypeptone 17 g Results
- Pancreatic digest of heart 3g
- Yeast extract 3g The colonies have the following
- Corn starch 1g appearance :
- Sodium chloride 5g
- Colistin 10 mg - Small white or gray beta hemolytic
- Nalidixic acid 15 mg colonies :
- Bacteriological agar 14 g Streptococcus pyogenes
- Small white or gray alpha hemolytic
pH of the ready-to-use medium at 25°C : 7.3 ± 0.2. colonies :
Streptococcus pneumoniae
Quality Control
- Grayish-blue beta or alpha hemolytic
colonies :
Enterococci
homogeneous.
- Large, white to yellow colonies, with
- Medium prepared with 5% defibrinated sheep blood : opaque
or without hemolysis :
red agar.
- Typical culture response after 24-48 hours of incubation at 37°C, Staphylococci
with 5% sheep blood : - Grayish-blue beta hemolytic colonies :
Listeria monocytogenes
Microorganisms Growth Type of hemolysis
Streptococcus pyogenes ATCC 19615
®
excellent beta
Streptococcus pneumoniae ATCC 6303 good to excellent alpha
Staphylococcus aureus ATCC 25923 excellent beta / gamma
Bacillus subtilis ATCC 6633 inhibited
106

- Media in plates (with sheep blood) : 1 month at 2-8°C.
Packaging
Dehydrated medium :
Bibliography
(1) Ellner, P.D., Stossel, C.I., Drakeford, E., and Vasi, F. 1966. A (2) Morello, J.A., and Ellner, P.D. 1969. A new medium for blood
new culture medium for medical bacteriology. Am. J. Clin. cultures. Appl. Microbiol., 17: 68.
Pathol., 45: 502.
Cooked Meat Medium

Target : Anaerobic microorganisms,
Cooked Meat Medium is recommended for the culture,
pathogenic Clostridia.
maintenance and storage of anaerobic microorganisms, in
particular pathogenic strains of Clostridium. Reconstitution : 1,25 g / 10 mL tube.
History
pH (25°C) : 7.2 ± 0.2.
Confirming the initial results of Smith, Tarozzi recognized the Supplement required : None.
usefulness of an autoclaved meat medium for the culture of Sterilization : 121°C / 15 minutes.
anaerobic bacteria. In 1916, following the work of Von Hibler on a
brain based medium, Robertson developed the use of beef heart Preparation
for the culture of anaerobes isolated from infected wounds. Henry
recommended boiled meat medium for differentiating between - Add 1.25 g of dehydrated granules (BK076)
proteolytic clostridia and those hydrolyzing sucrose. to a tube containing 10 mL of distilled or
deionized water.
Principles - Wait 15 minutes so that the particles are
well hydrated and form a suspension.
- Solid particles of beef confer detoxifying properties on the - Sterilize in an autoclave at 121°C for
medium and maintain the redox potential favorable for culturing 15 minutes.
anaerobes. This muscle tissue is rich in glutathione and sulfhydryl
groups, which are reducing substances that favor the growth of Instructions for Use
obligate anaerobes.
- Polypeptone and glucose are nutritive elements permitting - After cooling to 25°C, the supernatant
growth. broth should be clear.
- Sodium chloride maintains the osmotic balance. - The medium is used immediately after
- The addition of glucose (5 g/L), hemin (5 mg/L), vitamin K1 preparation. Otherwise, it must be
(1 mg/L) and yeast extract (5 g/L) to the basal medium enable regenerated for 15 minutes at 100°C in
particularly fastidious bacteria to develop. order to restore conditions of anaerobiosis.
- When growing obligate anaerobes, pour a
Typical Composition layer of sterile liquid paraffin on the
(can be adjusted to obtain optimal performance) surface or incubate the tube in an
anaerobiosis jar.
For 1 liter of medium : - After inoculating, incubate the tubes at
- Beef heart 98 g 37°C for 2 to 7 days depending on the
- Polypeptone 20 g bacteria to be cultivated.
- Glucose 2 g
- Sodium chloride 5 g
107
- Dehydrated medium : brownish granules. Bacteria hydrolyzing sucrose rapidly

- Prepared medium : amber medium, with a sedimented acidify the medium and produce gas
brownish insoluble mass and a clear supernatant. without digesting the meat (acid odor
- Typical culture response after 48 hours of incubation at 37°C : with reddening). Proteolytic strains
degrade the meat down to amino acids
Microorganisms Growth (tyrosine may precipitate as white
Clostridium perfringens ATCC® 13124 good to excellent crystals) causing a blackening and a
putrid odor due to the sulfur
Clostridium sporogenes ATCC 11437 good to excellent
compounds produced.

Packaging
Dehydrated medium :
Bibliography
(1) von Hibler, E. 1899. Beiträge zur Kenntnis der durch anaerobe (3) Quagliaro, D.A. 1977. An improved Cooked Meat Medium for
Spaltpilze erzeugten Infektions-krankheiten der Tiere und des the detection of Clostridium botulinum. J. Assoc. Anal. Chem.,
Menschen etc. Centr. Bakt., 25: 513-594,631. 60: 563-569.
(2) Tarozzi, G. 1905. Über ein leicht in aerober Weise ausführbares
Kulturmittel von einigen bis jetzt füu strenge Anaeroben
gehaltenen Keimen. Zentralb. Bakt., 38: 619-624.
Count-Tact Agar

Target : Total microorganisms.
Count-Tact Agar is used to enumerate microorganisms by the direct
application of the agar to the surfaces to test. The medium is Reconstitution : 52.2 g / L.
derived from Tryptone-Soy Agar and contains 4 neutralizers that Autonomy (500 g) : 505 plates
inactivate most disinfectants that may be present in trace quantities (19 mL by plate).
after cleaning. This combination facilitates the development of
pH (25°C) : 7.3 ± 0.2.
residual viable microorganisms. The use of this simplified technique
is a good verification of the state of cleanliness of equipment after Supplement required : None.
cleaning and disinfection. It can also be used to determine the Sterilization : 121°C / 15 minutes.
bacterial load on the hands and fingers of personnel.
Preparation
Principles
- The combination of Tryptone and soy peptone leads to an optimal (BK130) in 1 liter of distilled or deionized
growth due to the synergy between the proteins supplied by water.
casein and the carbohydrates supplied by soybeans. - Slowly bring to boiling, stirring until
- Sodium chloride maintains the osmotic balance. complete dissolution.
- The lecithin-polysorbate-histidine-thiosulfate combination - Dispense in tubes or flasks.
neutralizes most disinfectants. - Sterilize in an autoclave at 121°C for
- Polysorbate neutralizes hexachlorophene and phenols. 15 minutes.
- Lecithin neutralizes chlorhexidin and phenol.
- Lecithin and polysorbate combination inhibits quaternary
ammonium salts.
- Sodium thiosulfate neutralizes halogen derivatives.
108
For 1 liter of medium : - Pour the agar into sterile, 50 to 70 mm
- Tryptone 15.0 g diameter cross-ruled Petri dishes.
- Papaic digest of soybean meal 5.0 g - The quantity of medium poured should
- Sodium chloride 5.0 g create a well-formed meniscus.
- Lecithin 0.7 g - Let solidify on a cold surface, under
- Polysorbate (Tween 80) 5.0 g laminar flow and put the cover back on
- Sodium thiosulfate, 5H2O 0.5 g the plates.
- L-histidine 1.0 g - In order to evaluate the microbial
- Bacteriological agar 20.0 g contamination, apply the agar directly to
the surface to test, without excessive
pH of the ready to use medium at 25°C : 7.3 ± 0.2. pressure and without moving the plate in
order to preserve the integrity of the
Quality Control meniscus.
- Incubate at 30 or 37°C for 24 or 48 hours,
- Dehydrated medium : cream-white powder, homogeneous and depending on the protocol employed.
slightly clumpy.
- Prepared medium : amber agar. Results
- Typical culture response after 48 hours of incubation
at 37 or 30°C (1) : Enumerate the colonies. The cross-ruling on
the plates facilitates counting.
Escherichia coli ATCC® 8739 good to excellent
Staphylococcus aureus ATCC 6538 P good to excellent
Aspergillus niger (1) ATCC 16404 good to excellent

Packaging
Dehydrated medium :
Bibliography
(1) Rozier, J., et Pantaléon, J. 1969. Méthode simple et rapide (3) Drouin, P., et Toux J.Y. 1985. Méthode bactériologique pour
d'appréciation des flores microbiennes de surface. Bull. Acad. apprécier la désinfection des poulaillers. Bull. d'Inf. Station
Vet., XII: 119-125. Exp. d'Aviculture de Ploufragan, 25: 176-178.
(2) Desbordes, J. 1977. Biodégradation microbienne des (4) Singer, S. 1987. The Use of Preservative Neutralizers in
antiseptiques et conservateurs. Revue de l'Institut Pasteur de Diluents and Plating Media. Cosmetics and Toiletries, 102:
Lyon, 10 (4): 291-311. 55-60.
109
DCLS Agar

Target : Salmonella, Shigella.
Leifson's Desoxycholate Citrate Lactose Sucrose (DCLS) Agar is a
selective medium for the isolation of Salmonella and Shigella from Reconstitution : 58.0 g / L.
biological samples, dairy and other food products. Autonomy (500 g) : 575 plates.
pH (25°C) : 7.3 ± 0.2.
History
Leifson originally developed this medium by the use of chemically Sterilization : Heat to boiling.
defined products (citrates and sodium desoxycholate), enabling him Do not autoclave.
to better control its selectivity. The current modified formula is one
of the numerous variants of Desoxycholate Citrate Agar. It contains a Preparation
high concentration of inhibiting substances and differs from
Desoxycholate Citrate Lactose Agar by the inclusion of sucrose. - Suspend 58.0 g of dehydrated medium
Principles water.
- Gram-positive bacteria are inhibited by desoxycholate and by the complete dissolution.
sodium and iron citrates. - Do not autoclave.
- Coliform bacteria are generally inhibited, although some non-
inhibited strains may ferment sucrose more rapidly than lactose, Instructions for Use
which leads to the elimination of false positives for Salmonella.
The acidification resulting from the fermentation of sugars causes - Cool and maintain the medium at 47°C.
the precipitation of bile derivatives around the colonies and the - Pour into sterile Petri dishes.
red coloration of the pH indicator, neutral red. - Let solidify on a cold surface.
- Lactose-negative bacteria form colorless or very slightly pink - Dry in an incubator with the covers
colonies. partially removed.
- By reducing ferric ammonium citrate, bacteria producing H2S are - Inoculate by streaking from enrichment
revealed by a blackening due to the production of iron sulfide media used for the detection of Salmonella.
in the center of the colonies. - In parallel, transfer the inoculum to
another less selective medium :
Typical Composition MacConkey Agar (BK050) or Hektoen
(can be adjusted to obtained optimal performance) Enteric Agar (BK067).
- Incubate at 37°C for 24 and 48 hours.
- Lactose 5.0 g The colonies have the following appearance :
- Sucrose 5.0 g
- Sodium desoxycholate 5.0 g - Colorless colonies, with or without a black
- Sodium citrate 8.5 g center (depending on whether or not
- Sodium thiosulfate 8.5 g hydrogen sulfide is produced) :
- Ferric ammonium citrate 1.0 g Salmonella, Proteus mirabilis
- Neutral red 25 mg - Colorless or slightly pink colonies :
- Bacteriological agar 15.0 g Shigella, Salmonella
- Red colonies :
Bacteria fermenting lactose and/or sucrose.
Quality Control Suspicious colonies are transferred to Kligler
Iron Agar (BK034) or TSI Agar (BK059) for
- Dehydrated medium : pink-beige powder, free-flowing and subsequent identification.
homogeneous.
- Prepared medium : orange-red agar.

Salmonella Enteritidis ATCC® 13076 good to excellent colorless colonies with
a black center
Salmonella Typhimurium ATCC 14028 good to excellent colorless to pink
colonies
Escherichia coli ATCC 25922 partially inhibited red colonies
110

- Media in plates : 8 days at 2-8°C.
Packaging
Dehydrated medium :
Bibliography
(1) Leifson, E. 1935. New culture media based on sodium (3) NF EN 12824. February 1998. Microbiology of food and animal
desoxycholate for the isolation of intestinal pathogens and for feeding stuffs. Horizontal method for the detection of
the enumeration of colon bacilli in milk and water. J. Pathol. Salmonella.
Bacteriol., 40: 581.
(2) NF V 08-052. May 1997. Microbiology of food and animal
Desoxycholate Citrate Agar (DCA)

Target : Pathogenic enterobacteria,
Desoxycholate Citrate Agar is a selective medium for the isolation
Salmonella, Shigella.
and differentiation of pathogenic enterobacteria in biological
samples, water, dairy, and other food products. Reconstitution : 73.0 g / L.
History
pH (25°C) : 7.3 ± 0.2.
Leifson originally developed this medium by the use of chemically Supplement required : None.
defined products (citrates and sodium desoxycholate), enabling him Sterilization : Heat to boiling.
to better control its selectivity. The current modified formula is one Do not autoclave.
of the numerous variants of Desoxycholate Citrate Agar. It contains a
high concentration of inhibiting substances. Preparation

- Gram-positive bacteria are inhibited by desoxycholate and by the water.
sodium and iron citrates. - Slowly bring to boiling, stirring until
- The selectivity of the medium enables large inoculum to be used complete dissolution.
without fear that Salmonella or Shigella colonies will be masked - Do not autoclave.
by accompanying flora.
- Non-inhibited coliform bacteria ferment lactose. The resulting Instructions for Use
acidification causes precipitation of bile derivatives around the
colonies and the red coloration of pH indicator, neutral red. - Cool and maintain the medium at 47°C.
- Lactose-negative bacteria form colorless colonies.
- By reducing ferric ammonium citrate, bacteria producing H2S are
revealed by a blackening due to the production of iron sulfide in
partially removed.
the center of the colonies.
- Inoculate by streaking from enrichment
media used for the detection of
Salmonella.
- In parallel, transfer the inoculum to
another less selective medium :
MacConkey Agar (BK050), Hektoen Enteric
Agar (BK067), etc.
111
Typical Composition Results
The colonies have the following appearance :
- Meat infusion 10 g - Colorless colonies, with or without a black
- Peptic digest of meat 10 g center (depending on whether or not
- Lactose 10 g hydrogen sulfide is produced) :
- Sodium citrate 20 g Salmonella, Proteus mirabilis
- Sodium desoxycholate 5g - Colorless or slightly pink colonies :
- Ferric ammonium citrate 1g Shigella
- Neutral red 20 mg - Red colonies surrounded by a halo of
- Bacteriological agar 17 g precipitated bile salt :
Lactose fermenters
pH of the ready-to-use medium at 25°C : 7.3 ± 0.2. (Escherichia coli, Citrobacter….)
Quality Control Suspicious colonies are transferred to Kligler

Iron Agar (BK034) or TSI Agar (BK059) for
- Dehydrated medium : beige to pink-beige powder, free-flowing subsequent identification.
and homogeneous.
- Prepared medium : orange agar.

Salmonella Typhimurium ATCC® 14028 good to excellent colorless colonies with
a black center
Salmonella Enteritidis ATCC 13076 good to excellent colorless colonies
Shigella flexneri ATCC 29903 good colorless colonies
Escherichia coli ATCC 25922 partially inhibited pink colonies with a bile
precipitate at the edges

Packaging
Dehydrated medium :
Bibliography
(1) Leifson, E. 1935. New culture media based on sodium (3) Pharmacopée européenne. Addendum 2000. Contrôle
desoxycholate for the isolation of intestinal pathogens and for microbiologique des produits non stériles (recherché de
the enumeration of colon bacilli in milk and water. J. Pathol. microorganismes spécifiés). Solution et milieux de culture
Bacteriol., 40: 581-599. recommandés, 56-61.
(2) Hynes, M. 1942. The isolation of intestinal pathogens by
selective media. J. Path. Bact., 54: 193-207.
112
Desoxycholate Lactose Agar
Intended Use
Desoxycholate Lactose Agar is a selective medium for the

enumeration of coliform bacteria in water, milk, dairy and other
food products. The medium is also used for the differentiation and
isolation of enterobacteria from biological samples.
History
In 1935, Leifson developed the formulation of desoxycholate lactose

as a differentiation medium for enterobacteria. In contrast to prior
formulas using ingredients of unknown or variable composition, this
medium contained well defined chemical substances, such as
desoxycholate and sodium citrate, used as inhibitors. The degree of
inhibition was thus well controlled. The original formulation of
Leifson was slightly modified in terms of the use of better defined
peptones, as well as by non-negligible adjustments of the inhibitory Escherichia coli
substances.

Target : Enterobacteria, coliforms.
- Gram-positive bacteria are inhibited by desoxycholate, although Reconstitution : 42.5 g / L.
sodium citrate is also an effective inhibitor.
- The differentiation of enterobacteria is based on their capacity to
(16 mL per plate).
ferment lactose :
- Lactose-positive strains produce acid which leads to the pH (25°C) : 7.1 ± 0.2.
formation of red colonies in the presence of neutral red. Supplement required : None.
- Lactose-negative bacteria yield colorless colonies
(Salmonella or Shigella).
Do not autoclave.
Typical Composition
(can be adjusted to obtain optimal performance) Preparation

water.
- Lactose 10.00 g
- Sodium desoxycholate 0.50 g complete dissolution.
- Sodium chloride 5.00 g - Do not autoclave.
- Sodium citrate 2.00 g
- Neutral red 0.03 g Instructions for Use
pH of the ready-to-use medium at 25°C : 7.1 ± 0.2. - Transfer 1 mL of the product to analyze
and its serial tenfold dilutions into sterile
Quality Control Petri dishes.
- Pour in 12 mL of medium.
- Dehydrated medium : pinkish powder, free-flowing and - Homogenize by swirling.
homogeneous. - Let solidify on a cold surface.
- Prepared medium : reddish agar. - Overlay by pouring in an additional 4 mL
- Typical culture response after 24 hours of incubation at 30°C : of medium, so that a second layer is
formed.
Microorganisms Growth Characteristics - Let solidify.
Escherichia coli ATCC® 25922 good to excellent violet-red colonies with - Incubate at 30 or 37°C for 18 to 24 hours,
precipitated bile according to the experimental protocol
being used.
Citrobacter freundii ATCC 8090 good to excellent violet-red colonies with
precipitated bile
Enterobacter aerogenes ATCC 13048 good to excellent red colonies Results
Characteristic colonies are those which are
Staphylococcus aureus ATCC 25923 inhibited red and with a diameter equal to or greater
than 0.5 mm after 24 hours of incubation.
113

- Use on the day of preparation.
Packaging
Dehydrated medium
Bibliography
(1) Leifson, E. 1935. New culture media based on sodium (3) Journal Officiel (French) du 17 février 1985. Méthodes
desoxycholate for the isolation of intestinal pathogens and for d'analyses des laits pasteurisés (arrêté du 03 janvier 1985).
the enumeration of colon bacilli in milk and water. J. Pathol.
(2) American Public Health Association. 1960 (11th Ed). Standard
Methods for the examination of Dairy Products.
Desoxycholate (0.1%) Lactose Agar

Target : Coliforms, Shigella.
Desoxycholate (0.1%) Lactose Agar is a selective medium for the
enumeration of coliform bacteria in milk, dairy products and other Reconstitution : 45.0 g / L.
food products. The medium is also recommended for the isolation Autonomy (500 g) : 695 plates.
and culture of Shigella. (16 mL by plate).
pH (25°C) : 7.3 ± 0.2.
History
In 1935, Leifson developed the formulation of desoxycholate lactose Sterilization : Heat to boiling.
as a differentiation medium for enterobacteria. In contrast to prior Do not autoclave.
formulas using ingredients of unknown or variable composition, this
medium contained well defined chemical substances, such as Preparation
desoxycholate and sodium citrate, used as inhibitors. The degree of
inhibition was thus well controlled. The original formulation of (BK062) in 1 liter of distilled or deionized
Leifson was slightly modified by the use of better defined peptones, water.
as well as by non-negligible adjustments of the inhibitory substances. - Slowly bring to boiling, stirring until
Principles - Do not autoclave.
- Gram-positive bacteria are primarily inhibited by desoxycholate, Instructions for Use

although sodium citrate and ferric citrate are also effective
inhibitors. - Cool and maintain the medium at 47°C.
- The differentiation of enterobacteria is based on their capacity to - Transfer 1 mL of the product to analyze
ferment lactose : and its serial tenfold dilutions into sterile
- Lactose-positive strains produce acid which leads to the Petri dishes.
formation of red colonies in the presence of neutral red.
- Homogenize by swirling.
- Lactose-negative bacteria (Salmonella or Shigella) yield colorless
colonies.
- Overlay by pouring in an additional 4 mL
of medium, so that a second layer is
formed.
- Let solidify.
- Incubate at 30 or 37°C for 18 to 24 hours.
114
Lactose-positive enterobacteria form red
For 1 liter of medium : colonies with a diameter equal to or greater
- Peptic digest of meat 10.00 g than 0.5 mm after 24 hours of incubation.
- Lactose 10.00 g Lactose-negative enterobacteria form
- Sodium desoxycholate 1.00 g colorless colonies.
- Neutral red 0.03 g
Quality Control
- Dehydrated medium : beige to pinkish powder, free-flowing

and homogeneous.
- Prepared medium : orange-red agar.

Escherichia coli ATCC® 25922 good to excellent violet-red colonies
Citrobacter freundii ATCC 8090 good violet-red colonies

- Use on the day of preparation.
Packaging
Dehydrated medium :
Bibliography
(1) Leifson, E. 1935. New culture media based on sodium

desoxycholate for the isolation of intestinal pathogens and for
the enumeration of colon bacilli in milk and water. J. Pathol.
115
Dextrose Tryptone Agar
Intended Use
Dextrose Tryptone Agar is used to enumerate spores of mesophilic

and thermophilic Bacillus (especially Bacillus stearothermophilus,
responsible for flat sour) in sugar, sweet desserts, spices, aromatic
preparations and other food products.
History
The medium was developed by the National Canners Association

laboratories for the enumeration of mesophilic and thermophilic
aerobic bacteria in the sugar used in canned food.
Principles
- Soluble starch, a protective agent, favors spore germination. Bacillus stearothermophilus

- Bacteria which acidify the medium by metabolizing glucose result
in the pH indicator, bromcresol purple, turning yellow.
- Non-acidifying colonies are blue. Product Profile
Target : Bacillus spores,
Typical Composition Bacillus stearothermophilus.
(can be adjusted to obtain optimal performance) Reconstitution : 32.0 g / L.
For 1 liter of medium : Autonomy (500 g) : 1041 plates.

- Tryptone 10 g pH (25°C) : 7.0 ± 0.2.
- Glucose 5g Supplement required : None.
- Soluble starch 2g
- Bromcresol purple 40 mg Sterilization : 121°C / 15 minutes.
- Bacteriological agar 15 g
Preparation
Quality Control (BK042) in 1 liter of distilled or deionized
water.
- Dehydrated medium : beige green powder, free-flowing and - Slowly bring to boiling, stirring until
homogeneous. complete dissolution.
- Prepared medium : violet agar. - Dispense in tubes or flasks.
- Typical culture response after 48 hours of incubation : - Sterilize in an autoclave at 121°C for
15 minutes.
Microorganisms Growth Characteristics Instructions for Use
Bacillus stearothermophilus ATCC® 10149 good at 55°C yellow colonies
Bacillus subtilis ATCC 6633 good at 37°C yellow colonies - Heat the product to test in order to
destroy vegetative cells and activate spores.
- Transfer the inoculum to sterile Petri dishes.
Storage / Shelf Life - Pour in 15 mL of medium maintained at 47°C.
Dehydrated medium : 2-30°C. - Let solidify on a cold surface.
- The expiration date is indicated on the label. - Incubate :
Prepared medium (benchmark value) : - at 30°C for 5 days to enumerate
- Media in tubes or vials : 6 months at 2-8°C. mesophilic Bacillus spores ;
- at 55°C for 5 days to enumerate
Packaging thermophilic Bacillus spores after first
pouring several drops of sterile
Dehydrated medium :
paraffin oil in the cover of the plates
to obtain a tight seal.
Bibliography Results
(1) National Canners Association. 1933. Bacterial Standards for Sugar. For mesophilic and thermophilic Bacillus,
count separately the acidifying (yellow) and
non-acidifying (blue) colonies.
116
Drigalski Lactose Agar

Drigalski Lactose Agar is used for the selective isolation of Target : Enterobacteria (urine, feces).
enterobacteria from urine and stool samples. Bacteria are Reconstitution : 49.1 g / L.
differentiated on the basis of whether or not they can ferment lactose. Autonomy (500 g) : 678 plates.
Principles pH (25°C) : 7.4 ± 0.2.

- Crystal violet and sodium desoxycholate inhibit the development Sterilization : 115°C / 20 minutes.
of Gram-positive bacteria.
- Lactose fermentation leads to the production of acid, which make
Preparation
bromthymol blue turn yellow.
- This medium inhibits invasion by Proteus only partially. If their
presence is suspected, deposit 1 or 2 drops of alcohol in the cover
of the Petri dish just before inoculating. Alcohol vapors limit
water.
invasion but have no effect on the growth of enterobacteria.
Typical Composition
20 minutes.
- Tryptone 15 g
- Meat extract 3g
- Yeast extract 3g
- Sodium desoxycholate 1g
- Sodium thiosulfate 1g
- Lactose 15 g
- Crystal violet 5 mg
partially removed.
- Bromthymol blue 80 mg
- Inoculate.
pH of the ready-to-use medium at 25°C : 7.4 ± 0.2. NOTE
If simply lactose fermentation is sought using a
Quality Control previously purified culture, dispense as slants.
- Dehydrated medium : beige to slightly greenish powder, free- Results

flowing and homogeneous.
- Prepared medium : blue-green agar. Lactose-positive bacteria (Escherichia coli,
- Typical culture response after 24 hours of incubation at 37°C : Klebsiella, Enterobacter) form yellow
colonies.
Microorganisms Growth Characteristics Lactose-negative bacteria (Salmonella,
Escherichia coli ATCC® 25922 good to excellent yellow colonies Shigella, Proteus, Providencia,
Pseudomonas) form blue to blue-green
Salmonella Enteritidis ATCC 13076 good to excellent blue colonies
colonies.
Shigella sonnei ATCC 29930 good blue-green colonies
Proteus vulgaris ATCC 13315 good blue-green colonies

Packaging
Dehydrated medium
117
EE Broth (acc. to Mossel)

EE Broth proposed by Mossel is a selective enrichment medium for Target : Enterobacteria.
the detection of enterobacteriaceae in pharmaceutical and food Reconstitution : 43.5 g / L.
products. Autonomy (500 g) : 1150 tubes.
History pH (25°C) : 7.2 ± 0.2.

The original formula of the medium was developed by Mossel, Sterilization : 121°C / 15 minutes.
Visser and Cornelissen in 1963. Glucose favors the growth of
Enterobacteriaceae, in particular Shigella and Salmonella, both
Preparation
lactose-negative and lactose-positive strains. The technique of
enrichment in liquid medium demonstrating bacterial growth rather
than gas production, leads to the detection of enterobacteria that
would not systematically produce gas in other selective liquid
water.
media.
- Dispense in tubes at 10 mL per tube.
Principles
15 minutes.
- The simultaneous presence of ox bile and brilliant green results in
the inhibition of almost all Gram-positive bacteria and Gram-
negative bacteria other than coliforms.
- Pancreatic digest of gelatin and glucose are the nitrogen and
energy sources, respectively, for the development of
- Transfer 1 mL of inoculum from a pre-
enterobacteria.
enrichment medium : Buffered Peptone
- Sodium and potassium phosphates buffer the medium in order to
Water (BK018, BK131) or Lactose Broth
increase its recovery capacity.
(BK082) to a tube containing 10 mL of
medium.
Typical Composition
- Incubate for 18 to 24 hours at 37°C.
- Carry out isolation on a selective medium
such as VRBG Agar (BK011), inoculating
with a loop.
- Use the characteristic colonies obtained to
conduct biochemical confirmation tests
- Glucose 5.00 g
after purification of the cultures.
- Brilliant green 15 mg
Quality Control
- Dehydrated medium : beige to greenish powder, free-flowing

and homogeneous.
- Prepared medium : green solution, limpid.
- Typical culture response after 18-24 hours of incubation at 37°C,
followed by subculture on Violet Red Bile Glucose Agar :

Escherichia coli ATCC 25922 good to excellent
®
red-violet colonies
surrounded by a violet halo
Citrobacter freundii ATCC 8090 good to excellent pink-violet colonies
Salmonella Typhimurium ATCC 14028 good to excellent pink-violet colonies
118

- Media in tubes : 3 months at 2-8°C, shielded from light.
Packaging
Dehydrated media :
Bibliography
(1) Mossel, D.A.A., Visser, M., and Cornelissen, A.M.R. 1963. The (3) NF ISO 7402: December 1993. Microbiology. General guidance
examination of foods for Enterobacteriaceae using a test of the for the enumeration of Enterobacteriaceae without
type generally adopted for the detection of salmonellae. Jour. resuscitation. MPN technique and colony count technique.
of Appl. Bact., 24: 444-452. (4) Pharmacopée européenne. Addendum 2000. Contrôle
(2) NF ISO 8523: February 1992. Microbiology. General guidance microbiologique des produits non stériles (Recherche de
for the detection of Enterobacteriaceae with pre-enrichment. microorganismes spécifiés). Solution et milieux de culture
Elliker Broth
Intended Use / History

Product Profile
Elliker Broth, prepared according to the formula of Elliker, Anderson Target : Lactobacilli, lactic streptococci.
and Hannesson, contains nutrients required for the growth of Reconstitution : 48.5 g / L.
lactobacilli and lactic streptococci (Lactococcus) in dairy products. Autonomy (500 g) : 1030 tubes.
Typical Composition pH (25°C) : 6.8 ± 0.2.

(can be adjusted to obtain optimal performance) Supplement required : None.
- Tryptone 20.0 g Preparation
- Gelatin 2.5 g - Suspend 48.5 g of dehydrated
- Lactose 5.0 g medium (BK054) in 1 liter of distilled
- Sucrose 5.0 g or deionized water.
- Glucose 5.0 g - Slowly bring to boiling, stirring until
- Sodium acetate 1.5 g complete dissolution.
- Sodium chloride 4.0 g - Dispense in tubes.
- Ascorbic acid 0.5 g - Sterilize in an autoclave at 121°C
for 15 minutes.
Quality Control
- Inoculate tubes with 1 mL of the product
- Dehydrated medium : cream-white powder, free-flowing and to analyze and its serial tenfold dilutions.
homogeneous. - Incubate for 3 to 5 days at 37°C.
- Prepared medium : semi-solid medium, amber, very slightly
gelatinous, clear. Results
Carry out enumeration with the most
Microorganisms Growth probable number technique. In addition, a
Lactococcus lactis subsp. lactis ATCC® 11454 good to excellent rapid microscopic confirmation of the
Lactobacillus casei subsp. rhamnosus ATCC 7469 good to excellent bacteria grown is recommended.
119

Packaging
Dehydrated medium :
Bibliography
(1) McLauglin, C.B. 1946. Readily prepared medium for cultivation (3) Hausler, W.J. Ed. 1976. Standard Methods for the Examination
of lactobacilli. J. Bacteriol., 51: 560. of Dairy Products, 14th Ed. Washington D.C. American Public
(2) Elliker, P.R., Anderson, A.W., and Hannesson, G. 1956. An agar Health Association.
culture medium for lactic acid streptococci and lactobacilli. J.
Dairy. Sci., 39: 1611.
EMB Agar (Levine)

Intended Use
EMB Agar, originally recommended by Levine, is used to isolate and

identify Escherichia coli and Enterobacter, as well as Gram-negative
intestinal bacteria in pharmaceutical products, dairy and other food
products. It is also used as an isolation and identification medium in
tests of water quality, after culture in liquid medium (presumptive
tests). EMB Agar is also used to identify Candida albicans.
History
In 1916, Holt-Harris and Teague used the combination of eosin and

Methylene blue to differentiate microorganisms as a function of
whether or not they could ferment lactose. Levine subsequently
modified the formula by removing sucrose and increasing the
lactose concentration, which led to the easy differentiation between
Escherichia coli and Enterobacter aerogenes. Escherichia coli
Principles
Product Profile
- Eosin Y and Methylene blue have low selective capacities, since Target : Escherichia coli, Enterobacter,
they only partially inhibit the development of Gram-positive Candida albicans.
bacteria such as enterococci. Reconstitution : 37.5 g / L.
- The dyes allow to the differentiation between lactose-positive and
lactose-negative bacteria. Coliform strains form violet to brown
colonies, while salmonellae are colorless, transparent or amber. pH (25°C) : 7.0 ± 0.2.
Supplement required : Chlortetracycline
chlorydrate (for Candida albicans).
120
- Pancreatic digest of gelatin 10.0 g water.
- Lactose 10.0 g - Slowly bring to boiling, stirring until
- Dipotassium phosphate 2.0 g complete dissolution.
- Eosin Y 0.4 g - Dispense in tubes or flasks.
- Methylene blue 65 mg - Sterilize in an autoclave at 121°C for
pH of the ready-to-use medium at 25°C : 7.0 ± 0.2. Instructions for Use
Quality Control - Cool and maintain the medium at 47°C.

- Mix well to oxidize the methylene blue
- Dehydrated medium : violet powder, free-flowing and and insure the homogeneous suspension
homogeneous. of the precipitate.
- Prepared medium : claret agar, which may contain a slight - Pour into sterile Petri dishes.
precipitate after autoclaving. - Let solidify on a cold surface.
- Typical culture response after 24 hours of incubation at 37°C : - Dry in an incubator with the covers
partially removed.
Microorganisms Growth Characteristics - Inoculate by streaking.
®
good to excellent violet colonies with
greenish metallic sheen NOTE
Enterobacter aerogenes ATCC 13048 good to excellent violet-pink colonies For the culture of Candida albicans, add 100 mg
of chlortetracycline hydrochloride per liter of
Salmonella Typhimurium ATCC 14028 good to excellent colorless colonies medium before pouring in plates : the medium
Pseudomonas aeruginosa ATCC 27853 good colorless colonies will subsequently turn blue. Inoculated plates are
incubated for 24 to 48 hours at 37°C in an
Saccharomyces cerevisiae ATCC 9763 partially inhibited cream colonies anaerobiosis jar in a 10% CO2 atmosphere.
Results
Colonies have the following appearance :
- Dark violet colonies, convex, low
confluence, 2-3 mm in diameter with a black
center reaching more than 3/4 of the
- Media in plates : 1 month at 2-8°C, shielded from light.
diameter and which exhibit a greenish
metallic sheen in reflected light :
Packaging
Escherichia coli
Dehydrated medium : - Bluish flattened colonies, relatively
- 500 g bottle : BK056HA confluent, 4-6 mm in diameter with a dark
brown center, occasionally with
a metallic sheen :
Enterobacter aerogenes
- Violet colonies with slight metallic sheen :
Citrobacter
- Brownish mucous colonies :
Klebsiella
- Transparent amber colonies :
Salmonella and Shigella
- Cobweb or feather-shaped colonies :
Candida albicans
Bibliography
(1) Holt-Harris, J.E., and Teague, O. 1916. A new culture medium for the (4) Weld, J.T. 1952. Candida albicans. Rapide identification in pure
isolation of Bacillus typhosa from stools. J. Infect. Dis., 18: 596. cultures with carbon dioxyde on modified eosin methylene
(2) Levine, M. 1918. Differentiation of Escherichia coli and blue medium. Arch. Dermat. Syph., 66: 691-694.
Aerobacter aerogenes on a simplified eosin-methylene blue (5) Vogel, R.A., and Moses, M.R. 1957. Welds method for the rapid
agar. Jour. Inf. Dis., 23: 43-47. identification of Candida albicans in clinical materials.
(3) Levine, M. 1921. Bacteria fermenting lactose and the Am. J. Clin. Pathol., 28 (1): 103.
significance in water analysis. Bull Iowa State College. Eng. (6) United States Pharmacopoeia 24. 2000. Microbial Limit Tests,
Exp. Station. 1814-1818.
121
Endo Agar
Intended Use
Product Profile
Endo Agar is used to carry out confirmation tests for the detection Target : Coliforms, Thermotolerant coliforms.
and enumeration of coliform bacteria, after presumptive tests of Reconstitution : 41.5 g / L.
drinking water, as well as for the detection and isolation of coliform Autonomy (500 g) : 803 plates.
and fecal coliform bacteria in milk, dairy and other food products.
pH (25°C) : 7.5 ± 0.2.
In 1904, Endo worked on developing a bile salt-free medium
which could be used to differentiate enterobacteria as a function Preparation
of their capacity to ferment lactose. Gram-positive bacteria were
inhibited by sodium sulfite and basic fuchsin. Endo Agar was - Suspend 41.5 g of dehydrated medium
developed primarily to facilitate the isolation and identification (BK057) in 1 liter of distilled or deionized
of typhoid bacilli. water.
Principles complete dissolution.
- The selectivity of Endo Agar is due primarily to the simultaneous - Sterilize in an autoclave at 121°C for
presence of sodium sulfite and basic fuchsin which inhibit 15 minutes.
contaminating Gram-positive bacteria.
- In this medium, bacteria not fermenting lactose form colorless and Instructions for Use
shiny colonies, while coliform strains form red colonies. The color is
due to acetaldehyde produced which reacts with sulfited fuchsin - Cool and maintain the medium at 47°C.
to release fuchsin. The reaction is so intense in the case of - Homogenize thoroughly to disperse the
Escherichia coli that it imparts an iridescent green sheen to the precipitate.
colonies. - Pour into sterile Petri dishes.
Typical Composition - Dry in an incubator with the covers
(can be adjusted to obtain optimal performance) partially removed.
For 1 liter of medium : - Inoculate on the surface of the medium.
- Pancreatic digest of meat 10.0 g - Incubate at 37°C for 24 and 48 hours.
- Lactose 10.0 g
- Dipotassium phosphate 3.5 g Results
- Sodium sulfite 2.5 g
- Basic fuchsin 0.5 g Colonies have the following appearance :
- Pink to red colonies with green
pH of the ready-to-use medium at 25°C : 7.5 ± 0.2. metallic sheen :
Escherichia coli
Quality Control - Pink colonies :
Enterobacter aerogenes
- Dehydrated medium : violet powder, free-flowing and - Colorless colonies :
homogeneous. Proteus, Salmonella, Shigella,
- Prepared medium : orange-pink agar, may contain a slight Pseudomonas
precipitate after autoclaving.

Escherichia coli ATCC® 25922 good to excellent red colonies with
greenish metallic sheen
Enterobacter aerogenes ATCC 13048 good to excellent red colonies
Salmonella Typhimurium ATCC 14028 good colorless colonies
Shigella sonnei ATCC 29930 good colorless colonies
122

- As a result of the progressive penetration of oxygen into the
ready-to-use medium, sulfite is oxidized and progressively reddens
the medium, which becomes unusable.
Packaging
Dehydrated medium :
Bibliography
(1) Endo. 1904. Zentralbl. Bakteriol., Abt 1, orig., 35: 109. (3) Standard Methods for the Examination of Water and Wastewater.
(2) Standard Methods for the Examination of Dairy Products. 1967. 1975. Am. Public Health Association. Americ. Water Works
Am. Public Health Association N.Y., 12 th Ed. Association and Water Pollution Control Federation, 14th Ed.
Ethyl Violet Azide Broth

Ethyl Violet Azide Broth is used to carry out the confirmation test of Target : Intestinal enterococci.
the detection and enumeration of fecal streptococci (enterococci) in Reconstitution : 35.7 g / L.
drinking water and waste water, in frozen foods and other food Autonomy (500 g) : 1400 tubes.
products by the most probable number method. The procedure
involves two stages : pH (25°C) : 6.8 ± 0.2.
- presumptive test in Azide Dextrose Broth, Supplement required : None.
- confirmation in Ethyl Violet Azide Broth. Sterilization : 121°C / 15 minutes.
History
Preparation
Ethyl Violet Azide Broth was formulated according to the
recommendations of Litsky, Malmann and Fifield, who investigated - Dissolve 35.7 g of dehydrated medium
the action of a number of dyes and selective agents for the (BK061) in 1 liter of distilled or deionized
formulation of a confirmation medium for fecal streptococci. The water.
authors subsequently modified the original formula by reducing the - Stir slowly until complete dissolution.
glucose concentrations and increasing that of ethyl violet. The - Dispense in tubes at 10 mL per tube.
results indicated that the medium obtained was sufficiently specific - Sterilize in an autoclave at 121°C for
for enterococci and that the few strains of sporulated bacilli and 15 minutes.
Gram-positive cocci other than fecal streptococci which gave false
positives on Azide Dextrose Broth were inhibited by ethyl violet. Instructions for Use
Principles - Transfer one loop of a positive culture in

Azide Dextrose Broth to a tube of Ethyl
- The selectivity of the medium for enterococci is due to the Violet Azide Broth.
presence of ethyl violet and sodium azide, which inhibit - Incubate at 37°C for 24 and 48 hours.
the growth of Gram-negative bacilli and sporulated
Gram-positive species. Results
- Polypeptone and glucose supply the nutrient elements required
for the development of enterococci. The appearance of slight cloudiness and/or
- Phosphates buffer the medium and maintain the pH constant. the formation of a violet deposit at the
- Sodium chloride maintains the osmotic balance of the medium. bottom of the tube indicates the presence
of fecal streptococci. When the violet pellet
is deposited, the cloudiness of the medium
may become very slight.
Carry out enumeration with the most
123
Typical Composition
- Polypeptone 20.0 g
- Glucose 5.0 g
- Ethyl violet 0.5 mg
Quality Control

homogeneous.
- Prepared medium : amber solution, limpid.
Enterococcus faecalis ATCC® 33186 good to excellent
Enterococcus faecalis ATCC 29212 good to excellent
Streptococcus pyogenes ATCC 19615 inhibited
Bacillus subtilis ATCC 6633 inhibited

Packaging
Dehydrated medium :
Bibliography
(1) Litsky, W., Mallmann, W.L., and Fifield, C.W. 1953. A new (4) Rodier, J. 1984. L’analyse de l’eau. Dénombrement des
medium for the detection of enterococci in water. Am. J. streptocoques fécaux présumés (Méthode par ensemencement
Public Health, 43 (7): 873. en milieux liquides). Dunod 7ème Ed., 825-828.
(2) Litsky, W., Mallmann, W.L., and Fifield, C.W. 1955. Comparison (5) NF T 90-411: October 1989. Testing water. Detection and
of the most probable numbers of Escherichia coli and enumeration of group D streptococci. General method by
enterococci in river waters. Am. Jour. Public Health, culture in liquid media (MPN).
45 (2): 1049.
(3) Larkin, E.P., Litsky, W., and Fuller, J.E. 1955. Fecal
streptococci in frozen foods. I. A bacteriological survey
of some commercial frozen foods. App. Microbiol., 3: 98.
124
Eugon Agar

Recommended for the growth of a wide variety of microorganisms, Target : Total microorganisms, Lactobacilli.
Eugon Agar was especially designed for lactobacilli from meats and Reconstitution : 45.5 g / L.
other food products. After adding blood, it can be used to grow Autonomy (500 g) : 732 plates.
pathogenic molds (Nocardia, Blastomyces) and relatively fastidious
bacteria (Neisseria, Brucella). pH (25°C) : 7.0 ± 0.2.
Originally formulated by Vera in 1947, Harrison and Hansen

Preparation
successfully used this medium for the enumeration of intestinal
bacteria in turkeys. Frank also used it for enumerating the spores of
anaerobic bacteria.
water.
Principles
- As a result of its content of peptones, glucose and cystine, the
medium furnishes luxuriant (eugonic) and pigmented colonies.
- When used as the base for chocolate agar, it enables the growth
15 minutes.
of Neisseria gonorrhoea.
- Adding blood increases its nutritional capacities, but because of
the glucose content, Eugon Agar should not be used for the
detection of beta-type hemolysis.
- With the ready-to-use media (BM047) or if
the media has been prepared in advance
Typical Composition
from the dehydrated base, melt the
medium for the minimum amount of time
For 1 liter of medium : necessary to achieve total liquefaction.
- Tryptone 15.0 g - Cool and maintain the medium at 47°C.
- Papaic digest of soybean meal 5.0 g - Pour into sterile Petri dishes.
- Glucose 5.0 g - Let solidify on a cold surface.
- Sodium chloride 4.0 g - Dry in an incubator with the covers
- L-cystine 0.3 g partially removed.
- Sodium sulfite 0.2 g - Inoculate.
- Sodium citrate 1.0 g - Incubate at 37°C for 24 to 48 hours
- Bacteriological agar 15.0 g aerobically or anaerobically, depending on
the strain to be grown.
Quality Control

homogeneous.
- Typical culture response after 48 hours of incubation at 37 or 30°C (1) :
Lactobacillus plantarum ATCC® 8014 good
Aspergillus niger (1) ATCC 16404 good
125

Packaging
- 10 x 200 mL vials : BM04708
Dehydrated medium :
Bibliography
(1) Vera, H.D. 1947. The ability of peptones to support surface (3) Frank, H.A. 1955. The influence of various media on spore
growth of lactobacilli. J. Bacteriol., 54: 14. count determinations of a putrefactive anaerobe. J. Bacteriol.,
(2) Harrison, A.P., and Hansen, P.A. 1950. The bacterial flora of 70: 269.
the cecal feces of healthy turkeys. J. Bacteriol., 59: 197.
Eugon Broth

Target : Aerobic microorganisms.
Eugon Broth is used to obtain luxuriant cultures (eugonic) with a
wide variety of microorganisms including the most fastidious, with Reconstitution : 30.5 g / L.
yeasts and with molds. It is used for the detection and growth of Autonomy (500 g) : 1640 tubes.
lactic acid bacteria from meats and other food products. It is
pH (25°C) : 7.0 ± 0.2.
appropriate in industrial microbiology for antigen production.
Eugon Broth can also be used to detect microbial contamination in Supplement required : None.
packaging materials destined for food products, cosmetics and Sterilization : 121°C / 15 minutes.
pharmaceuticals.
Preparation
History
The medium was recommended by Vera for obtaining excellent
cultures of bacteria known to be difficult to grow, e.g. Neisseria,
water.
Haemophilus and Brucella.
Principles
- Eugon Broth is composed of a mixture of peptones, cystine,
15 minutes.
glucose and salts, which contribute to the growth of
microorganisms, whether or not they are fastidious.
- Inoculate the medium with purified

cultures or with other types of mixed
microflora inoculum.
- Incubate at the optimal required
temperature, aerobically or else in a CO2-
enriched atmosphere, depending on the
microorganisms to be cultured.
126
Growth results in turbidity, due to microbial
For 1 liter of medium : multiplication.
- Tryptone 15.0 g
- Papaic digest of soybean meal 5.0 g
- Glucose 5.0 g
- L-cystine 0.3 g
Quality Control

homogeneous.
- Typical culture response after 48 hours of incubation
at 30°C (1) or 37°C :
Neisseria meningitidis ATCC® 13090 good
Lactobacillus plantarum ATCC 8014 good to excellent
Aspergillus niger (1) ATCC 16404 good

Packaging
Dehydrated medium :
Bibliography
(1) Rosenthal, S.A., and Cox, C.D. 1933. The somatic antigens of (2) Vera, H.D. 1947. The ability of peptones to support surface
Corynebacterium michiganense and Corynebacterium insidiosum. growth of lactobacilli. J. Bacteriol., 54: 14.
J. Bacteriol., 65: 532.
127
Fraser Broth
Intended Use
Fraser Broth is used for the selective and differential enrichment of

Listeria monocytogenes in milk and other dairy products, food
products, as well as in other specimens which may contain it.
History
The complete medium, studied by Fraser et al. in 1988, is a

modification of the formulation of Donnelly and Baigent. The
composition of the base is identical to that of UVM II Broth and
was modified by the addition of lithium chloride as a selective
agent and of ferric ammonium citrate to visualize cultures that
hydrolyze esculin, by the resultant blackening of the medium.
Principles
Negative control Listeria monocytogenes
- The very good recovery of Listeria monocytogenes is assured by
the concentration differences in nalidixic acid and acriflavine Product Profile
between Half-Fraser and Fraser, as well as the two enrichment
Target : Listeria.
steps themselves.
- Polypeptone, yeast extract and meat extract furnish the nutrients Reconstitution : 54.9 g / L (BK115).
required for the growth of Listeria. 55.0 g / L (BK133).
- The high sodium chloride content increases the selectivity of the Autonomy (500 g) : 910 tubes.
medium. pH (25°C) : 7.2± 0.2.
- Phosphates buffer the pH of the medium.
- Esculin is hydrolyzed by Listeria to glucose and esculetin, the latter Supplement required : Ferric ammonium
compound forming a black complex with ferric ions supplied by citrate (for BK115).
Fraser Selective Supplement (for BK133).
ferric citrate, added just before use, which also favors the growth
of Listeria. Sterilization : 115°C / 15 minutes (for BK115).
- Lithium chloride inhibits the growth of most enterococci which 121°C / 15 minutes (for BK133).
can also hydrolyze esculin.
- Nalidixic acid blocks the DNA replication of bacteria sensitive to Preparation
this antibacterial agent.
- The growth of secondary Gram-positive microflora is inhibited by - Dissolve 54.9 g of the dehydrated base
acriflavine. medium (BK115) or 55.0 g of dehydrated
base medium Fraser Broth II (BK133) in
Typical Composition of complete medium 1 liter of distilled or deionized water.
- Dispense 10 mL in tubes.
- Polypeptone 10.00 g 15 minutes if Fraser Broth base BK115 is
- Yeast extract 5.00 g used, otherwise sterilize at 121°C for
- Meat extract 5.00 g 15 minutes if Fraser Broth base II (BK133)
- Sodium chloride 20.00 g is chosen.
- Disodium phosphate, anhydrous 9.60 g
- Monopotassium phosphate 1.35 g Instructions for Use
- Esculin 1.00 g
- Lithium chloride 3.00 g - Cool the medium to 25°C.
- Nalidixic acid 20 mg - To each tube of Fraser broth base BK115,
- Acriflavine 25 mg aseptically add 0.1 mL of a sterile 5%
- Ferric ammonium citrate 0.50 g solution of ferric ammonium citrate, or
0.1 mL if reconstituted Fraser Selective
pH of the ready-to-use medium at 25°C : 7.2 ± 0.2. Supplement (BS031) if the Fraser Broth
base II is used.
- Using tubes prepared as above, or the
ready-to-use tubes (BM013), transfer
0.1 mL of the culture obtained from the
primary enrichment broth (Half-Fraser).
- Mix well.
- Incubate the tubes for 24 to 48 hours at
37°C.
128
- Dehydrated media : yellowish powder, free-flowing and Blackening of the cultures is an indication
homogeneous. of the presumptive presence of Listeria.
- Prepared media (complete) : amber yellow solution, with bluish tint. Some microbial strains hydrolyzing esculin
- Typical culture response after 24 hours of incubation at 37°C, (enterococci) may also cause the medium to
followed by subculture on PALCAM Agar : blacken. Inoculate all tubes (with or
without blackening) on a selective medium :
Microorganisms Growth Oxford Agar (BK110 + BS003 or BM019) or
Listeria monocytogenes ATCC® 19115 good to excellent (with blackening) PALCAM Agar (BK145 + BS004/BS049 or
BM020). Identify suspected colonies and
Listeria monocytogenes ATCC 35152 good to excellent (with blackening)
subject them to biochemical identification
Escherichia coli ATCC 25922 inhibited tests.
NOTE
Storage / Shelf Life A minimum period of 24 hours is necessary to
permit the visualization of the black color.
Dehydrated base media (BK115, BK133) : 2-30°C, shielded from light.
Prepared medium BK115 (benchmark value) :
- Base media : 15 days at 2-8°C, shielded from light.
- Complete media in tubes : 8 days at 2-8°C, shielded from light.
Prepared medium BK133 (benchmark value) :
- Base media : 6 months at 2-8°C, shielded from light.
- Complete media in tubes : 8 days at 2-8°C, shielded from light.
Ready-to-use media in tubes,
Fraser Selective Supplement :
Packaging
- 50 x 10 mL tubes : BM01308
Dehydrated Fraser Broth base
(without ferric ammonium citrate) :
Dehydrated Fraser Broth base II
(without ferric ammonium citrate, nalidixic acid or acriflavine) :
Fraser Selective Supplement :
Bibliography
(1) Donnelly, C.W., and Baigent, G.J. 1986. Method for flow (3) NF EN ISO 11290-1. February 1997. Microbiology of food and
cytometric detection of Listeria monocytogenes in milk. App. animal feeding stuffs. Horizontal method for the detection and
Environ. Microbiol., 52: 689-695. enumeration of Listeria monocytogenes. Part 1: Detection method.
(2) Fraser, J.A., and Sperber, W.H. 1988. Rapid detection of (4) NF V08-055. August 1997. Microbiology of food and animal
Listeria spp. in food and environmental samples by esculin feeding stuffs. Detection of Listeria monocytogenes. Routine
hydrolysis. J. Food. Prot., 51: 762-765. method.
129
GC Agar
GC (also called “Chocolate agar” when supplemented) Agar is a Target : Neisseria, Haemophilus.
basic medium which after supplementing with blood, hemoglobin, Reconstitution : 36.0 g / L.
and diverse selective and nutritive agents, is intended for the Autonomy (500 g) : 925 plates.
growth and isolation of Neisseria and Haemophilus. It is used in
Thayer-Martin, Martin-Lewis and "Transgrow" media as well as for pH (25°C) : 7.2 ± 0.2.
the growth and transport of Neisseria gonorrhoeae from clinical Supplement required : Defibrinated sheep /
samples. horse blood.
History
Johnston developed a chocolate agar in 1945 that enabled the Preparation

observation of Neisseria gonorrhoeae growth in 24 hours. In 1949,
Carpenter and Morton described a modification which incorporated 1) Chocolate Agar
hemoglobin and yeast concentrate, which proved particularly - Suspend 36.0 g of dehydrated base
appropriate for the growth of this microorganism. Subsequently, medium (BK051) in 1 liter of distilled or
different authors contributed the use of polyvitamin nutritional deionized water.
supplements with well defined composition, as well as varied - Slowly bring to boiling, stirring until
selective additives which led to a large number of formulations. complete dissolution.
- Dispense 100 mL in 150 mL flasks.
Principles - Sterilize in an autoclave at 121°C for
15 minutes.
- The basic medium contains a highly nutritive polypeptone, - Cool to 47°C.
phosphate buffer to maintain the pH constant and corn starch to - Add 5 to 10% sterile defibrinated horse or
neutralize toxic fatty acids which may be present in the agar. sheep blood.
- The addition of hemoglobin contributes a source of hemin - Homogenize thoroughly.
- Heat to 80°C with constant stirring.
(factor X), required for the growth of Haemophilus and favors
- Maintain at this temperature for 10 minutes
that of gonococci.
until the color becomes chocolate brown.
- The addition of a polyvitamin supplement containing factor V
- Cool to 47°C.
(nicotinamide-adenine dinucleotide) enables the growth of
- Aseptically add 1 mL of restored Thayer-
Haemophilus, while other constituents (vitamins, amino acids,
Martin Growth Supplement (BS010) and, if
coenzymes, glucose, ferric ions and other factors) favor the growth
necessary, an antimicrobic supplement.
of Neisseria.
Typical Composition of base media
partially removed.
2) Thayer-Martin Medium
For 1 liter of medium : - Suspend 7.2 g of dehydrated medium in
- Polypeptone 15 g 100 mL of distilled or deionized water.
- Corn starch 1 g - Slowly bring to boiling, stirring until
- Dipotassium phosphate 4 g complete dissolution.
- Monopotassium phosphate 1 g - Dispense in 250 mL flasks.
- Sodium chloride 5 g - Sterilize in an autoclave at 121°C for
- Bacteriological agar 10 g 15 minutes.
pH of the ready-to-use medium at 25°C : 7.2 ± 0.2. - Add 100 mL of a sterile 2% solution of
hemoglobin, as well as 2 mL of
Quality Control reconstituted Thayer-Martin growth
supplement (BS010) and a reconstituted
- Dehydrated medium : off-white powder, free-flowing and antimicrobic supplement.
homogeneous.
- Prepared medium (base) : amber agar. 3) Modified Thayer-Martin Medium
- Typical culture response after 24 hours of incubation at 37°C In modified Thayer-Martin Medium, nystatin
(with Thayer-Martin Growth Supplement) : is replaced by amphotericin B which is more
stable. Trimethoprim is added to avoid
Microorganisms Growth culture invasion by Proteus.
Haemophilus influenzae ATCC 10211
®
good to excellent
Neisseria gonorrhoeae ATCC 43069 good to excellent 4) “Transgrow” Medium
”Transgrow” Medium is prepared following
Neisseria meningitidis ATCC 13090 good to excellent the same protocol as Thayer-Martin
Streptococcus pneumoniae ATCC 6305 good to excellent Medium to which 10 g of agar for 1 liter of
base are added.
130
Storage / Shelf Life Instructions for Use
Dehydrated base medium : 2-30°C. 1) Isolation and identification of Neisseria

- The expiration date is indicated on the label. - After inoculation, incubate the plates at
Prepared medium (benchmark value) : 37°C in a humid atmosphere enriched with
- Base media in vials : 6 months at 2-8°C. CO2 for 24 to 48 hours.
- Complete media in plates : 1 month at 2-8°C. - Colonies of gonococci are small, gray and
Thayer-Martin Growth Supplement : translucent (opaque in 48 hours) with
- Store between 2-8°C, shielded from light. smooth or scalloped edges.
- The expiration date is indicated on the label. - Colonies of meningococci are visible after
24 hours and are opaque, whitish,
Packaging sometimes yellowish, and with irregular
edges.
Dehydrated base medium : - Identify the bacteria encountered.
Thayer-Martin Growth Supplement : 2) Isolation and identification of Haemophilus
- 10 vial pack : - After inoculation, incubate at 37°C in a
(5 vials of freeze-dried supplement + 5 vials of diluent) : BS01008 humid atmosphere enriched with CO2
for 24 hours.
- Colonies of Haemophilus influenzae are
smooth, grayish, translucent and reach a
diameter of 0.5 to 1 mm.
- Identify the bacteria encountered.
Bibliography
(1) Lankford, C.E. 1942. Proceedings of Local Branches of the (6) Thayer, J.D., and Martin, J.E. Jr. 1966. Improved medium
Society of American Bacteriologists, Texas Branch: Some selective for cultivation of Neisseria gonorrhoeae and Neisseria
aspects of nutritional variation of the gonococcus (Abstracts). meningitidis. Public Health Rep., 81 (6): 559.
J. Bacteriol., 44 (1):139. (7) Martin, J.E. Jr., Billings, T.E., Hackney, J.F., and Thayer, J.D.
(2) Lankford, C.E., Scott, V., Cox, M.F., and Cooke, W. R. 1943. 1967. Primary isolation of Neisseria gonorrhoeae with a new
Some aspects of nutritional variation of gonococcus. J. commercial medium. Public Health Rep., 82 (4): 361.
Bacteriol., 45 (4): 321. (8) Martin, J.E., Jr, and Lester, A. 1971. Transgrow, a medium for
(3) Lankford, C.E., and Swell, E.E. 1943. Glutamine as a growth transport and growth of Neisseria gonorrhoeae and Neisseria
factor for certain strains of Neisseria gonorrhoeae. J. meningitidis. HSMHA Health Service Rep., 86 (1): 30.
Bacteriol., 45 (4): 410. (9) Martin, J.E., Jr., and Lewis, J.S. 1977. Anisomycin: improved
(4) Johnston, J. 1945. Comparison of gonococcus cultures read at anti-mycotic activity in modified Thayer-Martin medium. Public
24 and 48 hours. J. Vener. Dis. Inform., 26 (11): 239. Health Rep., 35 (2): 53.
(5) Thayer, J.D., and Martin, J.E., Jr. 1964. A selective medium for
the cultivation of N. gonorrhoeae and N. meningitidis. Public
Health Rep., 79 (1): 49.
Giolitti and Cantoni Broth

Intended Use
Product Profile
Giolitti and Cantoni Broth is a single strength enrichment base for Target : Coagulase positive staphylococci.
the detection of coagulase positive staphylococci in food products. Reconstitution : 54.2 g / L.
History (19 mL by tube).
Mossel, Harrewijn and Elzebrock recommended the medium pH (25°C) : 6.9 ± 0.2.
formulated by Giolitti and Cantoni in 1966 for the detection of Supplement required : 1% potassium
Staphylococcus aureus in powdered milk and baby formulas. tellurite solution.
131
- Pyruvate, glycine and especially mannitol favor the development - Dissolve 54.2 g of dehydrated medium
of staphylococci. (BK080) in 1 liter of distilled or deionized
- Gram-negative bacteria are inhibited by lithium chloride and water.
Gram-positive strains by tellurite. - Stir slowly until complete dissolution.
- The anaerobic environment suppresses the growth of micrococci. - Dispense 19 mL into appropriate
- The development of staphylococci is shown by the appearance of a sized tubes.
black color due to the reduction of tellurite to metallic tellurium. - Sterilize in an autoclave at 115°C for
20 minutes.
Typical Composition of the base medium
(can be adjusted to obtained optimal performance) Instructions for Use
For 1 liter of medium : - De-gas the basic medium before use by

- Tryptone 10.0 g heating for 15 minutes at 100°C, if
- Meat extract 5.0 g prepared in advance.
- Yeast extract 5.0 g - Rapidly cool to 25°C.
- Glycine 1.2 g - Add to each tube 0.1 mL of 1% potassium
- Mannitol 20.0 g tellurite solution previously sterilized by
- Sodium pyruvate 3.0 g filtration.
- Sodium chloride 5.0 g - Transfer 1 mL of inoculum to each tube.
- Lithium chloride 5.0 g
- Mix well, avoiding trapping air bubbles.
- Cover the medium in each tube with 2%
sterile, melted agar (type E Agar) cooled
to 47°C so as to obtain an airtight seal.
Quality Control
- Dehydrated medium : light beige powder, free-flowing and
homogeneous. Results
- Typical culture response after 24-48 hours of incubation at 37°C : Positive tubes are blackened or have a black
precipitate. Inoculate the resulting cultures
Microorganisms Growth by streaking on Baird-Parker Agar with Egg
Yolk Tellurite (BK055 + BS001 or BM018).
Staphylococcus aureus ATCC® 25923 good with blackening
Staphylococcus aureus ATCC 6538P good with blackening
Dehydrated base medium (without potassium tellurite) : 2-30°C.

- Base media in tubes : 6 months at 2-8°C.
- Complete media in tubes (with tellurite) : use on the day of
preparation.
Packaging
Dehydrated base medium (without potassium tellurite) :

Bibliography
(1) Giolitti, G., and Cantoni, C. 1966. A medium for the isolation (2) FIL-IDF provisional 60C: 1997. Dried dairy products.
of staphylococci from foodstuffs. J. App. Bacteriol., Enumeration of Staphylococcus aureus. MPN procedure.
29: 395-398.
132
Half-Fraser Broth
Intended Use
Product Profile
Half-Fraser Broth is used for the selective and differential Target : Listeria.
enrichment of Listeria monocytogenes in milk and other dairy Reconstitution : 55.0 g / L.
products, food products, as well as in other specimens which may Autonomy (500 g) : 40 vials.
contain it.
pH (25°C) : 7.2 ± 0.2.
History Supplement required : Half-Fraser
Selective Supplement.
The medium studied by Fraser et al. in 1988 is a modification of the Sterilization : 121°C / 15 minutes.
formulation of Donnelly and Baigent. The composition of the base
is identical to that of UVM Broth and was modified by the addition
of lithium chloride as selective agent and of ferric ammonium Preparation
citrate to visualize cultures that hydrolyze esculin, by the resultant
blackening of the medium. - Dissolve 55.0 g of dehydrated Fraser base II
medium (BK133) in 1 liter of distilled or
Principles deionized water.
- The very good recovery of Listeria monocytogenes is assured by - Dispense in flasks at 225 mL per flask.
the concentration differences in nalidixic acid and acriflavine - Sterilize in an autoclave at 121°C for
between Half-Fraser and Fraser, as well as the two enrichment 15 minutes.
steps themselves. Half-Fraser Broth allows the primary enrichment - Cool the medium to 25°C.
step, with secondary enrichment being performed in Fraser Broth. - Aseptically add 2.25 mL of reconstituted
- Polypeptone, yeast extract and meat extract furnish the nutrients Half-Fraser Selective Supplement BS030 or
required for the growth of Listeria. 2.0 mL of reconstituted Selective
- The high sodium chloride content increases the selectivity of the Supplement BS032.
medium.
- Phosphates buffer the pH of the medium. Instructions for Use
- Esculin is hydrolyzed by Listeria to glucose and esculetin, the latter
compound forming a black complex with ferric ions supplied by Primary enrichment
ferric citrate, added just before use, which also favors the growth - To media prepared as above, or to ready-
of Listeria. to-use vials BM016, aseptically add 25 g of
- Lithium chloride inhibits the growth of most enterococci which can the product to test.
also hydrolyze esculin. - Mix well.
- Nalidixic acid blocks the DNA replication of bacteria sensitive to - Incubate at 30°C.
this antibacterial agent. - After 24 hours of incubation, carry out
- The growth of secondary Gram-positive microflora is inhibited by isolation on the appropriate selective
acriflavine. media : Oxford Agar (BK110 + BS003
or BM019) or PALCAM Agar
Typical Composition of complete medium (BK145 + BS004/BS049 or BM020).
(can be adjusted to obtain optimal performance) - In addition, transfer 0.1 mL of the
resulting culture to 10 mL of Fraser Broth
For 1 liter of medium : (BK133 + BS031, BK115, BM013).
- Yeast extract 5.00 g NOTE
In certain detection protocols only, primary
enrichment broth with no supplement added can
- Sodium chloride 20.00 g be used as a preenrichment medium for the
- Disodium phosphate, anhydrous 9.60 g revitalization of stressed microorganisms. Once
- Monopotassium phosphate 1.35 g the resuscitation period has elapsed, the selective
- Esculin 1.00 g supplement must be added and the medium
- Lithium chloride 3.00 g mixed before continuing the incubation.
- Nalidixic acid 10.0 mg
Secondary enrichment
- Acriflavine (chlorhydrate) 12.5 mg
- After inoculation, incubate Fraser Broth at
37°C.
- After 24 and 48 hours of incubation,
conduct selective isolations in selective
solid media using a platinum loop.
133
- Dehydrated medium : yellowish powder, free-flowing and Identify suspected colonies and subject
homogeneous. them to identification tests.
- Prepared medium (complete) : amber yellow solution, limpid.
- Typical culture response after 24 hours of incubation at 30°C, NOTE
followed by subculture on PALCAM Agar : All the tubes, with or without blackening, must
be transferred to selective media. A minimum
period of 24 hours is necessary to permit the
visualization of the black color. Suspected
Listeria monocytogenes ATCC® 19115 good to excellent (with blackening) samples must systematically be transferred to
Listeria monocytogenes ATCC 35152 good to excellent (with blackening) selective isolation media, even in the absence
of a color change.
Dehydrated base medium : 2-30°C, shielded from light.

- Base media in vials : 6 months at 2-8°C, shielded from light.
- Complete medium in vials : 8 days at 2-8°C, shielded from light.
Ready-to-use media in vials,
Selective supplements for Half-Fraser Broth :
Packaging
- 10 x 225 mL vials : BM01608
Dehydrated Fraser base II medium
(without ferric ammonium citrate, nalidixic acid or acriflavine) :
Half-Fraser Selective Supplements (for base BK133) :
- 10 vial pack (for 500 mL) : BS03008
- 8 vial pack (for 2.25 liters) : BS03208
Bibliography
(1) Donnelly, C.W., and Baigent, G.J. 1986. Method for flow (3) NF EN ISO 11290-1. February 1997. Microbiology of food and
cytometric detection of Listeria monocytogenes in milk. App. animal feeding stuffs. Horizontal method for the detection and
Environ. Microbiol., 52: 689-695. enumeration of Listeria monocytogenes. Part 1 : Detection
(2) Fraser, J.A., and Sperber, W.H. 1988. Rapid detection of method.
Listeria spp. in food and environmental samples by esculin (4) NF V08-055. August 1997. Microbiology of food and animal
hydrolysis. J. Food. Prot., 51: 762-765. feeding stuffs. Detection of Listeria monocytogenes. Routine
method.
134
Hektoen Enteric Agar
Intended Use
Hektoen Enteric Agar is a selective medium for the isolation and

differentiation of pathogenic enterobacteria from biological
samples, water samples, dairy products and other food products.
The medium is particularly suited for the culture of Shigella, and
prevents invasion by Proteus.
History
Hektoen Enteric Agar was formulated in 1967 by King and Metzger

at the Hektoen Institute in order to increase the isolation frequency
of Shigella and Salmonella species in comparison to that obtained
with other selective isolation media. Although this medium enabled
a wide variety of pathogenic enterobacteria to be isolated, it was
less inhibitory towards non-pathogenic enteric bacteria. The current
formulation differs from the original by the suppression of
desoxycholate and the reduced concentration of bile salts. In
parallel, the concentration of peptones was increased to offset the Product Profile
inhibitory effect of bile salts.
Target : Pathogenic enterobacteria,
Salmonella, Shigella.
Principles
- Gram-positive flora are inhibited by bile salts which can also Autonomy (500 g) : 444 plates.
slightly inhibit the growth of a few Gram-negative
pH (25°C) : 7.6 ± 0.2.
microorganisms.
- The medium contains three carbohydrates : lactose, sucrose and Supplement required : None.
salicin. The high lactose concentration favors the visualization of Sterilization : Heat to boiling.
enterobacteria by avoiding the problem of late fermentations. The Do not autoclave.
other carbohydrates were added to insure a higher degree of
differentiation and to reduce the toxicity caused by colored
Preparation
indicators so as to obtain excellent recovery of Shigella.
- In the presence of sodium thiosulfate, H2S producing bacteria
reduce ferric ammonium citrate and are detected by darkening
due to the production of iron sulfide at the center of the colonies.
water.
- The color indicator system, composed of bromthymol blue and acid
fuchsin, yields orange yellow colonies for lactose-positive
enterobacteria and blue-green colonies in the case of lactose-
- Do not autoclave.
negative strains.
Typical Composition
partially removed.
- Lactose 12.0 g
- Inoculate by streaking enrichment media
- Sucrose 12.0 g
used for the detection of Salmonella.
- Salicin 2.0 g
- Simultaneously inoculate another selective
- Bile salts 9.0 g
medium.
- Sodium thiosulfate 5.0 g
- Acid fuchsin 40 mg
135
- Dehydrated medium : beige powder, free-flowing and The principle for reading the results is based
homogeneous. on the possible fermentation of the three
- Prepared medium : dark green agar. carbohydrates in the medium (lactose,
- Typical culture response after 24 hours of incubation at 37°C : sucrose, salicin). Strains fermenting at least
one of them form salmon colored colonies,
Microorganisms Growth Characteristics others forming blue or green colonies.
Salmonella Typhimurium ATCC® 14028 excellent green colonies with In the presence of sodium thiosulfate,
a black center hydrogen sulfide-producing bacteria give
colonies with a black center after reacting
Salmonella Enteritidis ATCC 13076 excellent green colonies with
with ferric citrate.
a black center
Shigella flexneri ATCC 29903 excellent green colonies The colonies have the following appearance :
Escherichia coli ATCC 25922 partially inhibited salmon-orange colonies
- Salmon-yellow colonies :
Escherichia coli, Citrobacter, Klebsiella,
Staphylococcus aureus ATCC 25923 inhibited Enterobacter, Arizona, Serratia
- Salmon-yellow colonies with a black center :
Storage / Shelf Life Proteus vulgaris
- Green colonies with a black center :
Dehydrated medium : 2-30°C. Proteus mirabilis, Salmonella
- The expiration date is indicated on the label. - Green or bluish colonies :
Prepared medium (benchmark value) : Shigella, Salmonella, Providencia, Proteus
- Media in plates : 8 days at 2-8°C. morganii, Proteus rettgeri
- Small blue or brownish colonies :
Packaging Pseudomonas (oxidase positive)
Dehydrated medium :
Bibliography
(1) King, S., and Metzger, W.I. 1968. A new plating medium for the (5) CNEVA Bactériologie animale : Pr 116/00/BA 60/00. December
isolation of enteric pathogens. Appl. Microbiol., 16: 577-578. 1996. (French) Isolement et identification des salmonelles
(2) King, S., and Metzger, W.I., 1968. Comparison of Hektoen Enteric chez les volailles.
Agar with SS and EMB Agar. Appl. Microbiol., 16: 579-581. (6) NF V 08-052. May 1997. Microbiology of food and animal
(3) Taylor, W.I., and Schelhaut, D. 1971. Isolation of Shigellae. feeding stuffs. Detection of Salmonella. Routine method.
Comparison of Xylose Lysine Desoxycholate Agar, Hektoen (7) NF EN 12824. February 1998. Microbiology of food and animal
Enteric Agar, Salmonella-Shigella Agar and Eosin Methylene feeding stuffs. Horizontal method for the detection of
Blue Agar with stool specimens. Appl. Microbiol., 21: 32-37. Salmonella.
(4) Bisciello, N.B., and Schrade, J.P. 1974. Evaluation of Hektoen (8) ISO/CD 21567. 2000. Microbiological examination of foods and
Enteric Agar for the detection of Salmonella in foods and animal feeding stuffs – Horizontal method for the detection of
feeds. Journ. of AOAC, 57: 992-996. Shigella species.
136
Karmali Agar
Intended Use
Karmali Agar is used for the selective isolation of Campylobacter in

food products and other samples that may contain it, after a prior
enrichment.
History
In 1985, the medium was first described and successfully used by

Karmali for the enumeration of Campylobacter jejuni and
Campylobacter coli, in comparison to media requiring the use of
horse blood. Karmali et al. showed that the medium, called CSM,
(Charcoal-based Selective Medium) more effectively inhibited
contaminating strains (Pseudomonas, Gram-positive bacteria,
yeasts), than Skirrow's medium. The formula of Karmali agar is
based on the following two basic observations :
- The discovery by Bolton et al. that charcoal could replace blood in Campylobacter jejuni
media for Campylobacter.
- The demonstration by Ahonkai et al. of the resistance of
Campylobacter jejuni to cefoperazone. Product Profile
Target : Campylobacter.
Principles Reconstitution : 46.7 g / L.
- Polypeptone favors the growth of Campylobacter colonies.
- Yeast extract is a source of the vitamin B complex. pH (25°C) : 7.4 ± 0.2.
- Starch is the energy source for development. Supplement required : Karmali
- Sodium chloride maintains osmotic balance. Selective Supplement.
- Activated charcoal, sodium pyruvate and hematin are compounds
that replace blood previously used in the media, and favor the
growth and aerotolerance of Campylobacter.
- Cycloheximide prevents the development of yeasts. Preparation
- Cefoperazone extends the spectrum of vancomycin action by more
thoroughly inhibiting contaminating bacteria (Pseudomonas and - Suspend 46.7 g of dehydrated medium
Enterobacteriaceae) than cefazolin, originally included in selective (BK117) in 1 liter of distilled or deionized
charcoal media. water.
Typical Composition of the complete medium complete dissolution.
(can be adjusted to obtain optimal performance) - Dispense 100 mL in flasks.
- Sterilize in an autoclave at 115°C
For 1 liter of medium : for 15 minutes.
- Yeast extract 3.0 g Instructions for Use
- Starch 1.0 g
- Sodium chloride 5.0 g - Melt the medium (if prepared in advance).
- Sodium pyruvate 0.1 g - Cool and maintain at 47°C.
- Activated charcoal 4.0 g - Aseptically add 1 mL of reconstituted
- Hematin 32 mg Karmali Selective Supplement (BS017) to
- Cycloheximide 0.1 g each flask.
- Vancomycin 20 mg - Homogenize thoroughly.
- Cefoperazone 32 mg - Pour into sterile Petri dishes.
- Bacteriological agar 13.5 g - Let solidify on a cold surface.
pH of the ready to use medium at 25°C : 7.4 ± 0.2. partially removed.
- Transfer 0.1 mL of the sample to analyze
(or of a selective enrichment broth) and its
serial tenfold dilutions to the dishes
containing medium.
- Spread the inoculum with a sterile
triangle.
- Incubate at 42°C in a microaerophilic
atmosphere for 24 and 48 hours.
137
- Dehydrated medium : gray powder, free-flowing and Campylobacter form shiny, smooth or
homogeneous. granular colonies, with a grayish-white
- Prepared medium (complete) : black agar. appearance. Suspected colonies are
- Typical culture response after 48 hours of incubation at 42°C : subjected to biochemical identification
tests.
Campylobacter jejuni ATCC® 33291 good to excellent
Campylobacter coli ATCC 33559 good

- Base media in flasks : 6 months at 2-8°C.
- Complete media in plates : 1 month at 2-8°C, shielded from light.
Karmali Selective Supplement :
- Store at 2-8°C, shielded from light.
Packaging
Dehydrated base medium (without supplement) :

Karmali Selective Supplement :
Bibliography
(1) Ahonkhai, V.I., Cherubin, C.E., Sierra, M.F., Bokhenheuser, V.D., (3) Karmali, M.A., Simor, A.E., Roscoe, M., Fleming, P.C., Smith,
Shulman, M.A., and Mosenthal, A.C. 1981. In vitro susceptibility S.S., and Lane, J. 1986. Evaluation of a Blood-Free, Charcoal-
of Campylobacter fetus subsp. jejuni to N-formimidoyl Based, Selective Medium for the Isolation of Campylobacter
thienamycin, rosaramicin, cefoperazone and other antimicrobial Organisms from Feces. Jour. of Clin. Microb., 23 (3) : 456-459.
agents. Antimicrob. Agents Chemother., 20 : 850-851. (4) NF ISO 10272. Jan. 1996. Microbiology of food and animal
(2) Bolton, F.J., and Coates, D. 1983. Development of a blood-free feeding stuffs. Horizontal method for detection of
Campylobacter medium : screening tests on basal media and thermotolerant Campylobacter.
supplements, and the ability of selected supplements to
facilitate aerotolerance. J. App. Bacteriol., 54 : 115-125.
138
KF Streptococcus Agar
Intended Use
KF (Kenner Fecal) Streptococcus Agar is a selective medium used for

the isolation and enumeration of fecal enteric cocci in drinking
water, beverages, waste water, lactic acid starters and food
products, using both the membrane filtration method and the
classical method of counting in Petri dishes.
History
KF Agar used by Kenner et al. in 1960 for the enumeration of fecal

streptococci in water samples was found to have an excellent
recovery power in comparison to other media used at that time. The
authors observed that the medium led to an excellent
characterization of enteric cocci as well as of microorganisms with
similar biochemical and antigenic properties : Streptococcus bovis Enterococcus faecalis
and Streptococcus equinus.
Principles
Product Profile
- The high nutritive capacity of the medium is due to the presence Target : Intestinal enterococci.
of a high proportion of polypeptone, yeast extract and Reconstitution : 68.4 g / L.
carbohydrates.
- Sodium chloride maintains the osmotic equilibrium. Autonomy (500 g) : 810 plates.
(membrane filtration).
- Lactose and maltose are energy sources for microorganisms that
can use them. pH (25°C) : 7.2 ± 0.2.
- The acidification of the medium is shown by bromcresol changing Supplement required : TTC Supplement
from purple to yellow. 50 mg.
- Sodium azide inhibits the growth of contaminating Gram-negative
bacteria.
Do not autoclave.
- TTC added just before use is an indicator of bacterial growth. It is
reduced to an insoluble formazan inside cells. This reaction is
visualized by the appearance of red to maroon colonies. Preparation

Typical Composition of the base medium (without TTC)
water.
- Continue boiling for 5 minutes without
exceeding this period of heating.
- Maltose 20.0 g
- Do not autoclave.
- Lactose 1.0 g
- Aseptically dispense 100 mL in sterile
flasks.
- Sodium glycerophosphate 10.0 g
- Bromcresol purple 15 mg
- Bacteriological agar 12.0 g Membrane filtration
pH of the ready-to-use medium at 25°C : 7.2 ± 0.2. - Aseptically add 1 mL of reconstituted
TTC 50 mg Supplement (BS027) per flask.
- Mix thoroughly.
- Aseptically filter a determined volume of
the sample to test through a membrane
filter.
- Place the membrane on the surface of the
agar, taking care to insure that membrane-
agar are in close contact.
- Incubate at 37°C for 48 hours.
139
Quality Control Inoculation in depth
- Transfer 1 mL of the product to test and its
- Dehydrated medium : whitish powder, free-flowing and tenfold serial dilutions to sterile Petri
homogeneous. dishes.
- Prepared medium : violet-blue agar. - Pour in 10 to 15 mL of complete medium
- Typical culture response after 24-48 hours of incubation at 37°C : (with TTC).
Microorganisms Growth Characteristics - Let solidify on a cold surface.
Enterococcus faecalis ATCC® 29212 good to excellent pink to red colonies, - Incubate at 37°C for 48 hours.
surrounded by a yellow halo
Results
Enterococcus faecium ATCC 19434 good to excellent pink to red colonies,
surrounded by a yellow halo Use membranes furnishing fewer than
Escherichia coli ATCC 25922 inhibited 100 colonies. Colonies that are red, maroon
or pink, surrounded by a yellow halo, are
considered characteristic.
Carry out the confirmation of typical
Storage / Shelf Life colonies using the appropriate confirmatory
medium (BK 158) and by screening for
Dehydrated base medium (without TTC) : 2-20°C. catalase.
- Complete media in plates : 8 days at 2-8°C.
TTC 50 mg Supplement :
Packaging
Dehydrated medium :
Bibliography
(1) Kenner, B.A., Clark, H.F., and Kabler, P.W. 1961. Fecal (3) FIL-IDF 149A: 1997. Dairy starter cultures of lactic acid
Streptococci. I. Cultivation and Enumeration of Streptococci in bacteria (LAB). Standard of identity.
Surface Waters. Appl. Microb., 9: 15-20.
(2) MacFaddin, J.F. 1985. Media for Isolation-Cultivation-
Maintenance of Medical Bacteria. Vol 1. Williams and Wilkins,
Baltimore, 395-398.
Kligler Iron Agar

Intended Use
Kligler Iron Agar is used for the identification of enterobacteria by the

rapid detection of lactose and glucose fermentation (with or without
gas production), as well as the production of hydrogen sulfide.
Identification of enterobacteria
140
History Product Profile
Target : Identification of Enterobacteria.
In 1911, Russell described a medium containing two sugars for the
isolation of typhoid bacilli in urine. Six years later, Kligler developed Reconstitution : 58.0 g / L.
a nutrient medium with glucose, Andrade's indicator and lead Autonomy (500 g) : 860 tubes.
acetate for the differentiation of bacilli from the typhi and pH (25°C) : 7.4 ± 0.2.
paratyphi groups. While experimenting this medium with other
combinations of ingredients, Kligler found that Russell's medium
containing Andrade's indicator and lead acetate led to an excellent Sterilization : 121°C / 15 minutes.
differentiation of salmonellae. Bailey and Lacey subsequently
recommended using phenol red as pH indicator, replacing Preparation
Andrade's indicator which was less adapted to this type of reaction.
Sulkin and Willett used sodium thiosulfate and ferrous sulfate to - Suspend 58.0 g of dehydrated medium
demonstrate hydrogen sulfide production. (BK034) in 1 liter of distilled or deionized
water.
- Dispense in tubes.
- The fermentations of lactose and glucose, used to differentiate
species of enterobacteria, result in acidification which makes
15 minutes.
phenol red (pH indicator) turn yellow.
- Incline the tubes so as to obtain a butt
- Microorganisms not fermenting lactose (Salmonella or Shigella) 3 cm in height and an oblique slant.
initially produce a yellow slant due to the acidification resulting
from glucose present in small quantities. When the glucose has NOTE
been exhausted in the aerobic portion of the slant, the reaction If the medium is not used within one week of its
becomes basic by oxidation of the acids produced, resulting in the preparation, it is recommended to regenerate it
phenomenon of a red color on the surface of the medium. This in a boiling water bath and to resolidify it in a
slanted position.
color does not appear in depth in the butt, where the color
remains yellow.
- Bacteria fermenting lactose and glucose make the slant and the
butt turn yellow because of the production of large quantities of - Using a suspected colony taken from a
acid. This is sufficient to maintain an acid pH on the surface. selective isolation medium, inoculate the
- Microorganisms which ferment neither of these two sugars do not butt by stabbing in the center and the
change the color of the medium. inclined surface by closely spaced streaks.
- The production of H2S is revealed in the base of the medium by - Pure cultures taken from the center of
the appearance of black iron sulfide, due to the reduction of well isolated colonies must be used to
thiosulfate in the presence of ferric citrate. avoid cross reactions which will make
- The production of gas (H2, CO2) resulting from sugar identification impossible.
fermentations is shown by the appearance of gas bubbles or by a - Incubate at 37°C for 24 hours with caps
fragmentation of the agar. loosely screwed to favor gas exchanges.

Kligler's medium supplies four types of
For 1 liter of medium : information :
- Tryptone 20.0 g
- Yeast extract 3.0 g (1) Glucose fermentation
- Meat extract 3.0 g - Red butt : glucose not fermented
- Yellow butt : glucose fermented
- Glucose 1.0 g
(2) Lactose fermentation
- Lactose 10.0 g
- Red slant : lactose not fermented
- Yellow slant : lactose fermented
(3) Gas production
- Ferric ammonium citrate 0.5 g - Production of gas bubbles in the butt
- Phenol red 25 mg (4) Formation of H2S
- Bacteriological agar 15.0 g - Formation of a black color between the butt
and the slant or along the inoculation stab.
141
Quality Control Typical reactions are listed
in the following table :
- Dehydrated medium : pinkish powder, free-flowing and SLANT BUTT
homogeneous. SPECIES Lactose Glucose Gas H2S
- Prepared medium : orange-red agar. Salmonella Typhi (2) - + - +
- Typical culture response after 24 hours of incubation at 37°C : Salmonella Paratyphi A (2) - + + -
Salmonella Choleraesuis (2) - + + -
Microorganisms Growth Slant Butt H2S Gas Salmonella Pullorum (2) - + + +
Salmonella Paratyphi B (2) - + + +
Escherichia coli ATCC® 25922 good yellow yellow + -
Salmonella Typhimurium (2) - + + +
Citrobacter freundii ATCC 8090 good yellow yellow + + Salmonella Enteritidis (2) - + + +
Proteus vulgaris ATCC 13315 good red yellow (+) + Salmonella Gallinarum (2) - + - +
Salmonella Enteritidis ATCC 13076 good red yellow + + Shigella dysenteriae - + - -
Shigella flexneri ATCC 29903 good red yellow - - Shigella flexneri - + - -
Shigella sonnei - + - -
Pseudomonas aeruginosa ATCC 27853 good red red - -
Shigella boydii - + - -
Proteus vulgaris - + (+) +
Storage / Shelf Life Proteus mirabilis - + + +
Proteus morganii - + + -
Dehydrated medium : 2-30°C. Proteus rettgeri - + - -
- The expiration date is indicated on the label. Serratia marcescens - + - -
Prepared medium (benchmark value) : Enterobacter hafniae - + + -
- Media in tubes non-slanted : 6 months at 2-8°C. Enterobacter aerogenes + + + -
- Media in slants : 8 days at 2-8°C. Enterobacter cloacae + + + -
Escherichia coli (1) + + + -
Packaging Citrobacter freundii + + + +
Klebsiella pneumoniae + + + -
Dehydrated medium : Alcaligenes faecalis - - - -
- 500 g bottle : BK034HA Pseudomonas aeruginosa - - - -
Yersinia enterocolitica - - - -
(1) Some strains of Escherichia coli ferment lactose

only very late in growth.
(2) In the case where interpretation may suggest
the presence of salmonellae, it is possible to use
Kligler cultures to detect -galactosidase,
urease and lysine-decarboxylase.
Bibliography
(1) Russell, F.F. 1911. The isolation of typhoid bacilli from urine (4) Buttiaux, R. 1951. L’analyse bactériologique des eaux de
and feces with the description of a new double sugar tube consommation. Flammarion-Paris.
medium. J. Med. Red. Res., 25: 217. (5) Buttiaux, R., Beerens, H., et Tacquet, A. 1962. Manuel des techniques
(2) Kligler, I.J. 1917. A simple medium for the differentiation of bactériologiques. Flammarion. Médecine-Sciences. 4ème Ed.
members of the typhoid-paratyphoid group. Am. J. Public (6) ISO 6340. December 1995. Water quality. Detection of
Health, 7: 1042. Salmonella.
(3) Kligler, I.J. 1918. Modifications of culture media used in the (7) NF V 08-052. May 1997. Microbiology of food and animal
isolation and differentiation of typhoid, dysentery, and allied feeding stuffs. Detection of Salmonella. Routine method.
bacilli. J. Exp. Med., 28: 319.
142
Lactose Broth

Lactose Broth is recommended by the United States Pharmacopoeia Target : Dilutions, Coliforms, Salmonella.
as a preenrichment medium for the detection of Salmonella and Reconstitution : 13.0 g / L.
Escherichia coli in pharmaceuticals. It also complies with the Autonomy (500 g) : 3846 tubes.
directives of the American Public Health Association for the
presumptive detection of Escherichia coli in dairy products and pH (25°C) : 6.9 ± 0.2.
drinking water. Supplement required : None.
Principles
Preparation
- This nutrient medium contains no inhibitors or indicators and
enables the growth of enterobacteriaceae.
- Lactose fermentation is shown by the production of gas
in 1 liter of distilled or deionized water.
in Durham tubes.
- Dispense in 16 x 160 mm tubes containing
Typical Composition
a Durham tube, or in flasks.
For 1 liter of medium : 15 minutes.
- Meat extract 3.0 g Instructions for Use
- Lactose 5.0 g
Samples of 1, 10 or 100 mL of liquid product
pH of the ready-to-use medium at 25°C : 6.9 ± 0.2. to analyze are mixed with measured
quantities of medium in order to respect
Quality Control the proportions of reconstitution. The
volumes and concentrations to used are
- Dehydrated medium : light beige powder, free-flowing and listed in the following table :
homogeneous.
- Prepared medium : amber solution, limpid. Inoculum Volume of Concentration
- Typical culture response after 24-48 hours of incubation at 30°C : volume (mL) medium (mL) of medium (g/L)
1 at least 10 13
Microorganisms Growth Gas production 10 10 26
Escherichia coli ATCC
®
25922 good to excellent positive 10 20 19.5
100 20 78
Enterobacter cloacae ATCC 13047 good to excellent positive
100 50 39
Citrobacter freundii ATCC 43864 good to excellent positive
Salmonella Typhimurium ATCC 14028 good negative Incubation
Enterococcus faecalis ATCC 29212 good negative - For total coliform bacteria, incubate for 24
and 48 hours at 30 or 37°C depending on
the analysis protocol followed.
Storage / Shelf Life - For heat-tolerant coliform bacteria,
incubate at 44.5°C for 48 ± 2 hours.
Dehydrated medium : 2-30°C. - Inoculate a loop from each positive
- The expiration date is indicated on the label. tube into :
Prepared medium (benchmark value) : - a tube of Brilliant Green Bile Broth
- Media in tubes or vials : 6 months at 2-8°C. (BK002) for coliforms.
- a tube of Brilliant Green Bile Broth
Packaging (BK002, BM011) or Schubert Broth
(BK120) for heat-tolerant coliforms.
Dehydrated medium :
- 500 g bottle : BK082HA Results
The presence of coliform bacteria is shown by

the production of gas in the Durham tubes.
Identification of Escherichia coli

One loop of each tube considered to be a
presumptive positive is transferred to
Brilliant Green Bile Broth (BK002, BM011),
and a tube of Peptone Water (BK084).
Incubate at 44 ± 0.5°C in a water bath.
143
simultaneously present the following
characteristics :
- growth on BGBB : positive with gas
production
- production of indole (Kovacs-Ehrlich
Bibliography
(1) Hausler, W.J. 1972. Standard Methods for the Examination of (4) United States Pharmacopoeia 24. 2000. Microbial Limit Tests,
Dairy Products. 13th Ed. Washington D.C. American Public 1814-1818.
Health Association. (5) Pharmacopée européenne. Addendum 2000. Contrôle
(2) American Public Health Association. 1975. Standard Methods microbiologique des produits non stéiles (Recherche de
for the Examination of Water and Wastewater. 14th Ed. microorganismes spécifiés). Solution et milieux de culture
Washington, D.C. recommandés, 56-61.
(3) Rodier, J. 1984. L’analyse de l’eau. Dénombrement des coliformes
fécaux et Escherichia coli présumés. Dunod 7ème Ed, 793-798.
Lactose-Sulfite Broth (LS)

Intended Use / History Product Profile
Lactose-Sulfite Broth is a confirmation medium allowing the Target : Clostridium perfringens.
selective detection of both the vegetative cells and spores of Reconstitution : 19.3 g / L.
Clostridium perfringens in food and biological samples, without the Autonomy (500 g) : 2878 tubes,
usual confirmatory tests. (9 mL per tube).
pH (25°C) : 7.1 ± 0.2.
History
Using the past work by Put (1961) relative to the sensitivity of Sterilization : 121°C / 15 minutes.
Clostridium to sulfite, the optimal growth of Clostridium
perfringens at 46°C, as well as its capacity to ferment lactose,
Preparation
Beerens et al. in 1982 successfully formulated and developed LS
broth. In particular, they recommended its use for the detection of
small quantities of Clostridium perfringens in food products in
which there was a heavy secondary contamination of other sulfito-
water.
reducing bacteria.
- Slowly stir until complete dissolution.
Principles
containing a Durham tube.
- The specificity of the medium to Clostridium perfringens is due
15 minutes.
principally to the sulfite resistance of the microorganism and its
ability to ferment lactose with gas production.
After cooling, the Durham tubes should not
- The concentration of metabisulfite inhibits the development of the
contain trapped air.
majority of clostridia other than Clostridium perfringens.
- Incubation at 46°C assures the specific culture of Clostridium
perfringens, which reduces the sodium metabisulfite to sulfide,
provoking with ferric citrate a black iron sulfide precipitate that
- From suspected black colonies isolated
settles in the bottom of the culture tube.
from an enumeration media for
Clostridium perfringens (TSC Agar, BK031),
inoculate into Thioglycollate Medium with
Resazurin (BK017).
- Incubate the tubes under anaerobic
conditions for 20 hours at 37°C.
- Inoculate 5 drops of the culture into a
tube of Lactose-Sulfite Broth.
- Incubate at 46°C for 24 hours in a
controlled water bath.
144
The fermentation of lactose is indicated by
the presence of gas in the Durham tubes
- Tryptone 4.44 g
(minimum volume equal to 1/4 of the volume
of the Durham tube itself) in 24 hours, as well
- Cysteine hydrochloride 0.27 g
as simultaneous appearance of a black iron
- Lactose 8.89 g
sulfide precipitate in the culture tubes
indicating the presence of Clostridium
- Sodium metabisulfite 0.67 g
perfringens.
Quality Control

homogeneous.
Microorganisms Growth Gas production Blackening

Clostridium perfringens ATCC® 13124 good to excellent positive positive
Clostridium bifermentans ATCC 19299 good negative positive

- Media in tubes : use on the day of preparation.
Packaging
Dehydrated medium :
Bibliography
(1) Beerens, H., Romond, C.H., Lepage, C., and Criquelion, J. (4) ISO 7937. 1997. Microbiology of food and animal feeding
1982. A Liquid Medium for the Enumeration of Clostridium stuffs. Horizontal method for enumeration of Clostridium
perfringens in Food and Faeces. Isolation and Identification perfringens. Colony count technique.
Methods for Foods Poisoning Organisms. Edited by Academic (5) Pharmacopée européenne. Addendum 2000. Contrôle
Press, London, 137-149. microbiologique des produits non stériles (recherche de
(2) NF V 59-107. Mars 1984. Gélatine alimentaire. Recherche des microorganisms spécifiés). Solution et milieux de culture
spores de Clostridium perfringens. Technique du nombre le plus recommandés, 56-61.
probable après incubation à 46°C.
(3) NF V08-056. Avril 1994. Microbiologie alimentaire.
Dénombrement des Clostridium perfringens par comptage des
colonies à 37°C. Méthode de routine.
145
Laurylsulfate-Tryptose Broth
Intended Use / History
Laurylsulfate-Tryptose Broth is a selective enrichment broth used for

the detection and enumeration of coliform bacteria in water, dairy
and other food products. The medium was formulated by Malmann
and Darby who showed, in 1941, that sodium laurylsulfate was the
best selective agent among a number of surfactants and did not
inhibit the growth of coliform bacteria. Levine later showed that
the medium reduced the number of false positives by inhibiting the
growth of sporulated gas-producing bacteria.
Principles
- Sodium laurylsulfate considerably inhibits the development of

accompanying microbial flora without inhibiting coliform species.
- Because of its excellent nutritive capacity, as well as the presence
of phosphate buffer, Laurylsulfate-Tryptose Broth enables coliform Escherichia coli Negative control
bacteria to rapidly develop and release large quantities of gas
from the fermentation of lactose, even with small inocula. Product Profile
Typical Composition Target : Coliforms.
(can be adjusted to obtain optimal performance) Reconstitution : 35.6 g / L.
- Tryptose 20.00 g pH (25°C) : 6.8 ± 0.2.
- Lactose 5.00 g Supplement required : MUG Supplement.
- Dipotassium phosphate 2.75 g Sterilization : 121°C / 15 minutes.
- Sodium chloride 5.00 g Preparation
- Sodium laurylsulfate 0.10 g
pH of the ready-to-use medium at 25°C : 6.8 ± 0.2. - Dissolve 35.6 g of dehydrated medium
Quality Control water.
- Dehydrated medium : off-white powder, free-flowing and - Dispense in 16 x 160 mm tubes containing
homogeneous. a Durham tube.
- Prepared medium : light amber solution, limpid. - Sterilize in an autoclave at 121°C for
- Typical culture response after 24-48 hours of incubation at 37°C : 15 minutes.
Microorganisms Growth Gas production not contain trapped air.
NOTE :
Klebsiella pneumoniae ATCC 13883 good to excellent positive MUG (4-methylumbelliferyl--D-glucuronide) can
Citrobacter freundii ATCC 43864 good to excellent positive be added to the medium in order to detect
Escherichia coli (Refer to the monograph on MUG
Enterococcus faecalis ATCC 29212 inhibited Supplement, BS024).
Double strength medium
Storage / Shelf Life - Dissolve 71.2 g of dehydrated medium
Dehydrated medium : 2-30°C. water.
- The expiration date is indicated on the label. - Stir slowly until complete dissolution.
Prepared medium (benchmark value) : - Dispense 10 mL in 20 x 200 mm tubes
- Media in tubes : 6 months at 15-30°C, shielded from light. containing a Durham tube.
- It is recommended not to refrigerate tubes since cloudiness and - Sterilize in an autoclave at 121°C for
precipitates may form, even though they disappear when the 15 minutes.
medium returns to room temperature. - After cooling, the Durham tubes should
MUG Supplement : not contain trapped air.
146
Packaging Instructions for Use
Dehydrated medium : Single strength medium

- 500 g bottle : BK010HA - Inoculate tubes with 1 mL of inoculum and
MUG Supplement : its serial tenfold dilutions.
- 10 vial pack : BS02408 Double strength medium
- Inoculate tubes with 10 mL of inoculum.
Incubation
Results
Lactose fermentation, shown by the

presence of gas in the Durham tubes in less
than 48 hours, indicates the presence of
coliform bacteria. Verification may be
carried out by isolating and identifying on
an appropriate medium.
Enumerate by using the most probable
number method.
Bibliography
(1) Malmann, W.L., and Darby, C.W. 1941. Use of a laurylsulphate (6) NF ISO 11866-1. September 1997. Milk and milk products.
tryptose broth for the detection of coliform organisms. Am. J. Enumeration of presumptive Escherichia coli. Part 1: most
Public Health, 31: 127-134. probable number technique.
(2) Hajna, A.A., and Perry, C.A. 1943. Comparative study of (7) NF ISO 11866-2. September 1997. Milk and milk products.
presumptive and confirmative media for bacteria of the Enumeration of presumptive Escherichia coli. Part 2: Most
coliform group and for fecal streptococci. Am. J. Public probable number technique using 4-methylumbelliferyl-beta
Health, 33: 550-556. D-glucuronide (MUG).
(3) NF T 90-413: October 1985. Testing water. Detection and (8) FIL-IDF provisional 73B: 1998. Milk and milk products.
enumeration of coliforms and thermotolerant coliforms. General Enumeration of coliforms. Part 2: Most probable number
method by culture in liquid media (MPN). technique at 30°C without resuscitation.
(4) NF ISO 4831: July 1991. Microbiology. General guidance for (9) FIL-IDF 170A: 1999. Milk and milk products. Enumeration of
the enumeration of coliforms. Most probable number presumptive Escherichia coli. Part 1: Most probable number
technique. technique.
(5) NF ISO 7251. September 1994. Microbiology. General guidance (10) NF V 08-600. October 2000. Microbiology of foad and
for enumeration of presumptive Escherichia coli. Most probable foadstuffs products. Enumeration of presemptive Escherichia
number technique. coli in living shell fishes MPN technique.
Lecithin-Polysorbate-Triton X Agar

Lecithin-Polysorbate-Triton X Agar is used for the enumeration of
total microorganisms in cosmetic products with or without Reconstitution : 52.4 g / L.
preservatives. It enables luxuriant colonies to form in the case of Autonomy (500 g) : 635 plates.
most microorganisms.
pH (25°C) : 7.0 ± 0.2.
Principles Supplement required : None.
- The medium is composed of a mixture of peptones, cystine,
glucose and salts which favor the growth of a wide variety of Preparation
microorganisms.
- Sodium chloride maintains osmotic pressure. - Suspend 52,4 g of dehydrated medium
- Lecithin and tween neutralize the antibacterial activity of most (BK138) in 1 liter of distilled or deionized
antiseptics or preservatives, such as phenolic derivatives, aldehydes water.
and quaternary ammonium salts. - Slowly bring to boiling, stirring until
- Triton X-100 favors the dispersion of cells and thus improves complete dissolution.
enumeration. - Dispense in tubes or flasks.
15 minutes.
147
Pour plate inoculation
For 1 liter of medium : - With the ready-to-use media in tubes
- Tryptone 15.0 g (BM023) or in vials (BM044, BM045), or if
- Papaic digest of soybean meal 5.0 g the media has been prepared in advance
- L-cystine 0.7 g from the dehydrated product as above,
- Glucose 5.5 g melt the medium for the minimum
- Sodium chloride 4.0 g amount of time needed to achieve total
- Sodium sulfite 0.2 g liquefaction.
- Lecithin 1.0 g - Cool and maintain at 47°C.
- Polysorbate (Tween 80) 5.0 g - Transfer 1 mL of the product to analyze
- Triton X-100 1.0 g and its serial tenfold dilutions in sterile
- Bacteriological agar 15.0 g Petri dishes.
- Pour in 15 to 20 mL of medium.
pH of the ready-to-use medium at 25°C : 7.0 ± 0.2. - Homogenize by swirling.
- Incubate at 30°C for 72 hours for the
Quality Control enumeration of mesophilic bacteria.
- Dehydrated medium : beige powder, homogeneous, slightly Surface inoculation

clumped. - On the surface of plates prepared from
- Prepared medium : amber agar. tubes or vials, or on prepoured ready-to-
- Typical culture response after 48 hours of incubation use plates (BM046) brought to room
at 30 (1) or 37°C : temperature, transfer 0.1 mL of the
product to analyze and its serial dilutions.
Microorganisms Growth Spread for isolation using a sterile loop or
Escherichia coli ATCC® 8739 good to excellent spreader.
Staphylococcus aureus ATCC 6538P good to excellent
Pseudomonas aeruginosa ATCC 9027 good to excellent Results
Aspergillus niger (1) ATCC 16404 good to excellent Only Petri dishes containing between
15 and 150 colonies are retained for
Storage / Shelf Life enumeration.

Ready-to-use media in tubes, vials or plates :
Packaging
- 20 plates : BM04608
- 50 x 15 mL tubes : BM02308
- 10 x 100 mL vials : BM04408
- 10 x 200 mL vials : BM04508
Dehydrated medium :
Bibliography
(1) Guisno, R., Gibby, I.W., and Foter, M.J. 1946. A neutralizing (3) Desbordes, J. 1977. Biodégradation microbienne des
medium for evaluation of the germicidal potency of the antiseptiques et conservateurs. Conséquences pour la qualité
quaternary ammonium salts. Amer. J. Pharm., 118: 320-323. microbiologique. Rev. de l'Inst. Pasteur de Lyon, 10, n° 4:
(2) Williamson, P., and Kligman, A.M. 1965. A new method for the 291-311.
quantitative investigation of cutaneous bacteria. J. Inv. (4) Singer, S. 1987. The use of Preservative Neutralizers in Diluents
Dermatol. 45: 498-503. and Plating Media. Cosmetics and Toiletries, 102: 55-60.
148
Lecithin-Polysorbate-Triton X Broth

Lecithin-Polysorbate-Triton X Broth is used for the enumeration
of total microorganisms in cosmetic products with or without Reconstitution : 37.4 g / L.
preservatives, by the use of the most probable number method. Autonomy (500 g) : 133 vials
(100 mL by vial).
Principles
pH (25°C) : 7.0 ± 0.2.
- The medium is composed of a mixture of peptones, cystine, Supplement required : None.
glucose and salts which favor the growth of a wide variety of Sterilization : 121°C / 15 minutes.
microorganisms.
- Sodium chloride maintains osmotic pressure. Preparation
- Lecithin and tween neutralize the antibacterial activity of most
antiseptics or preservatives, such as phenolic derivatives, aldehydes - Suspend 37.4 g of dehydrated medium
and quaternary ammonium salts. (BK137) in 1 liter of distilled or deionized
- Triton X-100 favors the dispersion of microorganisms and thus water.
improves enumeration. - Slowly bring to boiling, stirring until
Typical Composition - Dispense in tubes or flasks.
(can be adjusted to obtain optimal performance) - Sterilize in an autoclave at 121°C for
15 minutes.
For 1 liter of medium : - Cool rapidly.
- Tryptone 15.0 g
- Papaic digest of soybean meal 5.0 g NOTE
- L-cystine 0.7 g The medium is clear on cooling.
- Glucose 5.5 g
- Lecithin 1.0 g - Inoculate the medium prepared as above,
- Polysorbate (Tween 80) 5.0 g or the ready-to-use media (BM006, BM042,
- Triton X-100 1.0 g BM043) with the sample to test.
- Incubate at 30 or 37°C for 7 to 10 days,
pH of the ready-to-use medium at 25°C : 7.0 ± 0.2. depending on the analytical protocol
applied.
Quality Control - Enumerate using the most probable
number method.
- Dehydrated medium : yellowish powder, homogeneous, slightly
clumped.
- Prepared medium : amber solution, limpid after cooling.
- Typical culture response after 48 hours of incubation at 30 (1) or 37°C :
Staphylococcus aureus ATCC 6538 P good to excellent
Aspergillus niger (1) ATCC 16404 good to excellent

Ready-to-use media in tubes or vials :
149
Packaging
- 50 x 9 mL tubes : BM00608
- 10 x 50 mL vials : BM04208
- 10 x 100 mL vials : BM04308
Dehydrated medium :
Bibliography
(1) Guisno, R., Gibby, I.W., and Foter, M.J. 1946. A neutralizing (2) Williamson, P., and Kligman, A.M. 1965. A new method for the
medium for evaluation of the germicidal potency of the quantitative investigation of cutaneous bacteria. J. Inv.
quaternary ammonium salts. Amer. J. Pharm., 118: 320-323. Dermatol., 45: 498-503.
Listeria Enrichment Broth (LEB acc. to FDA)

LEB according to FDA (Food and Drug Administration) is used for Target : Listeria.
the selective enrichment of Listeria in milk, dairy products, meats, Reconstitution : 36.1 g / L.
poultry, other food products, as well as in other specimens which Autonomy (500 g) : 61 vials.
may contain it.
pH (25°C) : 7.3 ± 0.2.
The medium was formulated by Lovett et al. in 1985 for the selective
enrichment of Listeria monocytogenes, a contaminant of raw milk
Preparation
and pasteurized milk. Compared to prior techniques of enrichment
by cold treatment, which were slower, the Lovett method resulted in
an incubation at 30°C for not more than 48 hours, after which the (BK112) in 1 liter of distilled or deionized
culture was inoculated on the surface of a plate of MacBride Agar. water.
Principles - Dispense in flasks at 225 mL per flask.
- Tryptone, papaic digest of soybean meal and yeast extract furnish 15 minutes.
the nutrients required for the growth of Listeria.
- Sodium chloride provides osmotic balance. Instructions for Use
- Dipotassium phosphate buffers the pH of the medium.
- Cycloheximide inhibits the development of contaminating - Cool to 25°C.
saprophytic molds. - Aseptically add 25 g of the product to
- Nalidixic acid blocks the DNA replication of bacteria sensitive to analyze to a tared flask containing 225 mL
this antibacterial agent. of medium.
- Accompanying Gram-positive microflora are inhibited by - Homogenize thoroughly.
acriflavine. - Incubate at 30°C for 24 and 48 hours.
- Carry out an isolation on one or several
Typical Composition selective media : Oxford Agar (BK110 + BS003
(can be adjusted to obtain optimal performance) or BM019) or PALCAM Agar (BK145 +
BS004/BS049 or BM020), with a platinum loop.
For 1 liter of medium : - Locate suspected colonies and carry out
- Tryptone 17.0 g biochemical identification tests.
- Glucose 2.5 g
- Cycloheximide 50 mg
- Acriflavine hydrochloride 15 mg
- Nalidixic acid 40 mg

150
Quality Control

homogeneous.
- Prepared medium : amber yellow solution, limpid.
followed by subculture on PALCAM Agar :
Listeria monocytogenes ATCC® 19115 good to excellent
Saccharomyces cerevisiae ATCC 9763 inhibited
Dehydrated medium : 2-30°C, shielded from light.

- Media in tubes or vials : 15 days at 2-8°C, shielded from light.
Packaging
Dehydrated medium :
Bibliography
(1) Lovett, j. 1986. Protocole FDA de recherche des Listeria dans (3) Tiwari, N.P., and Aldenrath, S.G. 1989. Isolation of Listeria
les aliments. monocytogenes from Food Products on Four Selective Plating
(2) Lovett, J., Francis, D.W., and Hunt, J.M. 1987. Listeria Media. Jour. of Food Protec., 53: 382-385.
monocytogenes in raw milk: detection, incidence and (4) Bind, J.L. 1991. Mise en évidence et dénombrement des
pathogenicity. J. Food Protect., 50: 188-192. Listeria à partir de produits laitiers. Lait, 71: 99-105.
Listeria Enrichment Broth (LEB acc. to IDF)

LEB according to the IDF (International Dairy Federation) is used for Target : Listeria.
the selective enrichment of Listeria in milk and in dairy products. Reconstitution : 36.1 g / L.
Autonomy (500 g) : 61 vials.
History
pH (25°C) : 7.3 ± 0.2.
The medium was formulated by Lovett et al. in 1985 for the selective Supplement required : None.
enrichment of Listeria monocytogenes, a contaminant of raw and Sterilization : 115°C / 15 minutes.
pasteurized milk. Compared to prior techniques of enrichment by
cold treatment, which was slower, the Lovett method resulted in an Preparation
incubation at 30°C for not more than 48 hours, after which the
culture was inoculated on the surface of a plate of MacBride Agar. - Dissolve 36.1 g of dehydrated medium
Principles water.
- Tryptone, papaic digest of soybean meal and yeast extract furnish - Dispense in flasks at 225 mL per flask.
the nutrients required for the growth of Listeria. - Sterilize in an autoclave at 115°C for
- Sodium chloride provides osmotic balance. 15 minutes.
- Dibasic potassium phosphate buffers the pH of the medium.
- Cycloheximide inhibits the development of contaminating
saprophytic molds.
- Nalidixic acid blocks the DNA replication of bacteria sensitive to
- Secondary Gram-positive microflora are inhibited by acriflavine.
151
- Cool to 25°C.
For 1 liter of medium : - Aseptically add 25 g of the product to
- Tryptone 17.0 g analyze to a tared flask containing 225 mL
- Papaic digest of soybean meal 3.0 g of medium.
- Yeast extract 6.0 g - Homogenize thoroughly.
- Glucose 2.5 g - Incubate at 30°C for 24 and 48 hours.
- Dipotassium phosphate 2.5 g - Carry out an isolation on one or several
- Sodium chloride 5.0 g selective media, inoculating with a
- Cycloheximide 50 mg platinum loop Oxford Agar
- Acriflavine hydrochloride 10 mg (BK110 + BS003 or BM019) or PALCAM
- Nalidixic acid 40 mg Agar (BK145 + BS004/BS049 or BM020).
- Locate suspected colonies and carry out
pH of the ready-to-use medium at 25°C : 7.3 ± 0.2. biochemical identification tests.
Quality Control

homogeneous.
- Prepared medium : amber yellow solution, limpid.

Packaging
Dehydrated medium :
Bibliography
(1) Lovett, J., Francis, D.W., and Hunt, J.M. 1987. Listeria (3) ISO 10560.1993. Milk and milk products. Detection of Listeria
monocytogenes in raw milk : detection, incidence and monocytogenes.
pathogenicity. J. Food Protect., 50: 188-192. (4) FIL-IDF 143A: 1995. Milk and milk products. Detection of
(2) Tiwari, N.P., and Aldenrath, S.G. 1989. Isolation of Listeria Listeria monocytogenes.
monocytogenes from Food Products on Four Selective Plating
Media. Jour. of Food Protec., 53: 382-385.
152
Listeria Enrichment Broth
(UVM I and UVM II modified)
Target : Listeria monocytogenes.
UVM (University of Vermont) Enrichment Broths involve a two-step
process, leading to a higher degree of isolation of Listeria Reconstitution : 51.9 g / L.
monocytogenes in meat products (including poultry). Autonomy (500 g) : 42 vials (BK113)
963 tubes (BK114).
History
pH (25°C) : 7.2 ± 0.2.
UVM media were described by Donnelly and Baigent in 1986, based Supplement required : None.
on the Dominguez-Rodriguez et al. (1984) enrichment medium for Sterilization : 115°C / 15 minutes.
Listeria. In the modified formula currently used, the nalidixic acid
content has been reduced from 40 to 20 mg/liter.
Preparation
Principles - Dissolve 51.9 g of dehydrated medium
(BK113 or BK114) in 1 liter of distilled or
- The difference in acriflavine concentrations in UVM I and UVM II deionized water.
Broths, as well as the two-step enrichment procedure, lead to a - Stir slowly until complete dissolution.
very satisfactory recovery of Listeria monocytogenes. - Dispense in flasks at 225 mL
- Polypeptone, yeast extract and meat extract supply the essential per flask (UVM I) or in tubes
nutrients for the growth of Listeria. at 10 mL per tube (UVM II).
- The sodium chloride concentration increases selectivity. - Sterilize in an autoclave at 115°C for
- Phosphates buffer the pH of the medium. 15 minutes.
- Esculin is hydrolyzed to glucose and esculetin by Listeria.
- Nalidixic acid blocks the DNA replication of bacteria sensitive to Instructions for Use
- Accompanying Gram-positive microflora are inhibited by - Cool UVM I and UVM II to 25°C.
acriflavine.
Primary enrichment
Typical Composition - Aseptically add 25 g of the product to
(can be adjusted to obtained optimal performance) analyze to a tared flask containing 225 mL
of UVM I Broth.
For 1 liter of medium : - Homogenize thoroughly.
- Polypeptone 10.0 g - Incubate at 30°C.
- Yeast extract 5.0 g - After 24 hours of incubation of UVM I
- Meat extract 5.0 g Broth, carry out an isolation on selective
- Sodium chloride 20.0 g media and also transfer 0.1 mL of the
- Disodium phosphate anhydrous 9.6 g culture obtained to 10 mL of UVM II Broth.
- Monopotassium phosphate 1.3 g
- Esculin 1.0 g Secondary enrichment
- Nalidixic acid 20 mg - Incubate UVM II broth for 24 hours at
- Acriflavine hydrochloride 12 mg (1)
or 25 mg (2)
30°C.
(1)
UVM I - Carry out a final isolation on selective
(2)
UVM II medium : Oxford Agar (BK110 + BS003 or
BM019) or PALCAM Agar (BK145 +
pH of the ready-to-use media at 25°C : 7.2 ± 0.2. BS004/BS049 or BM020), using a loop to
inoculate.
Quality Control - Locate suspected colonies and carry out
biochemical identification tests.
- Dehydrated media (UVM I and UVM II) : beige powder, free-
flowing and homogeneous.
- Prepared media (UVM I and UVM II) : amber yellow solution,
with a bluish reflection, limpid.
153
Dehydrated media : 2-30°C, shielded from light.

Prepared media (benchmark value) :
Packaging
Dehydrated media :
UVM I Broth :
UVM II Broth :
Bibliography
(1) Dominguez-Rodriguez, L., Suarez-Fernandez, G., Fernandez- (3) Lee, W.H., and MacClain, D. 1986. Improved Listeria
Garayzabal, J.F., and Rodriguez-Ferri, E. 1984. New monocytogenes selective agar. Appl. Environ. Microbiol.,
methodology for the isolation of Listeria microorganisms from 52: 1215-1217.
heavily contaminated environments. Appl. Environ. Microbiol., (4) MacClain, D., and Lee, W.H. 1988. Development of USDA-FSIS
47: 1188-1190. method for isolation of Listeria monocytogenes from raw meat
(2) Donnelly, C.W., and Baigent, G.J. 1986. Method for flow and poultry. J. Assoc. Off. Anal. Chem., 71: 660-664.
cytometric detection of Listeria monocytogenes in milk. Appl.
Environ. Microbiol., 52: 689-695.
M17 Agar

Target : Lactococci.
M17 Agar is used for the enumeration of lactic streptococci Reconstitution : 57.2 g / L.
(especially Lactococcus lactis) in dairy products. It is also used to
study the sensitivity of these species to bacteriophages. It is well Autonomy (500 g) : 582 plates.
adapted to the enumeration of Streptococcus thermophilus in pH (25°C) : 7.1 ± 0.2.
natural or flavored yogurts, textured or not, and in yogurts Supplement required : None.
containing morsels of fruit.
History
Preparation
Terzhagi and Sandine showed that the incorporation of sodium
-glycerophosphate in M16 medium increased the buffering capacity - Suspend 57.2 g of dehydrated medium
of the medium. The new medium, named M17, led to an increase in (BK088) in 1 liter of distilled or deionized
the development of lactic streptococci, which are bacteria producing water.
large quantities of acid via the homofermentative metabolism of - Slowly bring to boiling, stirring until
lactose. complete dissolution.
20 minutes.
- Casein, meat and soybean peptones contain the carbon and
nitrogen sources required to grow lactic streptococci.
- Yeast extract is a source of B vitamins.
- Ascorbic acid stimulates growth.
- Lactose is fermented to lactic acid, which is buffered by
glycerophosphate.
154
For 1 liter of medium : - Transfer 1 mL of the product to analyze
- Tryptone 2.50 g and its serial tenfold dilutions to sterile
- Peptic digest of meat 2.50 g Petri dishes.
- Papaic digest of soybean meal 5.00 g - Pour in 15 mL of medium.
- Yeast extract 2.50 g - Homogenize by swirling.
- Meat extract 5.00 g - Let solidify on a cold surface.
- Lactose 5.00 g - Incubate :
- Sodium glycerophosphate 19.00 g - at 37°C for 48 hours for Streptococcus
- Magnesium sulfate 0.25 g thermophilus.
- Ascorbic acid 0.50 g - at 30°C for 48 to 72 hours for
- Bacteriological agar 15.00 g mesophilic streptococci.
pH of the ready-to-use medium at 25°C : 7.1 ± 0.2. Results
Quality Control Streptococcus thermophilus and mesophilic

streptococci form colonies 1-2 mm in
- Dehydrated medium : beige powder, free-flowing and diameter, depending on colony density in
homogeneous. the dish.
- Prepared medium : amber agar. Microscopic verification shows whether the
- Typical culture response after 48 hours of incubation at 37°C : Gram-positive cocci are in chains or are
diplococci.
Streptococcus thermophilus ATCC® 14485 good to excellent
Streptococcus lactis subsp. lactis ATCC 11454 good to excellent

Packaging
Dehydrated medium :
Bibliography
(1) Terzaghi, B.E., and Sandine, W.E., 1975. Appl. Microbiol., (3) FIL-IDF 117A: 1988. Yogurt. Enumeration of characteristic
29: 807-813. microorganisms. Colony count technique at 37°C.
(2) Journal Officiel (French) du 4 janvier 1978. Méthode officielle (4) FIL-IDF 149A: 1997. Dairy starter cultures of lactic acid
d'analyse pour le dénombrement de la flore spécifique du bacteria (LAB). Standard of identity.
yaourt ou yoghourt (arrêté du 25 novembre 1977).
155
M17 Broth

Target : Lactococci.
M17 Broth was developed for the growth and enumeration of lactic
streptococci (lactococci) in milk and dairy products. It favors the Reconstitution : 42.2 g / L.
growth of mutants unable to ferment lactose. It is well adapted to Autonomy (500 g) : 1185 tubes.
the culture of Lactococcus lactis (a particularly fastidious species).
pH (25°C) : 7.1 ± 0.2.
Terzhagi and Sandine showed that the incorporation of sodium
-glycerophosphate in M16 medium increased the buffering capacity Preparation
of the medium. The new medium, named M17, led to an increase
in the development of lactic streptococci, which are bacteria - Dissolve 42.2 g of dehydrated medium
producing large quantities of acid via the homofermentative (BK012) in 1 liter of distilled or deionized
metabolism of lactose. water.
Principles - Dispense in tubes or flasks.
- Casein, meat and soybean peptones contain the carbon and 20 minutes.
nitrogen sources required to cultivate lactococci.
- Yeast extract is a source of B vitamins. Instructions for Use
- Ascorbic acid stimulates growth.
- Lactose is fermented to lactic acid, which is buffered by - Inoculate the tubes with 1 mL of inoculum
glycerophosphate. and of its serial tenfold dilutions.
- Incubate :
Typical Composition - at 37°C for 48 hours for Streptococcus
(can be adjusted to obtain optimal performance) thermophilus.
- at 30°C for 72 hours for mesophilic
For 1 liter of medium : streptococci.
- Tryptone 2.50 g
- Lactose 5.00 g
- Sodium glycerophosphate 19.00 g
- Magnesium sulfate 0.25 g
- Ascorbic acid 0.50 g
Quality Control

homogeneous.
- Prepared medium : light brown solution, limpid.
Streptococcus thermophilus ATCC® 14485 good to excellent
Lactococcus lactis subsp. lactis ATCC 11454 good to excellent

156
Packaging
Dehydrated medium
Bibliography
(1) Terzaghi, B.E., and Sandine, W.E. 1975. Improved medium for (2) FIL-IDF 146A: 1998. Yoghurt. Identification of characteristic
lactic streptococci and their bacteriophages. Appl. Microbiol., microorganisms (Lactobacillus delbrueckii subsp. bulgaricus and
29: 807-813. streptococcus thermophilus).
MacConkey Agar
Intended Use
Product Profile
MacConkey Agar is a selective medium for the isolation of Target : Coliforms, Salmonella, Shigella.
Salmonella and Shigella, as well as for coliform bacteria in water, Reconstitution : 50.0 g / L.
food, pharmaceutical and biological products. Its formula is Autonomy (500 g) : 666 plates.
recommended in the European and United States Pharmacopoeias
for the control of microbial contamination. pH (25°C) : 7.1 ± 0.2.
History (optional).
The formulation of MacConkey for the isolation of enterobacteria
has been modified a number of times since its inception. The
present medium is the “classical” formula used for many years by Preparation
authors such as Block and Ferguson, who found the medium
satisfactory for the isolation of fastidious Shigella. - Suspend 50.0 g of dehydrated medium
Principles water.
- Bile salts and crystal violet inhibit the growth of Gram-positive complete dissolution.
bacteria. The dye inhibits primarily the development of enterococci - Dispense in tubes or flasks.
and staphylococci. - Sterilize in an autoclave at 121°C for
- The fermentation of lactose to acid is revealed in presence of 15 minutes.
neutral red by the formation of red or pink colonies.
NOTE :
- Lactose-negative colonies form colorless colonies.
MUG (4-methyl-umbelliferyl--D-glucuronide) can
be added to the medium in order to detect
Typical Composition Escherichia coli (Refer to the monograph on
(can be adjusted to obtain optimal performance) MUG Supplement, BS024).
For 1 liter of medium : Instructions for Use

- Tryptone 1.5 g - Cool and maintain the medium at 47°C.
- Peptic digest of meat 1.5 g - Pour into sterile Petri dishes.
- Lactose 10.0 g - Let solidify on a cold surface.
- Bile salts 1.5 g - Dry in an incubator with the covers
- Sodium chloride 5.0 g partially removed.
- Neutral red 30 mg - Inoculate by streaking on the surface of
- Crystal violet 1 mg the medium in order to obtain isolated
- Bacteriological agar 13.5 g colonies.
- Simultaneously inoculate a non-selective
pH of the ready-to-use medium at 25°C : 7.1 ± 0.2. medium such as Bromcresol Purple Lactose
Agar (BK023).
Results
Lactose-positive colonies are red and are

surrounded by a halo of precipitated bile
salts. Lactose-negative colonies are colorless.
157
Quality Control
- Dehydrated medium : pinkish-beige powder, free-flowing and

homogeneous.
- Prepared medium : violet-red agar.

Escherichia coli ATCC® 25922 good to excellent red colonies
Enterobacter aerogenes ATCC 13048 good to excellent red colonies
Proteus vulgaris ATCC 13315 good to excellent colorless colonies,
without swarming
Shigella dysenteriae ATCC 13313 moderate colorless colonies

MUG Supplement :
- Store at 2-8°C, shielded from light.
Packaging
Dehydrated medium :
MUG Supplement :
Bibliography
(1) MacConkey. 1905. Lactose-fermenting bacteria in faeces. J. (3) United States Pharmacopoeia 24. 2000. Microbial Limit Tests,
Hyg., 8: 333. 1814-1818.
(2) Pharmacopée européenne: Addendum 2000. Contrôle (4) ISO/CD 21567. 2000. Microbilogical examination of foods and
microbiologique des produits non stériles (recherche de animal feeding stuffs – Horizontal method for the detection of
microorganismes spécifiés). Solution et milieux de culture Shigella species.
158
MacConkey Broth Purple

MacConkey Broth Purple is used as a presumptive medium for the Target : Coliforms.
detection of coliform bacteria in water, milk, and seafood (oysters). Reconstitution : 35.0 g / L.
History
pH (25°C) : 7.3 ± 0.2.
MacConkey Broth Purple is a modification of the formula originally Supplement required : None.
described by MacConkey in 1901, which contained sodium Sterilization : 121°C / 15 minutes.
taurocholate as inhibitor and litmus as an indicator. In 1905,
MacConkey suggested the use of neutral red instead of litmus.
Preparation
Childs and Allen then showed the inhibitory effect of neutral red
and replaced it by bromcresol purple, which was less inhibitory.
Principles
water.
- Purified bile inhibits the growth of Gram-positive bacteria.
- Lactose fermentation by coliform bacteria results in the
- Dispense in 16 x 160 mm tubes containing
acidification of the medium, which makes the pH indicator
a Durham tube.
(bromcresol purple) turn yellow. The ensuing gas production is
revealed in Durham tubes.
15 minutes.
Typical Composition
not contain air.
- Tryptone 20.00 g
water.
- Lactose 10.00 g
- Bromcresol purple 0.01 g
(without a Durham tube).
15 minutes.
Quality Control
- Dehydrated medium : beige-green powder, free-flowing and
homogeneous.
- Prepared medium : violet solution, limpid.
- Inoculate tubes with 1 mL of inoculum and
its serial tenfold dilutions.
Microorganisms Growth Gas production Double strength medium

Escherichia coli ATCC
®
25922 good to excellent positive - Inoculate tubes with 10 mL of inoculum.
Enterobacter cloacae ATCC 13047 good to excellent positive - Cover with a plug of sterile agar (type E
Agar) previously maintained at 47°C.
Staphylococcus aureus ATCC 25923 inhibited Incubation
- For coliform bacteria, incubate for
Storage / Shelf Life 24 hours at 30 or 37°C, depending on the
analytical protocol applied.
Dehydrated medium : 2-30°C. - For heat-tolerant coliform bacteria,
- The expiration date is indicated on the label. incubate for 24 hours at 44.5°C.
Prepared medium (benchmark value) : - Transfer one loop from a double strength
- Media in tubes or vials : 6 months at 2-8°C, shielded from light. tube to a single strength tube and
incubate for an additional 24 hours
Packaging before reading.
159
Results
The fermentation of lactose results in the

production of gas in the Durham tube
(minimal volume equal to 1/10 of the
volume of the tube) within 24 hours and
indicates the presence of coliform bacteria.
Bacteria can be identified in the gas-
positive tubes using subcultures on
appropriate agar media :
- BCP Lactose Agar (BK023),
- EMB Agar (BK056).
Bibliography
(1) MacConkey, A., and Hill. 1901. Bile salt broth Thompson Yates. (3) MacConkey, A. 1908. Bile salt media and their advantages in
Lab. Rep VI/1. some bacterial examinations. J. Hyg., 8: 322-334.
(2) MacConkey, A. 1905. Lactose fermenting bacteria in faeces J. (4) Pharmacopée européenne. Addendum 2000. Contrôle
Hyg., 8: 333-379. microbiologique des produits non stériles (Recherche de
microorganisms specifies). Solution et milieux de culture
MacConkey Sorbitol Agar (CT-SMAC)

Intended Use
MacConkey Sorbitol Agar (CT-SMAC) is a selective medium for the

isolation and differentiation of Escherichia coli O157:H7 from milk,
ground beef and other food products, water and clinical samples.
History
Escherichia coli O157:H7 is recognized as an important human

pathogen, first associated with hemorrhagic colitis in 1982. Food
products of animal origin are the main source of human
contamination. Beef, pork, poultry and unpasteurized dairy
products have been incriminated in outbreaks due to Escherichia
coli O157:H7. This microorganism has also been isolated from
potatoes, apple cider and tap water. In 1985, Farmer and Davis
confirmed the works of Wells et al. (1983) while demonstrating that
Escherichia coli O157:H7 (colorless colonies)
the O157:H7 serotype does not ferment sorbitol, in contrast 80% of
Escherichia coli non O157:H7 (red colonies)
all other Escherichia coli ferment sorbitol. In 1986, March and
Ratman emphasized the advantage of using MacConkey-Sorbitol
Product Profile
Agar to distinguish Escherichia coli O157:H7 colonies. In 1991,
Chapman added cefixime to the medium to inhibit the growth of Target : Escherichia coli O157:H7.
Proteus. In 1993, Zadik combined the action of potassium tellurite Reconstitution : 50.0 g / L.
with cefixime, increasing the sensitivity for the detection of
Escherichia coli O157:H7 by differential inhibition of other
organisms. pH (25°C) : 7.1 ± 0.2.
Supplement required : Cefixime-Tellurite
Principles Selective Supplement for CT-SMAC Agar.
- Polypeptone favors the growth of Escherichia coli O157:H7.
- Sorbitol negative bacterial (in particular O157:H7) colonies are
colorless.
- Sorbitol positive bacteria give rise to red colonies, due to the
change of the color of the pH indicator (neutral red).
- Contaminating bacteria are inhibited by the association of bile
salts, crystal violet, cefixime and potassium tellurite.
160
Typical Composition of complete medium Preparation
- Tryptone 17.0 g deionized water.
- Peptic digest of meat 3.0 g - Bring to boiling slowly, stirring until
- D-Sorbitol 10.0 g complete dissolution.
- Bile salts n°3 1.5 g - Dispense 100 mL in flasks.
- Sodium chloride 5.0 g - Sterilize in an autoclave at 121°C for
- Neutral red 30 mg 15 minutes.
- Cefixime 0.05 mg Instructions for Use
- Potassium tellurite 2.5 mg
- Bacteriological agar 13.5 g - Melt the medium (if it was prepared in
advance).
pH of the ready-to-use medium at 25°C : 7.1 ± 0.2. - Cool and maintain at 47°C.
- Aseptically add 1 mL of reconstituted
Quality Control Cefixime-Tellurite Selective Supplement for
CT-SMAC Agar (BS037).
- Dehydrated medium : pinkish-beige powder, free-flowing and - Mix well.
- Prepared medium : violet-red agar. - Let solidify on a cold surface.
partially removed.
Microorganisms Growth Characteristics - Inoculate by streaking the enrichment
Escherichia coli O157:H7 ATCC® 43895 good to excellent colorless colonies medium used for the detection of
Escherichia coli O157:H7 ATCC 35150 good to excellent colorless colonies Escherichia coli O157:H7, in such a way as
Escherichia coli ATCC 25922 partially to completely red colonies to obtain isolated colonies.
(sorbitol-positive) inhibited - Incubate for 24 hours at 37°C.
Proteus mirabilis ATCC 29906 inhibited
Enterococcus faecalis ATCC 29212 inhibited Results
After 24 hours incubation, Escherichia coli
O157:H7 form smooth and colorless colonies,
which may present an orange-colored halo.
Due to incomplete specificity of the medium,
colonies suspected to be Escherichia coli
Prepared medium (benchmark value) : O157:H7 must be submitted to serological
- Base media in vials : 6 months at 2-8°C. confirmation.
- Complete media in plates : 8 days at 2-8°C, shielded from light.
Cefixime-Tellurite Selective Supplement :
Packaging

(without Cefixime-Tellurite Supplement) :
Cefixime-Tellurite Selective Supplement :
Bibliography
(1) Wells, J. G., B. R. Davis, I. K. Wachsmuth, L. W. Riley, R. S. (4) Chapman, P. A., C. A. Siddons, P. M. Zadik, and L. Jewes.
Remis, R. Sokolow, and G. K. Morris. 1983. Laboratory 1991. An improved selective medium for the isolation of
investigation of hemorrhagic colitis outbreaks associated with a Escherichia coli O157. J. Med. Microbiol. 35 : 107-110.
rare Escherichia coli serotype. J. Clin. Microbiol. 18 : 512-520. (5) Zadik, P. M., P. A. Chapman, and C. A. Siddons. 1993. Use
(2) Farmer, J. J. III, and B. R. Davis. 1985. H7 antiserum-sorbitol tellurite for the selection of verocytotoxigenic Escherichia coli
fermentation medium : a single tube screening medium for O157. J. Med. Microbiol. 39 : 155-158.
detecting Escherichia coli O157:H7 associated with (6) prNF EN ISO 16654. March 1999. Microbiology of food and
hemorrhagic colitis. J. Clin. Microbiol. 22 : 620-625. animal feeding stuffs. Horizontal method for the detection of
(3) March, S. B., and S. Ratnam. 1986. Sorbitol-MacConkey Escherichia coli O157.
medium for detection of Escherichia coli O157 : H7 associated
with hemorrhagic colitis. J. Clin. Microbiol. 23 : 869-872.
161
Malt Agar
Intended Use
Product Profile
Malt Agar is used for the enumeration of yeasts and molds in foods Target : Yeasts, Molds.
and pharmaceuticals. It can also be used for the isolation and Reconstitution : 45.0 g / L.
maintenance of strains. Autonomy (500 g) : 740 plates.
History pH (25°C) : 4.5 ou 3.5 (depending on use).

In 1919, Reddish, followed by Fullmer and Grimes, used malt extract Sterilization : 121°C / 15 minutes.
to promote the development of yeasts in a synthetic medium. In
1926, Thom and Church used the Reddish medium successfully for Preparation
the study of Aspergillus species.
water.
Malt extract is rich in carbohydrates. In an acid medium, it supplies - Slowly bring to boiling, stirring until
all the nutritive elements required for the metabolism of yeasts and complete dissolution.
molds. In addition, the acidity of the medium inhibits most - Dispense in tubes or flasks.
contaminating bacteria. - Sterilize in an autoclave at 121°C for
15 minutes.
Typical Composition
(can be adjusted to obtain optimal performance) Instructions for Use
- Malt extract 30 g - Cool and maintain the medium at 47°C.
- Bacteriological agar 15 g - Adjust the pH to 4.5 by adding a sterile
10% solution of lactic acid.
Quality Control - Pour into sterile Petri dishes.
- Dehydrated medium : light beige powder, free-flowing and - Dry in an incubator with the covers
homogeneous. partially removed.
- Prepared medium : amber agar. - Transfer 0.1 mL of the sample to analyze
- Typical culture response after 72 hours of incubation at 25-30°C : and its serial tenfold dilutions to the plates.
- Spread the inoculum on the surface with a
Microorganisms Growth sterile triangle.
- Incubate at 20-25°C for 3 to 5 days in the light.
Saccharomyces cerevisiae ATCC® 9763 good
Candida albicans ATCC 10231 good NOTE
Aspergillus niger ATCC 16404 good In order to obtain a more selective utilization,
the pH can be adjusted to 4.5 or 3.5 with sterile
lactic acid. Never heat the medium after adding
Storage / Shelf Life acid in order to avoid diminishing the gelling
properties of the agar.
- The expiration date is indicated on the label. Results
- Media in tubes or vials : 6 months at 2-8°C. Count yeasts and molds separately.
- Media in plates : 1 month at 2-8°C. Carry out a rapid microscopic confirmation
test for each type of colony encountered.
Packaging
Dehydrated medium :
Bibliography
(1) Thom, and Church. 1926. The Aspergilli.

(2) NF V 18-301: March 1983. Animal feeding stuffs. Enumeration
of moulds.
162
Mannitol Salt Agar
Intended Use
Mannitol Salt Agar is used for the selective isolation, detection and
enumeration of pathogenic staphylococci in milk, meat products,
fish and seafood, other food products, pharmaceuticals, cosmetics,
and biological samples.
History
The experiments of Koch showed that staphylococci could tolerate

hypersalted media at 7.5%. Chapman confirmed these initial results
and observed that staphylococci which coagulated rabbit plasma
formed yellow colonies on this medium, while most other bacteria
were inhibited.
Principles
Staphylococcus aureus Staphylococcus epidermidis
- The high sodium chloride concentration inhibits the growth of
most bacteria other than staphylococci.
- Mannitol fermentation, shown by the color change of the pH Product Profile
indicator (phenol red) to yellow orients the diagnosis. Target : Pathogenic staphylococci.
- The demonstration of pathogenic staphylococci is confirmed by a Reconstitution : 111.0 g / L.
coagulase test and, optionally, deoxyribonuclease and phosphatase tests.
Typical Composition pH (25°C) : 7.4 ± 0.2.
(can be adjusted to obtain optimal performance) Supplement required : None.
For 1 liter of medium : Sterilization : 121°C / 15 minutes.
- Tryptone 5.0 g
- Peptic digest of meat 5.0 g Preparation
- Mannitol 10.0 g - Suspend 111.0 g of dehydrated medium
- Sodium chloride 75.0 g (BK030) in 1 liter of distilled or deionized
- Phenol red 25.0 mg water.
- Bacteriological agar 15.0 g - Slowly bring to boiling, stirring until
pH of the ready-to-use medium at 25°C : 7.4 ± 0.2. - Dispense in tubes or flasks.
Quality Control 15 minutes.
- Dehydrated medium : pinkish powder, free-flowing and Instructions for Use

homogeneous.
- Prepared medium : red agar. - Melt the medium (if prepared in advance).
- Typical culture response after 24-48 hours of incubation at 37°C : - Cool and maintain at 47°C.
Microorganisms Growth Characteristics - Let solidify on a cold surface.
Staphylococcus aureus ATCC® 6538P good to Yellowish colonies sur- - Dry in an incubator with the covers
excellent rounded by a yellow halo partially removed.
- Transfer 0.1 mL of the product to analyze
Staphylococcus aureus ATCC 25923 good to Yellowish colonies sur-
and its serial tenfold dilutions to the Petri
excellent rounded by a yellow halo
dishes.
Staphylococcus epidermidis ATCC 12228 good Pinkish colonies surrounded - Spread the inoculum on the surface of the
by a red zone medium with a sterile triangle.
Escherichia coli ATCC 25922 inhibited - Incubate at 37°C for 24 and 48 hours.
163
Dehydrated medium : 2-30°C. Pathogenic staphylococci form luxuriant

- The expiration date is indicated on the label. pigmented colonies, surrounded by a yellow
Prepared medium (benchmark value) : ring due to the fermentation of mannitol.
- Media in vials : 6 months at 2-8°C. Non-pathogenic staphylococci generally
- Media in plates : 1 month at 2-8°C. give rise to small red colonies which do not
change the color of the medium.
Packaging Several strains of Staphylococcus
epidermidis can ferment mannitol.
Dehydrated medium : After 48 hours of incubation, several strains
- 500 g bottle : BK030HA of enterococci, Bacillus, Micrococcus and
- 5 kg drum : BK030GC Serratia may grow.
Bibliography
(1) Chapman, G.H. 1945. The significance of sodium chloride in (4) NF T 90-421: October 1989. Testing water. Bacteriological
studies of staphylococci. J. Bacteriol., 50: 201. examinations of swimming pool water.
(2) Chapman, G.H. 1948. An improved Stone medium for the (5) United States Pharmacopoeia 24. 2000. Microbial Limit Tests,
isolation and testing of food poisoning staphylococci. Food 1814-1818.
Research, 13: 100-105.
(3) Journal Officiel (French) du 21 septembre 1968 (arrêté du 30
août 1968). Méthodes officielles de prélèvement et d'analyse
bactériologiques des glaces et crèmes glacées.
Meat-Liver Glucose Agar
Intended Use
Product Profile
Meat-Liver Glucose Agar is used to enumerate spores of sulfite- Target : Spores of sulfite-reducing Clostridia.
reducing clostridia in water, dairy and other food products. Reconstitution : 49.0 g / L (BK092)
48.0 g / L (BK157).
Principles Autonomy (500 g) :
510 tubes (20 mL by tube) for BK092.
- Meat-liver peptone assures the growth of most microorganisms, 520 tubes (20 mL by tube) for BK157.
particularly that of anaerobic bacteria.
pH (25°C) : 7.6 ± 0.2.
- Glucose is the energy source for growth.
- Starch favors spore germination. Supplement required : Sodium sulfite +
- Anaerobic bacteria reduce sulfite to sulfide, which in presence of Ferric ammonium sulfate (BK092).
ferric citrate causes the blackening of the colonies due to the Sterilization : 121°C / 15 minutes.
formation of iron sulfide.
Preparation
Typical Composition of base medium (BK092)
(can be adjusted to obtain optimal performance) - Suspend 49.0 g of dehydrated base
medium (BK092) or 48.0 g of complete
For 1 liter of medium : dehydrated medium (BK157) in 1 liter of
- Meat-liver peptone 30.0 g distilled or deionized water.
- Soluble starch 2.0 g complete dissolution.
- Bacteriological agar 15.0 g - Dispense 20 mL in 20 x 200 mm tubes.
pH of the ready-to-use (complete) medium at 25°C : 7.6 ± 0.2. 15 minutes.
164
Typical Composition of complete medium (BK157) Instructions for Use
Base medium (BK092) :
For 1 liter of medium : - Cool and maintain the medium at 47°C.
- Meat-liver peptone 30.0 g - Successively add the following two
- Glucose 2.0 g solutions to each tube :
- Soluble starch 2.0 g - 0.5 mL of sterile 10% sodium sulfite
- Sodium sulfite 2.5 g solution.
- Ferric ammonium citrate 0.5 g - 2 to 3 drops of sterile 5% ferric
- Bacteriological agar 11.0 g ammonium sulfate solution.
- Heat the product to analyze in order to
pH of the ready-to-use medium at 25°C : 7.6 ± 0.2. destroy vegetative cells and activate spores.
- Transfer the medium to a tube containing
Quality Control 5 mL of inoculum or its serial tenfold
dilutions, carefully avoiding the
- Dehydrated media : off-white powder, free-flowing and incorporation of air.
homogeneous. - Homogenize by inverting the tubes.
- Prepared (complete) media : amber agar. - Cool in an ice-water bath.
- Typical culture response after 24 hours of incubation in - Incubate at 37°C for 24 to 48 hours.
complete medium at 37°C :
Complete medium (BK157) :
- Heat the product to analyze in order to
Clostridium perfringens ATCC® 13124 good to excellent black colonies destroy vegetative cells and activate spores.
Clostridium bifermentans ATCC 19299 good to excellent black colonies - Transfer the medium to a tube containing
5 mL of inoculum or its serial tenfold
Storage / Shelf Life dilutions, carefully avoiding the
incorporation of air.
Dehydrated media : 2-30°C. - Homogenize by inverting the tubes.
- The expiration dates are indicated on the labels. - Cool in an ice-water bath.
Prepared media (benchmark value) : - Incubate at 37°C for 24 to 48 hours.
- Base media in tubes : 6 months at 2-8°C.
- Complete media in tubes : 1 month at 2-8°C.
Regenerate at 100°C for 20 minutes before inoculation. Results
Do not repeat the operation more than once.
Count colonies surrounded by a black halo.
Packaging

Dehydrated complete medium :
Bibliography
(1) NF T 90-415: October 1985. Testing water. Detection and

enumeration of the spores of sulfite-reducing anaerobies and
of sulfite-reducing Clostridia. General method by the standing
tube technique.
165
Meat-Liver Glucose - 0.6% Agar

Meat-Liver Glucose - 0.6% Agar is a medium specially designed for
Clostridia spores.
the growth and isolation of anaerobic bacteria growing in the
depth of the medium. It can also be used to elucidate the type of Reconstitution : 38.0 g / L.
bacterial respiration and is suitable for sterility tests of Autonomy (500 g) : 1315 tubes.
pharmaceutical products and canned foods.
pH (25°C) : 7.3 ± 0.2.
- Meat-liver peptone favors the growth of most microorganisms,
particularly that of anaerobic bacteria.
- Glucose is the energy source for growth. Preparation
Typical Composition - Suspend 38.0 g of dehydrated medium

(can be adjusted to obtain optimal performance) (BK024) in 1 liter of distilled or deionized
water.
- Meat-liver peptone 30.0 g - Dispense in 9 x 180 mm tubes (detection of
- Glucose 2.0 g type of respiration and isolation in depth)
- Bacteriological agar 6.0 g or in 20 x 200 mm tubes (sterility testing).
pH of the ready-to-use medium at 25°C : 7.3 ± 0.2. 15 minutes.
- Dehydrated medium : beige powder, free-flowing and - Cool and maintain the medium at 47°C.
homogeneous.
- Prepared medium : amber, semi-solid agar. Respiration determination
- Typical culture response after 24 hours of incubation at 37°C : - Immerse the tapered tip of a Pasteur
pipette in the culture to examine.
Microorganisms Growth - Plunge the inoculum to the bottom of the
tube.
Clostridium perfringens ATCC® 13124 good to excellent
- Raise the tip to the surface in a helicoid
Clostridium bifermentans ATCC 19299 good to excellent movement.
- Cool in an ice bath.
Storage / Shelf Life - Incubate at the optimal growth
temperature of the strain in question.
Dehydrated medium : 2-30°C. - Read the results of the 4 main types of
- The expiration date is indicated on the label. respiration :
Prepared medium (benchmark value) : - Growth in the upper zone : obligate
- Media in tubes : 6 months at 2-8°C. aerobes.
- The medium must be regenerated at 100°C for 20 minutes if it is - Growth in the deep zone : obligate
not used just after autoclaving. Do not repeat this operation more anaerobes.
than once. - Growth throughout the height of the
tube : facultative anaerobes.
Packaging - Growth as a ring in the intermediate
zone : microaerophilic bacteria.
Dehydrated medium :
- 500 g bottle : BK024HA Isolation of anaerobic bacteria
- Transport the inoculum successively in
several tubes of medium (as above) until
exhaustion.
- Cool in an ice-water bath.
- Incubate at the desired temperature.
- Isolated colonies appear in the last tubes of
the series.
- Transfer to another suitable medium for
identification.
166
Sterility tests
- Transfer the inoculum to the medium.
- Incubate for 10 days :
- at 30°C for mesophilic bacteria.
- at 55°C for thermophilic bacteria.
- Read daily.
Meat-Yeast Agar
Product Profile
Intended Use
Target : Spores of sulfite-reducing Clostridia.
Meat-Yeast Agar is used to enumerate spores of sulfite-reducing Reconstitution : 45.3 g / L.
anaerobic bacteria in gelatin and other food products. Autonomy (500 g) : 551 tubes
(20 mL by tube).
Principles
pH (25°C) : 7.6 ± 0.2.
- Tryptone and extracts of meat and yeast favor the development of Supplement required : None.
cultures. Sterilization : 115°C / 20 minutes.
- Glucose is the energy source for growth.
- Starch favors spore germination.
- Anaerobic bacteria reduce sulfite to sulfide, which in presence Preparation
of ferric citrate causes the blackening of the colonies due to the
formation of iron sulfide. - Suspend 45.3 g of dehydrated medium
(can be adjusted to obtain optimal performance) - Slowly bring to boiling, stirring until
For 1 liter of medium : - Dispense 20 mL in 20 x 200 mm tubes.
- Tryptone 10.0 g - Sterilize in an autoclave at 115°C for
- Meat extract 3.0 g 20 minutes.
- Glucose 2.0 g Instructions for Use
- Cysteine hydrochloride 0.3 g - Cool and maintain the medium at 47°C.
- Soluble starch 5.0 g - Heat the product to analyze in order to
- Sodium metabisulfite 1.0 g destroy vegetative cells and activate spores.
- Ferric ammonium citrate 1.0 g - Transfer the medium to a tube containing
- Bacteriological agar 12.0 g 5 mL of inoculum or its serial tenfold
pH of the ready-to-use medium at 25°C : 7.6 ± 0.2. dilutions, carefully avoiding the
incorporation of air.
Quality Control - Homogenize by inverting the tubes.
- Dehydrated medium : beige powder, free-flowing and - Incubate at 37°C.
homogeneous.
- Prepared medium : amber agar. Results
- Typical culture response after 24 hours of anaerobic incubation
at 37°C : Read after 24 hours of incubation, then
daily for 5 days, counting all black
Microorganisms Growth Characteristics colonies.
Clostridium perfringens ATCC® 13124 good to excellent black colonies
Clostridium bifermentans ATCC 19299 good to excellent black colonies

- Not recommended ; use immediately after preparation.
167
Packaging
Dehydrated medium :
Bibliography
(1) NF V 59-106: October 1982. Edible gelatin. Enumeration of

sulfito-reducing anaerobic micro-organisms spores. Anaerobic
colony count technique at 37°C.
Meat-Yeast Glucose - 0.6% Agar

Meat-Yeast Glucose - 0.6% Agar is used to detect the type of
Clostridia spores.
respiration and to isolate anaerobic bacteria, as well as for the
enumeration of spores of thermophilic clostridia in food products. Reconstitution : 30.3 g / L.
It is also suitable for sterility tests of pharmaceutical products and Autonomy (500 g) : 1650 tubes.
preserved foods.
pH (25°C) : 7.2 ± 0.2.
Typical Composition Supplement required : None.
(can be adjusted to obtain optimal performance) Sterilization : 121°C / 15 minutes.

Preparation
- Tryptone 10.0 g
- Yeast extract 5.0 g (BK052) in 1 liter of distilled or deionized
- Cysteine hydrochloride 0.3 g water.
- Sodium chloride 5.0 g complete dissolution.
- Bacteriological agar 6.0 g - Dispense in 9 x 180 mm tubes (detection
of type of respiration and isolation in
depth) or in 20 x 200 mm tubes (sterility
pH of the ready-to-use medium at 25°C : 7.2 ± 0.2. testing).
Quality Control 15 minutes.
- Dehydrated medium : cream-white powder, free-flowing and Instructions for Use

homogeneous.
- Prepared medium : semi-solid agar, amber. - Cool and maintain the medium at 47°C.
Respiration determination
Microorganisms Growth - Immerse the tapered tip of a Pasteur
Clostridium perfringens ATCC® 13124 good to excellent pipette in the culture to examine.
- Plunge the inoculum to the bottom of the tube.
Clostridium bifermentans ATCC 19299 good to excellent
- Raise the tip to the surface in a helicoid
Escherichia coli ATCC 25922 good movement.
Staphylococcus aureus ATCC 25923 good - Cool in an ice bath.
- Incubate at the optimal growth
Storage / Shelf Life temperature of the strain in question.
- Reading results: 4 main types of
Dehydrated medium : 2-30°C. respiration :
- The expiration date is indicated on the label. - Growth in the upper zone : obligate
Prepared medium (benchmark value) : aerobes.
- Media in tubes : 6 months at 2-8°C. - Growth in the deep zone : obligate
- The medium must be regenerated at 100°C for 20 minutes before anaerobes.
inoculating samples. Do not carry out this operation more than - Growth throughout the height of the
once. tube : facultative anaerobes.
- Growth as a ring in the intermediate
zone : microaerophilic bacteria.
168
Packaging Isolation of anaerobic bacteria
- Transport the inoculum successively in
Dehydrated medium : several tubes of medium (as above) until
- 500 g bottle : BK052HA exhaustion.
- Incubate at the desired temperature.
- Isolated colonies appear in the last tubes
of the series.
- Transfer to another suitable medium for
identification.
Sterility tests
- Transfer the inoculum to the medium.
- Incubate for 10 days :
- at 30°C for mesophilic bacteria.
- at 55°C for thermophilic bacteria.
- Read daily.
Bibliography
(1) Journal Officiel (French) du 21 septembre 1968 (arrêté du 30 (2) NF V 03-456 expérimentale (French). Mai 1971. Dénombrement
août 1968). Méthodes officielles de prélèvement et d'analyses des spores de Clostridium thermophiles.
bactériologiques des glaces et crèmes glacées.
mEndo LES Agar

Intended Use
mEndo LES Agar is used for the enumeration of coliforms in water

by membrane filtration.
History
mEndo LES (Laurence Experimental Station) Agar was formulated in

1961 by MacCarthy, Delaney and Grasso for the detection of
coliforms in water. The authors demonstrated that a 2 step
technique combining an enrichment in Laurylsulfate-Tryptose Broth
and isolation on mEndo LES Agar allowed a greater recovery of
coliforms than the one step method using mEndo Broth. Other
procedures have subsequently recommended the use of mEndo LES
Agar without prior enrichment.
Principles Escherichia coli
- Acid hydrolysate of casein, peptic digest of meat, Tryptone and Product Profile
yeast extract favor the growth of coliforms.
Target : Coliforms.
- Phosphates provide buffering action.
- The association between sodium desoxycholate and sodium Reconstitution : 48.0 g / L.
laurylsulfate assure the inhibition of secondary Gram-positive flora. Autonomy (500 g) : 1157 plates
- Sodium sulfite allows the decolorization of basic fuchsin. (9 mL by plate).
- Through the fermentation of lactose by coliforms, the resulting
pH (25°C) : 7.2 ± 0.2.
acetaldehyde produced acts upon the sulfite-fuchsin complex
liberating the fuchsin. This imparts a red coloration to the Supplement required : 95% non-denatured
colonies. In microorganisms that rapidly ferment lactose, the ethanol.
reaction is of an intensity that it produces colonies with a Sterilization : Heat to boiling.
metallic sheen. Do not autoclave.
Microorganisms that do not ferment lactose produce pink to
colorless colonies.
169
- Suspend 48.0 g of dehydrated media
For 1 liter of medium : (BK155) in 1 liter of distilled or
- Yeast extract 1.20 g demineralized water containing 20 mL
of 95% non-denatured ethanol.
- Acid hydrolysate of casein 3.70 g
- Mix well.
- Peptic digest of meat 3.70 g - Slowly bring to boiling, stirring until
- Tryptose 7.50 g complete dissolution.
- Lactose 9.40 g - Do not autoclave.
- Dipotassium phosphate 3.30 g Instructions for Use
- Sodium desoxycholate 0.10 g - Cool and maintain the medium at 47°C.
- Sodium laurylsulfate 0.05 g - Pour into sterile Petri dishes.
- Sodium sulfite 1.60 g - Let solidify on a cool surface.
- Basic fuchsin 0.80 g - Aseptically filter on the appropriate
- Bacteriological agar 12.00 g membrane a determined volume of
sample to test.
pH of the medium ready-to-use at 25°C : 7.2 ± 0.2. - Place the membrane on the surface of the
agar, filtered side up, assuring the proper
Quality Control contact with the agar.
- Dehydrated medium : violet powder, free-flowing and
homogeneous. Results
- Prepared medium : rose-fuchsia agar, which may contain a
slight precipitate. Red colonies presenting a metallic sheen are
- Typical cultural response after 24 hours incubation at 37°C : characteristic of coliforms.
Certain coliforms, however, may produce
Microorganisms Growth Characteristics red colonies without the metallic sheen.
®
good to excellent red colonies with A heavy inoculum may also prevent
a metallic sheen the formation of the characteristic metallic
sheen.
Enterobacter cloacae ATCC 13047 good to excellent red colonies
Salmonella Typhimurium ATCC 14028 good pink colonies

Packaging
Dehydrated medium :
Bibliography
(1) McCarthy, J.A., J.E. Delaney, and R.J. Grasso. 1961. Measuring (3) Eaton, A.D., L.S. Clesceri, and A.E. Greenberg (ed), 1995.
coliforms in water. Water and Sewage Works. 108: 238-243. Standard Methods for the Examination of Water and
(2) Cowman, S., and R. Kelsey. 1992. Bottled water, p 1031-1036. Wastewater. 19th ed. American Public Health Association,
In: C. Vanderzant, and D.F. Splittstoesser (ed). Compendium of Washington, D.C.
methods for the microbiological examination of foods, 3rd ed.
American Public Health Association, Washington, D.C.
170
mFC Agar
Intended Use
mFC Agar is used for the detection and enumeration of

thermotolerant coliforms in water or foods by membrane filtration.
History
The medium was formulated in 1965 by Geldreich and his

collaborators for the enumeration of thermotolerant coliforms in
water by membrane filtration. The authors demonstrated that the
use of mFC Agar allowed the elimination of preliminary enrichment
phases and biochemical identification tests necessary up until that
time, in order to achieve results more rapidly.
Principles
Escherichia coli
- Tryptose and enzymatic digest of meat favor the growth of
coliforms.
- Yeast extract is a source of vitamin B complex. Product Profile
- Bile salts inhibit the growth of Gram-positive secondary flora. Target : Thermotolerant coliforms.
- Lactose fermentation by thermotolerant coliforms leads to an
acidification which is revealed by the blue coloration of the pH Reconstitution : 49.1 g / L.
indicators, comprised of aniline blue and rosolic acid. Autonomy (500 g) : 1131 plates
(9 mL by plate).
Typical Composition of the complete medium pH (25°C) : 7.6 ± 0.2.
Supplement required : Rosolic Acid
For 1 liter of medium : Selective Supplement.
- Tryptose 10.0 g
- Enzymatic digest of meat 5.0 g
Do not autoclave.
- Lactose 12.5 g
Preparation
- Bile salts 1.5 g
- Aniline blue 0.1 g
- Rosolic acid 0.1 g
deionized water.
- Add 10 mL of reconstituted Rosolic Acid
pH of the complete medium at 25°C : 7.6 ± 0.2. Selective Supplement (BS040)
- Mix well.
Quality Control - Slowly bring to boiling, stirring until
- Dehydrated medium : beige powder, free-flowing and - Do not autoclave.
homogeneous.
- Prepared medium (complete) : pinkish-purple agar. Instructions for Use
- Typical cultural response after 24 hours incubation at 44°C :
Microorganisms Growth Characteristics - Pour into sterile Petri dishes.
Escherichia coli ATCC® 25922 good to excellent blue colonies - Let solidify on a cool surface.
Enterobacter cloacae ATCC 13047 good to excellent blue colonies - Aseptically, filter on the appropriate
Salmonella Typhimurium ATCC 14028 good colorless colonies membrane a determined volume of the
Enterococcus faecalis ATCC 29212 inhibited sample to test.
Staphylococcus aureus ATCC 25923 inhibited - Place the membrane on the surface of the
agar, filtered side up, assuring the proper
Storage / Shelf Life contact with the agar.
Dehydrated base medium (without Rosolic Acid) : 2-30°C.
Rosolic Acid Selective Supplement :
171
Packaging Results
Dehydrated base medium (without Rosolic Acid) : Retain those membranes with colonies
- 500 g bottle : BK154HA numbering under 100.
Rosolic Acid Selective Supplement : The thermotolerant coliforms present a blue
- 1 g vial : BS04008 to blue-gray coloration. A blue halo in the
medium under the membrane is observed
during an intense fermentation of lactose.
Other bacteria that do not ferment lactose
produce pink or colorless colonies.
Bibliography
(1) Geldreich, E.E., H.F. Clarck, C.B. Huff, and L.C. Best. 1965. (4) G.R. Acuff. 1992. Media, reagents, and stains. In: C. Vanderzant,
Fecal-coliform-organism medium for the membrane filter and D.F. Splittstoesser (ed). Compendium of methods for the
technique. J. Am. Water Works Assoc. 57: 208-214. microbiological examination of foods, 3rd ed. American Public
(2) Cowman, S., and R. Kelsey. 1992. Bottled water,. In: Health Association, Washington D.C. p 1093-1208.
C. Vanderzant, and D.F. Splittstoesser (ed). Compendium of (5) Andrews, W. 1995. Microbial methods. In Official methods of
methods for the microbiological examination of foods, 3rd ed. analysis of AOAC International, 16th ed. AOAC International.
American Public Health Association, Washington, D.C. Arlington, VA 17.1-17.119.
p 1031-1036.
(3) Eaton, A.D., L.S. Clesceri, and A.E. Greenberg (ed), 1995.
Standard Methods for the Examination of Water and
Wastewater. 19th ed. American Public Health Association,
Washington, D.C.
Minerals Glutamate Medium

Target : Total coliforms,
Minerals Glutamate Medium is used for the detection and Thermotolerant coliforms.
enumeration of heat-tolerant coliform bacteria in gelatin. It can Reconstitution : 17.7 g / L.
also be used for the presumptive identification and enumeration of
presumed total and heat-tolerant coliform bacteria, as well as Autonomy (500 g) : 2825 tubes.
Escherichia coli in water, by the most probable number method. pH (25°C) : 6.7 ± 0.1.
Supplement required : Ammonium chloride.
History
In 1948, Folpmers was the first to develop a chemically defined
medium based on the use of glutamic acid for the enumeration of Preparation
coliform bacteria in water. The medium was subsequently modified
by Gray and found to be better than MacConkey broth for the Double strength medium
detection of Escherichia coli in chlorinated and non-chlorinated - Dissolve 35.4 g of dehydrated medium
water. (BK005) in 1 liter of distilled or deionized
water.
Principles - Add 5 g of ammonium chloride.
- The presence of coliform bacteria is shown by gas production from complete dissolution.
lactose, as well as the color change of bromcresol purple to yellow. - Dispense in 20 x 200 mm tubes or in flasks
- Enterococci are inhibited by the absence of nutrients essential for containing one or several Durham tubes.
their growth. - Sterilize in an autoclave at 115°C for
15 minutes.
- Verify that the pH after cooling to 25°C is
6.7 ± 0.1.
- Verify that the Durham tubes do not
contain trapped air.
172
Typical Composition of base medium (without ammonium chloride) Single strength medium
(can be adjusted to obtain optimal performance) - Dissolve 17.7 g of dehydrated medium
For 1 liter of medium : water.
- Sodium glutamate 6.35 g - Add 2.5 g of ammonium chloride.
- Sodium formate 0.25 g - Slowly bring to boiling, stirring until
- L(-) cystine 20.0 mg complete dissolution.
- L(-) aspartic acid 24.0 mg - Dispense in 16 x 160 mm tubes containing
- L(+) arginine hydrochloride 20.0 mg a Durham tube.
- Lactose 10.00 g - Sterilize in an autoclave at 115°C for
- Thiamine 1.0 mg 15 minutes.
- Nicotinic acid 1.0 mg - Verify that the pH after cooling to 25°C
- Pantothenic acid 1.0 mg is 6.7 ± 0.1.
- Magnesium sulfate 0.10 g - Verify that the Durham tubes do not
- Ferric ammonium citrate 10.0 mg contain trapped air.
- Calcium chloride 10.0 mg
- Dipotassium phosphate 0.90 g Instructions for Use
- Bromcresol purple 10.0 mg
Quality Control - Inoculate the tubes or flasks with a volume
of inoculum equal to the volume of
- Dehydrated medium : bluish-gray powder, free-flowing and medium.
homogeneous. Single strength medium
- Prepared medium : blue-violet solution, limpid. - Inoculate one loop of presumed positive
- Typical culture response after 24-48 hours of incubation at 30°C : double strength medium into tubes of
single strength medium and incubate
Microorganisms Growth Gas production again.
Escherichia coli ATCC ®
25922 good to excellent positive Incubation
Enterobacter aerogenes ATCC 13048 good to excellent positive - For total coliform bacteria, incubate for
24 and 48 hours at 30 or 37°C, depending
Citrobacter freundii ATCC 8090 good to excellent positive
on the analytical protocol applied.
Enterococcus faecalis ATCC 29212 inhibited - For heat-tolerant coliform bacteria,
Staphylococcus aureus ATCC 25923 inhibited incubate at 44.5°C for 48 ± 2 hours.
Results
Dehydrated base medium (without ammonium chloride) : 2-30°C. The presence of coliform bacteria is shown
- The expiration date is indicated on the label. by gas production in the Durham tubes as
Prepared medium (benchmark value) : well as the fact that the indicator turns
- Complete media in tubes : 6 months at 2-8°C. yellow.
Enumerate with the most probable number
Packaging method.
Dehydrated base medium (without ammonium chloride) : IDENTIFICATION OF ESCHERICHIA COLI

- 500 g bottle : BK005HA One loop of each presumptive positive tube
is transferred to Brilliant Green Bile Broth
(BK002, BM011) and to a tube of Peptone
Water (BK084).
Incubate at 44.5 ± 0.5°C in a water bath for
24 and 48 hours.
presents the following characteristics :
- growth in BGBB : positive and gas
production.
- indole production (Ehrlich-Kovacs
Bibliography
(1) Folpmers, T. 1948. Ant. V. Leenwenhoek. J. Microbi. Serol., 14: (5) NF V 59-103: October 1982. Edible gelatin. Determination of
58-64. fecal coliforms. 44.5°C culture method on liquid selective
(2) Gray, R. D. 1959. J. Hyg. Camb., 57: 249-265. medium.
(3) Gray, R.D. 1964. J. Hyg. Camb., 62: 495-508. (6) Rodier, J. 1984. L’analyse de l’eau. Dénombrement des
(4) NF V 59-102: October 1982. Edible gelatin. Detection of coliformes, coliformes fécaux et Escherichia coli présumés.
coliforms. 30°C culture method on liquid selective medium. Dunod 7ème Ed : 793-798.
173
Modified EC Broth (mEC)

Target : Escherichia coli O157:H7.
Modified EC Broth (mEC) is an enrichment medium destined for the
detection of pathogenic serotypes of Escherichia coli, in particular Reconstitution : 36.6 g / L.
the O157:H7 serotype, in food products and other potentially Autonomy (500 g) : 60 vials.
contaminated samples.
pH (25°C) : 6.9 ± 0.2.
History Supplement required : Novobiocin Selective
Supplement.
The medium is a modification of EC Broth used for the detection and Sterilization : 121°C / 15 minutes.
enumeration of coliforms and Escherichia coli. In 1986, Szabo et al.
noted that Modified EC Broth, in which the bile salts concentration
Preparation
had been reduced to 1.12 g/L, allowed for full recovery of
Escherichia coli O157:H7, while the majority of other non-
enterobacteria were inhibited. In 1987, Doyle et al. demonstrated
the advantage of selective enrichment using novobiocin at 20 mg/L.
water.
In 1990, Okrend et al. successfully employed and proposed the use of
Modified EC Broth with novobiocin for the enrichment of Escherichia
- Dispense in flasks, 225 mL per flask.
coli O157:H7.
15 minutes.
Principles
- The combined presence of bile salts and novobiocin ensures the
inhibition of Gram-positive microorganisms and certain Gram-
- Cool to 25°C.
negative microorganisms such as Proteus mirabilis.
- Aseptically add 2.25 mL of reconstituted
- Tryptone and lactose, respectively, provide nitrogen and energy
Novobiocin Selective Supplement (BS033)
sources for the growth of Escherichia coli O157:H7.
per flask.
- The phosphates buffer the medium to increase recovery.
- Aseptically add 25 g of the product to be
analyzed.
Typical Composition
- Mix well.
- Incubate at 37°C or 41.5°C for 18 to
24 hours, depending on the analysis
protocol followed.
- Tryptone 20.0 g
- Isolate on CT-SMAC Agar (BK147 + BS037).
- Lactose 5.0 g
- Bile salts 1.12 g NOTE
- Sodium chloride 5.0 g After 6 hours incubation, it is recommended to
- Dipotassium phosphate 4.0 g perform an immunomagnetic enrichment on an
- Potassium dihydrogen phosphate 1.5 g aliquot and isolate on CT-SMAC Agar (BK147 +
- Novobiocin 20 mg BS037).
Quality Control

homogeneous.
- Prepared medium : amber solution, clear.
- Typical culture response after 24 hours of incubation at 41.5°C,
followed by subculture on CT-SMAC Agar :
Escherichia coli O157:H7 ATCC
®
43895 good to excellent
Escherichia coli O157:H7 ATCC 35150 good to excellent
174

- Base media in vials : 3 months at 2-8°C, shielded from light.
- Complete media in vials : 30 days at 2-8°C, shielded from light.
Novobiocin Selective Supplement :
Packaging
Dehydrated base medium (without novobiocin) :

Bibliography
(1) Szabo, R.A., ECD Todd, and A. Jean. 1986. Method to isolate (3) Okrend, A.J.G., B.E. Rose, and B. Bennett. 1990. A screening
Escherichia coli O157:H7 from food. J. Food Prot. 49: 768-772. method for isolation of Escherichia coli O157:H7 from ground
(2) Doyle, M.P., and J.L. Schoeni. 1987. Isolation of Escherichia beef. J. Food Prot. 53: 249-252.
coli O157:H7 from retail fresh meats and poultry. Appl. Envir.
Microbiol. 53: 2394-2396.
Modified Tryptone-Soy Broth (mTSB)
Intended Use
Product Profile
Modified Tryptone-Soy Broth (mTSB ) is an enrichment medium Target : Escherichia coli O157:H7.
destined for the detection of pathogenic serotypes of Escherichia Reconstitution : 33.0 g / L.
coli, in particular the O157:H7 serotype, in food products and other Autonomy (500 g) : 67 vials.
potentially contaminated samples.
pH (25°C) : 7.4 ± 0.2.
History Supplement required : Novobiocin Selective
Supplement.
Many authors have successfully used modified Tryptone-Soy Broth Sterilization : 121°C / 15 minutes.
for the selective enrichment of Escherichia coli O157:H7. The base
formula of Tryptone-Soy Broth was originally modified by addition
Preparation
of 1.5 g/L of dipotassium phosphate and 1.5 g/L of bile salts. In
1987, Doyle et al. proposed the addition of 20 mg/L of novobiocin - Dissolve 33.0 g of dehydrated medium
to reinforce selectivity and to enhance detection of Escherichia coli (BK150) in 1 liter of distilled or deionized
O157:H7. water.
- Stir slowly, until complete dissolution.
Principles - Dispense in flasks, 225 mL per flask.
- The nutrient base combining Tryptone, papaic digest of soybean 15 minutes.
meal and glucose provides for optimal growth of Escherichia coli
O157:H7.
- The combined presence of bile salts and novobiocin ensures the
inhibition of Gram-positive microorganisms and certain Gram-
negative microorganisms such as Proteus.
- The dipotassium phosphate maintains pH and thus increases
recovery capacity.
175
Typical Composition of complete medium Instructions for Use
- Cool to 25°C.
For 1 liter of medium : - Aseptically add 2.25 mL of reconstituted
- Tryptone 17.0 g Novobiocin Selective Supplement (BS033)
- Papaic digest of soybean meal 3.0 g per flask.
- Glucose 2.5 g - Aseptically add 25 g of the product to be
- Bile salts n°3 1.5 g analyzed.
- Sodium chloride 5.0 g - Mix well.
- Dipotassium phosphate 4.0 g - Incubate at 41.5°C.
- Novobiocin 20 mg - After 6 hours of incubation, perform an
immunomagnetic separation on an aliquot
pH of the ready-to-use medium at 25°C: 7.4 ± 0.2. and isolate on CT-SMAC Agar
(BK147 + BS037).
Quality Control - After 24 hours incubation, isolate again on
CT-SMAC Agar.
- Dehydrated medium : cream white powder, free-flowing and
homogeneous.
- Prepared medium (complete) : amber solution, clear.
followed by subculture on CT-SMAC Agar :
Escherichia coli O157:H7 ATCC® 43895 good to excellent
Escherichia coli O157:H7 ATCC 35150 good to excellent

- Base medium : 3 months at 2-8°C, shielded from light.
- Complete medium in flasks : 30 days at 2-8°C, shielded from light.
Packaging
Dehydrated base medium (without novobiocin) :

Bibliography
(1) Doyle, M.P., and J.L. Schoeni. 1987. Isolation of Escherichia (3) prNF EN ISO 16654. March 1999. Microbiology of food and
coli O157:H7 from retail fresh meats and poultry. Appl. Envir. animal feeding stuffs. Horizontal method for the detection of
Microbiol. 53: 2394-2396. Escherichia coli O157.
(2) Feldsine, P.T., M.T. Falbo-Nelson, S.L. Brunelle, and R.L. Forgey.
1997. Assurance Enzyme Immunoassay for the Detection of
Enterohemorrhagic Escherichia coli O157:H7 in Selected Foods:
Collaborative Study. Journal of AOAC International.
80: 530-543.
176
MRS Agar
Intended Use
MRS Agar is used for the growth and enumeration of cultures of

Lactobacillus in dairy and other food products. The medium can be
used to culture slowly-growing lactobacilli such as Lactobacillus
brevis and Lactobacillus fermentum. Acidified to low pH, it can be
used to enumerate Lactobacillus bulgaricus in yogurts.
History
De Man, Rogosa and Sharpe developed a formulation in 1960 for a

medium specifically designed for the culture of lactobacilli in dairy
products, without the need to add tomato juice (an ingredient of
highly variable composition).
Principles
Lactobacillus casei
- The different peptones, glucose, magnesium and manganese salts
supply the nutritive elements required for the growth of
lactobacilli. Product Profile
- Tween 80 is composed of a mixture of oleic esters and is a source Target : Lactobacilli.
of fatty acids essential for the growth of these bacteria. Reconstitution : 70.3 g / L.
- Dipotassium phosphate stabilizes the pH during bacterial growth.
- Ammonium citrate and sodium acetate inhibit the development of
most contaminants, including streptococci and molds. pH (25°C) : Variable depending on sample.
Typical Composition
For 1 liter of medium : Preparation

- Meat extract 10.00 g - Suspend 70.3 g of dehydrated medium
- Yeast extract 5.00 g (BK089) in 1 liter of distilled or deionized
- Glucose 20.00 g water.
- Tween 80 1.08 g - Slowly bring to boiling, stirring slowly
- Dipotassium phosphate 2.00 g until complete dissolution.
- Sodium acetate 5.00 g - Adjust the pH, depending on the product
- Ammonium citrate 2.00 g to analyze with acetic acid.
- Magnesium sulfate 0.20 g - Dispense in tubes or flasks.
- Manganese sulfate 0.05 g - Sterilize in an autoclave at 121°C for
- Dehydrated medium : cream powder, slightly clumped - Cool and maintain the medium at 47°C.
and brittle. - Transfer 1 mL of the product to analyze
- Prepared medium : amber agar. and its serial tenfold dilutions to sterile
- Typical culture response after 48 hours of incubation Petri dishes.
at 37°C in 5% CO2 : - Pour in 15 mL of medium.
Microorganisms Growth - Let solidify on a cold surface.
Lactobacillus casei subsp. rhamnosus ATCC 7469
®
good to excellent - Place the inoculated plates in an
anaerobiosis jar in an atmosphere
of 5% CO2.
Lactobacillus delbrueckii subsp. lactis ATCC 4797 good to excellent - Incubate at 37°C for 2 to 3 days.
177
Dehydrated medium : 2-20°C. Lactobacillus delbrueckii subsp. bulgaricus

- The expiration date is indicated on the label. forms lens-shaped colonies, often
Prepared medium (benchmark value) : polylobate, 1 to 3 mm in diameter, after
- Media in vials : 6 months at 2-8°C. 3 days of incubation.
Verify under the microscope that the cells
Packaging are Gram-positive, non-sporulated bacilli.
Dehydrated medium :
Bibliography
(1) De Man, J.C., Rogosa, M., and Sharpe, M.E. 1960. A medium (4) ISO 13721. December 1995. Meat and meat products.
for the cultivation of lactobacilli. J. App. Bacteriol., 23, (1): Enumeration of lactic acid bacteria. Colony-count technique at
130-135. 30°C.
(2) Journal Officiel (French) du 4 janvier 1978. Méthode officielle (5) FIL-IDF 117B: 1997. Yogurt. Enumeration of characteristic
d’analyse pour le dénombrement de la flore spécifique du microorganisms. Colony count technique at 37°C.
yaourt ou yoghourt (arrêté du 25 Novembre 1977). (6) NF ISO 15214. Sept. 1998. Microbiology of food and animal
(3) NF V 04-503: September 1988. Meat and meat products. feeding stuffs. Horizontal method for the enumeration of
Enumeration of lactic bacteria. mesophilic lactic acid bacteria. Colony-count technique at
30°C.
MRS Broth
Intended Use
Product Profile
MRS Broth is used for the growth and enumeration of lactobacilli in Target : Lactobacilli.
food products. By transferring isolated colonies, luxuriant Reconstitution : 55.3 g / L.
subcultures can be obtained. The medium can also be used to
culture slowly-growing lactobacilli such as Lactobacillus brevis and
Lactobacillus fermentum. pH (25°C) : 6.4 ± 0.2.
De Man, Rogosa and Sharpe developed a formulation in 1960 for Preparation

the growth of lactobacilli, which could be used for dairy products
without the need to add tomato juice. - Dissolve 55.3 g of dehydrated medium
Principles water.
- Stir slowly until complete dissolution,
- The different peptones, glucose, manganese and magnesium heating if necessary.
salts supply the nutritive elements required for the growth - Dispense in tubes or flasks.
of lactobacilli. - Sterilize in an autoclave at 121°C for
- Tween 80 is composed of a mixture of oleic esters and is a source 15 minutes.
of fatty acids essential for the growth of these bacteria.
- Dipotassium phosphate stabilizes the pH during bacterial growth. Instructions for Use
- Ammonium citrate and sodium acetate inhibit the development
of most contaminants, including streptococci and molds. - Transfer 1 mL of the product to analyze
and its serial tenfold dilutions to one or
several tubes of medium.
- Incubate for 48 hours to 5 days at 30 or
37°C, depending on the species being
studied.
178
Examine the tubes containing characteristic
For 1 liter of medium : cloudiness of microbial growth.
- Polypeptone 10.00 g In addition to lactobacilli, Leuconostoc and
- Meat extract 10.00 g Pediococcus may also develop.
- Yeast extract 5.00 g It is recommended to prepare subcultures
- Glucose 20.00 g on an appropriate media. Depending on
- Tween 80 1.08 g the qualitative results obtained, use the
- Dipotassium phosphate 2.00 g most probable number method for
- Sodium acetate 5.00 g enumeration.
- Ammonium citrate 2.00 g
- Manganese sulfate 0.05 g
Quality Control
- Dehydrated medium : cream powder, slightly clumped.

Lactobacillus casei subsp. rhamnosus ATCC® 7469 good to excellent
Lactobacillus delbrueckii subsp. lactis ATCC 4797 good to excellent

Packaging
Dehydrated medium :
Bibliography
(1) de Man, J.C., Rogosa, M., and Sharpe, M.E. 1960. A medium (3) FIL-IDF 146A: 1998. Yoghurt. Identification of characteristic
for the cultivation of lactobacilli. J. App. Bacteriol., 23, (1): mircoorganisms (Lactobacillus delbrueckii subsp. bulgaricus and
130-135. Streptococcus thermophilus).
(2) MacFaddin, J.F. 1985. Media for Isolation-Cultivation-
Identification-Maintenance of Medical Bacteria. vol I, Williams
and Wilkins Baltimore, 543-545.
179
MSE Agar

This medium, developed by Mayeux, Sandine and Elliker in 1962, is Target : Leuconostoc.
an elective medium for the detection and enumeration of Reconstitution : 138.5 g / L.
Leuconostoc in milk, dairy products and sweet foods.
Principles pH (25°C) : 6.9 ± 0.2.
- Sodium azide inhibits Gram-negative bacteria and lactic
streptococci coexisting with Leuconostoc in dairy products.
- Leuconostoc mesenteroides and Leuconostoc dextranicum utilize
the sucrose in the medium to synthesize polysaccharides (dextrans) Preparation
which impart a gelatinous appearance to the colonies.
Typical Composition (BK087) in 1 liter of distilled or deionized
- Tryptone 10.0 g - Dispense in tubes or flasks.
- Gelatin 2.5 g - Sterilize in an autoclave at 110°C for
- Yeast extract 5.0 g 20 minutes.
- Sucrose 100.0 g
- Glucose 5.0 g Instructions for Use
- Sodium azide 75 mg - Cool and maintain the medium at 47°C.
- Bacteriological agar 15.0 g - Pour into sterile Petri dishes.
partially removed.
Quality Control - Transfer 0.1 mL of the product to analyze
and its serial tenfold dilutions to the plates.
- Dehydrated medium : off-white powder, free-flowing and - Spread the inoculum on the surface of the
homogeneous. agar with a sterile triangle.
- Prepared medium : cream-white agar. - Incubate at 21°C and examine daily for
- Typical culture response after 72 hours of incubation at 21°C : 4 days.
Microorganisms Growth Results

Leuconostoc dextranicum ATCC®19255 good to excellent
Leuconostoc mesenteroides ATCC 14935 good Colorless, mucous, gelatinous colonies,
Leuconostoc mesenteroides subsp. cremoris ATCC 19254 good 1 to 5 mm in diameter : Leuconostoc
dextranicum.
Escherichia coli ATCC 25922 inhibited Small colorless colonies, 0.5 to 2 mm in
Lactococcus lactis subsp. lactis ATCC11454 inhibited diameter : Leuconostoc citrovorum or
Leuconostoc kefir.
Storage / Shelf Life Under these conditions, the species must be
carefully isolated and identified.
Dehydrated medium : 2-30°C. After 4 days of incubation, lactic
- The expiration date is indicated on the label. streptococci may develop on the medium
Prepared medium (benchmark value) : as small, opaque, white or yellowish-white
- Media in vials : 6 months at 2-8°C. colonies.
Packaging
Dehydrated medium :
Bibliography
(1) Mayeux, J.V., Sandine, W.E., and Elliker, P.R. 1962. A selective (3) Devoyod, J.S., et Muller, M. 1969. La flore microbienne du
medium for detecting Leuconostoc in mixed-strain starter fromage de Roquefort. Les streptocoques lactiques et les
cultures. J. Dairy Science, 45: 655-656. Leuconostoc. Le lait, 49: 369-399.
(2) Buttiaux, R., Beerens, H., et Tacquet. 1962. Manuel des (4) FIL-IDF 149A: 1997. Lactic acid starters. Standard of identity.
techniques bactériologiques. 4ème Ed. Flammarion, 455-458.
180
MSRV Medium
Intended Use
Modified Semi-Solid Rappaport-Vassiliadis Medium (MSRV) is a

selective formula used for the isolation of Salmonella in chocolate
and other food products.
History
The composition of the medium, developed by De Smedt et al., is

derived from that of Rappaport-Vassiliadis Broth, made semi-solid
by adding a small quantity of agar.
Principles Salmonella Typhimurium
- Bacteriological efficacy is based on the capacity of salmonellae to

selectively migrate in the medium.
- The high concentration of magnesium chloride and the presence
of malachite green inhibit contaminating bacteria.
- The addition of novobiocin inhibits most Gram-positive bacteria
and avoids the development of Proteus.
- Salmonella produce an opaque halo, a reflection of growth.
- The medium can be inoculated directly from a preenrichment
medium or after 8 hours in a selective liquid medium.
- The medium is not appropriate for immobile salmonellae
(Salmonella Gallinarum, Salmonella Pullorum). If the presence of
these strains is suspected, the cultures obtained with
preenrichment media must be transferred to appropriate media
using conventional standardized procedures.
Klebsiella pneumoniae
Typical Composition of the complete media

(can be adjusted to obtain optimal performance) Product Profile
- Tryptone 4.59 g Reconstitution : 31.6 g / L.
- Acid hydrolysate of casein 4.59 g Autonomy (500 g) : 1055 plates.
pH (25°C) : 5.2 ± 0.2.
- Magnesium chloride anhydrous 10.93 g Supplement required : Novobiocin
- Malachite green oxalate 37 mg Selective Supplement.
- Novobiocin 20 mg Sterilization : Heat to boiling.
- Bacteriological agar 2.70 g Do not autoclave.

Preparation
Quality Control
- Dehydrated medium : blue powder, free-flowing and (BK134) in 1 liter of distilled or deionized
homogeneous. water.
- Prepared (complete) medium : blue semi-solid agar. - Slowly bring to boiling, stirring until
- Typical culture response after 24 hours of incubation at 42°C : complete dissolution.
- Continue boiling for 2 minutes.
Microorganisms Growth Characteristics - Aseptically dispense 100 mL in sterile
Salmonella Typhimurium ATCC® 14028 good to whitish opaque culture with flasks.
excellent a diameter of at least 30 mm - Do not autoclave.
surrounding the point of
inoculation Instructions for Use
Salmonella Enteritidis ATCC 13076 good to whitish opaque culture with
excellent a diameter of at least 30 mm - Cool and maintain the medium at 47°C.
surrounding the point of - Aseptically add 1 mL of sterile
inoculation reconstituted Novobiocin Selective
Escherichia coli ATCC 25922 inhibited Supplement (BS033) per flask.
Staphylococcus aureus ATCC 25923 inhibited - Mix well.
181
Storage / Shelf Life - Let solidify on a cold surface.
- Inoculate 0.1 mL of inoculum obtained from
Dehydrated medium : 2-25°C, shielded from light. a preenrichment or selective enrichment
- The expiration date is indicated on the label. medium in the center of the plate.
Prepared medium (benchmark value) : - Incubate at 42°C for 20 to 24 hours
- Complete media in plates : 15 days at 2-8°C, shielded from light. maximum without inverting the plates.
The presence of an opaque halo centered
Packaging on the point of inoculation is a presumption
for Salmonella. Subcultures can be prepared
Dehydrated base medium (without novobiocin) : by removing a fraction of culture from the
- 500 g bottle : BK134HA outer edge of the halo to confirm purity
Novobiocin Selective Supplement : and conduct additional biochemical and
- 10 vial pack : BS03308 serological tests.
Bibliography
(1) De Smedt, J.M., Bolderdijk, R.F., Rappold, H., and (3) Bolderdijk, R.F., and Millas, J.E. 1996. Food biological
Lautenschlaeger, D.1986. Rapid Salmonella Detection in Foods contaminants. Salmonella Detection in Dried Milk Products by
by Motility Enrichment on a Modified Semi-Solid Rappaport- Motility Enrichment on Modified Semisolid Rappaport-
Vassiliadis Medium. J. of Food. Prot., 49: 510-514. Vassiliadis Medium: Collaborative Study. Journ. of AOAC Intern.
(2) De Smedt, J.M., and Bolderdijk, R.F., 1987. Dynamics of 79 (2): 441-450.
Salmonella Isolation with modified Semi-Solid Rappaport-
Vassiliadis Medium. J. of Food Prot., 50: 658-661.
Mueller Hinton Agar
Intended Use
Mueller Hinton Agar is recognized by all experts as being the

reference medium for the study of the susceptibility of bacteria to
antibiotics and sulfamides. It is also useful for the isolation of
Neisseria and is an excellent base medium for the preparation of
blood agars.
History
In work concerning the development of a transparent medium capable

of resisting autoclaving, Mueller and Hinton selected the complex
medium of Gordon and Hine in an attempt to determine the essential
components. The authors found that starch could replace pea extract
in terms of nutritive value as well as protective agent acting against
toxic substances present in the medium. They subsequently found that
Antibiotic susceptibility testing
pancreatic digest of meat could be replaced by acid hydrolysate of
casein, thereby favoring the growth of gonococci and meningococci. In
1966, Bauer, Kirby, Shervis and Turck recommended Mueller Hinton Product Profile
medium for the study of the antibiotic susceptibility of bacteria using
Target : Neisseria - Antibiotic
the disk method. Finally, a standardized control method was published
susceptibility testing.
by the National Committee for Clinical Laboratory Standards for the
Kirby-Bauer method. Reconstitution : 38.0 g / L.
pH (25°C) : 7.3 ± 0.2.
182
- The choice of ingredients is determined in order to obtain : - Suspend 38.0 g of dehydrated medium
- a very low quantity of thymine and thymidine, substances (BK048) in 1 liter of distilled or deionized
known to inhibit the antibacterial activity of trimethoprim, water.
- a very low quantity of para-aminobenzoic acid (PABA) and its - Slowly bring to boiling, stirring until
structural analogues, which antagonize the activity of complete dissolution.
sulfonamides. - Dispense in tubes or flasks.
- As a result of the influence of calcium and magnesium on the - Sterilize in an autoclave at 115°C for
sensitivity of Pseudomonas strains to aminoglycosides, Reller et al. 15 minutes.
recommended that the ion concentrations be included within the
following limits : NOTE
The water used to prepared the medium must be
- calcium : 50-100 mg/liter
of high quality, since the levels of calcium and
- magnesium : 20-35 mg/liter magnesium in the medium are precisely adjusted.
- The Kirby-Bauer method is based on the diffusion of antibiotics
impregnated in previously dried paper disks, deposited on the Instructions for Use
surface of the agar. When applied to the surface of the agar, the Antibiotic susceptibility testing
disks absorb a sufficient quantity of water to dissolve the
antibiotic, which then diffuses into the medium according to The medium :
physical laws of diffusion of molecules through a gel. In this way, a - Cool and maintain at 47°C.
concentration gradient of antibiotic forms around each disk. At - Pour into sterile Petri dishes.
the same time as the antibiotics diffuse, the bacteria inoculated on - The agar must be 4 mm thick.
the surface of the agar multiply. During the logarithmic phase of - Let solidify on a cold surface.
growth, bacterial multiplication is more rapid than the diffusion of - Dry in an incubator with the covers
the antibiotic and uninhibited bacterial cells continue to multiply partially removed in order to avoid the
until growth can be visualized. No growth appears when the formation of water droplets on the surface
antibiotic is present at inhibiting concentrations. It now becomes of the agar, a phenomenon which
possible to measure the diameter of the inhibition zone, which is can deteriorate the diffusion qualities
indirectly proportional to the minimal inhibitory concentrations of the medium.
found by the dilution method. Tables exist for interpreting the
results in order to determine if the bacteria are sensitive or The inoculum :
resistant to the antibiotic tested. Standard Kirby Bauer method
- The antibiotic spectrum must be
Typical Composition determined with a pure pathologic strain.
(can be adjusted to obtain optimal performance) - Transfer 4 to 5 colonies in an appropriate
broth (Tryptone Soy Broth : BK046).
For 1 liter of medium : - Place in a 37°C incubator (in general 2 to 5
- Acid hydrolysate of casein 17.5 g hours) until an opacity is obtained which is
- Beef infusion 2.0 g equivalent to the standard opacity of a
- Soluble starch 1.5 g barium sulfate suspension (density of 0.5
- Bacteriological agar 17.0 g on the MacFarland scale).
pH of the ready-to-use medium at 25°C : 7.3 ± 0.2. The inoculation :

Standard Kirby and Bauer method
Quality Control - Add a sterile swab to the inoculum
adjusted to the opacity standard, and
- Dehydrated medium : whitish powder, free-flowing and drain excess broth by pressing the swab on
homogeneous. the walls of the tube. Inoculate the agar.
- Prepared medium : amber agar. The swab should be passed 2 or 3 times
- Typical culture response (*) after 24 hours of incubation at 37°C : over the entire surface in order to obtain a
homogeneous inoculum.
- Allow the plates to dry for 10 minutes
before depositing the disks.
Staphylococcus aureus ATCC 25923 good to excellent NOTE
The Kirby and Bauer method is recognized as
Pseudomonas aeruginosa ATCC 27853 good to excellent furnishing the most reliable and most
Enterococcus faecalis ATCC 29212 good to excellent reproducible results. Other methods may also be
used, however, provided that inoculum and
(*)
Refer to NCCLS standards for antibiotic susceptibility tests. inoculation method are first studied and
standardized.
183
Storage / Shelf Life Disk placement and incubation
- Place the disks using slight pressure to
Dehydrated medium : 2-30°C. insure good adhesion to the agar.
- The expiration date is indicated on the label. - They should be situated at least 15 mm
Prepared medium (benchmark value) : from the edge of the dish and sufficiently
- Media in tubes or vials : 6 months at 2-8°C. far apart so the inhibition zones do not
- Media in plates : 1 month at 2-8°C. overlap.
Packaging Results
Dehydrated medium : Measure the inhibition zone with a

- 500 g bottle : BK048HA compass.
- 5 kg drum : BK048GC Refer to the table for interpreting inhibition
zones furnished by the suppliers of antibiotic
disks in order to establish the correlation
between the inhibition zone and the
minimal inhibitory concentration (MIC).
Bibliography
(1) Mueller, J.H., and Hinton, J. 1941. Proc. Soc. Exp. Biol. Med., (5) Acar, J.F. 1976. Journées Nationales de Biologie.
48: 330-333. Grenoble-Lyon.
(2) Bauer, A.W., Kirby, W.M.M., Sherris, J.C., and Turck, M. 1966. (6) National Committee for Clinical Laboratory Standards. 1984.
Antibiotic susceptibility testing by a standardized single disk Approved standard : M2 A3. Performance standards for
method. Am. J. Clin Pathol., 45: 493-496. antimicrobial disk susceptibility tests, 3rd Ed, NCCLS, Villanova,
(3) Daguet, G.L., et Chabbert, Y.A. 1972. Techniques en Pa.
bactériologie. 3. Sérologie bactérienne, antibiotiques en (7) Courvalin, P., Goldstein, F., Philippon, A., et Sirot, J. 1985.
bactériologie médicale. Ed. Flammarion, Paris. L'antibiogramme. MPC. Bruxelles.
(4) Reller, L.B., Schoenknecht, F.D., Kenny, M.A., and Sherris, J.C. (8) National Committee for Clinical Laboratory Standard. 1987.
1974. Antibiotic susceptibility testing of Pseudomonas Second informational supplement : M100-S2. Performance
aeruginosa : selection of a control strain and criteria for standards for antimicrobial susceptibility testing, NCCLS,
magnesium and calcium content in media. J. Infect. Dis., 130: Villanova, Pa.
454-463.
Mueller Hinton Broth

Mueller Hinton Broth is a non-selective medium for the culture of a Target : Total microorganisms - Antibiotic
susceptibility testing.
large number of microorganisms of varied origins, as well as for
determining minimal inhibitory concentrations by the dilution Reconstitution : 21.0 g / L.
method. Autonomy (500 g) : 2380 tubes.
pH (25°C) : 7.4 ± 0.2.
History
In work concerning the development of a medium capable of resisting Sterilization : 121°C / 15 minutes.
autoclaving, Mueller and Hinton selected the complex medium of
Gordon and Hine in an attempt to determine the essential Preparation
components. The authors found that starch could replace pea extract
in terms of nutritive value and also as a protective agent acting against - Dissolve 21.0 g of dehydrated medium
toxic substances present in the medium. They subsequently found that (BK108) in 1 liter of distilled or deionized
pancreatic digest of meat could be replaced by acid hydrolysate of water.
casein, thereby favoring the growth of gonococci and meningococci. - Slowly stir until complete dissolution.
15 minutes.
184
- The choice of ingredients is determined in order to obtain : Culture in tubes

- a very small quantity of thymine and thymidine, substances - Inoculate the medium with purified
known to inhibit antibacterial activity of trimethoprim, cultures or with other inocula containing
- a very small quantity of para-aminobenzoic acid (PABA) and its mixed microflora.
structural analogues, which antagonize the activity of - Incubate at the optimal temperature
sulfonamides. required in aerobic conditions or else in an
- Beef infusion and acid hydrolysate of casein are sources of amino anaerobiosis jar, depending on the
acids and other nitrogenous substances favoring the growth of bacteria to be cultured.
microorganisms.
- Starch is a detoxifying agent. Determination of minimal inhibitory
concentrations
Typical Composition - Add serial dilutions of the stock solution
(can be adjusted to obtain optimal performance) of antibiotic to test to tubes containing
the medium.
For 1 liter of medium : - Inoculate with a defined quantity of the
- Acid hydrolysate of casein 17.5 g culture of the microorganism to test.
- Beef infusion 2.0 g - Incubate for 24 hours at 37°C.
- Soluble starch 1.5 g
Results
Growth is determined by turbidity resulting
Quality Control from microbial division.
The last tube with no microbial development
- Dehydrated medium : light beige powder, free-flowing and contains antibiotic at a concentration which
homogeneous. is the minimal inhibitory concentration.
- Prepared medium : amber solution, limpid. Multiplying this concentration by the
- Typical culture response after 24 hours of incubation at 37°C : corresponding dilution factor furnishes
the concentration of active antibiotic.
Staphylococcus aureus ATCC ®
Streptococcus pyogenes ATCC 19615 good

Packaging
Dehydrated medium :
Bibliography
(1) Mueller, J.H., and Hinton, J., 1941. Proc. Soc. Exp. Biol. Med., (2) National Committee for Clinical Laboratory Standards. 1985.
48: 330-333. Approved standard: M7 A. Method for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically. NCCLS,
Villanova, Pa.
185
Müller-Kauffmann Broth

Müller-Kauffmann Broth is used as a selective enrichment medium
for salmonellae in pathological products, pharmaceutical products, Reconstitution : 88.3 g / L.
water samples, dairy products and other food products. Autonomy (500 g) : 56 vials (100 mL per vial).
pH (25°C) : 7.3 ± 0.2.
History
Supplement required : Iodine-iodide solution
The medium was described by Müller in 1923 to favor the inhibition Novobiocine Selective Supplement (optional).
of coliform bacteria at the same time as permitting the Sterilization : Heat to boiling.
development of typhoid and paratyphoid bacilli. Kauffmann Do not autoclave.
modified the formula and obtained a greater number of positive
results with this enrichment method than with the direct method of
Preparation
isolating on selective media poured into plates. The formulation of
the medium complies with the specifications described in the
international standard on the detection for Salmonella in meats and
meat-based products.
water.
- Slowly bring to boiling with stirring.
Principles
- Do not autoclave.
- Ox bile and brilliant green inhibit principally the development of
Gram-positive bacteria.
- The production of tetrathionate resulting from the action of the
iodine-iodide solution on sodium thiosulfate, inhibits coliform
bacteria and most intestinal bacteria.
- Dissolve 4 g of iodine in 20 mL of a
- The development of Proteus can be suppressed by adding 40 mg
solution containing 5 g of potassium iodide
of novobiocin per liter of medium.
in a sterile flask.
- Calcium carbonate neutralizes the sulfuric acid produced when
- Add the iodine-iodide solution to the
tetrathionate is reduced. The resultant effect is to maintain pH at
medium.
a constant level.
- Optionally add 40 mg of Novobiocin
Selective Supplement (BS033) per liter of
Typical Composition of the base medium,
medium.
(without iodine or novobiocin)
- Mix thoroughly.
- Do not heat the medium once prepared.
- Aseptically dispense 100 mL in sterile flasks.
- To each flask, transfer 10 mL of inoculum
- Tryptone 8.45 g
from the preenrichment medium used :
Buffered Peptone Water (BK018, BK131,
BM010) or Lactose Broth (BK082).
- Verify that the pH of the medium
- Calcium carbonate 38.04 g
is 7.3 ± 0.2.
- Sodium thiosulfate anhydrous 30.27 g
- Incubate 24 to 48 hours at 42-43°C or at
- Brilliant green 9.5 mg
37°C, depending on the analysis procedure
to follow.
Quality Control
- Isolate on several selective media with a
platinum loop.
- Dehydrated medium : whitish powder, free-flowing and
- Using well formed colonies, inoculate
homogeneous.
Kligler Iron Agar (BK034) or TSI Agar
- Prepared (complete) medium : bluish opaque suspension, with
(BK059) which are the starting points
abundant precipitate when left standing.
for identification.
- Typical culture response after enrichment for 24 hours at 43°C,
followed by subculture on MacConkey Agar :

Salmonella Typhimurium ATCC 14028
®
good colorless colonies
Salmonella Enteritidis ATCC 13076 good colorless colonies
Escherichia coli ATCC 25922 partially to totally red colonies
inhibited
186

- Base media in tubes or vials : 1 month at 2-8°C, shielded
from light.
- Complete media in tubes or vials : use immediately after adding
the iodine-iodide solution and novobiocin.
Packaging
Dehydrated base medium (without iodine or novobiocin) :

Bibliography
(1) Müller, L. 1923. Un nouveau milieu d’enrichissement pour la (5) NF V59-104: October 1982. Edible gelatin. Detection of
recherche du bacille typhique et des paratyphiques. Compt. Salmonella.
rend. Soc. biol., 89: 434-437. (6) CNEVA Bactériologie animale (French) : Pr 116/00/BA 70/00.
(2) Kauffmann, F. 1935. Weitere Erfahrungen mit dem Decembre. 1996. Isolement et identification des salmonelles
Kombinierten Anreicherungsverfahren für Salmonellen bacillen. en élevage avicole.
Z. Hyg. Infekt. Krkh. 117: 26-32. (7) CNEVA Bactériologie animale (French) : Pr 116/00/BA 50/00.
(3) Jeffries, L. 1959. Novobiocin - tetrathionate broth: A medium Decembre 1996. Isolement et identification des salmonelles
of improved selectivity for the isolation of Salmonellae from chez les mammifères.
feces. J. Clin. patho., 12: 568-571. (8) CNEVA Bactériologie animale (French) : Pr 116/00/BA 60/00.
(4) ISO 3565. 1975. Meat and meat products. Detection of Decembre 1996. Isolement et identification des salmonelles
Salmonellae (reference method). chez les volailles.
Nutrient Agar

Nutrient Agar is used for the growth and enumeration of microorganisms
in water, beverages and biological products. It is useful for Reconstitution : 35.0 g / L.
microorganisms without particular nutritional requirements, as well as for Autonomy (500 g) : 952 plates.
the detection of the intrinsic inhibitory capacity of cosmetic products.
pH (25°C) : 7.1 ± 0.2.
Relatively simple, the formula supplies the nutritive elements
required for the growth of a wide variety of non-fastidious
microorganisms. Preparation

water.
For 1 liter of medium : - Slowly bring to boiling, stirring slowly
- Tryptone 10 g until complete dissolution.
- Meat extract 5 g - Dispense in tubes or flasks.
- Sodium chloride 5 g 15 minutes.
187
- Dehydrated medium : cream powder, free-flowing and - Cool and maintain the medium at 47°C.
- Prepared medium : amber agar. - Let solidify on a cold surface.
partially removed.
Microorganisms Growth - Inoculate.
Salmonella Enteritidis ATCC 13076 good to excellent
Bacillus subtilis ATCC 6633 good to excellent

Packaging
Dehydrated medium :
Bibliography
(1) Journal Officiel (French) du 8 Août 1972. Méthode officielle

d’analyse bactériologique pour la recherche d’un pouvoir inhibiteur
intrinsèque des produits cosmétiques. Annexe III : 8552-8553.
Nutrient Broth

Target : Aérobic microorganisms.
Nutrient Broth is a general usage medium for a large variety of
microorganisms without particular nutritional requirements. It is Reconstitution : 20.0 g / L.
used in alimentary bacteriology as a preenrichment medium for the Autonomy (500 g) : 2500 tubes.
detection of Salmonella. Its formula complies with the directives of
pH (25°C) : 7.2 ± 0.2.
the French J.O. of August 8, 1972 for the detection of the intrinsic
inhibitory capacity of cosmetic products. Supplement required : None.
Principles
- Nutrient Broth is composed of a mixture of peptones, which aids Preparation

microbial growth.
- Sodium chloride is included to maintain osmotic equilibrium. - Dissolve 20.0 g of dehydrated medium
15 minutes.
- Tryptone 10.0 g
188
- Dehydrated medium : cream powder, free-flowing and - Inoculate the medium with purified
homogeneous. cultures or with other type of mixed
- Prepared medium : amber solution, limpid. microflora inocula.
- Typical culture response after 24-48 hours of incubation at 37°C : - Incubate at the required optimal
temperature, aerobically or in a CO2-
Microorganisms Growth enriched atmosphere, depending on the
Escherichia coli ATCC® 25922 good to excellent bacteria to be cultured.
Salmonella Typhimurium ATCC 14028 good to excellent
Results
Shigella sonnei ATCC 29930 good
Enterococcus faecalis ATCC 29212 good Growth is shown by turbidity resulting from
Staphylococcus aureus ATCC 25923 good microbial multiplication.

Packaging
Dehydrated medium :
Bibliography
(1) Journal Officiel (French) du 8 Août 1972. Méthode officielle

d’analyse bactériologique pour la recherche d’un pouvoir
inhibiteur intrinsèque.
Önöz Agar

Önöz Agar is a selective medium used to isolate Salmonella in Reconstitution : 80.3 g / L.
biological samples and food products. It has the advantage of being
easily capable of differentiating contaminating Proteus and Citrobacter. Autonomy (500 g) : 415 plates.
pH (25°C) : 7.0 ± 0.2.
Developed by Önöz and Hoffmann in 1977, the medium leads to a Sterilization : Heat to boiling.
Do not autoclave.
better and more rapid recovery of salmonellae from stool samples
than SS Agar and Leifson Agar.
Preparation
Principles
- The inhibitory effect of bile salts and brilliant green on (BK032) in 1 liter of distilled or deionized
contaminating Gram-positive flora makes the medium well water.
adapted to the detection of Salmonella. - Slowly bring to boiling, stirring until
- The addition of phenylalanine neutralizes chloramphenicol so that complete dissolution.
identification is made easier during antibiotic treatments. - Do not autoclave.
- The growth of lactose or sucrose-positive bacteria is limited. In
addition, colonies can be easily differentiated by the colors resulting
from the effect of neutral red, aniline blue and metachrome yellow.
- Proteus deaminates phenylalanine to phenylpyruvate, which forms
a brownish complex in the presence of ferric ions in the medium,
so that the colonies formed are easily recognizable.
189
- Peptic digest of meat 6.8 g - Let solidify on a cold surface.
- Yeast extract 3.0 g - Dry in an incubator with the covers
- Meat extract 6.0 g partially removed.
- Lactose 11.5 g - Inoculate by streaking enrichment media
- Sucrose 13.0 g used for the detection of Salmonella.
- Bile salts 3.825 g - Incubate at 37°C for 24 to 48 hours.
- Sodium thiosulfate 4.25 g Results
- L-phenylalanine 5.0 g
- Ferric ammonium citrate 0.5 g Colonies have the following appearance :
- Disodium phosphate 1.0 g - Small yellow colonies with a black center
- Brilliant green 1.66 mg after 2 to 3 days of incubation. The
- Neutral red 22 mg medium is yellowish around the colonies :
- Aniline blue 250 mg Salmonella Typhi
- Metachrome yellow 470 mg - Yellow colonies with a black center. The
- Bacteriological agar 15.0 g medium becomes yellow at the periphery
after 2 days of incubation :
pH of the ready-to-use medium at 25°C : 7.0 ± 0.2. Other Salmonella
- Bluish to greenish colonies within 2 days
of incubation :
Quality Control Shigella
- Small violet-blue colonies with a blue halo :
- Dehydrated medium : beige powder, free-flowing and Escherichia coli
homogeneous. - Violet-red colonies with a blue center :
- Prepared medium : green agar. Citrobacter
- Typical culture response after 48 hours of incubation at 37°C : - Large mucilaginous colonies, bluish to
pinkish, with a small precipitation zone at
Microorganisms Growth Characteristics the periphery :
Salmonella Typhi ATCC® 19430 good yellow colonies Enterobacter
Salmonella Enteritidis ATCC 13076 good yellow colonies with a - Large mucilaginous colonies, gray-blue,
black center with a bluish to mauve precipitation zone
at the periphery :
Salmonella Typhimurium ATCC 14028 good yellow colonies with a Klebsiella
black center - Rust colored colonies becoming black
Escherichia coli ATCC 25922 partially inhibited violet-blue colonies when growth is intense :
Proteus, Providencia
- Brilliant greenish-yellow colonies :
Staphylococcus aureus ATCC 25923 inhibited Pseudomonas

Packaging
Dehydrated medium :
Bibliography
(1) Önöz, E., and Hoffmann, K. 1978. Erfahrungen mit einem (3) NF EN 12824. February 1998. Microbiology of food
neuen Nährboden für die Salmonella - Diagnostik. Zbl. Bakt. and animal feeding stuffs. Horizontal method
Hyg., I. Abt. Orig., A 240: 16-21. for the detection of Salmonella.
(2) NF V 08-052. May 1997. Microbiology of food and animal
190
Orange Serum Agar
Intended Use
Orange Serum Agar is used for the growth, isolation and

enumeration of yeasts, molds and acid-tolerant bacteria (Bacillus,
lactobacilli, Leuconostoc, Streptococcus, Clostridium), which are
responsible for deteriorations in fruit juices and citrus concentrates.
It has also been used for hygiene controls of industrial equipment
used to prepare fruit-based beverages.
History
In 1951, the medium was described by Hayes for the enumeration

and isolation of microorganisms causing alteration in frozen orange
juice concentrates and subsequently by Murdock et al. for lemon
juice concentrates. Yeasts

Target : Yeasts, Molds,
The addition of clarified orange juice to the peptones and extracts acid-tolerant bacteria.
in the formula leads to a satisfactory recovery of microorganisms
which can resist the acidity of the fruit juices they contaminate. Reconstitution : 42.0 g / L.
Typical Composition
pH (25°C) : 5.5 ± 0.2.
For 1 liter of medium : Sterilization : 115°C / 15 minutes.
- Tryptone 10.0 g
- Yeast extract 3.0 g Preparation
- Orange extract 5.0 g
- Glucose 4.0 g - Suspend 42.0 g of dehydrated medium
- Dipotassium phosphate 3.0 g (BK103) in 1 liter of distilled or deionized water.
pH of the ready-to-use medium at 25°C : 5.5 ± 0.2. - Dispense in tubes or flasks.
Quality Control
15 minutes.
homogeneous.
- Typical culture response after 72 hours of incubation at 25-30°C : - Cool and maintain the medium at 47°C.
- Transfer 1 mL of the product to analyze
Microorganisms Growth and its serial tenfold dilutions to sterile
Lactobacillus plantarum ATCC® 8014 good to excellent Petri dishes.
Leuconostoc mesenteroides - Pour in 15 mL of medium.
subsp. mesenteroides ATCC 14935 good to excellent - Homogenize by swirling.
Saccharomyces cerevisiae ATCC 9763 good to excellent - Let solidify on a cold surface.
Aspergillus niger ATCC 16404 good to excellent - Incubate at 30°C for 3 to 5 days.
NOTE
Storage / Shelf Life Do not overheat the medium in order to avoid
browning and the loss of the gelling properties
Dehydrated medium : 2-30°C. of the agar. The medium should be used
- The expiration date is indicated on the label. preferentially the day of its preparation.
- Use on the same day of preparation. Results
Packaging Separately enumerate yeasts, molds and

bacteria. Carry out a microscopic
Dehydrated medium : examination and identification tests on
- 500 g bottle : BK103HA each type of colony found.
Bibliography
(2) Murdock, D.I., Folinazzo, J.F., and Troy, V.S. 1951. Evaluation of
(1) Hays, G.L. 1951. The isolation, cultivation and identification of plating media for citrus concentrates. Food Tech., 6: 181-185.
organisms which have caused spoilage in frozen concentrated (3) Hays, G.L., and Reister, D.W. 1952. The control of “off-odor” spoilage
orange juice. Proc. Florida State Hort. Soc., 54: 135. in frozen concentrated orange juice. Food Tech., 6: 386-389.
191
Oxford Agar
Intended Use
Oxford Agar is a selective medium used for the differentiation and

isolation of Listeria monocytogenes in pathologic specimens, milk
and cheese, as well as in other samples, even highly contaminated.
History
The medium was prepared by Curtis et al. in 1988 for the isolation of
Listeria monocytogenes from clinical samples containing
considerable contaminating microflora. In most cases, the authors
observed that Listeria colonies appeared within 24 hours of
incubation and that associated microorganisms were inhibited. The
studies were based on the work of Rodriguez (1984), who was the
first to use esculin and iron salts to visualize Listeria monocytogenes
by its esculinase-positive character. Many selective media for Listeria
Listeria monocytogenes
containing esculin, however, also enable enterococci to grow. Curtis
et al. showed that secondary microflora were inhibited by lithium
chloride, acriflavine, cycloheximide, colistin, cefotetan and Product Profile
fosfomycin. Target : Listeria monocytogenes.

- Polypeptone favors the excellent growth of Listeria. pH (25°C) : 7.0 ± 0.2.
- Yeast extract is a source of vitamin B complex.
- Starch is the energy source for microbial development. Supplement required : Oxford Selective
- Sodium chloride maintains osmotic balance. Supplement (CCCFA).
- Listeria hydrolyze esculin to glucose and esculetin, the latter Sterilization : 121°C / 15 minutes.
compound forming a black complex with ferric ions supplied by
ferric citrate. Preparation
- Contaminating microflora is inhibited by lithium chloride,
cycloheximide, colistin, cefotetan, fosfomycin and acriflavine. - Suspend 58.5 g of dehydrated base
Typical Composition of the complete medium deionized water.
(can be adjusted to obtain optimal performance) - Slowly bring to boiling, stirring until
For 1 liter of medium : - Dispense 100 mL in flasks.
- Polypeptone 20.0 g - Sterilize in an autoclave at 121°C for
- Yeast extract 3.0 g 15 minutes.
- Starch 1.0 g
- Esculin 1.0 g
- Ferric ammonium citrate 0.5 g - Melt the medium (if it was prepared in
- Lithium chloride 15.0 g advance).
- Cycloheximide 400 mg - Cool and maintain the at 47°C.
- Colistine (sulfate) 20 mg - Per 100 mL of base, aseptically add 1 mL of
- Cefotetan 2 mg restored Oxford Selective Supplement
- Fosfomycin 10 mg (BS003).
- Acriflavine 5 mg - Mix well.
partially removed.
- For enumeration, transfer 0.1 mL of the
sample to analyze and its serial dilutions
to the surface of plates prepared as above,
or to ready-to-use plates (BM019)
previously brought to room temperature.
Spread the inoculum on the surface of the
agar with a sterile triangle.
192
- Dehydrated medium : beige powder, free-flowing and After 24 hours of incubation, Listeria
homogeneous. monocytogenes forms olive-green colonies
- Prepared (complete) medium in plates : yellow-greenish agar surrounded by a black halo. After 48 hours,
with bluish reflections. they become darker with a hollow black
- Typical culture response after 48 hours of incubation in center and are surrounded by black zones.
complete medium at 37°C : Oxford Agar is a highly selective medium
but it is sometimes possible to observe
Microorganisms Growth Characteristics colonies of staphylococci or enterococci
Listeria monocytogenes ATCC® 19115 good to excellent olive-green colonies (which grow slowly, giving a weak yellow
surrounded by a black halo or black color, generally after 30 to 40 hours
Listeria monocytogenes ATCC 35152 good to excellent olive-green colonies of incubation).
surrounded by a black halo Suspected colonies are subjected to
Listeria innocua ATCC 33090 good olive-green colonies
surrounded by a black halo
Staphylococcus aureus ATCC 25293 partially inhibited yellow colonies

Ready-to-use media in plates,
Oxford Selective Supplement (CCCFA) :
Packaging
Ready-to-use (complete) media in plates :

- 20 plate kit : BM01908
Dehydrated base medium (without Cycloheximide, Colistine,
Cefotetan, Fosfomycin, Acriflavin) :
Oxford Selective Supplement (CCCFA) :
Bibliography
(1) Curtis G.D.W., Mitchell R.G., King A.F., and Griffin E.J. 1989. A (5) ISO 10560. 1993. Milk and milk products. Detection of Listeria
selective differential medium for the isolation of Listeria monocytogenes.
monocytogenes. Letters in Appl. Microb., 8: 95-98. (6) FIL-IDF 143A: 1995. Milk and milk products. Detection of
(2) Curtis G.D.W., Nichols W.W., and Falla T.J. 1989. Selective Listeria monocytogenes.
agents for Listeria can inhibit their growth. Letters in Appl. (7) NF EN ISO 11290-1. Feb. 1997. Microbiology of food and
Microb., 8: 169-172. animal feeding stuffs. Horizontal method for the detection and
(3) Tiwari, N.P. and Aldenrath S.G. 1990. Isolation of Listeria enumeration of Listeria monocytogenes. Part 1: Detection
monocytogenes from Food Products on Four Selective Plating method.
Media. Journ. of Food Protection, 53: 382-385. (8) XP V08-062. Octobre 2000. Microbiologie des aliments.
(4) Journal Officiel (French) du 7 Avril 1992. Contrôle Méthode de dénombrement de Listeria monocytogenes. Méthode
microbiologique des produits végétaux ou d’origine végétale. de routine.
(arrêté du 13 mars 1992).
193
Oxytetracycline Glucose Agar (OGA)
Intended Use
Oxytetracycline Glucose Agar is used for the detection and

enumeration of yeasts and molds in food products and cosmetics.
History
In 1965, Buttiaux and Catsaras recommended the use of this

medium for the detection of yeasts and molds in beer. Subsequently,
Sainclivier and Roblot recommended it for the analysis of butter.
Mossel also showed that neutral pH led to a better recovery
than acid pH medium with several types of foods (pH being
the only selective mechanism against the growth of bacteria in this
particular medium).
Principles
Aspergillus niger
- The growth of yeasts and molds is favored by the presence
of glucose and yeast extract. Product Profile
- The addition of oxytetracycline or chloramphenicol,
or chloramphenicol + gentamicin, or oxytetracycline + gentamicin Target : Yeasts, Molds.
just before use inhibits most bacteria, including lactobacilli Reconstitution : 40.0 g / 1.1 L.
(acidophilic bacteria which may be the dominant flora in certain Autonomy (500 g) : 915 plates.
food products).
pH (25°C) : 6.6 ± 0.2.
Typical Composition of the complete medium with Oxytetracycline Supplement required : Oxytetracycline
(can be adjusted to obtain optimal performance) Selective Supplement.
For 1.1 liter of medium :
- Yeast extract 5 g
Preparation
- Glucose 20 g
- Oxytetracycline 0.1 g - Suspend 40.0 g of dehydrated base
- Bacteriological agar 15 g medium (BK053) in 1.1 liters of distilled or
deionized water.
pH of the ready-to-use medium at 25°C : 6.6 ± 0.2. - Slowly bring to boiling, stirring until
Quality Control - Dispense 110 mL per flask.
- Dehydrated medium : beige powder, free-flowing and 15 minutes.
homogeneous.
- Prepared medium : amber agar. Instructions for Use
- Typical culture response after 72 hours of incubation at 25°C on
complete medium with oxytetracycline : - With the ready-to-use base medium (or if
the media has been prepared in advance
Microorganisms Growth from the dehydrated powder, as above),
Saccharomyces cerevisiae ATCC 9763
®
good to excellent melt the agar for the minimum amount of
Aspergillus niger ATCC 16404 good to excellent time necessary to achieve total
liquefaction.
- Under aseptic conditions, add 1 mL of
Storage / Shelf Life restored Oxytetracycline Selective
Supplement (BS008) to each flask
Dehydrated base medium : 2-30°C. containing 110 mL of base.
- The expiration date is indicated on the label. - Mix well.
Prepared medium (benchmark value) : - Pour into sterile Petri dishes.
- Base media in vials : 6 months at 2-8°C. - Let solidify on a cold surface.
- Complete media in plates : 15 days at 2-8°C. - Dry in an incubator with the covers
Ready-to-use base media in vials, partially removed.
Oxytetracycline Selective Supplement, - Transfer 0.1 mL of the product to analyze
Gentamicin Selective Supplement, and its serial tenfold dilutions to the plates.
Chloramphenicol Selective Supplement : - Spread the inoculum on the surface of the
- Store between 2-8°C, shielded from light. agar with a sterile triangle.
- The expiration dates are indicated on the labels. - Incubate at 25°C for 3 to 5 days.
194
Packaging NOTE
Oxytetracycline (BS008) can be replaced by
Ready-to-use base media (without Oxytetracycline) : Chloramphenicol (BS021) or Chloramphenicol +
- 10 x 110 mL vials : BM02208 Gentamicin (BS021 + BS009) combination or can
Dehydrated base medium (without Oxytetracycline) : be supplemented with Gentamicin (BS009) alone.
Refer to corresponding monographs and
standards concerning the products to analyze.
Oxytetracycline Selective Supplement :
Results
- 10 vial pack : BS00908 After incubation separately count yeasts
Chloramphenicol Selective Supplement : and molds.
- 10 vial pack : BS02108 Carry out a rapid microscopic confirmation
test for each type of colony found.
Bibliography
(1) Mossel, D.A.A., Visser, M., and Mengerink, W.H.J. 1962. A (6) NF ISO 7954. August 1988. Microbiology. General guidance for
comparison of media for the enumeration of moulds and yeasts enumeration of yeasts and moulds. Colony count technique at 25°C.
in foods and beverages. Lab. Pract., 11: 109-112. (7) FIL-IDF 94 B: 1991. Milk and milk products. Yeasts and
(2) Buttiaux, R., et Catsaras, M. 1965. L’analyse bactériologique moulds.
des bières. Ann. Inst. Pasteur, 16: 167. (8) XP V08-059. November 1995. Microbiology of food and animal
(3) Sainclivier, M., et Roblot, A.M. 1966. Choix d’un milieu de feeding stuffs. Enumeration of yeasts and moulds by colony-
culture pour le dénombrement des levures et moisissures dans count technique at 25°C. Routine method.
le beurre. Ann. Inst. Pasteur, 17: 181. (9) NF ISO 13681. April 1996. Meat and meat products.
(4) Journal Officiel (French) du 8 août 1972. Dénombrement des Enumeration of yeasts and moulds. Colony count technique.
levures et des moisissures dans les produits cosmétiques.
8553-8554.
(5) NF V 03-454. December 1981. Agricultural food products.
Spices and condiments. Enumeration of yeasts and moulds.
PALCAM Agar
Intended Use
PALCAM Agar is a selective medium used for the differentiation and

isolation of Listeria monocytogenes in pathologic specimens, milk
and cheese, as well as in other samples, even highly contaminated.
History
The medium was formulated by van Netten et al. in 1989 to

overcome the insufficient selectivity of media used in the past for
the detection and enumeration of Listeria. The studies were based
on the work of Rodriguez (1984), who was the first to use esculin
and iron salts to visualize Listeria monocytogenes by its esculinase-
positive character. Many selective media for Listeria containing
esculin, however, also enable the growth of several group D
streptococci so that the use of esculin was only of limited value.
Based on the work of Rocourt (1987), van Netten supplemented Listeria monocytogenes
the medium with D-mannitol in order to differentiate mannitol-
positive enterococci from mannitol-negative Listeria. Based on
these two principles, and using the ecometric evaluation method,
the authors showed that the combined action of ceftazidim and
lithium chloride was more effective in terms of selectivity than
that obtained by using 2-phenylethanol. PALCAM Agar is a
combination of ALPAMY medium (van Netten et al., 1988 b) and
Oxford Medium (Curtis et al., 1989).
195
The authors showed that among the 13 selective media tested, Product Profile
PALCAM Agar gave satisfactory results, producing highly typical
Target : Listeria monocytogenes.
Listeria colonies at the same time as inhibiting almost all other
contaminating bacteria. Reconstitution : 68.9 g / L.
Principles
pH (25°C) : 7,2 ± 0,2.
- Peptones and yeast extract favor the excellent growth of Listeria. Supplement required : PALCAM
- Glucose and starch are the energy sources for microbial Selective Supplement.
development. Sterilization : 121°C / 15 minutes.
- Sodium chloride maintains osmotic balance.
- Listeria hydrolyze esculin to glucose and esculetin, the latter Preparation
compound forming a black complex with ferric ions supplied by
ferric citrate. - Suspend 68.9 g of dehydrated base
- Accompanying microflora are inhibited by lithium chloride, medium (BK145) in 1 liter of distilled or
ceftazidim, polymyxin and acriflavine. deionized water.
- The fermentation of mannitol by contaminating bacteria that may - Slowly bring to boiling, stirring until
grow causes phenol red to turn yellow, thereby orienting the complete dissolution.
diagnosis. - Dispense 100 mL in flasks.
Typical Composition of the complete medium 15 minutes.
- Peptones 23.00 g - Melt the medium (if it was prepared in
- Yeast extract 3.00 g advance).
- Glucose 0.50 g - Cool and maintain the medium at 47°C.
- Per 100 mL of base, aseptically add 1 mL
- Starch 1.00 g of reconstituted PALCAM Selective
- D-mannitol 10.00 g Supplement (BS004 or BS049).
- Esculin 0.80 g - Mix well.
- Ferric ammonium citrate 0.50 g - Pour into sterile Petri dishes.
- Lithium chloride 15.00 g - Dry in an incubator with the covers
- Polymyxin B (sulfate) 10 mg partially removed.
- For enumeration, transfer 0.1 mL of the
- Ceftazidime 20 mg sample to analyze (or of a selective
- Acriflavine 5 mg enrichment broth) and its serial tenfold
- Phenol red 0.08 g dilutions to the plates prepared as above
- Bacteriological agar 10.00 g or to ready-to-use plates (BM020) that
have been brought to room temperature.
pH of the ready-to-use medium at 25°C : 7.2 ± 0.2. Spread the inoculum on the surface of the
agar with a sterile triangle.
- For detection, isolate individual colonies
Quality Control by inoculating a loop of selective
enrichment broth.
- Dehydrated medium : pinkish powder, free-flowing and - Incubate at 37°C for 24 and 48 hours.
homogeneous.
- Prepared (complete) media : red agar. Results
After 24 hours of incubation, Listeria
Microorganisms Growth Characteristics monocytogenes forms olive-green colonies
Listeria monocytogenes ATCC® 19115 good to excellent olive-green colonies with a hollow black center and surrounded
surrounded by a black halo by black zones. When the colonies reach
Listeria monocytogenes ATCC 35152 good to excellent olive-green colonies confluence, the medium becomes brown-
surrounded by a black halo black. PALCAM Agar is highly selective, but
Listeria innocua ATCC 33090 good olive-green colonies it is sometimes possible to observe colonies
surrounded by a black halo of staphylococci or enterococci (which
Staphylococcus aureus ATCC 25923 inhibited ferment mannitol and produce yellow
Enterococcus faecalis ATCC 29212 inhibited colonies with a yellow halo, thereby being
easily distinguished from Listeria).
Suspected colonies are subjected to
196

Ready-to-use (complete) media in plates,
PALCAM Selective Supplement :
Packaging
Ready-to-use (complete) media :

- 20 plates : BM02008
Dehydrated base medium (without Polymyxin, Ceftazidime,
Acriflavine) :
PALCAM Selective Supplement (QS 500 mL) :
PALCAM Selective Supplement (QS 2.5 liters) :
Bibliography
(1) van Netten, P., van de Ven, A., Perales, I., and Mossel D.A.A. (6) FIL-IDF 143A: 1995. Milk and milk products. Detection of
1988. A selective and diagnostic medium for use in the Listeria monocytogenes.
enumeration of Listeria spp. in foods. Int. Jour. of Food (7) NF EN ISO 11290-1. February 1997. Microbiology of food and
Microb., 6: 187-198. animal feeding stuffs. Horizontal method for the detection and
(2) van Netten, P., Perales, I., and Mossel, D.A.A. 1988. An enumeration of Listeria monocytogenes. Part 1: Detection
improved selective and diagnostic medium for isolation and method.
counting of Listeria spp. in heavily contaminated foods. (8) NF V08-055. August. 1997. Microbiology of food and animal
Letters in Applied Microbiology, 7: 17-21. feeding stuffs. Detection of Listeria monocytogenes.
(3) van Netten, P., Perales, I., van de Moosdijk., Curtis G.D.W., Routine method.
and Mossel D.A.A. 1989. Liquid and solid selective differential (9) NF EN ISO 11290-2. August. 1998. Microbiology of food and
media for the detection and enumeration of Listeria animal feeding stuffs. Horizontal method for the detection
monocytogenes and other Listeria spp. International Journal of and enumeration of Listeria monocytogenes. Part 2:
Food Microbiology, 8: 299-316. Enumeration method.
(4) Bind, J.L. 1991. Mise en évidence et dénombrement des (10) XP V08-062. Octobre 2000. Microbiologie des aliments.
Listeria à partir de produits laitiers. Lait, 71: 99-105. Méthode de dénombrement de Listeria monocytogenes.
(5) Journal Officiel (French) du 7 Avril 1992. Contrôle Méthode de routine.
microbiologique des produits végétaux ou d’origine végétale,
5144. (arrêté du 13 mars 1992).
197
Peptone Water
Intended Use
Product Profile
Peptone Water is used to promote the growth of bacteria with no Target : Escherichia coli.
particular requirements. The medium is used primarily in the Reconstitution : 15,0 g / L.
Mackenzie test for the identification of Escherichia coli by indole
production.
pH (25°C) : 7.2 ± 0.2.
Principles Supplement required : Kovacs reagent.
When grown aerobically, Escherichia coli degrades tryptophan to
indole by a tryptophanase. The indole produced is detected with
the Kovacs reagent. Preparation
Typical Composition - Dissolve 15.0 g of dehydrated medium

water.
For 1 liter of medium : - Stir slowly until complete dissolution.
- Tryptone 10.0 g - Dispense 10 mL in 16 x 160 mm tubes.
- Sodium chloride 5.0 g - Sterilize in an autoclave at 121°C for
15 minutes.
Quality Control
- For the identification of Escherichia coli
- Dehydrated medium : cream white powder, free-flowing and with the Mackenzie test, transfer one loop
homogeneous. of gas-producing culture in BGBB (BK002)
- Prepared medium : clear amber solution, limpid. or Minerals Glutamate Medium (BK005) to
- Typical culture response after 24-48 hours of incubation at 44°C : a tube of Peptone Water.
- Incubate at 44 ± 0.5°C in a water bath for
Microorganisms Growth Indole production 24 and 48 hours.
Escherichia coli ATCC® 25922 good positive
Escherichia coli ATCC 8739 good positive Results
Salmonella Typhimurium ATCC 14028 good negative
After incubation of the inoculated tubes,
the presence of indole is shown by a red
Storage / Shelf Life color in the alcohol phase of the Kovacs
reagent, added at 0.5 mL per tube.
NOTE
- The expiration date is indicated on the label. Kovacs reagent :
Prepared medium (benchmark value) : - Para-aminodimethylbenzaldehyde 5g
- Media in tubes : 6 months at 2-8°C. - Amyl alcohol 75 mL
- Hydrochloric acid 25 mL
Packaging
Dissolve the para-aminodimethylbenzaldehyde in
the amyl alcohol in a 60°C water bath. Cool and add
Dehydrated medium : the hydrochloric acid dropwise, maintaining the
- 500 g bottle : BK084HA recipient in ice-water. Store at 2-8°C in an inactinic
recipient with ground glass stopper. The reaction is
immediate :
- Indole-positive reaction : red ring.
- Indole-negative reaction : brownish ring (original
color of reagent).
Bibliography
(1) NF V 08-017: June 1980. General guidance for the enumeration (5) NF ISO 7251. September 1994. Microbiology. General guidance
of faecal coliforms and Escherichia coli. for enumeration of presumptive Escherichia coli. Most probable
(2) Rodier, J. 1984. L’analyse de l’eau. Dénombrement des number technique.
coliformes, coliformes fécaux et Escherichia coli présumés. (6) NF V08-600. October 2000. Microbiology of foods and
Dunod 7ème Ed., 793-798. foodstuffs products. Enumeration of presumptive Escherichia
(3) NF V 04-015: February 1984. Dried milk and sweetened coli in living shell fishes - MPN technique.
(4) Marchal, N., Bourdon, J.L., et Richard, Cl. 1987. Les milieux de
culture pour l’isolement et l’identification biochimique des
bactéries. Doin Ed, 144-147.
198
Plate Count Agar (PCA)

Plate Count Agar containing glucose and yeast extract is used in Reconstitution : 20.5 g / L.
alimentary bacteriology to enumerate aerobic bacteria in milk,
meats, meat-based products, other food products, as well
as for the analysis of pharmaceuticals, cosmetics and their associated pH (25°C) : 7.0 ± 0.2.
raw materials. Supplement required : None.
History
Plate Count Agar is prepared with the same ingredients as those Preparation
originally used by Buchbinder et al. These authors compared several
batches of yeast extract and showed that the results obtained - Suspend 20.5 g of dehydrated medium
(without milk added to the medium) were satisfactory for the (BK144) in 1 liter of distilled or deionized
enumeration of bacteria that were contaminating samples of raw water.
and pasteurized milk. The transparency of the medium and the - Slowly bring to boiling, stirring until
relative size of colonies formed led to uncomplicated enumeration. complete dissolution.
15 minutes.
The nutrients supplied by Tryptone, vitamins from yeast extract, and
glucose used as energy source favor the growth of most bacteria. Instructions for Use
Typical Composition - With the ready-to-use media BM015 or

(can be adjusted to obtain optimal performance) BM033 (or if the media has been prepared
in advance from the dehydrated base),
For 1 liter of medium : melt the medium for the minimum
- Tryptone 5.0 g amount of time necessary to achieve total
- Yeast extract 2.5 g liquefaction.
- Glucose 1.0 g - Cool and maintain at 47°C.
- Bacteriological agar 12.0 g - Transfer 1 mL of the product to analyze
and its serial tenfold dilutions to sterile
pH of the ready-to-use medium at 25°C : 7.0 ± 0.2. Petri dishes.
- Pour 10 to 15 mL of medium.
Quality Control - Homogenize by swirling.
- Dehydrated medium : cream powder, free-flowing and - If necessary, overlay with 4 mL of sterile
homogeneous. agar (Agar type A at 15 g/L).
- Prepared medium : light amber agar. - Let solidify again.
- Typical culture response after 72 hours of incubation at 30°C : - Incubate :
- at 30°C for 72 hours for the detection
of mesophilic bacteria.
Escherichia coli ATCC® 25922 good to excellent - at 55°C for thermophilic bacteria.
Staphylococcus aureus ATCC 25923 good to excellent - at 6.5°C for 10 days for psychrophilic
Pseudomonas aeruginosa ATCC 10145 good to excellent bacteria.
Storage / Shelf Life NOTE

In dairy bacteriology, it is recommended to add
1 g of powdered skimmed milk per liter of
Dehydrated medium : 2-30°C. reconstituted medium.
Prepared medium (benchmark value) : Results
- Media in plates : 1 month at 2-8°C
Only plates containing less than 300 colonies
are used for enumeration.
When grown on PCA containing milk,
caseinolytic bacteria form a clear halo
around each colony (proteolysis of casein in
the milk).
199
Packaging
- 10 x 100 mL vials : BM01508
- 10 x 200 mL vials : BM03308
Dehydrated medium :
Bibliography
(1) Buchbinder, Baris, Alff, Reynolds, Dillon, Pessin, Pincus and (5) ISO 2293: 1988. Meat and meat products. Enumeration of
Strauss., 1951. Public Health Reports, 66: 327. micro-organisms. Colony count technique at 30°C. (Reference
(2) FIL-IDF 57: 1970. Detection of penicillin in milk by a disk method).
assay technique. (6) NF ISO 4833. July 1991. Microbiology. General guidance for
(3) Méthode (French) officielle pour le dénombrement des germes the enumeration of micro-organisms. Colony count technique
aérobies mésophiles. Ministère de l’Agriculture. Commission at 30°C.
XXX. Cosmétologie. (7) NF V 08-051. Feb. 1999. Microbiology of food and animal
(4) NF T 72-151. November 1987. Water miscible, antiseptics and feeding stuffs. Enumeration of microorganisms by colony-count
disinfectants used in liquid form. Determination of the technique obtained at 30°C. Routine method.
bactericidal activity. Membrane filtration method.
Plate Count Agar with skimmed milk

Plate Count Agar with skimmed milk is used in food and dairy
bacteriology to enumerate aerobic bacteria in milk powders and Reconstitution : 21.5 g / L.
whey, as well as in ice cream, according to the standards of the Autonomy (500 g) : 1550 plates.
International Dairy Federation.
pH (25°C) : 7.0 ± 0.2.
The nutrients supplied by skimmed milk and Tryptone, vitamins from
yeast extract and glucose used as energy source favor the growth of
Preparation
most bacteria.
Typical Composition (BK161) in 1 liter of distilled or deionized
- Tryptone 5.0 g - Dispense in tubes or flasks.
- Yeast extract 2.5 g - Sterilize in an autoclave at 121°C for
- Glucose 1.0 g 15 minutes.
- Dried skimmed milk (free of inhibiting substances) 1.0 g
- Bacteriological agar 12.0 g Instructions for Use
pH of the ready-to-use medium at 25°C : 7.0 ± 0.2. - Melt the medium (if prepared in advance).
Petri dishes.
200
Quality Control - Pour 10 to 15 mL of medium.
- Dehydrated medium : beige powder, free-flowing and - Let solidify on a cold surface.
homogeneous. - Incubate :
- Prepared medium : amber agar. - at 30°C for 72 hours for the detection
- Typical culture response after 72 hours of incubation at 30°C : of mesophilic bacteria,
- at 55°C for thermophilic bacteria,
Microorganisms Growth - at 6.5°C for 10 days for psychrophilic
bacteria.
Lactobacillus casei subsp. rhamnosus ATCC ®
Staphylococcus aureus ATCC 25923 good to excellent Results
Storage / Shelf Life Only plates containing less than 300 colonies
are used.
Dehydrated medium : 2-30°C. Caseinolytic bacteria form a clear halo
- The expiration date is indicated on the label. around each colony (proteolysis of milk
Prepared medium (benchmark value) : casein).
Packaging
Dehydrated medium :
Bibliography
(1) NF V 04-015: February 1984. Dried milk and sweetened (5) FIL-IDF 101 A: 1991. Liquid milk. Psychrophilic
condensed milk. Microbiology. microorganisms. Colony count at 6.5°C.
(2) Journal Officiel (French) du 17 Février 1985: Méthodes (6) FIL-IDF (provisional) 169 : 1994. Quality Control in the
d'analyses des laits pasteurisés (arrêté du 3 janvier 1985). microbiological laboratory – Analyst performance assessment
(3) NF V 04-016: August 1985. Milk. Enumeration of for colony count.
microorganisms. Colony count technique at 30°C.
(4) FIL-IDF 100 B: 1991. Milk and milk products. Microorganisms.
Colony count at 30°C.
Potato Dextrose Agar

Potato Dextrose Agar is used for the isolation, culture and Target : Yeasts, Molds.
enumeration of yeasts and molds in dairy and other food products. Reconstitution : 39.0 g / L.
History
pH (25°C) : 4.5 ou 3.5 (depending on use).
In the course of a comparative test on several culture media in 1938, Supplement required : None.
Shadwick found that Potato Dextrose Agar gave good results for the Sterilization : 121°C / 15 minutes.
enumeration of yeasts and molds in butter. This medium is also
recommended by the United States Pharmacopeia for the control of Preparation
pharmaceutical products.
water.
- The concentrations of glucose and potato extract favor the growth - Slowly bring to boiling, stirring until
of yeasts and molds. complete dissolution.
- The acid pH inhibits most bacteria. - Dispense in tubes or flasks.
15 minutes.
201
For 1 liter of medium : - Adjust the pH to 4.5 or 3.5 by adding
- Potato extract 4.0 g sterile lactic or tartaric acid.
- Glucose 20.0 g - Pour into sterile Petri dishes.
- Bacteriological agar 15.0 g - Let solidify on a cold surface.
Quality Control partially removed.
- Transfer 0.1 mL of the product to analyze
- Dehydrated medium : whitish powder, free-flowing and and its serial tenfold dilutions to the
homogeneous. plates.
- Prepared medium : amber agar. - Spread the inoculum on the surface of the
- Typical culture response after 72 hours of incubation at 30°C : agar with a sterile triangle.
- Incubate at 25-30°C for 3 to 5 days.
NOTE
Saccharomyces cerevisiae ATCC ®
9763 good to excellent Never heat the medium after adding acid in
Candida albicans ATCC 10231 good to excellent order to avoid the loss of the gelling properties
of the agar.
Aspergillus niger ATCC 16404 good
Results
Separately enumerate yeasts and molds.
Dehydrated medium : 2-30°C. Carry out a rapid microscopic confirmation
- The expiration date is indicated on the label. test on each type of colony encountered.
Packaging
Dehydrated medium :
Bibliography
(1) Beever, R.E., and Bollard, E.G. 1970. The nature of the (2) United States Pharmacopoeia 24. 2000. Microbial Limit tests,
stimulation of fungal growth by potato extract. J. Gen. 1814-1818.
Microbiology, 60: 273-279.
Rappaport-Vassiliadis Broth

Rappaport-Vassiliadis Broth is used for the selective enrichment of Target : Salmonella.
Salmonella in food products, in water and in other samples which Reconstitution : 26.5 g / L.
may contain the species. Autonomy (500 g) : 1885 tubes.
pH (25°C) : 5.2 ± 0.2.
History
The composition of the medium was developed by Rappaport Sterilization : 115°C / 15 minutes.
following the observation that Salmonella was more resistant to
hypertonic media than most other enterobacteria. In his experiments, Preparation
Rappaport showed that magnesium chloride was the most effective of
all the salts tested. Selectivity of the medium was increased still further - Dissolve 26.5 g of dehydrated medium (BK136)
by the addition of malachite green. Vassiliadis subsequently showed in 1 liter of distilled or deionized water.
that a larger number of Salmonella could be recovered by reducing the - Stir slowly until complete dissolution.
malachite green content and incubating at 43°C instead of 37°C. - Dispense in tubes or flasks.
15 minutes.
202
Subsequent studies by Peterz et al. showed the effects of incubation Instructions for Use
temperature and magnesium chloride concentration on the recovery
performance of the medium. - Cool the medium to 25°C.
- Transfer 0.1 mL of inoculum from a
Principles preenrichment medium (Lactose Broth
BK082 or Buffered Peptone Water BK018,
- The high concentration of magnesium chloride and the presence BK131 or BM 010) to 10 mL of medium
of malachite green reduce the growth of bacteria other than (respect the 1/100 ratio) prepared as above
salmonellae. or into ready-to-use tubes of BM012.
- Growth of Salmonella Typhi and Shigella are inhibited by - Incubate for 24 hours at 42°C.
malachite green. - Carry out an isolation in several selective
- The use of Selenite Broth in parallel to Rappaport-Vassiliadis Broth media for Salmonella, using a platinum loop.
is recommended for samples contaminated by Salmonella Typhi,
Salmonella Cholerasuis and other salmonella serotypes.
Typical Composition

- Tryptone 4.50 g
- Magnesium chloride anhydrous 13.3 g
- Malachite green oxalate 36 mg
Quality Control
- Dehydrated medium : bluish powder, free-flowing and

homogeneous.
- Prepared medium : blue solution, limpid.

Salmonella Typhimurium ATCC® 14028 good colorless colonies
inhibited

Ready-to-use media in tubes :
Packaging
- 50 x 10 mL tubes : BM01208
Dehydrated medium :
203
Bibliography
(1) Rappaport, F., Konforti, N., and Navon, B. 1956. A new (5) Peterz, M., Wiberg, C., and Norberg, P. 1989. The effect of
enrichment medium for certain Salmonella. J. Clin. Path., incubation temperature and magnesium chloride concentration
9: 261-266. on growth of salmonella in home-made and in commercially
(2) Vassiliadis, P., Kalapothaki, V., Trichopoulos, D., Papadakis, available dehydrated Rappaport-Vassiliadis broths. Journ. of
J.A., and Serie, C.H. 1979. Recent experience on the use of Appl. Bact., 66: 523-528.
modified Rappaport’s medium (R 10/43°C) for the isolation of (6) ISO 6579: 1993. Microbiology. General guidance or methods for
Salmonella. Quality assurance and quality control of the detection of Salmonella.
microbiological culture media. Dr Janet E.L. Corry. London, (7) FIL-IDF 93B: 1995. Milk and milk products. Detection of
141-145. Salmonella.
(3) Harvey, R.W.S., and Price, T.H. 1979. Comparison of Selenite F, (8) NF V08-052. May 1997. Microbiology of food and animal
Müller-Kauffmann Tetrathionate and Rappaport’s medium for the feeding stuffs. Detection of Salmonella. Routine method.
isolation of Salmonella from sewage polluted natural water (9) NF EN 12824. February 1998. Microbiology of food and animal
using a pre-enrichment technique. J. Hyg. Comb., 83: 451-459. feeding stuffs. Horizontal method for the detection of
(4) Harvey, R.W.S., and Price, T.H. 1981. Comparison of Selenite F, Salmonella.
Müller-Kauffmann Tetrathionate and Rappaport’s medium for
Salmonella isolation from chicken gibbets after pre-enrichment
in buffered peptone water. J. hygiene, 87: 219-224.
Rappaport-Vassiliadis Soja Broth (RVS)
Intended Use
Product Profile
Rappaport-Vassiliadis Soja Broth is used for the selective enrichment Target : Salmonella.
of Salmonella in food products, in water and in other samples which Reconstitution : 26.5 g / L.
may contain the species. Autonomy (500 g) : 188 vials
(100 mL per vial).
History
pH (25°C) : 5.2 ± 0.2.
The composition of the medium was developed by Rappaport Supplement required : None.
following the observation that Salmonella was more resistant to Sterilization : 115°C / 15 minutes.
hypertonic media than most other enterobacteria. In his experiments,
Rappaport showed that magnesium chloride was the most effective of
Preparation
all the salts tested. Selectivity of the medium was increased still further
by the addition of malachite green. Vassiliadis subsequently showed
that a larger number of Salmonella could be recovered by reducing the
malachite green content and incubating at 43°C instead of 37°C. water.
Subsequent studies by Peterz et al. showed the effects of incubation - Stir slowly until complete dissolution.
temperature and magnesium chloride concentration on the recovery - Dispense in tubes or flasks.
performance of the medium. Finally, van Schothorst and Renaud - Sterilize in an autoclave at 115°C for
modified Rappaport-Vassiliadis Broth by replacing the casein peptone 15 minutes.
with a soy peptone and by incorporating a potassium
hydrogenophosphate buffer into the formula, resulting in a greater Instructions for Use
stability of the medium over time.
Principles - Transfer 1 mL of inoculum from a
preenrichment medium : Lactose Broth
- The high concentration of magnesium chloride and the presence (BK082) or Buffered Peptone Water
of malachite green reduce the growth of bacteria other than (BK018, BK131, or BM010) to 100 mL of
salmonellae. medium (respect the 1/100 ratio).
- Growth of Salmonella Typhi and Shigella are inhibited by - Incubate for 24 hours at 41.5°C.
malachite green. - Carry out an isolation in several selective
- The use of Selenite broth in parallel to Rappaport-Vassiliadis Soja media for Salmonella, using a platinum loop.
Broth (RVS) is recommended for samples contaminated by
Salmonella Typhi, Salmonella Cholerasuis and other salmonella
serotypes.
204
Typical Composition

- Magnesium chloride anhydrous 13.4 g
- Malachite green oxalate 36 mg
Quality Control
- Dehydrated medium : bluish powder, free-flowing and

homogeneous.
- Prepared medium : blue solution, limpid.

Salmonella Typhimurium ATCC® 14028 good colorless colonies
inhibited

Packaging
Dehydrated medium :
Bibliography
(1) Rappaport, F., Konforti, N., and Navon, B. 1956. A new (5) Peterz, M., Wiberg, C., and Norberg, P. 1989. The effect of
enrichment medium for certain Salmonella. J. Clin. Path., incubation temperature and magnesium chloride concentration
9: 261-266. on growth of Salmonella in home-made and in commercially
(2) Vassiliadis, P., Kalapothaki, V., Trichopoulos, D., Papadakis, available dehydrated Rappaport-Vassiliadis broths. Journ. of
J.A., and Serie, C.H. 1979. Recent experience on the use of Appl. Bact., 66: 523-528.
modified Rappaport’s medium (R 10/43°C) for the isolation of (6) ISO 6340. December 1995. Water Quality. Detection of
Salmonella. Quality assurance and quality control of Salmonella.
microbiological culture media. Dr Janet E.L. Corry. London, (7) NF EN 12824. Feb. 1998. Microbiology of food and animal
141-145. feeding stuffs. Horizontal method for the detection of
(3) van Schothorst, M., and Renaud, A.M. 1983. Dynamics of Salmonella.
Salmonella isolation with modified Rappaport’s medium (R10). (8) ISO/CD 6579: 1998. Microbiology of food and animal feeding
Journ. Appl. Bacteriol., 54, 209-215. stuffs. Horizontal method for the detection of Salmonella.
(4) van Schothorst, M, Renaud, A., and van Beek. 1987.
Salmonella isolation using RVS broth and MLCB agar. Food
Microb., 4, 11-18.
205
Reinforced Clostridial Agar

Reinforced Clostridial Agar is a non-selective medium used for the
Clostridium, Lactobacilli.
growth, isolation and enumeration of Clostridium, other anaerobic
bacteria and lactobacilli in biological samples, dairy, and other food Reconstitution : 52.5 g / L.
products. Autonomy (500 g) : 635 plates.
pH (25°C) : 6.8 ± 0.2.
History
This medium was described by Hirsch and Grinsted for the isolation Sterilization : 121°C / 15 minutes.
of Clostridium butyricum in agar medium. Barnes used it for the
enumeration of clostridia in foods. Attenborough and Scarr used it
for the enumeration of Clostridium saccharolyticum in sugar. Preparation

- This non-selective medium also enables streptococci and water.
lactobacilli to grow. - Slowly bring to boiling, stirring until
- Nutritive factors are supplied by Tryptone, meat and yeast extracts, complete dissolution.
glucose and cysteine, the latter also acting as a reducing substance. - Dispense in tubes or flasks.
- Starch favors spore germination. - Sterilize in an autoclave at 121°C for
- Sodium chloride maintains the osmotic equilibrium. 15 minutes.
- In order to inhibit the growth of Gram-negative bacteria, Hirsch
and Grinsted recommended using polymyxin B in the Instructions for Use
concentration range of 15 to 20 mg/liter.
- Melt the medium (if prepared in advance).
Typical Composition - Cool and maintain at 47°C.
(can be adjusted to obtain optimal performance) - Transfer 1 mL of the product to analyze
and its serial tenfold to sterile Petri dishes.
For 1 liter of medium : - Pour in 15 mL of medium.
- Tryptone 10.0 g - Homogenize by swirling.
- Meat extract 10.0 g - Let solidify on a cold surface.
- Yeast extract 3.0 g - Place the inoculated plates in an
- Cysteine hydrochloride 0.5 g anaerobiosis jar.
- Glucose 5.0 g - Incubate between 30 and 55°C for
- Soluble starch 1.0 g 1 to 10 days, depending on the bacteria
- Sodium chloride 5.0 g to enumerate.
- Sodium acetate 3.0 g
- Bacteriological agar 15.0 g Results
pH of the ready-to-use medium at 25°C : 6.8 ± 0.2. Only plates containing between 15 and
150 colonies are used.
Quality Control

homogeneous.
- Typical culture response after 48 hours of anaerobic incubation at 37°C :
Clostridium perfringens ATCC® 13124 good to excellent
Clostridium bifermentans ATCC 19299 good to excellent
Clostridium tyrobutyricum CNRZ 608 good to excellent

206
Packaging
Dehydrated medium :
Bibliography
(1) Hirsch, A., and Grinsted, E. 1954. Methods for the growth and (3) Munoa, F.J., and Pares, R. 1988. Selective medium for isolation
enumeration of anaerobic sporeformers from cheese with and enumeration of Bifidobacterium spp. App. Environ.
observations on the effect of nisin. J. Dairy Res., 21: 101-110. Microbiol., 54: 1715-1718.
(2) Barnes, E.M., and Ingram, M. 1956. The effect of redox
potential on the grown Clostridium welchii strain isolated from
horse muscle. J. Appl. Bacteriol., 19 (1): 177-178.
Reinforced Clostridial Medium

Target : Spores of total gas producing
Reinforced Clostridial Medium is a non-selective medium used for the
Clostridia.
growth and enumeration of spores of total gas producing Clostridia in
dairy products, canned foods, semi-preserved foods and other food Reconstitution : 38.0 g / L.
products. It can also be used for the growth of lactobacilli and most other Autonomy (500 g) : 1315 tubes.
anaerobic bacteria.
pH (25°C) : 6.8 ± 0.2.
This medium was described by Hirsch and Grinsted for the
enumeration of anaerobes, especially Clostridium butyricum, using
the most probable number method. Preparation

- The medium is non-selective, enabling streptococci and lactobacilli water.
to grow. - Slowly bring to boiling, stirring until
- Nutritive factors are supplied by Tryptone, meat and yeast complete dissolution.
extracts, glucose and cysteine, the latter also acting as a reducing - Dispense 10 mL in 16 x 160 mm tubes.
substance. - Sterilize in an autoclave at 121°C for
- Starch favors spore germination. 15 minutes.
- Sodium chloride maintains the osmotic equilibrium.
- In order to inhibit the growth of Gram-negative bacteria, Hirsch Instructions for Use
and Grinsted recommended using polymyxin B in the
concentration range of 15 to 20 mg/liter. - Regenerate the medium by heating for
20 minutes at 100°C (if it was prepared in
Typical Composition advance).
(can be adjusted to obtain optimal performance) - Cool and maintain at 25°C.
For 1 liter of medium : - Heat the inoculum at 75°C for 15 minutes
- Tryptone 10.0 g to destroy vegetative cells and activate
- Meat extract 10.0 g spores.
- Yeast extract 3.0 g - Inoculate 1 mL of the inoculum and each
- Cysteine hydrochloride 0.5 g of its serial tenfold dilutions.
- Glucose 5.0 g - Pour 2 mL of melted paraffin (58-60°C)
- Soluble starch 1.0 g previously sterilized at 121°C for
- Sodium chloride 5.0 g 20 minutes.
- Sodium acetate 3.0 g - Incubate at 37°C for 7 days.
207
- Dehydrated medium : cream-white powder, free-flowing and Read daily after 48 hours of incubation.
homogeneous. Positive tubes are those with growth
- Prepared medium : amber semi-solid medium. and release of gas which raises the
- Typical culture response after 72 hours of anaerobic incubation at 37°C : paraffin plug.
Enumerate with the most probable number
Microorganisms Growth method.
Clostridium perfringens ATCC®13124 good to excellent
Clostridium bifermentans ATCC19299 good to excellent
Clostridium tyrobutyricum CNRZ 608 good to excellent
Storage / Preservation

Packaging
Dehydrated medium :
Bibliography
(1) Hirsch, A., and Grinsted, D.E. 1954. “Methods for the growth and (3) Pharmacopée européenne. Addendum 2000. Contrôle
enumeration of anaerobic sporeformers from cheese with microbiologique des produits non stériles (Recherche de
observations on the effect of nisin”. Jour. Dairy Res., 21: 101-110. microorganismes spécifiés). Solution et milieux de culture
(2) Gibbs, B.M., and Freame, B. 1965. Methods for the recovery of recommandés, 56-61.
Clostridia from foods. Jour. Appl. Bacteriol., 28 (1): 95.
Rogosa Agar
Target : Lactobacilli.
Rogosa Agar is a selective medium used for the enumeration of
lactobacilli in meat, food products and biological samples. Reconstitution : 74.7 g / L.
History
pH (25°C) : 5.5.
Rogosa and his coworkers showed that this medium was more Supplement required : None.
selective for lactobacilli than the tomato juice medium previously Sterilization : Heat to boiling.
used. The growth of Streptococci, Proteus and molds were Do not autoclave.
considerably reduced.
- In addition to Tryptone and yeast extract rich in B vitamins, Tween - Suspend 74.7 g of dehydrated medium
80 is a source of fatty acids required for the development of (BK033) in 1 liter of distilled or deionized
lactobacilli. water.
- Ammonium citrate and sodium acetate inhibit the development - Slowly bring to boiling, stirring until
of most contaminants, including streptococci and molds. complete dissolution.
- The acidic pH resulting from the addition of acetic acid favors the - Adjust the pH to 5.5 by adding sterile
growth of lactobacilli and inhibits that of other bacteria. glacial acetic acid (about 1.3 mL).
- Do not autoclave.
208
For 1 liter of medium : - Transfer 1 mL of the product to analyze
- Tryptone 10.0 g and its serial tenfold dilutions to Petri
- Yeast extract 5.0 g dishes.
- Glucose 20.0 g - Pour in 15 mL of medium.
- Sodium acetate 15.0 g - Homogenize by swirling.
- Ammonium citrate 2.0 g - Let solidify on a cold surface.
- Potassium dihydrogen phosphate 6.0 g - Place the inoculated plates in an
- Magnesium sulfate 575 mg anaerobiosis jar.
- Manganese sulfate 120 mg - Incubate at 37°C for 72 hours in an
- Ferrous sulfate 34 mg atmosphere enriched with 5-10% CO2.
- Tween 80 1.0 g
- Bacteriological agar 15.0 g Results
Quality Control Lactobacilli produce large white colonies.

Prepare the subcultures required for the
- Dehydrated medium : cream-white powder, slightly clumped, brittle. biochemical identification of the species
- Prepared medium : amber agar. having grown.
- Typical culture response after 48 hours of incubation at 37°C The medium is not suitable for the isolation
in 5% CO2 : of several dairy lactobacilli, such as
Lactobacillus delbrueckii subsp. lactis or
Microorganisms Growth Lactobacillus delbrueckii subsp. bulgaricus
Lactobacillus casei subsp. rhamnosus ATCC® 7469 good to excellent because of the excessive salt concentration.

Prepared medium in flasks (benchmark value) :
Packaging
Dehydrated medium :
Bibliography
(1) Rogosa, M., Mitchell, J.A., and Wiseman, R.F. 1951. A selective (3) Sharpe, M.E. 1960. Selective media for the isolation and
medium for the isolation of oral and faecal lactobacilli. J. enumeration of lactobacilli. Lab. Pract., 9: 223-227.
Bact., 62: 132-133. (4) Sabine, D.B., and Vaselekos, J. 1965. Isolation of Lactobacillus
(2) Rogosa, M., Mitchell, J.A., and Wiseman, R.F. 1951. A selective acidophilus from fecal material. Nature, 206: 960.
medium for the isolation and enumeration of oral lactobacilli.
J. Dental Res., 30 (5): 682.
209
Rose Bengal Chloramphenicol Agar
Intended Use
Rose Bengal Chloramphenicol Agar is recommended for the selective

isolation and enumeration of yeast and molds in food products,
environmental, and any other potentially contaminated samples.
History
In 1962, work done by Mossel on the detection of yeast and molds in

foods demonstrated the superiority of media at a neutral pH
containing an antibacterial agent over those based on an acid pH. In
1973, Jarvis developed and used with success Rose Bengal
Chlortetracycline. During the course of subsequent work in 1978 by
Korburger and Rogers, and in 1981 by Baggerman, chloramphenicol
or gentamicin replaced chlortetracycline. Current formulations
contain chloramphenicol.
Aspergillus niger
Principles
- Papaic digest of soybean meal and glucose assure the growth of Product Profile
yeast and molds. Target : Yeasts, Molds.
- Rose bengal inhibits the development of bacteria and prevents the Reconstitution : 29.7 g / L.
invasion of molds on the Petri dish, by limiting their proliferation.
Assimilated by yeasts, it facilitates their enumeration by coloring
the colonies pink. pH (25°C) : 7.2 ± 0.2.
- The presence of chloramphenicol, a thermostable antibiotic, allows Supplement required : None.
a reinforcement of the selectivity against the majority of
contaminating bacteria.

(may be adjusted to obtain optimal performances)
- Papaic digest of soybean meal 5.0 g water.
- Monopotassium phosphate 1.0 g complete dissolution.
- Magnesium sulfate, 7H2O 0.5 g - Dispatch 100 mL aliquots into vials.
- Rose bengal 50 mg - Sterilize by autoclaving at 121°C for
- Chloramphenicol 0.1 g 15 minutes.
- Melt the media (if it has been prepared in
Quality Control advance).
- Dehydrated medium : cream to pinkish powder, fluid and - Pour into sterile Petri dishes.
homogeneous. - Let solidify on a cool surface.
- Prepared medium : pink agar. - Dry the plates in an incubator with the
- Typical cultural response after 72 hours incubation at 25°C : covers partially removed.
- Transfer 0.1 mL of the sample to be tested
Microorganisms Growth or its serial dilutions to Petri dishes.
Saccharomyces cerevisiae ATCC ®
9763 good to excellent - Spread the inoculum over the surface of
Candida albicans ATCC 10231 good to excellent the plate using a sterile triangle.
- Incubate at 20-25°C for 3 to 5 days,
Aspergillus niger ATCC 16404 good to excellent
shielded from light.
210
Dehydrated medium : 2-30°C. Enumerate separately yeasts and molds.

- The expiry date is indicated on the label. Yeast colonies appear pink due to the
Prepared medium (benchmark value) : assimilation of the rose bengal dye. Yeasts
- Media in vials : 3 months at 2-8°C, shielded from light. and non-inhibited bacteria can be mistaken
- Media in plates : 7 days at 2-8°C, shielded from light. for one another. It is necessary to confirm
identity under the microscope.
Packaging
Dehydrated medium :
Bibliography
(1) Mossel, D..A.A., Vasser, M., and Mengerink, W.J.H. 1962. A (4) Baggerman W.I. 1981. A modified rose bengal medium for the
comparison of media for the enumeration of moulds and yeasts enumeration of yeasts and moulds from foods. Eu. J. Appl.
in foods and beverages. Lab. Pract., 11: 109-112. Microbiol. Biotechnol., 12: 242-247.
(2) Jarvis, B. 1973. Comparison of an improved rose-bengal- (5) MacFaddin, J.F. 1985. Media for isolation-cultivation-
chlortetracycline agar with other media for the selective identification-maintenance of medical bacteria. Williams &
isolation and enumeration of moulds and yeasts in food., J. Wilklins, Baltimore, volume 1: 681-682.
Appl. Bacteriol., 36: 723-727. (6) Mislivec, P. B., L. R. Beuchat, and M. A. Cousin. 1992. Yeasts
(3) Korburger, J.A., and Rodgers, M.F. 1978. Single or multiple and Molds. In Compendium of methods for the microbiological
antibiotic-amended media to enumerate yeasts and molds. J. examination of foods, 3rd Ed. American Public Healh Assoc.,
Food Prot., 41: 367-369. Washington D.C: 239-249.
Rose-Gal BCIG Agar
Intended Use
Rose-Gal BCIG Agar is a selective medium used for the simultaneous

and specific enumeration of all Escherichia coli and thermotolerant
coliforms in water, milk and dairy products and other foods.
History
Coliform classification is traditionally based on their capacity to

ferment lactose, producing acid. Lactose fermentation successively
involves two enzymes : initially a permease responsible for entry of
the sugar into the bacterium and subsequently a -galactosidase
which cleaves glucose from galactose allowing the fermentation to
proceed. In 1962, Le Minor and Ben Hamida demonstrated the
advantages of the -galactosidase test over lactose fermentation for
the identification of enterobacteria. Strains which are slow
Escherichia coli + coliforms
fermenters or lactose negative are found in all species of coliforms.
Traditional media ignore -galactosidase positives biotypes which are
permease negative, even though they are of equal hygienic Product Profile
importance. In 1989, Leclerc and Mossel proposed the presence of Target : Thermotolerant coliforms,
-galactosidase as a classification criterion for coliforms. Utilization Escherichia coli.
of a synthetic chromogenic substrate, insensitive to variations in
lactose permeation, permits detection of this enzyme by means of a Reconstitution : 24.0 g / L.
color reaction. Rose-galactopyranoside (6-chloro-3-indolyl--D- Autonomy (500 g) : 1302 plates
galactopyranoside) is one of these substrates. Hydrolysis by - (16 mL by plate).
galactosidase produces a pink coloration. Buehler et al., in 1949, pH (25°C) : 7.2 ± 0.2.
were the first to discover the presence of -D-glucuronidase in
Escherichia coli. Since then, studies have shown that 94 % to 97 % of Supplement required : None.
all Escherichia coli possess -D-glucuronidase and that this enzyme is Sterilization : 121°C / 15 minutes.
only rarely encountered in other species : small numbers of
Citrobacter, Enterobacter, Klebsiella, Salmonella, Shigella and Yersinia.
211
In 1990, Restaino successfully used the chromogenic substrate Preparation
5-bromo-4-chloro-3-indolyl--D-glucuronate (BCIG) which, added to
agar medium allowed enumeration of Escherichia coli in meat - Suspend 24.0 g of dehydrated medium
products within 24 hours. (BK142) in 1 liter of distilled or deionized
water.
- The simultaneous presence of these two substrates enables the - Dispense in tubes or flasks.
detection of the two specific enzyme activities : -galactosidase - Sterilize in an autoclave at 121°C for
and -glucuronidase. Coliforms are distinguished by the 15 minutes.
production of -galactosidase (-gal).
- This enzyme reacts with rose-galactopyranoside to form a pink Instructions for Use
precipitate by the following mechanism :
and its tenfold dilutions to sterile Petri
dishes.
- Mix well.
- Overlay the solidified agar with 4 mL of
medium.
- Let solidify.
- Incubate at 44.0 ± 1.0°C for 18 to 24 hours.
Results
All Escherichia coli possess -galactosidase and 94 % to 97 % are Coliforms produce pink colonies.
also positive for -glucuronidase (GUD). This enzyme cleaves BCIG Escherichia coli is characterized by the
to form a blue coloration illustrated by the following reaction : formation of purple colonies.
Colonies of other enterobacteria able to
grow at 44°C are white.
- The simultaneous action of the two enzymes makes the colonies

of Escherichia coli appear purple.
Microorganisms Typical phenotype Colony color

Escherichia coli GUD +/ -gal + purple
Non Escherichia coli coliforms GUD -/ -gal + pink
Other Gram-negative bacteria GUD -/ -gal - white
- Polypeptone favors excellent coliform growth.

- Yeast extract is a source of B vitamins.
- Bile salts inhibit growth of Gram-positive bacteria.
- Phosphate buffer maintain optimal pH for the enzymatic reactions.
- Sodium chloride and magnesium sulfate facilitate color
development.
NOTE
Escherichia coli strains which are -glucuronidase negative, notably the
enterohemorrhagic serotype O157:H7 produce pink colonies when grown on
this medium. Other Gram-negative bacteria able to grow at 44.0 ± 0.5°C,
such as some strains of Salmonella produce white colonies.
212
Typical Composition

- Bile salts 0.80 g
- BCIG 0.05 g
- Rose-galactopyranoside 0.15 g
pH of ready-to-use medium at 25°C : 7.2 ± 0.2.
Quality Control
- Dehydrated medium : white-cream powder, free-flowing and

homogeneous.
- Prepared medium : whitish agar.

Escherichia coli ATCC® 25922 good to excellent purple colonies
Escherichia coli ATCC 15224 good to excellent purple colonies
Escherichia coli ATCC 43895 good to excellent pink colonies
(-D-glucuronidase negative)
Enterobacter cloacae ATCC 13047 good to excellent pink colonies
Klebsiella pneumoniae ATCC 23357 moderate pink colonies

- Media in vials or tubes : 1 month at 2-8°C, shielded from light.
Packaging
Dehydrated medium :
- 100 g bottle : BK142HM
Bibliography
(1) Le Minor, L., and Ben Hamida, F. 1962. Avantages de la (4) Manafi M., Kneifel W., and Bascomb, S. 1991. Fluorogenic and
recherche de la -galactosidase sur celle de la fermentation du chromogenic substrates used in bacterial diagnostics.
lactose en milieu complexe dans le diagnostic bactériologique, Microbiol. Rev 55 : 335-348.
en particulier des Enterobacteriaceae. An. Inst. Pasteur (Paris) (5) Coiffier, O. 1992. Les bactéries coliformes dans : Les groupes
102 : 267-277. microbiens d’intérêt laitier, CEPIL, Paris : 303-323.
(2) Kilian, M., and Bülow, P. 1976. Rapid diagnostic of
Enterobacteriaceae. I. Detection of bacterial glycosidases. Acta
Pathol. Microbiol. Scand. Sect. B 84 : 245-251.
(3) Adams, M.R., Grubb, S.M., Hamer, A., and Clifford M.N. 1990.
Colorimetric enumeration of Escherichia coli based on -
glucuronidase activity. Appl. Environ. Microbiol. 56 : 2021-
213
Sabouraud Chloramphenicol Agar

Sabouraud Chloramphenicol Agar is recommended for the isolation Target : Yeasts, Molds.
of yeasts and molds, particularly dermatophytes, especially when Reconstitution : 45.5 g / L.
the samples are highly contaminated with bacteria. Autonomy (500 g) : 732 plates.
Principles pH (25°C) : 5.7 ± 0.2.

- Peptic digest of meat is the nitrogen source for growth. Sterilization : 121°C / 15 minutes.
- Glucose is an energy source.
- Chloramphenicol is a heat-stable, broad spectrum antibiotic which
inhibits the development of contaminating microflora. Preparation

water.
For 1 liter of medium : - Slowly bring to boiling, stirring until
- Peptic digest of meat 10.0 g complete dissolution.
- Glucose 20.0 g - Dispense in tubes or flasks.
- Chloramphenicol 0.5 g - Sterilize in an autoclave at 121°C for
NOTE
Excessive heating of the medium will denature
the agar in an acid pH, thus resulting in a
Quality Control medium which is too soft.
- Dehydrated medium : cream-white powder, free-flowing and Instructions for Use

homogeneous.
- Prepared medium : amber agar. - Cool and maintain the medium at 47°C.
- Typical culture response after 72 hours of incubation at 25-30°C : - Pour into sterile Petri dishes.
Microorganisms Growth - Dry in an incubator with the covers
Candida albicans ATCC ®
10231 good partially removed.
Aspergillus niger ATCC 16404 good - Transfer the sample to analyze to the
medium.
Trichophyton megninii ATCC 12106 good - Spread the inoculum on the surface of the
Trichophyton rubrum ATCC 28188 good agar with a sterile triangle.
Escherichia coli ATCC 25922 inhibited - For the isolation of dermatophytes which
Proteus vulgaris ATCC 13315 inhibited may be present in the tested samples,
inoculate a non-selective medium
(Sabouraud Dextrose Agar, BK025) in
parallel.
- Incubate at 20-25°C for 3 to 5 days.
- The expiration date is indicated on the label. Results
- Media in vials : 6 months at 2-8°C. Examine the plates weekly for 5 weeks
- Media in plates : 1 month at 2-8°C. before concluding on the absence
of cultures.
Packaging
Dehydrated medium :
214
Sabouraud Dextrose Agar
Intended Use
Sabouraud Dextrose Agar is a classical medium for the culture,

isolation and identification of saprophytic or pathogenic molds and
yeasts. It is recommended primarily for the isolation of molds from
samples with low bacterial loads, sterility tests of pharmaceutical,
cosmetic or food products, and the culture of molds for their
identification. If samples are highly contaminated, it is preferable to
use Sabouraud Chloramphenicol Agar. Sabouraud Dextrose Agar can
be used as a base for preparing special media, obtained by the
addition of antibiotics (chloramphenicol, gentamicin), blood or
vitamins. When triphenyltetrazolium chloride (TTC) is added at
100 mg per liter, it can be used to differentiate Candida.
History
The medium was recommended by Sabouraud for the growth of Trichophyton spp
pathogenic fungi in skin infections.
Product Profile
Principles Target : Sterility testing, Yeasts, Molds.
- Sabouraud Dextrose Agar is a peptone glucose medium enabling Reconstitution : 60.0 g / L.

the growth of yeasts and molds, in particular dermatophytes. Autonomy (500 g) : 555 plates.
pH (25°C) : 5.7± 0.2.
Typical Composition of the base medium
(can be adjusted to obtain optimal performance) Supplement required : Gentamicin Selective
Supplement and/or Chloramphenicol
Selective Supplement (optional).
- Peptic digest of meat 10.0 g Sterilization : 121°C / 15 minutes.
- Glucose 35.0 g
- Bacteriological agar 15.0 g Preparation
pH of the ready-to-use medium at 25°C : 5.7 ± 0.2. - Suspend 60.0 g of dehydrated medium
Quality Control water.
- Dehydrated medium (base) : cream-white powder, free-flowing complete dissolution.
and homogeneous. - Dispense in tubes or flasks.
- Prepared medium : light amber agar. - Sterilize in an autoclave at 121°C for
- Typical culture response after 72 hours of incubation at 25-30°C : 15 minutes.
Microorganisms Growth NOTE

Excessive heating of the medium will denature
Saccharomyces cerevisiae ATCC® 9763 good to excellent the agar in an acid pH, thus resulting in a
Candida albicans ATCC 10231 good to excellent medium which is too soft.
Aspergillus niger ATCC 16404 good, with production of
characteristic spores Instructions for Use
Trichophyton rubrum ATCC 28188 good - With the ready-to-use media (BM052,
Trichophyton megninii ATCC 12106 good BM053), or if the media has been prepared
Escherichia coli ATCC 25922 partially inhibited in advance from the dehydrated base,
melt the medium for the minimum
amount of time necessary to achieve total
liquefaction.
- If necessary, add 1 mL of a reconstituted
Gentamicin Selective Supplement (BS009)
and/or Chloramphenicol Selective
Supplement (BS021) to 100 mL of medium.
- Mix well
215
Storage / Shelf Life - Dry in an incubator with the covers
partially removed.
Dehydrated base medium : 2-30°C. - Transfer the sample to analyze to the
- The expiration date is indicated on the label. medium.
Prepared medium (benchmark value) : - Spread the inoculum on the surface of the
- Base media in vials or tubes : 6 months at 2-8°C. agar.
- Complete media in plates : 1 month at 2-8°C. - Incubate at 20-25°C for 3 to 5 days.
TTC 50 mg Supplement, Results
Gentamicin Selective Supplement,
Chloramphenicol Selective Supplement : Examine the plates weekly, over a period of
- Store between 2-8°C, shielded from light. several weeks, before concluding on the
- The expiration dates are indicated on the labels. absence of cultures.
Separately count yeasts and molds.
Packaging Carry out a confirmation test under the
microscope on each type of colony
Ready-to-use media encountered.
- 10 x 100 mL vials : BM05208
- 10 x 200 mL vials : BM05308
Dehydrated base medium (without supplements) :
Bibliography
(1) Sabouraud, R. 1900. Les teignes, Paris : Masson et Cie: 553. (4) Pharmacopée européenne. Addendum 2000. Contrôle
(2) Ajello, L. 1957. Cultural methods for human pathogenic fungi. microbiologique des produits non stériles (Recherche de
J. Chronic Dis., 5: 545. microorganismes spécifiés). Solution et milieux de culture
(3) Pagano, J., Levin, J.D., and Trejo, W. 1957 / 1958. Diagnostic recommandés, 56-61.
medium for differentiation of species of Candida. Antibiot. (5) United States Pharmacopoeia 24. 2000. Microbial Limit Tests,
Annu., 137. 1814-1818.
Sabouraud Dextrose Broth

Intended Use
Product Profile
Sabouraud Dextrose Broth is recommended for the detection of Target : Sterility testing, Yeasts, Molds.
yeasts and molds, as well as in the pharmaceutical and cosmetic Reconstitution : 30.0 g / L.
industries to comply with sterility tests.
Autonomy (500 g) : 166 vials
(100 mL by vial).
Principles
pH (25°C) : 5.7± 0.2.
- Meat and casein digests, sources of nitrogen and carbohydrates, Supplement required : None.
are factors for the development of yeasts, molds and acidophilic Sterilization : 121°C / 15 minutes.
bacteria.
- Glucose is an energy source.
216
- Tryptone 5.0 g water.
- Peptic digest of meat 5.0 g - Stir slowly until complete dissolution.
- Glucose 20.0 g - Dispense in tubes or flasks.
pH of the ready-to-use medium at 25°C : 5.7 ± 0.2. 15 minutes.
- Dehydrated medium : cream-white powder, free-flowing and - Inoculated medium is incubated at 25°C
homogeneous. for 3 to 15 days.
- Prepared medium : light amber solution, limpid.
- Typical culture response after 72 hours of incubation at 25-30°C : Results
Microorganisms Growth Growth is reflected by turbidity in the

Saccharomyces cerevisiae ATCC® 9763 good to excellent medium. Subcultures must be prepared to
Candida albicans ATCC 10231 good to excellent purify the strains to be identified.
Lactobacillus casei subsp. rhamnosus ATCC 7469 good to excellent

- Media in tubes or vials : 6 months at 2-8°C, shielded from light.
Packaging
Dehydrated medium :
Schaedler Agar

Schaedler Agar is a base intended for the formulation of a variety Target : Blood agar,
of media for the growth and enumeration of anaerobic bacteria in Anaerobic microorganisms.
food products, pharmaceuticals and biological samples. When Reconstitution : 41.9 g / L.
enriched with 5% sheep blood, it favors the growth of bacteria such
as Bacteroides, Peptococcus and Peptostreptococcus. The addition of
nalidixic acid and colistin leads to the selective isolation of Gram- pH (25°C) : 7.6 ± 0.2.
positive anaerobic cocci. The addition of kanamycin and vancomycin Supplement required : Vitamin K1 (optional),
make it a selective medium for the isolation of Gram-negative Defibrinated sheep blood (optional).
anaerobes. When neomycin and sodium chloride are added, a
selective isolation medium for anaerobic lactobacilli and
streptococci is obtained.
History
The original version of the base without inhibitors was formulated

by Schaedler, Dubos and Castello to favor the growth of fastidious
anaerobic bacteria such as lactobacilli, streptococci, clostridia and
bacteroides. Subsequently, Mata and his coworkers examined a
number of new formulations, obtained by adjusting the
concentration of peptones and by increasing the sodium chloride
concentration. At the same time, the glucose concentration was
217
decreased in order to obtain more characteristic hemolytic reactions. Preparation
The concentration of yeast extract was similarly reduced to avoid
the browning of the medium after adding blood. The addition of - Suspend 41.9 g of dehydrated medium
vitamin K1 was recognized as favoring the development of a few (BK063) in 1 liter of distilled or deionized
strains of Prevotella melaninogenica and Gram-positive sporulated water.
species. - Add 1 mL of 1% vitamin K1 in ethanol, if
Schaedler Agar with vitamin K1 and sheep blood is the base for required.
different media containing selective agents : - Slowly bring to boiling, stirring until
- Schaedler CNA Agar (colistin, nalidixic acid) complete dissolution.
- Schaedler KV Agar (kanamycin, vancomycin) prepared by - Do not overheat the medium.
Finegold for the isolation of Gram-negative anaerobes, - Dispense in tubes or flasks.
particularly Bacteroides. - Sterilize in an autoclave at 121°C for
15 minutes.
Principles
- The medium is rendered highly nutritive by the presence of plant
and animal peptones, yeast extract and glucose.
- Defibrinated sheep blood and hemin stimulate the growth of
- Add 5% sterile defibrinated sheep blood if
fastidious bacteria.
required.
- Vitamin K1 favors the growth of Bacteroides.
- Beta-hemolytic streptococci give a greenish reaction which may be - Add the other constituents as a function
confused with alpha hemolysis due to the presence of high of usage :
concentrations of carbohydrates. - colistin (10 mg/L) + nalidixic acid
- The addition of colistin renders the medium selective by causing (10 mg/L) to obtain Schaedler CNA Agar
the rupture of the membranes of contaminating Gram-negative - kanamycin (100 mg/L) + vancomycin
bacteria. (7.5 mg/L) to obtain Schaedler KV Agar
- Nalidixic acid blocks DNA replication in Gram-negative bacteria, - Homogenize thoroughly without
which are sensitive to this antimicrobial agent. introducing air.
- When kanamycin and vancomycin are added, the medium - Pour into sterile Petri dishes.
becomes selective for the growth of Gram-negative bacteria. - Let solidify on a cold surface.
- Inoculate.
Typical Composition - Incubate at 37°C for 18 to 48 hours in the
(can be adjusted to obtain optimal performance) aero/anaerobic conditions required by the
species being studied.
- Casein-soy peptone 10.0 g Results
- Polypeptone 5.0 g
- Yeast extract 5.0 g Pathogenic aerobes which may be present in
- Glucose 5.0 g the inoculum should be incubated in an
- Tris (hydroxymethyl) aminomethane 3.0 g atmosphere enriched with CO2.
- Hemin 10 mg The growth of Gram-positive obligate
- L-cystine 0.4 g anaerobes is considerably favored by
- Bacteriological agar 13.5 g conditions of strict anaerobiosis, which
should be obtained in less than 3 hours.
pH of the ready-to-use medium at 25°C : 7.6 ± 0.2. If cultures do not appear within 48 hours,
prolong the incubation up to 7 days before
Quality Control considering the results as negative. Bacteria
which do not grow in aerobic conditions are
- Dehydrated medium : beige powder, free-flowing and presumed to be obligate anaerobes which
homogeneous. are then studied in broth(s) appropriate for
- Prepared medium : amber agar. their optimal conditions of development.
with 5% sheep blood :
Bacteroides fragilis (1) ATCC® 23745 good to excellent
Clostridium perfringens (1)
ATCC 13124 good to excellent
(1)
incubated anaerobically
218

Packaging
Dehydrated medium :
Bibliography
(1) Gibbons, R.L., and MacDonald, J.B. 1960. Hemin and vitamin K (4) de Waart, J., and Pouw, H. 1970. Studies on the suitability of
compounds as required factors for the cultivation of certain blood-free media for the enumeration of clostridia. Zentralb.
strains of Bacteroides melaninogenicus. J. Bacteriol., 80: 164. Bakteriol. 1 Abt. Orig. A, 214: 551.
(2) Schaedler, R.W., Dubos, R., and Costello, R. 1965. The (5) Stalons, D.R., Thornsberry, C., and Dowell, V.R. Jr. 1974. Effect
development of the bacterial flora in the gastrointestinal tract of culture medium and carbon dioxide concentration on growth
of mice. J. Exp. Med., 122: 59. of anaerobic bacteria commonly encountered in clinical
(3) Mata, L.J., Carillo, C., and Villatoro, E. 1969. Fecal microflora specimens. Appl. Microbiol., 27: 1098.
in healthy persons in the preindustrial region. App. Microbiol., (6) MacFaddin, J.F. 1985. Media for Isolation-Cultivation-
17 (4): 596. Identification-Maintenance of medical bacteria, vol 1. Williams
and Wilkins. Baltimore, 695-699.
Schaedler Broth

Target : Sterility testing, Aerobic
Schaedler Broth is used as a non-selective medium for the growth
and anaerobic microorganisms.
and enumeration of particularly fastidious aerobic and anaerobic
bacteria. Reconstitution : 28.4 g / L.
History
pH (25°C) : 7.6 ± 0.2.
Schaedler and his coworkers developed the composition of this Supplement required : Vitamine K1 (optional).
medium in order to obtain an abundant culture of anaerobic bacteria : Sterilization : 121°C / 15 minutes.
lactobacilli, streptococci, clostridia and bacteroides. The current
formula results from the modifications of Mata, Carillo and Villatoro, Preparation
who adjusted the quantities of peptones, sodium chloride, glucose and
yeast extract in order to optimize the cultures. - Suspend 28.4 g of dehydrated medium
Principles water.
- The high nutrient capacity of the medium is due primarily to the complete dissolution.
quantities of peptones of different origins, to glucose - Add the vitamin supplement depending
and to yeast extract. on the protocol : 1 mL of 1% vitamin K1 in
- Hemin is used to supplement the medium with factor X required ethanol.
for the growth of bacteria requiring its presence for luxuriant - Dispense in tubes or flasks.
cultures. - Sterilize in an autoclave at 121°C for
- The addition of vitamin K1 favors the development of Prevotella 15 minutes.
melaninogenica, other Bacteroides and Gram-positive
sporulated species.
219
For 1 liter of medium : - If the medium is not used immediately
- Casein-soy peptone 10.0 g after its preparation, it must be
- Polypeptone 5.0 g regenerated for 20 minutes at 100°C in
- Yeast extract 5.0 g order to restore conditions of
- Glucose 5.0 g anaerobiosis.
- Tris(hydroxymethyl)aminomethane 3.0 g - Do not repeat this operation more than once.
- Hemin 10 mg - Inoculate the sample to analyze.
- L-cystine 0.4 g - Incubate at 37°C for 18 to 48 hours in the
aero/anaerobic conditions required by the
pH of the ready-to-use medium at 25°C : 7.6 ± 0.2. species being studied.
Quality Control

homogeneous.
- Prepared medium : amber solution, clear, may contain a fine
precipitate.
Bacteroides fragilis (1) ATCC® 23745 good to excellent
Clostridium perfringens (1) ATCC 13124 good to excellent
(1)
incubated anaerobically

Packaging
Dehydrated medium :
Bibliography
(1) Gibbons, R.L., and MacDonald, J.B. 1960. Hemin and vitamin K (3) Mata, L.J., Carillo, C., and Villatoro, E. 1969. Fecal microflora
compounds as required factors for the cultivation of certain in healthy persons in the preindustrial region. App. Microbiol.,
strains of Bacteroides melaninogenicus. J. Bacteriol., 80: 164. 17 (4): 596.
(2) Schaedler, R.W., Dubos, R., and Costello, R. 1965. The (4) Stalons, D.R., Thornsberry, C., and Dowell, V.R. 1974. Effect of
development of the bacterial flora in the gastrointestinal tract culture medium and carbon dioxide concentration on growth of
of mice. J. Exp. Med., 122: 59. anaerobic bacteria commonly encountered in clinical
specimens. Appl. Microbiol., 27: 1098.
220
Schubert Broth (modified)

Schubert Broth modified by Fennel is used as a confirmation Target : Thermotolerant coliforms.
medium for the detection for thermotolerant coliform bacteria in Reconstitution : 26.2 g / L.
water samples. Autonomy (500 g) : 1908 tubes.
Principles pH (25°C) : 7.6 ± 0.2.

- The detection of thermotolerant coliform bacteria usually involves Sterilization : 115°C / 10 minutes.
two steps :
- Inoculation of a presumptive medium : Bromcresol Purple Preparation
Lactose Broth or Laurylsulfate Tryptose Broth.
- Transfer of positive cultures to a confirmation medium : - Dissolve 26.2 g of dehydrated medium
Brilliant Green Bile Broth or Schubert Broth. (BK120) in 1 liter of distilled or deionized
- The development of coliform bacteria is shown by the appearance water.
of turbidity combined with gas production in the Durham tube, - Slowly bring to boiling, stirring until
resulting from the fermentation of mannitol. Numerous results complete dissolution.
have shown the concordance of the results with the fermentation - Dispense in 16 x 160 mm tubes containing
of lactose present in most other media for coliform bacteria. a Durham tube.
(can be adjusted to obtain optimal performance) - After cooling, the Durham tubes should
not contain trapped air.
- Tryptone 10.00 g Instructions for Use
- Tryptophan 0.20 g
- Glutamic acid 0.20 g - Inoculate one tube of medium with the
- Mannitol 7.50 g contents of each tube of presumptive
- Magnesium sulfate 0.70 g medium having given a positive culture.
- Ammonium sulfate 0.40 g - Incubate at 44°C for 24 hours in a
- Sodium citrate 0.50 g thermostated water bath.
- Disodium phosphate 4.12 g Results
Tubes presenting turbidity combined with
pH of the ready to use medium at 25°C : 7.6 ± 0.2. the release of gas are considered as
positive.
Quality Control Carry out enumeration using the most
- Dehydrated medium : cream-white powder, free-flowing and The production of indole, shown by adding
homogeneous. the Kovacs reagent, guides the analysis
- Prepared medium : amber solution, limpid. towards the search for Escherichia coli.

Escherichia coli ATCC 8739 good to excellent positive

- Media in tubes : 1 month at 2-8°C.
Packaging
Dehydrated medium :
221
Bibliography
(1) Rodier. J. 1984. L'analyse de l'eau. Dénombrement des (2) NF T 90-413: October 1985. Testing water. Detection and
coliformes, coliformes fécaux et Escherichia coli présumés. enumeration of coliforms. General method by culture in liquid
Méthode de dénombrement par inoculation en milieux liquides. media (MPN).
Dunod 7ème Ed., 793-798.
Selenite Broth (Leifson)
Intended Use
Product Profile
Selenite Broth of Leifson is used for the selective enrichment of Target : Salmonella, Shigella sonnei.
Salmonella and may also be used for Shigella sonnei in pathological Reconstitution : 23.0 g / L.
samples, water and food products. Autonomy (500 g) : 217 vials
(100 mL by vial).
History
pH (25°C) : 7.0 ± 0.2.
Guth, confirming the initial observations of Handel and Thodorascu, Supplement required : None.
used sodium selenite as a selective agent in an enrichment broth for Sterilization : Heat to boiling.
Salmonella Typhi, after demonstrating the toxicity of the substance Do not autoclave.
towards Escherichia coli. Leifson extended the work of Guth by
developing the formula of a selenite broth for the selective enrichment Preparation
of Salmonella Typhi and Paratyphi from pathological samples by
showing that the number of coliform bacteria decreased during the - Dissolve 23.0 g of dehydrated medium
first 12 hours of incubation, while the number of typhoid bacilli rapidly (BK079) in 1 liter of distilled or deionized
increased in parallel. water.
Principles complete dissolution.
- The quantity of selenite inhibits microorganisms other than - Do not autoclave.
salmonellae, especially coliform bacteria and enterococci. - Cool rapidly.
Pseudomonas and Proteus are not inhibited. - Dispense in sterile tubes of flasks by filling
- Dipotassium phosphate maintains the pH constant and reduces the to 2/3 of the volume of the recipients.
toxicity of selenium in order to increase the recovery capacity of
NOTE
the medium. - If overheated, a brick red selenium precipitate
may form denaturing the medium, which
Typical Composition should be discarded.
(can be adjusted to obtain optimal performance) - In order to increase the shelf life of the ready-
to-use medium, it is recommended to utilize the
membrane filtration method for sterilization.
- Polypeptone 5.0 g
- Lactose 4.0 g
- Sodium selenite 4.0 g - Add 1 or 2 g of pathologic sample to a
prepared tube, or transfer 10 mL of
pH of the ready-to-use medium at 25°C : 7.0 ± 0.2. inoculum from a preenrichment culture :
Lactose Broth (BK082) or Buffered Peptone
Quality Control Water (BK018, BK131 or ready-to-use
BM010) to 100 mL of medium.
- Dehydrated medium : cream-white powder, free-flowing and - Incubate for a maximum of 24 hours at
homogeneous. 37 or 43°C, depending on the analytical
- Prepared medium : very light amber solution, limpid, may protocol applied.
contain a very slight precipitate. - Isolate on several selective media by
- Typical culture response after 18-24 hours of incubation at 37°C, streaking with a loop.
followed by subculture on MacConkey Agar : - Using well formed colonies, inoculate
(BK059) which are the starting points for
identification.
222
Microorganisms Growth Characteristics NOTE
Salmonella Enteritidis ATCC® 13076 good colorless colonies The maximum incubation time of 24 hours is
required because of the decreased inhibitor
Salmonella Typhimurium ATCC 14028 good colorless colonies effect after the first 12 hours of incubation.
Escherichia coli ATCC 25922 slow pink-red colonies Salmonella are rapidly destroyed beyond that
time when in the presence of powerful
Staphylococcus aureus ATCC 25923 inhibited competitors such as Proteus.

- Media in tubes or vials : 8 days at 2-8°C, shielded from light
and after having verified stability and bacteriological efficacy.
Packaging
Dehydrated medium :
Bibliography
(1) Leifson, E. 1936. New selenite selective enrichment medium (2) Banffer, J. 1971. Comparison of the isolation of Salmonellae
for the isolation of typhoid and paratyphoid (Salmonella) from human faeces by enrichment at 37°C and at 43°C.
bacilli. Am. J. Hyg., 24. Zentrabl. Bakt.I.Orig., 217: 35-40.
Selenite-Cystine Broth
Intended Use
Product Profile
Selenite-Cystine Broth is used for the selective enrichment of Target : Salmonella.
salmonellae in pharmaceutical, dairy and other food products. Reconstitution : 23.0 g / L.
Autonomy (500 g) : 217 vials
History (100 mL by vial).
pH (25°C) : 7.0 ± 0.2.
Guth, confirming the initial observations of Handel and Thodorascu,
used sodium selenite as a selective agent in an enrichment broth for Supplement required : None.
Salmonella Typhi, after demonstrating the toxicity of the substance Sterilization : Heat to boiling.
towards Escherichia coli. Leifson extended the work of Guth by Do not autoclave.
developing the formula of a selenite broth for the selective
enrichment of Salmonella Typhi and Paratyphi from pathological
samples by showing that the number of coliform bacteria decreased Preparation
during the first 12 hours of incubation, while the number of
typhoid bacilli rapidly increased in parallel. This broth is a - Suspend 23.0 g of dehydrated
modification of the original formula of Leifson. This formulation medium (BK009) in 1 liter of distilled
containing cystine was proposed by the Food and Drug or deionized water.
Administration as being one specifically destined for the detection - Slowly bring to boiling, stirring until
of Salmonella in food products. The composition complies with the complete dissolution.
United States Pharmacopoeia. - Continue boiling for 2 minutes.
- Do not autoclave.
Principles - Cool rapidly.
- Dispense in sterile tubes of flasks by
- The quantity of selenite inhibits microorganisms other than filling to 2/3 of the volume of the
salmonellae, especially coliform bacteria and enterococci. recipients.
Pseudomonas and Proteus are only partially inhibited.
- Dipotassium phosphate maintains the pH constant and reduces the
toxicity of selenium in order to increase the recovery capacity of
the medium.
223
Typical Composition NOTE
(can be adjusted to obtain optimal performance) - If overheated, a brick red selenium
precipitate may form, denaturing the
medium, which should be discarded.
- In order to increase the shelf life of the
- Tryptone 5.0 g ready-to-use medium, it is recommended
- Lactose 4.0 g to utilize the membrane filtration method
- Disodium phosphate 10.0 g for sterilization.
- Sodium selenite 4.0 g
- L-cystine 10.0 mg
Quality Control - Transfer 10 mL of inoculum from a
preenrichment culture : Lactose Broth
- Dehydrated medium : cream-white powder, free-flowing and (BK082) or Buffered Peptone Water
homogeneous. (BK018, BK131 or ready-to-use BM010) to
- Prepared medium : light amber to pinkish solution, may contain 100 mL of medium.
a very slight precipitate. - Incubate for a maximum of 24 hours at
- Typical culture response after 18-24 hours of incubation at 37°C, 37 or 43°C, depending on the analytical
followed by subculture on Brilliant Green Agar : protocol applied.
- Isolate on several selective media.
Microorganisms Growth Characteristics - Using well formed colonies, inoculate
Salmonella Typhimurium ATCC ®
14028 good colorless colonies (BK059) which are the starting points for
Salmonella Enteritidis ATCC 13076 good colorless colonies identification.
Escherichia coli ATCC 25922 partially inhibited pink-red colonies
Staphylococcus aureus ATCC 25923 inhibited NOTE :
The maximum incubation time of 24 hours is
required because of the decreased inhibitor
Storage / Shelf Life effect after the first 12 hours of incubation.
Salmonella are rapidly destroyed beyond that
Dehydrated medium : 2-20°C. time when in the presence of powerful
competitors such as Proteus.
On the other hand, a 48 hour incubation is
Prepared medium (benchmark value) : required for the selective enrichment of
- Media in tubes or vials : 8 days at 2-8°C, shielded from light and Salmonella Pullorum.
after having verified stability and bacteriological efficacy.
Packaging
Dehydrated medium :
Bibliography
(1) Leifson, E. 1936. New selenite selective enrichment medium (6) FIL-IDF 93B: 1995. Milk and milk products. Detection of
for the isolation of typhoid and paratyphoid (Salmonella) Salmonella.
bacilli. Am. J. Hyg., 24. (7) ISO 6340. 1995. Water Quality. Detection of Salmonella.
(2) Food and Drug Administration, Ottawa. 1962. The (8) NF V 08-052. May 1997. Microbiology of food and animal
Determination of Salmonellae in Foods. feeding stuffs. Detection of Salmonella. Routine method.
(3) Am. Publ. Health Assoc., New York. 1967. (12th Ed.) Standard (9) NF EN 12824. February 1998. Microbiology of food and
Methods for the Examination of Foods. animal feeding stuffs. Horizontal method for the detection of
(4) NF V 59-104: October 1982. Edible gelatin. Detection of Salmonella.
Salmonella. (10) United States Pharmacopoeia 24. 2000. Microbial Limit Tests,
(5) NF V 04-015: February 1984. Dried milk and sweetened 1814-1818.
224
Slanetz and Bartley Agar
Intended Use
Slanetz and Bartley Agar is a selective medium used for the

enumeration of enterococci in drinking water, beverages, waste
water and various biological products, both by the membrane
filtration method and the classical technique of enumeration in
Petri dishes.
History
The medium was formulated by Slanetz et al. in order to enumerate

enterococci in water and beverages by the membrane filtration
method. A modification of the method, involving the addition of
TTC (triphenyltetrazolium chloride) leads to better counts when the
membranes are placed directly on the surface of the agar. The
current formula produces results comparable to those of the Enterococcus faecalis
method developed by Litsky, Mallmann and Fifield for the detection
of fecal streptococci.
Product Profile
Principles Target : Intestinal enterococci
Reconstitution : 41.5 g / L (BK037).
- Sodium azide inhibits the growth of Gram-negative bacteria. 41.4 g / L (BK129).
- TTC is an indicator of bacterial growth. It is reduced to an Autonomy (500 g) : 1340 plates
insoluble formazan inside the cells. This reaction is seen by the (9 mL by plate).
formation of red to maroon colonies.
pH (25°C) : 7.2 ± 0.2.
Typical Composition of the complete medium Supplement required : TTC Supplement 50 mg.
(can be adjusted to obtain optimal performance) Sterilization : 110°C / 20 minutes
(if necessary).
- Tryptose 20.0 g Preparation
- Glucose 2.0 g From complete dehydrated medium :
- Dipotassium phosphate 4.0 g - Suspend 41.5 g of complete dehydrated
- Sodium azide 0.4 g medium (BK037) in 1 liter of distilled or
- 2,3,5-triphenyltetrazolium chloride 0.1 g deionized water.
- Avoid excessive heating.
- Do not autoclave.
- Cool and maintain the medium at 47°C (do
Quality Control not remelt a medium having previously
solidified).
- Dehydrated media : cream-white powder, free-flowing and - Pour into sterile Petri dishes (the agar
homogeneous. layer should be 5 mm thick).
- Prepared medium : amber to orange-pink agar. - Let solidify on a cold surface.
From the dehydrated base medium
(without TTC) :
Microorganisms Growth Gas production - Suspend 41.4 g of dehydrated base
Enterococcus faecalis ATCC® 29212 good to excellent pink to red-brown colonies medium (BK129) in 1 liter of distilled or
Enterococcus faecalis ATCC 33186 good to excellent pink to red-brown colonies deionized water.
Staphylococcus aureus ATCC 25923 inhibited - Dispense 100 mL in sterile flasks (if
necessary, the base medium can be
autoclaved for 20 minutes at 110°C).
- Aseptically add 1 mL of reconstituted TTC
50 mg Supplement (BS027) per flask.
- Pour into sterile Petri dishes (the agar
layer should be 5 mm thick).
225
Storage / Shelf Life Instructions for Use
Dehydrated media (BK037, base BK129) : 2-30°C. - Aseptically filter a known volume of the
- The expiration dates are indicated on the labels. sample to test through a membrane.
Prepared media (benchmark value) : - Deposit the membrane, filtered side up, on
- Base media without TTC (BK129) in vials : 6 months at 2-8°C. the surface of the agar prepared as above,
- Complete media in plates (base BK129) : 8 days at 2-8°C, shielded or on ready-to-use media (BM026), making
from light. sure that the membrane and agar are in
- Complete media in plates (BK037) : 3 days at 2-8°C, shielded from light. close contact.
Ready-to-use media in plates - Incubate at 37°C for 48 hours.
Retain only membranes containing fewer
Packaging than 100 colonies.
Red, maroon or pink colonies are considered
Ready-to-use (complete) media : as characteristic. Carry out confirmation of
- 10 plates (55 mm) kit : BM02608 typical colonies using Bile Esculin Azide Agar
Dehydrated (complete) medium : (BK158) or Ethyl Violet Azide Broth (BK061).
Dehydrated (base ; without TTC) medium :
Bibliography
(1) Slanetz, L.W., Bent, D.F., and Bartley, C.H. 1955. Use of the (3) Rodier, J. 1984. L’analyse de l’eau. Dénombrement des
membrane filter technique to enumerate enterococci. Public streptocoques fécaux présumés. (Méthode par filtration sur
Health. Rep., 70: 67. membrane). Dunod 7è Ed., 828-829.
(2) Slanetz, L.W., and Bartley, C.H. 1957. Numbers of enterococci in (4) NF EN ISO 7899-2. Aug. 2000. Water Quality. Detection and
water, sewage and faeces, determined by the Membrane Filter enumeration of intestinal enterococci - Part 2: Membrane
Technique with an improved medium. J. Bacteriol., 74 (5): 591. filtration method.
SS Agar

Target : Salmonella, Shigella.
Salmonella-Shigella (SS) Agar is used for the isolation of salmonellae and
shigellae after prior enrichment, from food products and other samples Reconstitution : 63.0 g / L.
which are suspected of harboring them. Autonomy (500 g) : 530 plates.
History pH (25°C) : 7.0 ± 0.2.

A number of authors, including Hormaeche, Surraco, Hardy, Rose,
Mayfiels, Goeber, Pots and Caudill, successfully used SS agar for the Do not autoclave.
isolation of Salmonella and Shigella. Schaub showed that the
medium was useful for the production of hydrogen sulfide by the
bacteria sought. Preparation

water.
- Do not autoclave.
226
- SS Agar is a moderately selective medium in which Gram-positive - Cool and maintain the medium at 47°C.
bacteria are inhibited by bile salts, brilliant green and sodium - Pour into sterile Petri dishes.
citrate. - Let solidify on a cold surface.
- The high concentrations of citrate and sodium thiosulfate limit the - Dry in an incubator with the covers
development of coliform bacteria and prevent the invasion of the partially removed.
medium by Proteus. - Inoculate by streaking the enrichment
- Fermentation of lactose to acid is revealed by the formation of media used on the surface of the medium.
red colonies in the presence of neutral red. Lactose-negative - In parallel, inoculate another selective
bacteria form colorless colonies. medium.
- In the presence of thiosulfate and ferric citrate, hydrogen sulfide - Incubate at 37°C for 24 to 48 hours.
producers form colonies with a black center.
Results
It is particularly recommended to first enrich in Selenite Broth or Selenite-
Cystine Broth and to simultaneously inoculate other less selective media :
MacConkey Agar, Hektoen Enteric Agar, XLD Agar, etc... Salmonella species not fermenting lactose
form colorless, transparent colonies, with or
Typical Composition without a black center (H2S production).
(can be adjusted to obtain optimal performance) Colonies of Shigella are colorless.
Colonies of coliform bacteria are red or
For 1 liter of medium : pink. Suspected colonies are transferred to
- Pancreatic digest of meat 5.0 g Kligler Iron Agar (BK034) or TSI Agar
- Meat extract 5.0 g (BK059) for subsequent identification.
- Lactose 10.0 g
- Bile salts 8.5 g
- Neutral red 25 mg
- Brilliant green 0.33 mg
Quality Control
- Dehydrated medium : pinkish powder, free-flowing and

homogeneous.
- Prepared medium : orange-pink agar.

®
Salmonella Typhimurium ATCC 14028 good to excellent colorless colonies with

a black center
Shigella flexneri ATCC 29903 good colorless colonies
Shigella sonnei ATCC 29930 good colorless colonies
Escherichia coli ATCC 25922 partially inhibited pink to red colonies

- Media in vials : 1 month at 2-8°C.
Packaging
Dehydrated medium :
227
Bibliography
(1) Leifson, E. 1935. New culture media based on sodium (4) Horwitz,W. 1980. Official methods of analysis of the
desoxycholate for the isolation of intestinal pathogens and for Association of Official Analytical Chemists. Washington DC.
the enumeration of colon bacilli in milk and water. J. Pathol. (5) CNEVA Bactériologie animale: Pr 116/00/BA 50/00. December
Bacteriol., 40: 581. 1996. Isolement et identification des salmonelles chez les
(2) Taylor, W.I., and Harris, B. 1965. Isolation of Shigellae. II. mammifères.
Comparison of plating media and enrichment broths. Am. J. (6) NF V 08-052. May 1997. Microbiology of food and animal
Clin. Pathol., 44 (4): 476. feeding stuffs. Detection of Salmonella. Routine method.
(3) Isenberg, H.D., Kominos, S., and Siegel, M. 1969. Isolation of (7) NF EN 12824. February 1998. Microbiology of food and animal
Salmonellae and Shigellae from an artificial mixture of fecal feeding stuffs. Horizontal method for the detection of Salmonella.
bacteria. Appl. Microbiol., 18 (4): 656.
Sugar-Free Agar (SFA)

Sugar-Free Agar for the enumeration of contaminants in dairy
products is a carbohydrate-free medium used for the detection and Reconstitution : 34.0 g / L.
enumeration of microorganisms that do not undergo specific Autonomy (500 g) : 980 plates.
fermentation processes during the preparation of dairy products.
pH (25°C) : 7.5 ± 0.2.
The result of the enumeration furnishes an indication of the level of
contamination of the sample tested. Supplement required : None.
Principles
Preparation
- The presence of the relatively non-nutritive substances supplied by
peptones (Tryptone and pancreatic digest of gelatin), as well as the
absence of fermentable carbohydrates, leads to a substantial
reduction in the growth of specific microorganisms, in particular
water.
lactobacilli. This favors the growth of microorganisms that alter
the quality of the product.
- Sodium chloride maintains the osmotic balance.
Typical Composition
15 minutes.
- Tryptone 7.5 g
- Melt the medium (if prepared in advance).
- Transfer 1 mL of inoculum or of extraction
liquid from the sample to test and its
tenfold dilutions to sterile Petri dishes.
- Pour 10 to 15 mL of medium.
Quality Control
- Incubate for 72 hours at 30°C.
- Dehydrated medium : off-white powder, free-flowing and
- Retain only plates containing less than
homogeneous.
150 colonies.
- Prepared medium : very light amber agar.
Results
Small “pinhead” colonies are not counted,
Escherichia coli ATCC® 25922 good to excellent since they represent lactic acid flora.
Staphylococcus aureus ATCC 25923 good to excellent In case of doubt, carry out a catalase test
Aspergillus niger ATCC 16404 good with a significant number of colonies.
The production of gas bubbles confirms
Lactobacillus casei subsp. rhamnosus ATCC 7469 very slow
the presence of a contaminant.
Lactococcus lactis subsp. lactis ATCC 11454 very slow
228

Packaging
Dehydrated media :
Bibliography
(1) Circulaire (French) DQ/SVHA/N86/N° 8163 du 25 Novembre (3) ISO/DIS 13559. 2000. Butter, fermented milks and fresh
1986: Méthodes d'analyse des beurres. cheese. Enumeration of contaminating microorganisms.
(2) FIL-IDF provisional 153: 1991. Butter, fermented milks and Colony count technique at 30°C.
fresh cheese. Enumeration of contaminating microorganisms.
Colony count technique at 30°C.
TBX Agar
Intended Use
TBX Agar is a selective medium for the enumeration of

-D-glucuronidase-positive Escherichia coli in food products.
The result is obtained directly by counting characteristic colonies after
only 24 hours of incubation and no confirmation step is required.
History
In 1949, Buehler et al. were the first to report the presence of a

-D-glucuronidase in Escherichia coli. Since then, most studies have
shown that 94 to 97% of Escherichia coli of human or environmental
origin possess -D-glucuronidase. This enzyme could also be detected
in Citrobacter, Enterobacter, Klebsiella, Salmonella, Shigella and
Yersinia, but its presence involves only a limited number of strains of
each of these species. -D-glucuronidase can thus be considered to
be a valid marker for the detection of Escherichia coli in food Escherichia coli
products and in water samples. In 1990, Restaino successfully used a
new chromogenic substrate : BCIG. When incorporated into Tergitol Product Profile
agar, it enables Escherichia coli in meat products to be counted in
Target : Escherichia coli.
24 hours. In contrast, TBX agar has been specifically formulated
without using tergitol as a selective agent. In its place are Reconstitution : 30.6 g / L.
substituted bile salts which confer similar selective properties. Autonomy (500 g) : 1090 plates.
pH (25°C) : 7.2 ± 0.2.
Principles
- Bile salts inhibit the growth of Gram-positive bacteria and favors Sterilization : 121°C / 15 minutes.
the recovery of Escherichia coli.
- BCIG (5-bromo-4-chloro-3-indolyl--D-glucuronic acid) is a
Preparation
chromogenic substrate. Most Escherichia coli strains possess
a -D-glucuronidase that cleaves BCIG, causing the colonies to
become blue according to the following reaction mechanism :
water.
15 minutes.
229
- It is important to note that not all Escherichia coli possess Instructions for Use
-D-glucuronidase, in particular enterohemorrhagic serotype
O157:H7, which forms white colonies. - Cool the medium to 47°C.
- Incubation at 44°C and imperatively limited to 24 hours enables - Transfer 1 mL of the product to analyze or
the growth of most contaminating bacteria to be reduced. its tenfold serial dilutions to sterile Petri
dishes.
Typical Composition - Pour in 15 mL of medium.
(may be adjusted to obtain optimal performances) - Mix thoroughly.
- Bile salts n° 3 1.5 g
- BCIG 75 mg Count characteristic blue colonies in plates
- Bacteriological agar 9.0 g containing not more than 150 colonies.
pH of ready to use medium at 25°C : 7.2 ± 0.2.
Quality Control

homogeneous.
- Prepared medium : whitish agar.

Escherichia coli ATCC® 25922 good to excellent blue colonies
Escherichia coli ATCC 8739 good to excellent blue colonies
Escherichia coli ATCC 43895 good to excellent white colonies

- Media in vials : 1 month at 2-8°C, shielded from light.
Packaging
Dehydrated medium :
230
Bibliography
(1) Buehler, H.J., Katzman, P.A., and Doisey, E.A. 1949. Bacterial (4) prNF EN ISO 16649-1. 1999. Microbiology of food and animal
glucuronidase. Federation Proceedings., 8: 189. feeding stuffs. Horizontal method for the enumeration of
(2) Adams, M.R., Grubb, S.M., Hamer, A., and Clifford, M.N. 1990. presumptive Escherichia coli. Part 1: Colony-count technique at
Colorimetric enumeration of Escherichia coli based on 44°C using membranes and 5-bromo-4-chloro-3-indolyl--D-
-Glucuronidase Activity. App. and Envir. Microb., 56 glucuronic acid.
(7): 2021-2024. (5) prNF EN ISO 16649-2. 1999. Microbiology of food and animal
(3) Restaino, L., Frampton, E.W., and Lyon, R.H. 1990. Use of the feeding stuffs. Horizontal method for the enumeration of
Chromogenic Substrate 5-bromo-4-chloro-3 Indolyl--D- presumptive Escherichia coli. Part 2: Colony-count technique at
glucuronide (X-GLUC) for Enumerating Escherichia coli in 24 h 44°C using 5-bromo-4-chloro-3-indolyl--D-glucuronic acid.
from ground beef. Jour. of Food Protect., 53 (6): 508-510.
TCBS Agar

Thiosulfate-Citrate-Bile-Sucrose Agar is a selective medium for the Target : Enterophatologic Vibrio,
Vibrio cholerae.
isolation of Vibrio cholerae and other enteropathologic Vibrio (in
particular Vibrio parahaemolyticus) in fish, seafood and biological Reconstitution : 88.1 g / L.
samples. Autonomy (500 g) : 378 plates.
pH (25°C) : 8.6 ± 0.2.
History
The original formula developed by Nakanishi was subsequently Sterilization : Heat to boiling.
modified by Kobayashy et al. for the selective isolation of Do not autoclave.
pathogenic Vibrio species.
Preparation
Principles
- The high concentrations of thiosulfate and sodium citrate, as well (BK040) in 1 liter of distilled or deionized
as the alkalinity of the medium, considerably inhibit the growth of water.
enterobacteria. - Slowly bring to boiling, stirring until
- Ox bile and sodium cholate slow the growth of enterococci and complete dissolution.
inhibit the development of Gram-positive bacteria. - Do not autoclave.
- The acidification of the medium resulting from the fermentation
of sucrose by Vibrio makes bromthymol blue turn yellow. Instructions for Use
- Using thiosulfate as a sulfur source, the production of hydrogen
sulfide is visualized in the presence of ferric citrate. All Vibrio are - Cool and maintain the medium at 47°C.
H2S-negative. - Pour into sterile Petri dishes.
Typical Composition of the complete medium - Dry in an incubator with the covers
(can be adjusted to obtain optimal performance) partially removed.
- Inoculate by spreading on the surface
For 1 liter of medium : of the medium.
- Polypeptone 10.0 g - Incubate at 37°C for 18 to 24 hours
- Yeast extract 5.0 g in darkness.
- Sucrose 20.0 g
- Sodium cholate 3.0 g
- Thymol blue 40 mg
231
- Dehydrated medium : greenish-beige powder, free-flowing and The colonies have the following appearance :
homogeneous.
- Prepared medium : dark green agar. - Flat yellow colonies, 2-3 mm in diameter :
- Typical culture response after 24 hours of incubation at 37°C : Vibrio cholerae.
- Large yellow colonies :
Microorganisms Growth Characteristics Vibrio alginolyticus.
Vibrio cholerae ATCC® 9459 good yellow colonies - Yellow or translucent colonies :
Vibrio parahaemolyticus ATCC 17802 good colorless colonies Vibrio fluvialis, Vibrio vulnificus.
with blue-green center - Colorless colonies with a green center :
Escherichia coli ATCC 25922 inhibited Vibrio parahaemolyticus.
Enterococcus faecalis ATCC 29212 partially inhibited - Blue colonies :
Pseudomonas aeruginosa ATCC 10145 partially inhibited Pseudomonas, Aeromonas.
- Tiny transparent colonies :
Storage / Shelf Life Enterobacteria or others.
Additional tests must be carried out to

identify doubtful colonies.
Packaging
Dehydrated medium :
Bibliography
(1) Nakanishi, Y. 1963. An isolation agar medium for cholera and (5) NF V 45-111: July 1985. Fish products. Detection of Vibrio
enteropathogenic halophilic vibrios. Modern media, 9: 246. parahaemolyticus in shellfish breeding waters and in living
(2) Kobayashi, T., Enomoto, S., Sakazaki, R., and Kuwahara, S. marine shellfish.
1963. A new selective isolation medium for pathogenic vibrios (6) Circulaire (French) DGAL/SUHA/C88/N° 8003 du 28 avril 1988.
: T.C.B.S. Agar. Jap. Journ. Bact., 18: 387-391. Méthodes d’analyses bactériologiques pour le contrôle des
(3) Kampelmacher, E.H., Mossel, D.A.A., van Noorle-Jansen, and coquillages.
Vicentie, H. 1970. A survey on the occurence of Vibrio (7) NF ISO 8914: May 1991. Microbiology. General guidance for the
parahaemolyticus in the Netherlands. J. Hyg. Camp., 68: 189-196. detection of Vibrio parahaemolyticus.
(4) Lennette, E.H., Balows, A., Hausler, Jr, W.J., and Shadomy,
H.J. 1985. Manual of Clinical Microbiology. 4th Ed. American
Society for Microbiology. Washington, D.C.
232
Tergitol 7 Agar

Tergitol 7 Agar is a selective medium used for the enumeration of Target : Coliforms, Escherichia coli.
coliform bacteria and for the early diagnosis of Escherichia coli in Reconstitution : 33.1 g / L.
drinking water and food products. Autonomy (500 g) : 1678 plates
(9 mL per plate).
History
pH (25°C) : 6.9 ± 0.2.
In 1946, Pollard demonstrated the bactericidal action of Tergitol 7 Supplement required : TTC Supplement
on Gram-positive bacteria. Chapman subsequently developed a 12.5 mg.
medium on which the enumeration of coliform bacteria was about Sterilization : 121°C / 15 minutes.
30% higher than that in other selective media. The formula was
then modified by the author by the incorporation of TTC
(triphenyltetrazolium chloride) which is useful for the rapid Preparation
recognition of Escherichia coli which produce yellow colonies
surrounded by a yellow halo, while other Gram-negative bacteria - Suspend 33.1 g of dehydrated medium
(including Proteus and Pseudomonas) form dark red colonies. (BK038) in 1 liter of distilled or deionized
water.
- Tergitol 7 inhibits Gram-positive bacteria, limits invasion by Proteus - Dispense 100 mL in vials.
and favors the recovery of coliform bacteria. - Sterilize in an autoclave at 121°C for
- Lactose-positive bacteria form yellow colonies surrounded by a 15 minutes.
yellow halo, since acidification makes the pH indicator,
bromthymol blue, turn color. Lactose-negative bacteria form Instructions for Use
colonies surrounded by a blue halo.
- TTC is reduced to insoluble red formazan by most bacteria except - Cool and maintain the medium at 47°C.
Escherichia coli which forms yellow to yellow-orange colonies - Aseptically add 1.6 mL of reconstituted
surrounded by a yellow halo. TTC 12.5 mg Supplement (BS026) per vial.
Typical Composition of the base medium - Pour into sterile Petri dishes (the agar
(can be adjusted to obtain optimal performance) layer should be 5 mm thick).
For 1 liter of medium : - Under sterile conditions, filter on the
- Pancreatic digest of meat 5.0 g appropriate membrane a determined
- Yeast extract 3.0 g volume of the sample to test.
- Lactose 10.0 g - Place the membrane on the surface of the
- Tergitol 7 0.1 g agar, filtered side up, assuring the proper
- Bromthymol blue 25 mg contact with the agar.
- Bacteriological agar 15.0 g - Incubate at 37°C for 24 hours for coliform
bacteria and at 44°C for heat-tolerant
pH of the ready-to-use medium at 25°C : 6.9 ± 0.2. coliform species.
Results
Quality Control
The bacterial strains have the following
- Dehydrated base medium : cream-white powder, free-flowing
characteristics :
and homogeneous.
- Yellow to orange-yellow colonies
- Prepared (complete) medium : green agar.
surrounded by a yellow halo :
Escherichia coli.
- Dark red colonies, surrounded by a
yellow halo : other coliforms.
Escherichia coli ATCC® 25922 good yellow orange colonies - Dark red colonies with a bluish halo :
Enterobacter aerogenes ATCC 13048 good red colonies other lactose-negative bacteria.
Staphylococcus aureus ATCC 25923 inhibited In order to identify coliform bacteria,
microscopic examinations and transfer to
specific media must be carried out for
confirmation.
233

TTC 12,5 mg Supplement :
- Store between 2 and 8°C, shielded from light.
Packaging
Dehydrated base medium (without TTC) :

TTC 12,5 mg Supplement :
Bibliography
(1) Pollard, A.L. 1946. A useful selective bactericidal property of (4) Mossel, D.A.A. 1962. An ecological investigation on the
Tergitol 7. Science, 103: 758-759. usefulness of two specific modifications of Eijkman's test as an
(2) Chapman, G.H. 1947. A superior culture medium for the element of the methods for the detecting of faecal
enumeration and differentiation of coliforms. J. Bacteriol., contamination of foods. J. Appl. Bact., 25: 20-29.
53: 504. (5) Rodier, J. 1984. L'analyse de l'eau. Dénombrement des
(3) Chapman, G.H. 1951. A culture medium for detecting and coliformes fecaux, et Escherichia coli présumés. Méthode de
confirming Escherichia coli in ten hours. Am. J. Publ. Health, dénombrement par filtration sur membrane. Dunod 7ème Ed.,
41: 1381. 798-803.
Tergitol 7 Agar (acc. to NF EN ISO)
Intended Use
Tergitol 7 Agar (ISO) is used for the detection and enumeration of

coliform and heat-tolerant coliform bacteria in drinking water,
swimming water and surface water with the membrane filtration
method.
History
In 1946, Pollard showed the bactericidal action of Tergitol 7 against

Gram-positive bacteria. The medium was then developed by
Chapman, whose enumeration of coliform bacteria were about 30%
higher than those done on other selective media. The formula was
then modified by the same author by adding TTC
(triphenyltetrazolium chloride), which was found to be useful for
the rapid recognition of Escherichia coli and Enterobacter
aerogenes, which produce yellow colonies surrounded by a yellow Escherichia coli Salmonella spp.
halo, while other Gram-negative bacteria (including Proteus and
Pseudomonas) give dark red colonies. This medium is recommended
by the NF EN ISO 9308-I for the detection and enumeration of Product Profile
Escherichia coli and coliforms in water samples, using the membrane Target : Coliforms, Thermotolerant coliforms.
filtration method.
Autonomy (500 g) : 1087 plates
(9 mL per plate).
pH (25°C) : 7.2 ± 0.2.
Supplement required : TTC Supplement 12.5 mg.
234
- Tergitol 7 inhibits Gram-positive bacteria, limits invasion by Proteus - Suspend 51.1 g of dehydrated medium
and favors recovery of coliform bacteria. (BK123) in 1 liter of distilled or deionized
- Coliform bacteria form colonies that are yellow or orange inside a water.
yellow halo. The halo results from the acidification of lactose - Slowly bring to boiling, stirring slowly
under the membrane filter in the presence of a colored indicator, until complete dissolution.
bromthymol blue. - Dispense 100 mL in flasks.
- Other strains form colonies whose red color is due to the reduction - Sterilize in an autoclave at 121°C for
of TTC to insoluble formazan. 15 minutes.
- Bacteria that do not ferment lactose form colonies surrounded by
a blue halo. Instructions for Use
Typical Composition of the complete media - Cool and maintain the medium at 47°C.
(can be adjusted to obtain optimal performance) - Aseptically add 1 mL of reconstituted TTC
12.5 mg Supplement (BS026) per vial.
For 1 liter of medium : - Homogenize thoroughly.
- Pancreatic digest of meat 10.0 g - Pour into sterile Petri dishes (the agar
- Meat extract 5.0 g layer should be 5 mm thick).
- Yeast extract 6.0 g - Let solidify on a cold surface.
- Lactose 20.0 g - Aseptically filter a known volume of the
- Tergitol 7 0.1 g sample to test through a membrane.
- Bromthymol blue 50 mg - To the surface of plates prepared as above,
- 2,3,5 triphenyltetrazolium chloride (TTC) 25 mg or using ready-to-use plates (BM024)
- Bacteriological agar 10.0 g brought to room temperature, deposit the
membrane on the surface of the agar,
pH of the ready to use medium at 25°C : 7.2 ± 0.2. filtered side up and making sure that the
membrane and agar are in close contact.
Quality Control - Incubate at 37°C for 21 and 44 hours (if
necessary).
- Dehydrated medium : beige to greenish powder, free-flowing
and homogeneous. Results
- Prepared medium (complete) : blue-green agar.
- Typical culture response after 21 hours of incubation at 37°C : - Examine the membranes and consider as
typical all lactose-positive bacteria,
Microorganisms Growth Characteristics regardless of colony size, if a
Escherichia coli ATCC® 25922 good yellow-orange corresponding yellow halo in the medium
Enterobacter aerogenes ATCC 13048 good yellow-orange under the membrane is present.
Salmonella Typhimurium ATCC 14028 good red - Count as coliforms all characteristic
colonies that are oxidase-negative.
Shigella flexneri ATCC 29903 good red
- All colonies having a negative oxidase
Staphylococcus aureus ATCC 25923 inhibited reaction, but indole positive, should be
considered as Escherichia coli.

Ready-to-use media in plates,
TTC 12.5 mg Supplement :
Packaging
Ready-to-use (complete) media :

- 10 plates (55 mm) : BM02408
Dehydrated base medium (without TTC) :
TTC 12.5 mg Supplement :
235
Bibliography
(1) Pollard, A.L. 1946. A useful selective bactericidal property of (5) Rodier, J. 1984. L’analyse de l’eau. Dénombrement des
Tergitol 7. Science, 103: 758-759. coliformes, coliformes fécaux, et Escherichia coli présumés.
(2) Chapman, G.H. 1947. A superior culture medium for the Méthode de dénombrement par filtration sur membrane. Dunod
enumeration and differentiation of coliforms. J. Bacteriol., 53: 504. 7ème Ed., 798-803.
(3) Chapman, G.H. 1951. A culture medium for detecting and (6) NF EN ISO 9308-1. Sept. 2000. Water quality - Detection and
confirming Escherichia coli in ten hours. Am. J. Publ. Health, enumeration of Escherichia coli and coliform bacteria. Part 1 :
41: 1381. Membrane filtration method.
(4) Mossel, D.A.A. 1962. An ecological investigation on the
usefulness of two specific modifications of Eijkman’s test as an
element of the methods for the detecting of faecal
contamination of foods. J. Appl. Bact., 25: 20-29.
Tergitol BCIG Agar
Intended Use
Tergitol BCIG Agar is a selective medium for the enumeration of

-D-glucuronidase-positive Escherichia coli in food products. The
result is obtained directly by counting characteristic colonies after
only 24 hours of incubation and no confirmation step is required.
History
Buehler et al., in 1949, were the first to report the presence

of a -D-glucuronidase in Escherichia coli. Since then, most studies
have shown that 94 to 97% of Escherichia coli of human or
environmental origin possess -D-glucuronidase. This enzyme could
also be detected in Citrobacter, Enterobacter, Klebsiella, Salmonella,
Shigella and Yersinia, but its presence involves only a limited
number of strains of each of these species.
-D-glucuronidase can thus be considered to be a valid marker for the
detection of Escherichia coli in food products and in water samples. In Escherichia coli
1990, Restaino successfully used a new chromogenic substrate : BCIG.
When incorporated in Tergitol Agar, it enables Escherichia coli in
meat products to be counted in 24 hours. Product Profile
Target : Escherichia coli.
- Tergitol 7 inhibits the growth of Gram-positive bacteria, limits Autonomy (500 g) : 1634 plates.
invasion by Proteus and favors the recovery of Escherichia coli. pH (25°C) : 7.2 ± 0.2.
BCIG (5-bromo-4-chloro-3-indolyl--D-glucuronic acid) is a
chromogenic substrate. Most Escherichia coli strains possess Supplement required : None.
a -D-glucuronidase that cleaves BCIG, causing the colonies to Sterilization : 121°C / 15 minutes.
become blue according to the following reaction mechanism :
Preparation

water.
15 minutes.
It is important to note that not all Escherichia coli possess

-D-glucuronidase, in particular enterohemorrhagic serotype O157:H7,
which forms white colonies.
236
- Incubation at 44°C and imperatively limited to 24 hours enables Instructions for Use
the growth of most contaminating bacteria to be reduced.
- With the ready-to-use medium BM025 (or if
Typical Composition the medium has been prepared in advance
(can be adjusted to obtain optimal performance) from the dehydrated powder, as above)
melt the medium for the minimum amount
For 1 liter of medium : of time necessary to achieve total
- Peptic digest of meat 5.0 g liquefaction.
- Yeast extract 3.0 g - Cool the medium to 47°C.
- Dipotassium phosphate 0.3 g - Transfer 1 mL of the product to analyze or
- Tergitol 7 95 mg its tenfold serial dilutions to sterile Petri
- BCIG 50 mg dishes.
- Bacteriological agar 12.0 g - Pour in 15 mL of medium.
- Mix thoroughly.
Quality Control
Results
Count characteristic blue colonies in plates
homogeneous.
- Prepared medium : whitish agar. containing not more than 150 colonies.

Escherichia coli ATCC® 25922 good to excellent blue colonies
Escherichia coli ATCC 8739 good to excellent blue colonies
Escherichia coli ATCC 43895 good to excellent white colonies

- Media in tubes or vials : 1 month at 2-8°C, shielded from light.
Packaging
- 10 x 100 mL vials : BM02508
Dehydrated medium :
Bibliography
(1) Buehler, H.J., Katzman, P.A., and Doisey, E.A. 1949. Bacterial (3) Restaino, L., Frampton, E.W., and Lyon, R.H. 1990. Use of the
glucuronidase. Federation Proceedings., 8: 189. Chromogenic Substrate 5-bromo-4-chloro-3 Indolyl--D-
(2) Adams, M.R., Grubb, S.M., Hamer, A., and Clifford, M.N. 1990. glucuronide (X-GLUC) for Enumerating Escherichia coli in 24 h
Colorimetric enumeration of Escherichia coli based from ground beef. Jour. of Food Protect., 53 (6): 508-510.
on -Glucuronidase Activity. App. and Envir. Microb., (4) NF V 08-053: December 1993. Food microbiology. Enumeration
56 (7): 2021-2024. of -glucuronidase-positive Escherichia coli by colony count
technique at 44°C. Routine method.
237
Tetrathionate Broth (USP)

Tetrathionate Broth is used for the selective enrichment of
salmonellae from pathological products, pharmaceutical, dairy and Reconstitution : 35.0 g / L.
other food products. Autonomy (500 g) : 142 vials
(100 mL by vial).
History
pH (25°C) : 7.4 ± 0.2.
This medium was described in 1923 by Müller for the inhibition Supplement required : Iodine-iodide
of coliform bacteria while enabling typhoid and paratyphoid bacilli to solution, 0.1 % Brillant green solution.
develop. Kauffmann modified the formula and obtained a larger Sterilization : Heat to boiling.
number of positive results with this method of prior enrichment in Do not autoclave.
comparison to the method of direct isolation on selective media in
Petri dishes. Preparation
Principles - Suspend 35.0 g of dehydrated base

- Bile salts and brilliant green (added just before use) inhibit deionized water.
primarily the development of Gram-positive bacteria. - Slowly bring to boiling with stirring.
- Tetrathionate production results from the action of iodine-iodide - Continue boiling for 2 minutes.
solution on sodium thiosulfate. This compound inhibits the growth - Do not autoclave.
of coliform bacteria and of most intestinal flora. The development
of Proteus is unaffected. Instructions for Use
- Calcium carbonate neutralizes the sulfuric acid produced when
tetrathionate is reduced. The resultant effect is to maintain the pH. - Cool the medium to 25°C.
- Dissolve 6 g of iodine in 20 mL of a
Typical Composition of the base medium solution containing 5 g of potassium
(without iodide or brilliant green) iodide in a sterile flask.
(can be adjusted to obtain optimal performance) - Add the iodine-iodide solution to the
medium.
For 1 liter of medium : - Also add 10 mL of a sterile aqueous 0.1%
- Tryptone 2.5 g solution of brilliant green.
- Peptic digest of meat 2.5 g - Mix well.
- Bile salts 1.0 g - Do not heat this medium once prepared.
- Calcium carbonate 10.0 g - Aseptically dispense 100 mL in sterile vials.
- Sodium thiosulfate 19.0 g - Immediately transfer 10 mL of inoculum
from the preenrichment medium used
Quality Control (Buffered Peptone Water : BK018, BK131,
BM010 or Lactose Broth BK082) to the
- Dehydrated medium : white powder, free-flowing and medium.
homogeneous. - Verify that the pH of the medium is 7.4 ± 0.2.
- Incubate for a maximum of 24 hours at 37,
- Prepared complete medium : bluish-white suspension, opaque,
42 or 43°C, depending on the analytical
with substantial precipitate when left standing.
protocol applied.
- Typical culture response after enrichment for 18 hours at 37°C,
- Isolate on several selective media, by
streaking with a loop.
- Using well formed colonies, inoculate
Microorganisms Growth Characteristics Kligler Iron Agar (BK034) or TSI Agar
Salmonella Typhimurium ATCC® 14028 good colorless colonies (BK059) which are the starting points
Salmonella Enteritidis ATCC 13076 good colorless colonies for identification.
Escherichia coli ATCC 25922 limited red colonies

- Base media in tubes or vials : 1 month at 2-8°C, shielded from light.
- Complete media in tubes or vials : use immediately after adding the
iodine-iodide and brilliant green solutions.
238
Packaging

(without iodine-iodide solution and brilliant green) :
Bibliography
(1) Müller, L. 1923. Un nouveau milieu d’enrichissement pour la (3) United States Pharmacopoeia 24. 2000. Microbial Limit Tests,
recherche du bacille typhique et des paratyphiques. 1814-1818.
C.R. Soc. Biol. (Paris), 89: 434.
(2) Kauffmann, F. 1930. Ein kombiniertes Anreichrungsverfahren
für Typhus-und-Paratyphusbazillen. Zentralb. Bakteriol.
Parasitenkd. Infectionskr. Hyg. Abt I Orig., 113: 148.
Thioglycollate Medium with Resazurin

Thioglycollate Medium with Resazurin is used for sterility tests of Target : Sterility testing,
Clostridium perfringens.
biological products and for the culture of aerobic, anaerobic and
microaerophilic bacteria. The formulation of the medium complies Reconstitution : 29.7 g / L.
with the requirements of the United States Pharmacopoeia and the Autonomy (500 g) : 168 vials
AOAC for the bacteriological analysis of antibiotics and the (100 mL by vial).
determination of the sporicidal effect of disinfectants.
pH (25°C) : 7.1 ± 0.2.

Brewer demonstrated the value of this medium, containing a small
quantity of agar and of a reducing substance, for the culture of Preparation
anaerobic bacteria in the presence of sodium thioglycollate.
Nungester, Hood and Warren then showed that sodium - Suspend 29.7 g of dehydrated medium
thioglycollate neutralized the inhibitory effect of mercuric (BK017) in 1 liter of distilled or deionized
compounds present in the samples analyzed. Malin and Flynn water.
observed that in the presence of carbohydrates, thioglycollate was - Slowly bring to boiling, stirring until
slightly inhibitory for several species. complete dissolution.
15 minutes.
- Thioglycollate Medium with Resazurin formulated with pancreatic
digest of casein, yeast extract, cystine and glucose assures the
growth of a large variety of aerobic and anaerobic bacteria.
- Sodium thioglycollate at the concentration of 0.05% decreases the - Inoculate with the sample to analyze
redox potential without having a toxic effect. It also neutralizes into tubes or vials prepared as above,
the antibacterial power of mercuric derivatives used as or the ready-to-use media BM031 (tubes)
preservatives in biological products. or BM007 (vials).
- The presence of agar favors the development of anaerobic - Incubate at least 2 weeks at the desired
bacteria by stabilizing the medium against convection currents in temperature.
such a way as to maintain anaerobiosis in the lower part of the
recipients.
- Resazurin, less toxic than methylene blue, is used as a redox
indicator : it is colorless in a reducing medium and becomes pink in
an oxidized medium.
239
Typical Composition

- Tryptone 15.00 g
- Glucose 5.50 g
- Sodium thioglycollate 0.50 g
- L-cystine 0.50 g
- Resazurin 1 mg
Quality Control

homogeneous.
- Prepared medium : semi-solid medium, slightly opalescent, light
amber with a pink ring on the surface.
Bacillus subtilis ATCC® 6633 good to excellent
Micrococcus luteus ATCC 9341 good to excellent
Bacteroides vulgatus ATCC 8482 good
Clostridium sporogenes ATCC 11437 good to excellent

- Media in tubes or vials : 6 months at 2-8°C, shielded from light.
- If after conservation, the media presents a rose coloration (a sign
of oxidation) greater than 1/3 of the tube measuring from the
surface downward, then restore anaerobiosis by heating at 100°C
for 10 minutes. Do not carry out this operation more than once.
Ready-to-use media in tubes or vials :
Packaging
- 50 x 14 mL tubes : BM03108
- 10 x 100 mL vials : BM00708
Dehydrated medium :
240
Bibliography
(1) Brewer, J.H. 1940. Clear liquid medium for the “aerobe” (5) XP V 08-056. April 1994. Food microbiology. Enumeration of
cultivation of anaerobes. JAMA, 115: 598. Clostridium perfringens by colony count technique at 37°C.
(2) Journal Officiel (French) du 25 octobre 1978. Essai de stérilité, Routine method.
8233-8237. (6) ISO 7937: 1997. Microbiology of food and animal feeding
(3) NF V 08-019. December 1985. Microbiology of food products. stuffs. Horizontal method for enumeration of Clostridium
General guidance for the enumeration of Clostridium perfringens. Colony count technique.
perfringens. Colony count technique. (7) Pharmacopée européenne. 1997. Méthodes biologique.
(4) MacFaddin, J.F. 1985. Media for Isolation. Cultivation. Stérilité. Milieux de culture proposés, 72-76.
Identification. Maintenance of Medical Bacteria. Vol 1. (8) United States Pharmacopoeia 24. 2000. Sterility Tests,
Williams and Wilkins. Baltimore, 755-762. 1818-1823.
Todd Hewitt Broth
Intended Use
Product Profile
Todd Hewitt Broth is used to culture hemolytic streptococci for Target : Hemolytic streptococci.
serological typing. Reconstitution : 30.0 g / L.
History
pH (25°C) : 7.7 ± 0.2.
The medium was originally described by Todd and Hewitt for the Supplement required : None.
production of hemolysin from streptococci. Updycke and Nickle Sterilization : 121°C / 15 minutes.
subsequently modified the medium to obtain satisfying growth of
beta-hemolytic streptococci for serological typing based on the Preparation
production of the type M-specific protein, since proteinase is not
produced in this medium. - Suspend 30.0 g of dehydrated medium
Principles water.
- The high nutrient power of the medium is due to its rich - Dispense in tubes.
composition in peptones, glucose and salts. - Sterilize in an autoclave at 121°C
- Glucose favors hemolysin production. for 15 minutes.
- Sodium phosphate and sodium carbonate act as buffers to
neutralize the acidity produced by the fermentation of glucose, Instructions for Use
which protects hemolysin from the inactivation occurring in acid
medium. - Incubate samples in tightly closed tubes at
37°C in a carbon dioxide-enriched
Typical Composition atmosphere for 24 hours.
- Isolate on blood agar.
(can be adjusted to obtain optimal performance) - Pure cultures can be prepared on the same
medium to carry out serological typing.
- Beef heart infusion 3.1 g
- Glucose 2.0 g
- Sodium carbonate 2.5 g
241
Quality Control

homogeneous.
Streptococcus pneumoniae ATCC ®

- Media in tubes : 24 hours at 2-8°C.
Packaging
Dehydrated medium :
Bibliography
(1) Todd, E.W., and Hewitt, L.F. 1932. A new culture medium for (3) Fenton, L.J., and Harper, M.H. 1979. Evaluation of colistin and
the production of antigenic streptococcal haemolysin. J. nalidixic acid in Todd Hewitt broth for selective isolation of
Pathol. Bacteriol., 35: 973. group B streptococci. J. Clin. Microbiol., 9 (2): 167.
(2) Updyke, E.L., and Nickle, M.I. 1954. A dehydrated medium for (4) Lennette, E.H., Balows, A., Hausler, Jr., W.J., and Shadomy,
the preparation of type specific extracts of group A H.J. 1985. Manual of Clinical Microbiology. 4th Ed. American
streptococci. Appl. Microbiol., 2: 117. Society for Microbiology. Washington. D.C.
Tryptone-Salt Broth
Intended Use
Product Profile
Tryptone-Salt Broth is a diluent used to prepare stock solutions of Target : Dilutions, Suspensions.
powdered milk and concentrates, of dairy products and other food Reconstitution : 9.5 g / L.
products for microbiological analysis. It is also used to prepare Autonomy (500 g) : 5847 tubes
tenfold dilutions. (9 mL per tube).
pH (25°C) : 7.0 ± 0.2.
Principles
- Tryptone assures the resuscitation of microorganisms having Sterilization : 121°C / 15 minutes.
undergone sublethal treatments.
- Sodium chloride provides an isotonic solution.
Preparation
Typical Composition
(BK014) in 1 liter of distilled or
deionized water.
- Tryptone 1.0 g
for 15 minutes.
242
- Dehydrated medium : white powder, free-flowing and - Cool the medium to 25°C.
homogeneous.
- Prepared medium : colorless solution, limpid. Preparation of stock solutions :
- Aseptically add 10 or 25 g of the product
to analyze to a tared flask containing 90
Microorganisms Growth or 225 mL of medium for the preparation
Escherichia coli ATCC® 25922 good of 1:10 dilutions.
Staphylococcus aureus ATCC 25923 good - Mix well.
Salmonella Typhimurium ATCC 14028 good
Preparation of 1:10 dilutions :
Storage / Shelf Life - Add 1 mL of stock solution to a tube
containing 9 mL of medium prepared as
Dehydrated medium : 2-30°C. above or to ready-to-use media BM008.
- The expiration date is indicated on the label. - Mix well.
Prepared medium (benchmark value) : - Repeat the operation until the desired
- Media in tubes or vials : 6 months at 2-25°C. dilution is obtained.
Ready-to-use media in tubes :
Packaging
- 50 x 9 mL tubes : BM00808
Dehydrated medium :
Bibliography
(1) Journal Officiel (French) du 19 janvier 1980. Critères (4) FIL-IDF 122C: 1996. Milk and milk products. Preparation
microbiologiques auxquels doivent satisfaire certaines denrées of samples and dilutions for microbiological examination.
animales ou d’origine animale. Méthodes générales d'analyse (5) NF EN ISO 6887-1: September 1999. Microbiology of food and
bactériologique (arrêté du 21 décembre 1979 modifié). animal feeding stuffs. Preparation of test samples, initial
(2) NF V 04-015: February 1984. Dried milk and sweetened suspension and decimal dilutions for microbiological
condensed milk. Microbiology. examination. Part 1: General rules for preparation of the initial
(3) NF V 04-016. August 1985. Milk. Enumeration of suspension and decimal dilutions.
microorganisms. Colony count technique at 30°C.
243
Tryptone-Soy Agar

Tryptone-Soy Agar is a universal nutrient medium suitable for a wide
range of uses. In light of its excellent nutritive value, it can be used Reconstitution : 40.0 g / L.
for the growth and isolation of both aerobic and anaerobic bacteria Autonomy (500 g) : 833 plates.
and to favor the development of the most fastidious microorganisms.
pH (25°C) : 7.3 ± 0.2.
Its formula is that listed in the United States Pharmacopoeia (Soybean
Casein Digest Agar Medium) for the enumeration of total bacteria. Supplement required : None.
Poured in plates or on strips, it is useful in rapid tests for examining Sterilization : 121°C / 15 minutes.
the contamination of surfaces.
Preparation
Principles
- The combination of Tryptone and papaic digest of soybean meal (BK047) in 1 liter of distilled or deionized
leads to optimal growth, due to the synergy between the protein water.
supply of casein and the carbohydrate supply of soybeans, favoring - Slowly bring to boiling, stirring until
the growth of most fastidious or non-fastidious microorganisms. complete dissolution.
- Sodium chloride maintains osmotic balance. - Dispense in tubes or flasks.
for 15 minutes.
Typical Composition
- With the ready-to-use base media BM017
or BM049 (or if the media is prepared in
For 1 liter of medium : advance from the dehydrated product),
- Tryptone 15.0 g melt the agar for the minimum amount
- Papaic digest of soybean meal 5.0 g of time necessary to achieve total
- Sodium chloride 5.0 g liquefaction.
Use in prepoured plates
Quality Control - Cool and maintain the medium at 47°C.
- Pour in sterile Petri dishes.
- Dehydrated medium : cream-white powder, free-flowing and - Let solidify on a cold surface.
homogeneous. - Dry in an incubator with the covers
- Prepared medium : amber agar. partially removed.
- Typical culture response after 48 hours of incubation at 30-35°C : - To the surface of plates prepared as above,
or to ready-to-use plates (BM050) brought
to room temperature, inoculate 0.1 mL of
Microorganisms Growth the test sample and its serial dilutions.
Escherichia coli ATCC® 25922 good to excellent - Incubate at the optimal temperature for
Staphylococcus aureus ATCC 6538 good to excellent the development of the species inoculated
for the time required to obtain luxuriant
colonies.
Use in plates for enumeration
Dehydrated medium : 2-30°C. - Transfer 1 mL of the product to analyze
- The expiration date is indicated on the label. and its tenfold dilutions to sterile Petri
Prepared medium (benchmark value) : dishes.
- Media in tubes or vials : 6 months at 2-25°C. - Pour in 10 to 15 mL of medium.
- Media in plates : 1 month at 2-8°C. - Homogenize by swirling.
Ready-to-use media in plates : - Let solidify on a cold surface.
- Store between 2-8°C, shielded from light. - Incubate according to the analysis protocol
- The expiration date is indicated on the label. applied to the product being examined.
Ready-to-use media in vials : - When reading the results, only plates
- Store between 2-25°C, shielded from light. containing between 15 and 150 colonies
- The expiration date is indicated on the label. are retained.
244
Packaging
- 20 plates (90 mm) : BM05008
- 10 x 100 mL vials : BM01708
- 10 x 200 mL vials : BM04908
Dehydrated medium :
Bibliography
(1) NF T72-151: November 1987. Water miscible, antiseptics and (3) Pharmacopée européenne. Addendum 2000. Contrôle
disinfectants used in liquid form. Determination of the microbiologique des produits non-stériles (Recherche de
bactericidal activity. Membrane filtration method. microorganismes spécifiés). Solution et milieux de culture
(2) United States Pharmacopoeia 24. 2000. Microbial Limit Tests, recommandés, 56 – 61.
1814 - 1818. (4) NF EN ISO 9308-1. Water Quality. Dectection and enumeration
of Escherichia coli and coliform bacteria. Part. 1 : membrane
filtation method.
Tryptone-Soy Agar (Blood Agar)

Tryptone-Soy Agar, used as a base to be supplemented with blood, is Target : Blood agar, Neisseria,
Hemolytic Streptococci.
prepared with selected starting materials which do not turn brown. It
was specially designed to detect beta-hemolytic reactions of Reconstitution : 40.0 g / L.
streptococci in Lancefield groups A and B, and to favor the growth of Autonomy (500 g) : 833 plates.
particularly fastidious aerobic and anaerobic bacteria. It is also used in
pH (25°C) : 7.3 ± 0.2.
the CAMP test.
Supplement required : Defibrinated sheep /
Principles horse blood, selective agents.
- The combination of casein digest and papaic digest of soybean
meal leads to optimal growth due to synergy between the protein Preparation
supply of casein and the carbohydrate supply of soybeans.
- Sodium chloride maintains osmotic balance. - Suspend 40.0 g of dehydrated medium
- Sterile defibrinated sheep blood used to enrich the medium (BK028) in 1 liter of distilled or deionized
demonstrates the beta-hemolysis characteristic of Streptococcus water.
pyogenes and Streptococcus agalactiae. - Slowly bring to boiling, stirring until
- The addition of 5% horse blood supplies factors X and V, necessary complete dissolution.
for the growth of Haemophilus influenzae. - Dispense in tubes or flasks.
- When gentamicin (aminoglycoside antibiotic) is added at 2.5 - Sterilize in an autoclave at 121°C for
mg/mL of medium containing sheep blood, the resulting medium 15 minutes.
facilitates the detection of Streptococcus pneumoniae in clinical
samples while inhibiting contaminating bacteria such as
staphylococci and Gram-negative bacilli. - Melt the medium (if prepared in advance).
Typical Composition of the base medium - Aseptically add the supplements required
(can be adjusted to obtain optimal performance) for the growth of the bacteria to be
detected (sterile defibrinated sheep or
For 1 liter of medium : horse blood, growth accelerators, selective
- Tryptone 15.0 g agents).
- Papaic digest of soybean meal 5.0 g - Pour into sterile Petri dishes.
- Bacteriological agar 15.0 g - Dry in an incubator with the covers
partially removed.
pH of the ready-to-use medium at 25°C : 7.3 ± 0.2. - Inoculate.
- Incubate at 37°C for 24 to 48 hours
aerobically or in CO2-enriched atmosphere
for Brucella and Neisseria.
245
Results
Quality Control
Group A hemolytic streptococci are small
- Dehydrated medium : cream-white powder, free-flowing and gray colonies, translucent or opaque,
homogeneous. surrounded by a zone of beta type
- Prepared medium (with 5% defibrinated sheep blood) : opaque hemolysis. Other bacteria may exhibit the
red agar. same type of hemolysis : Listeria, hemolytic
- Typical culture response after 48 hours of incubation at 37°C : staphylococci, Escherichia coli and
Pseudomonas. It is thus necessary to carry
Microorganisms Growth Type of hemolysis out a Gram stain to confirm the results.
Streptococci exhibiting the following
Streptococcus pyogenes ATCC® 19615 good beta
characteristics : Bacitracin-negative, CAMP-
Streptococcus pneumoniae ATCC 6303 good alpha positive, beta-hemolytic, are considered to
Escherichia coli ATCC 25922 good be presumptively belonging to group B. The
Enterococcus faecalis ATCC 29212 good CAMP test can be carried out with the
medium supplemented with 5% sheep
Storage / Shelf Life blood as follows :
- Inoculate a 10 hour culture of beta-
Dehydrated base medium : 2-30°C. hemolytic Staphylococcus aureus
- The expiration date is indicated on the label. ATCC® 33862 as a single median streak.
Prepared medium (benchmark value) : - Perpendicular to the initial streak, streak
- Base media in vials : 6 months at 2-8°C. a culture of the streptococcus to identify,
- Complete media in plates (with sheep blood) : 1 month at 2-8°C. approaching the first as closely as possible
(2-3 mm) without touching it.
Group B streptococci produce a heat-
Packaging resistant extracellular substance (CAMP
factor) which leads to a triangle of total
Dehydrated medium : hemolysis in the zone of incomplete
- 500 g bottle : BK028HA hemolysis of the Staphylococcus, at the
junction of the two cultures. This procedure
should be done with known collection
strains in parallel with the unknown strains
to be identified.
Group B streptococci generally present
smaller hemolytic zones than those observed
with Streptococcus pyogenes.
Pneumococci appear as flat, smooth, grayish
and sometimes mucous colonies, surrounded
by a narrow greenish zone of alpha type
hemolysis.
Staphylococci form opaque golden yellow or
white colonies, with or without beta type
hemolysis.
Listeria form small beta hemolysis zones.
Other non-pathogenic bacteria may also
grow on this non-selective medium.
Bibliography
(1) Facklam, R.R., and Carey, R.B. 1985. Streptococci and Aerococci (2) MacFaddin, J.F. 1985. Media for Isolation-Cultivation-
in Lennette, E.H., Balows, A., Hausler, Jr., W.J., and Shadomy, Identification-Maintenance of Medical Bacteria. Vol 1.
H.J. (ed). Manual of Clinical Microbiology 4th Ed., ASM Williams, and Wilkins, Baltimore, 794-806.
Washington DC, 154-175.
246
Tryptone-Soy Broth

Target : Aerobic microorganisms,
Tryptone-Soy Broth is a universal nutrient medium suitable for a wide
Staphylococcus aureus.
range of uses. In light of its excellent nutritive value, it favors the growth
of most fastidious microorganisms. Reconstitution : 30.0 g / L.
It is used in the pharmaceutical industry to satisfy sterility tests and its Autonomy (500 g) : 1666 tubes.
formula is that listed in the United States Pharmacopoeia under the
pH (25°C) : 7.3 ± 0.2.
monograph “Fluid Soybean Casein Digest Medium”. It is also suitable as
an enrichment medium for the detection of Staphylococcus aureus. Supplement required : None.
Principles
- The combination of Tryptone and papaic digest of soybean meal Preparation

leads to a synergy between the protein supply of casein and the
carbohydrate supply of soybeans. This leads to the optimal growth - Dissolve 30.0 g of dehydrated medium
of a large number of species, both fastidious or not. (BK046) in 1 liter of distilled or deionized
- Glucose is an energy source. water.
- Sodium chloride maintains osmotic balance. - Stir slowly until complete dissolution.
- Dipotassium phosphate acts as a buffer to maintain a constant pH. - Dispense in tubes or flasks.
- Tryptone 17.0 g - Media inoculated from the dehydrated
- Papaic digest of soybean meal 3.0 g base or from ready-to-use references
- Glucose 2.5 g BM030 (tubes) or BM009 (vials) are
- Dipotassium phosphate 2.5 g incubated at 30 or 37°C for 3 to 15 days.
Results
pH of the ready-to-use medium at 25°C: 7.3 ± 0.2.
Growth results in turbidity of the medium.
Quality Control Subcultures are prepared in order to purify
strains to be identified.
homogeneous.
Bacillus subtilis ATCC® 6633 good to excellent
Micrococcus luteus ATCC 9341 good to excellent

Ready-to-use media in vials or tubes :
247
Packaging
- 50 x 10 mL tubes : BM03008
- 10 x 100 mL vials : BM00908
Dehydrated medium :
Bibliography
(1) Ministère de l’Agriculture (French). Cosmétologie. Commission (4) United States Pharmacopeia 24. 2000. Sterility Tests,
XXX. Recherche de Pseudomonas aeruginosa dans les produits 1818-1823.
cosmétiques. (5) Pharmacopée européenne. Addendum 2000. Contrôle
(2) Journal Officiel (French) du 25 octobre 1978. Essai de stérilité, microbiologique des produits non obligatoirement stériles
8233-8237. (recherche de microorganismes spécifiés). Solution et milieux
(3) Pharmacopée européenne. 1997. Méthodes biologiques. de culture recommandés, 56-61.
Stérilité. Milieux de culture proposes, 72-76.
TSC Agar
Intended Use
Tryptone Sulfite Cycloserine Agar was described by Harmon for the

selective isolation and enumeration of Clostridium perfringens in
water, foods and other biological samples. The medium was
recommended for the enumeration of sulfite-reducing anaerobes
from foods of animal origin.
Principles
- Sulfur reducing microorganisms reduce the sodium sulfite to

sulfide, which with ferric citrate forms a black iron sulfide
precipitate around the colonies.
- For enumeration of Clostridium perfringens, it is recommended to
incubate the inoculated medium at 37°C and to confirm
characteristic colonies.
- Contaminating flora are inhibited by D-Cycloserine which also Clostridium perfringens
reduces the size of the black halos around the colonies.
Product Profile
Typical Composition of the complete medium Target : Clostridium perfringens.
For 1 liter of medium : Autonomy (500 g) : 595 tubes
- Tryptone 15.0 g (20 mL by tube).
- Papaic digest of soybean meal 5.0 g pH (25°C) : 7.6 ± 0.2.
- Yeast extract 5.0 g Supplement required : D-Cycloserine
- Sodium metabisulfite 1.0 g Selective Supplement.
- D-cycloserine 0.4 g Sterilization : 121°C / 15 minutes.
Preparation
- Suspend 42.0 g of dehydrated medium (BK031)
in 1 liter of distilled or deionized water.
- Dispense 20 mL in 20 x 200 mm tubes or
100 mL per flask.
15 minutes.
248
- Dehydrated medium : beige powder, free-flowing and Use in tubes

homogeneous.
- Prepared medium : amber agar. - Cool and maintain the medium at 47°C.
- Typical culture response after 24 hours of anaerobic incubation - Add 0.2 mL of reconstituted Selective
at 37°C : Supplement D-Cycloserine (BS006) to each
tube. Mix well.
Microorganisms Growth Characteristics - Heat the product to analyze in order to
Clostridium perfringens ATCC® 13124 good to excellent black colonies destroy vegetative cells and activate
Bacillus cereus ATCC 11778 inhibited spores.
- Transfer 1 mL of the inoculum and its
serial tenfold dilutions to the tubes,
avoiding the incorporation of air in the
Storage / Shelf Life medium.
- Homogenize thoroughly by inversion.
Dehydrated base medium (without D-cycloserine) : 2-30°C. - Cool in an ice water bath.
- The expiration date is indicated on the label. - Incubate at 46°C or 37°C, depending on
Prepared medium (benchmark value) : the analytical protocol applied.
- Complete medium : use immediately after preparation. NOTE
D-Cycloserine Selective Supplement, Avoid heating the tubes after inoculating.
Egg Yolk Enrichment :
- Store between 2-8°C, shielded from light. Use in Petri dishes
BASE MEDIUM + CYCLOSERINE
Packaging - Inoculated plates are incubated in an
anaerobiosis jar in the presence of a
Dehydrated base medium (without D-Cycloserine) : mixture of hydrogen and carbon dioxide.
- 500 g bottle : BK031HA - The plates are read immediately after
D-Cycloserine Selective Supplement : opening the jar, since the colonies may
- 10 vial kit : BS00608 become pale by the oxidation of iron
Egg Yolk Enrichment : sulfide by air.
- 50 mL vial : BS00208 - Carry out the tests for the identification of
Clostridium perfringens : Gram-staining,
catalase, nitrate reduction test, mobility,
gelatin hydrolysis, growth in LS Broth
(BK140), etc...
BASE MEDIUM + CYCLOSERINE + EGG YOLK

The medium can be supplemented with a
suspension of Egg Yolk (BS002) at 8 mL per
100 mL of melted base medium held at
47°C, in order to detect the lecithinase
produced by Clostridium perfringens.
Results

When grown in the presence of egg yolk,
colonies of Clostridium perfringens are
surrounded by an opaque halo due to the
production of lecithinase.
249
Bibliography
(1) Harmon, S.M., Kanter, D.A., and Peeler, J.T. 1971. Comparison (7) NF EN 26461-2. July 1993. Water quality. Detection and
of media for enumeration of Clostridium perfringens. Appl. enumeration of the spores of sulfite-reducing anaerobes
Microb., 21: 922-927. (Clostridia). Part 2: Method by membrane filtration.
(2) Hauschild, A.H.W., Hilsheimer, R., and Griffith, D.W. 1974. (8) V08-056. April 1994. Food microbiology. Enumeration of
Enumeration of faecal Clostridium perfringens spores in Clostridium perfringens by colony count technique at 37°C.
eggyolk-free Tryptose-Sulfite-Cycloserine Agar. Appl. Microb., Routine method.
27: 527-530. (9) XP V08-061. October 1996. Microbiology of food and animal
(3) Orth, D.S. 1977. Comparison of sulfite-polymyxin-sulfadiazine feedings stuffs. Anaerobic enumeration of sulfito-reducing
medium and tryptose-sulfite-cycloserine medium without egg bacteria by colony count technique. Routine method.
yolk for recovering Clostridium perfringens. Appl. Envir. (10) ISO 7937: 1997. Microbiology of food and animal feeding
Microbiol., 33: 986-988. stuffs. Horizontal method for enumeration of Clostridium
(4) Journal Officiel (French) du 19 janvier 1980. Critères perfringens. Colony-count technique.
microbiologiques auxquels doivent satisfaire certaines denrées (11) prEN 13401. 1998. Food Microbiology. Horizontal method for
animales ou d’origine animale. Méthodes générales d'analyse enumeration of Clostridium perfringens. Colony-count
bactériologique (arrêté du 21 décembre 1979 modifié par technique (ISO 7937 : 1997 modified).
l'arrêté du 13 Mars 1989 paru au J.O. du 20 avril 1989). (12) ISO/CD 15213. 1999. Horizontal method for the enumeration
(5) NF V 08-019. December 1985. Microbiology of food products. of sulphite-reducing bacteria growing under anaerobic
General guidance for the enumeration of Clostridium conditions.
perfringens. Colony count technique.
(6) NF T 90-415. October 1985. Water testing. Detection and
enumeration of the spores of sulfite-reducing anaerobes and of
sulfite-reducing Clostridia. General method by the standing
tube technique.
TSI Agar

TSI (Triple Sugar Iron) Agar is used for the identification of Target : Enterobacteria.
enterobacteria by the rapid detection of the fermentation of Reconstitution : 60.1 g / L.
lactose, glucose (with or without gas production) and of sucrose, as Autonomy (500 g) : 830 tubes.
well as the production of hydrogen sulfide.
pH (25°C) : 7.4 ± 0.2.
Hajna developed the formulation of this agar containing three
sugars by adding sucrose to a two-sugar (lactose and glucose)
medium of Kligler. The addition of sucrose increases the sensitivity of Preparation
the medium by leading to the early detection of coliform bacteria
which ferment lactose slowly and degrade sucrose more rapidly. The - Suspend 60.1 g of dehydrated medium
medium also favors the differentiation of certain Proteus strains (BK059) in 1 liter of distilled or deionized
water.
(lactose-negative) which ferment sucrose within 24 hours of
incubation.
- Dispense in tubes.
15 minutes.
- Sugar fermentation results in an acidification which makes phenol - Incline the tubes to obtain a butt 3 cm
red (pH indicator) turn yellow. high and a slant.
- The detection of bacteria fermenting only glucose is facilitated by
decreasing the concentration of the sugar to 1/10 of that of NOTE
lactose or sucrose, so that the small quantity of acid produced on When the medium is not used within 8 days of its
the slant during fermentation is rapidly oxidized. This causes a preparation, it is recommended to regenerate it
rapid return to the red color or else a more pronounced re- in a boiling water bath and solidify it again in
alkalinization. The acid reaction (yellow color), on the contrary, is the correct position.
maintained in the depth of the agar, in the butt of the tube.
- Bacteria fermenting lactose or sucrose will make the slant of the
tube turn yellow.
250
- Bacteria fermenting none of these sugars will not change the color Instructions for Use
of the medium.
- The production of H2S is revealed in the butt by the appearance of - Inoculate the butt by stabbing and the
black iron sulfide, due to the reduction of thiosulfate in the slant by streaking close together, using a
presence of ferric citrate. suspected colony taken from a selective
- The production of gas (H2, CO2), resulting from sugar isolation medium.
fermentation, is revealed by the appearance of bubbles or by the - Pure cultures taken from the center of
fragmentation of the agar. well isolated colonies must be used, since
otherwise cross reactions will make
Typical Composition identification impossible.
- Incubate at 37°C for 24 hours
(caps loosened) to favor gas exchanges.
Results
- Tryptone 14.0 g
- Yeast extract 3.0 g The use of one of the sugars in the medium
- Meat extract 3.0 g results in acidification (phenol red turns yellow).
- Glucose 1.0 g Alkalinization is shown by a dark red color.
- Lactose 10.0 g Hydrogen sulfide production from thiosulfate
- Sucrose 10.0 g is detected by a black color due to the
- Sodium chloride 5.0 g formation of iron sulfide in the presence of
- Sodium thiosulfate 0.3 g ferric citrate.
- Ferric ammonium citrate 0.3 g TSI Agar supplies four types of information :
- Phenol red 24 mg (1) Glucose fermentation
- Bacteriological agar 13.5 g - Butt red : glucose not fermented
- Butt yellow : glucose fermented
pH of the ready-to-use medium at 25°C : 7.4 ± 0.2. (2) Lactose and/or sucrose fermentation
- Slant red : lactose and sucrose not fermented
- Slant yellow : lactose and/or sucrose fermented
Quality Control (3) Gas production
- Production of gas bubbles in the butt
- Dehydrated medium : pinkish powder, free-flowing and (4) Formation of H2S
homogeneous. - Formation of a black color between the butt
- Prepared medium : orange red agar. and the slant or along the inoculation stab.
- Typical culture response after 24 hours of incubation at 37°C : - Typical reactions are in the following table :
Species Slant Butt H2S
Microorganisms Growth Slant Butt H2S Gas Lactose/Sucrose Glucose Gas
Escherichia coli ATCC® 25922 good yellow yellow - + Salmonella Typhi (2) - + - +
Citrobacter freundii ATCC 8090 good yellow yellow + + Salmonella Paratyphi A (2) - + + -
Salmonella Choleraesuis (2) - + + -
Proteus vulgaris ATCC 13315 good yellow yellow + +
Salmonella Pullorum (2) - + + +
Salmonella Enteritidis ATCC 13076 good red yellow + + Salmonella Paratyphi B (2) - + + +
Shigella flexneri ATCC 29903 good red yellow - - Salmonella Typhimurium (2) - + + +
Pseudomonas aeruginosa ATCC 27853 good red red - - Salmonella Enteritidis (2) - + + +
Salmonella Gallinarum (2) - + - +
Shigella dysenteriae - + - -
Shigella flexneri - + - -
Shigella sonnei - + - -
Dehydrated medium : 2-30°C. Shigella boydii - + - -
- The expiration date is indicated on the label. Proteus vulgaris + + {+} +
Prepared medium (benchmark value) : Proteus mirabilis - + + +
- Media in non-slanted tubes : 6 months at 2-8°C. Proteus morganii - + + -
- Media in slanted tubes : 8 days at 2-8°C. Proteus rettgeri - + - -
Serratia marcescens - + - -
Packaging Enterobacter hafniae - + + -
Enterobacter aerogenes + + + -
Enterobacter cloacae + + + -
Dehydrated medium :
Escherichia coli (1) + + + -
- 500 g bottle : BK059HA Citrobacter freundii + + + +
Klebsiella pneumoniae + + + -
Alcaligenes faecalis - - - -
Pseudomonas aeruginosa - - - -
Yersinia enterocolitica - - - -
(1) Some strains of Escherichia coli ferment lactose only very late in
growth.
(2) In the case where interpretation may suggest the presence of
salmonellae, it is possible to use TSI medium to detect -galactosidase,
urease and lysine decarboxylase.
251
Bibliography
(1) Sulkin, E.S., and Willet, J.C. 1940. A Triple Sugar Ferrous (6) NF EN 12824. February 1998. Microbiology of food and animal
Sulfate Medium for use in Identification of Enteric Organisms. feeding stuffs. Horizontal method for the detection of Salmonella.
J. Lab. Clin. Med., 25: 649-653. (7) Pharmacopée européenne. Addendum 2000. Contrôle
(2) Hajna, A.A. 1945. Triple Sugar Iron Medium for the microbiologique des produits non-stériles (Recherche de
Identification of the Intestinal Groups of Bacteria. J. Bact. microorganismes spécifiés). Solution et milieux de culture
49: 516-517. recommandés, 56-61.
(3) NF V 04-015: February 1984. Dried milk and sweetened (8) United States Pharmacopoeia. 24. 2000. Microbial Limit Tests,
condensed milk. Microbiology. 1814-1818.
(4) FIL-IDF 93A: 1985. Milk and milk products. Detection of
Salmonella.
(5) NF ISO 10272. January 1996. Microbiology of food and animal
feeding stuffs. Horizontal method for detection of
thermotolerant Campylobacter.
TSN Agar

Tryptone Sulfite Neomycin Agar was recommended by Mossel and Target : Clostridium perfringens.
developed by Marschall et al. in 1965 for the selective isolation and Reconstitution : 40.0 g / L.
enumeration of Clostridium perfringens in food products and other Autonomy (500 g) : 625 tubes
samples, primarily when they are contaminated by considerable (20 mL by tube).
accompanying microflora. The detection of Clostridium perfringens
pH (25°C) : 7.2 ± 0.2.
is based on the following characteristics :
- optimal growth at 46°C, Supplement required : None.
- tolerance to neomycin and polymyxin, Sterilization : 121°C / 15 minutes.
- high capacity to reduce sulfite, as shown by Wilson and Blain,
Mossel and Beerens, Angelotti, Hall and Foter.
Preparation
Principles
- The simultaneous presence of neomycin and polymixin inhibit the (BK001) in 1 liter of distilled or deionized
growth of enterobacteria. water.
- Neomycin inhibits the growth of most strains of Clostridium - Slowly bring to boiling, stirring until
bifermentans. complete dissolution.
- Incubation at 46°C assures the specificity of the medium for - Dispense 20 mL in 20 x 200 mm tubes, or
Clostridium perfringens. in flasks.
- This species reduces sulfite to sulfide, which with ferric citrate - Sterilize in an autoclave at 121°C for
causes a black iron sulfide precipitate around the colonies. 15 minutes.

Use in tubes
For 1 liter of medium : - Cool and maintain the medium at 47°C.
- Tryptone 15.0 g - Heat the product to analyze in order to
- Yeast extract 10.0 g destroy vegetative cells and activate spores.
- Sodium sulfite 1.0 g - Transfer 1 mL of the inoculum and its
- Ferric ammonium citrate 0.5 g serial tenfold dilutions to the tubes,
- Neomycin sulfate 50 mg avoiding the incorporation of air into the
- Polymixin B sulfate 20 mg medium.
- Bacteriological agar 13.5 g - Homogenize thoroughly by inversion.
- Cool in an ice water bath.
pH of the ready-to-use medium at 25°C : 7.2 ± 0.2. - Incubate at 46°C for 24 hours.
NOTE
Do not overheat the medium. Avoid heating the
tubes after inoculating.
252
Quality Control Use in Petri dishes
- Inoculated plates are incubated in an
- Dehydrated medium : beige powder, free-flowing and anaerobiosis jar in the presence of a
homogeneous. mixture of hydrogen and carbon dioxide.
- Prepared medium : amber agar. - The plates are read immediately after
- Typical culture response after 24 hours of anaerobic incubation opening the jar, since the colonies may
at 46°C : become pale by the oxidation of iron
sulfide by the outside air.
Clostridium perfringens ATCC® 13124 good to excellent black colonies Results

- Not recommended ; use immediately after preparation.
Packaging
Dehydrated medium :
Bibliography
(1) Mossel, D.A.A. 1959. Enumeration of sulphite reducing (4) Marschall, R.S., Steenbergen, J.F., and McClung, L.S. 1965.
clostridia occuring in foods. J. Sci. Food Agr., 662-669. Rapid technique for the enumeration of Clostridium perfringens.
(2) Arbuckle, R. E. 1960. The influence of temperature on the Appl. Microb., 13: 559-563.
growth of Clostridium perfringens M.A. Thesis, Indiana, Univ. (5) J.O. du 19 janvier 1980. (French). Critères microbiologiques
Bloomington. auxquels doivent satisfaire certaines denrées animales ou
(3) Collee, J.G., Knowlden, J. A., and Hobbs, B. C. 1961. Studies d’origine animale. Méthodes générales d’analyse
on the growth, sporulation and carriage of Clostridium welchii bactériologique. (arrêté du 21 décembre 1979 modifié).
with special reference to food poisoning strains. J. Appl. (6) Rodier, J. 1984. L’analyse de l’eau. Recherche et dénombrement
Bacteriol., 24: 326-339. des Clostridium perfringens. Dunod 7ème Ed., 855-857.
Violet Red Bile Agar (VRBL)
Intended Use
Violet Red Bile Agar (VRBL) is a selective medium used for the
detection and enumeration of coliform bacteria in water, milk,
dairy and other food products.
History
A large number of researchers have studied this medium. MacCrady

in 1932 for the Committee of Standard Methods of Milk Analysis of
the American Public Health Association, Bartram and Black for the
isolation of coliform bacteria in raw and pasteurized milk, and also
Miller and Prickett in a note concerning the recontamination of
milk. All these authors found the medium satisfactory since
complete results were obtained within 24 hours of incubation.
Escherichia coli
253
- The simultaneous presence of crystal violet and bile salts inhibit Target : Coliforms.
Gram-positive bacteria. Reconstitution : 38.5 g / L.
- Lactose fermentation results in acidification of the medium, shown Autonomy (500 g) : 811 plates
by the red color of the pH indicator (neutral red) and by the (16 mL by plate).
precipitation of bile salts around the colonies.
pH (25°C) : 7.4 ± 0.2.
Typical Composition Supplement required : MUG Supplement
(can be adjusted to obtain optimal performance) (optional).
For 1 liter of medium : Sterilization : Heat to boiling.
- Peptic digest of meat 7.0 g Do not autoclave.
- Lactose 10.0 g Preparation
- Bile salts 1.5 g
- Sodium chloride 5.0 g - Suspend 38.5 g of dehydrated medium
- Neutral red 30 mg (BK152) in 1 liter of distilled or deionized
- Crystal violet 2 mg water.
pH of the ready-to-use medium at 25°C : 7.4 ± 0.2. complete dissolution.
- Do not overheat.
Quality Control - Do not autoclave.
- Dehydrated medium : beige to pinkish-beige powder, NOTE

MUG (4-methylumbelliferyl--D-glucuronide) can
free-flowing and homogeneous.
be added to the medium in order to detect
- Prepared medium : reddish agar. Escherichia coli (refer to the monograph on MUG
- Typical culture response after 24 hours of incubation at 30°C : Supplement, BS024).
Microorganisms Growth Characteristics - With the ready-to-use base media (BM034
Escherichia coli ATCC ®
25922 good to excellent violet-red colonies or BM035), melt the medium with the
Citrobacter freundii ATCC 8090 good to excellent pinkish colonies minimum amount of time necessary in
Enterobacter aerogenes ATCC 13048 good to excellent violet-pink colonies order to achieve total liquefaction.
Storage / Shelf Life - Transfer 1 mL of the product to analyze
and its tenfold dilutions to Petri dishes.
Dehydrated medium : 2-30°C. - Pour in 12 mL of medium.
- The expiration date is indicated on the label. - Homogenize by swirling.
Prepared medium (benchmark value) : - Let solidify on a cold surface.
- Use on the day of preparation. - Overlay the solidified agar with 4 mL of
Ready-to-use media in vials, medium.
MUG Supplement :
- Let solidify.
- Incubate :
- at 30°C for 24 hours for the detection
and enumeration of coliform bacteria,
Packaging
- at 44°C for heat-tolerant coliform
bacteria.
- 10 x 100 mL vials : BM03408
Results
- 10 x 200 mL vials : BM03508
Dehydrated medium :
- 500 g bottle : BK152HA Coliform bacteria form violet colonies
- 5 kg drum : BK152GC whose diameter is equal to or greater
MUG Supplement : than 0.5 mm after 24 hours of incubation.
- 10 vial pack : BS02408 Lactose-negative enterobacteria are
colorless.
Bibliography
(1) NF ISO 4832: July 1991. Microbiology. General guidance for (3) NF V 08-050. February 1999. Microbiology of food and animal
the enumeration of coliforms. Colony count technique. feeding stuffs. Enumeration of coliforms by colony-count
(2) NF V 08-060. March 1996. Microbiology of food and animal technique at 30°C. Routine method.
feeding stuffs. Enumeration of thermotolerant coliforms by (4) FIL-IDF provisional 73B: 1998. Milk and milk products.
colony-count technique at 44°C. Routine method. Enumeration of coliforms. Part 1: Colony count technique at
30°C without resuscitation.
254
Violet Red Bile Glucose Agar (VRBG)

Bile Glucose Agar containing crystal violet and neutral red (VRBG Target : Enterobacteria.
Agar) was used by Mossel for the detection and enumeration of Reconstitution : 39.5 g / L.
enterobacteria in dairy products, meat, prepared pork products and
other food products.
(16 mL by plate).
Principles pH (25°C) : 7.4 ± 0.2.
- The simultaneous presence of crystal violet and bile salts inhibit (optional).
Gram-positive bacteria.
- The degradation of glucose to acid is shown by the red color of Sterilization : Heat to boiling.
the pH indicator, neutral red. Do not autoclave.

- Peptic digest of meat 7.0 g water.
- Yeast extract 3.0 g - Slowly bring to boiling, stirring until
- Glucose 10.0 g complete dissolution.
- Bile salts 1.5 g - Do not overheat.
- Sodium chloride 5.0 g - Do not autoclave.
- Neutral red 30 mg
- Crystal violet 2 mg NOTE
- Bacteriological agar 13.0 g MUG (4-methylumbelliferyl--D-glucuronide)
can be added to the medium in order to detect
pH of the ready-to-use medium at 25°C : 7.4 ± 0.2. Escherichia coli (refer to the monograph
on MUG Supplement, BS024).
Quality Control
- Dehydrated medium : slightly pink powder, free-flowing and
homogeneous. - Cool and maintain the medium at 47°C.
- Prepared medium : reddish agar. - Transfer 1 mL of the product to analyze
- Typical culture response after 24 hours of incubation at 37°C : and its tenfold dilutions to Petri dishes.
Microorganisms Growth Characteristics - Homogenize by swirling.
Salmonella Typhimurium ATCC 14028 good to excellent
®
violet-red colonies - Let solidify on a cold surface.
surrounded by a violet halo - Overlay the solidified agar with 4 mL of
medium.
Escherichia coli ATCC 25922 good to excellent violet-pink colonies - Let solidify.
Enterobacter aerogenes ATCC 13048 good to excellent violet-pink colonies - Incubate at 37°C for 24 hours.
Citrobacter freundii ATCC 8090 good to excellent violet-pink colonies
Enterococcus faecalis ATCC 29212 inhibited Results
Staphylococcus aureus ATCC 25923 inhibited Enterobacteria form violet colonies whose
diameter is equal to or greater than 0.5 mm,
Storage / Shelf Life surrounded by a halo of precipitated
bile salts.
- Not recommended ; use on the day of preparation.
MUG Supplement :
Packaging
Dehydrated medium :
MUG Supplement :
255
Bibliography
(1) Mossel, D.A.A., Mengerink, W.H.J., and Scholts, H.H. 1962. Use (4) NF ISO 7402. December 1993. Microbiology. General guidance
of a modified MacConkey agar medium for the selective growth for the enumeration of Enterobacteriaceae without
and enumeration of all Enterobacteriaceae. J. Bact., 84: 381. resuscitation. MPN technique and colony count technique.
(2) Mossel, D.A.A., Wisser, M., and Cornelissen, A.M.R. 1963. The (5) ISO 5552. December 1997. Meat and meat-based products.
examination of foods for Enterobacteriaceae using a test of the Detection and enumeration of Enterobacteriaceae without
type generally adopted for the detection of Salmonellae. J. resuscitation. MPN technique and colony count technique.
Appl. Bact., 24: 444-452. (6) NF V08-054. February 1999. Microbiology of food and animal
(3) NF ISO 8523. February 1992. Microbiology. General guidance feeding stuffs. Enumeration of the enterobacteria by colony
for the detection of Enterobacteriaceae with pre-enrichment. count technique at 30°C. Routine method.
Wilkins Chalgren Agar

Target : Antibiotic susceptibility testing,
Wilkins Chalgren Agar is used to determine the minimal inhibitory
Anaerobic microorganisms.
concentrations of antibiotics for anaerobic bacteria by the dilution
method. It can also be used for the abundant growth and isolation Reconstitution : 48.0 g / L.
of anaerobic bacteria of various origins. Autonomy (500 g) : 694 plates.
pH (25°C) : 7.1 ± 0.2.
History
Wilkins and Chalgren established the formula of the medium in 1976. Sterilization : 121°C / 15 minutes.
They showed that it had the advantage of not requiring blood to
obtain satisfactory growth of anaerobic bacteria of clinical importance. Preparation
The modalities of utilization are described in the NCCLS Collaborative
Study of the Proposed Reference Dilution Method of Antimicrobial - Suspend 48.0 g of dehydrated medium
Susceptibility Testing of Anaerobic Bacteria. (BK101) in 1 liter of distilled or deionized
water.
- The combination of casein and gelatin peptones, yeast extract and - Dispense in tubes or flasks.
glucose makes the medium very nutritious. - Sterilize in an autoclave at 121°C for
- Hemin stimulates the growth of fastidious bacteria. 15 minutes.
- Sodium chloride maintains osmotic equilibrium. Instructions for Use
Typical Composition - Cool and maintain the medium at 47°C.

(can be adjusted to obtain optimal performance) - Add the antibiotics to test.
- Mix well.
- Tryptone 10.0 g - Let solidify on a cold surface.
- Pancreatic digest of gelatin 10.0 g - Dry in an incubator with the covers
- Yeast extract 5.0 g partially removed.
- L-arginine 1.0 g - Inoculate.
- Glucose 1.0 g - Incubate anaerobically at 37°C for
- Hemin 5 mg 24 to 48 hours.
- Vitamin K1 0.5 mg - Read the results.
- Sodium pyruvate 1.0 g
256
Quality Control
- Dehydrated medium : white-cream powder, free-flowing

and homogeneous.
- Typical culture response after 48 hours of anaerobic incubation
at 37°C :
Bacteroides fragilis ATCC® 23745 good to excellent
Clostridium perfringens ATCC 13124 good to excellent

Packaging
Dehydrated medium :
Bibliography
(1) Wilkins, T.D., and Chalgren, S. 1976. Medium for use in (3) Lennette, E.H., Balows, A., Hausler, Jr., W.J., and Shadomy,
antibiotic susceptibility testing of anaerobic bacteria. H.J. 1985. Manual of Clinical Microbiology. A.S.M. Washington.
Antimicrob. Agents Chemother., 10 (6): 926. 4th Ed., 988-990.
(2) N.C.C.L.S. 1982. Tentative Standard Reference Agar Dilution
Procedure for Susceptibility Testing of Anaerobic Bacteria , 2, n°3.
Wilkins Chalgren Broth

Wilkins Chalgren Broth is used as a non-selective medium for the Target : Antibiotic susceptibility testing,
Anaerobic microorganisms.
growth and enumeration of aerobic and anaerobic microorganisms
of varied origins, as well as for the determination of the minimal Reconstitution : 33.0 g / L.
inhibitory concentrations of antibiotics by the dilution method in Autonomy (500 g) : 1515 tubes.
the case of anaerobic species.
pH (25°C) : 7.1 ± 0.2.
- The combination of casein and gelatin peptones, yeast extract and
glucose makes the medium very nutritious.
- Hemin stimulates the growth of fastidious bacteria.
Preparation
- Sodium chloride maintains osmotic equilibrium. - Suspend 33.0 g of dehydrated medium
water.
15 minutes.
257
Growth in tubes
For 1 liter of medium : - Inoculate the medium with purified
- Tryptone 10.0 g cultures or with other types of mixed
- Pancreatic digest of gelatin 10.0 g microflora inoculum.
- Yeast extract 5.0 g - Incubate at the optimal required
- L-arginine 1.0 g temperature, aerobically or in an
- Glucose 1.0 g anaerobiosis jar, depending on the
- Hemin 5 mg bacteria to be grown.
- Vitamin K1 0.5 mg
- Sodium pyruvate 1.0 g Determining minimal inhibitory
concentrations for anaerobes
- Add a series of dilutions of the stock
solution of the antibiotic to test to tubes
of medium.
- Inoculate with a defined quantity of
Quality Control culture of the species to control.
- Incubate in an anaerobiosis jar for
- Dehydrated medium : cream powder, free-flowing and 48 hours.
homogeneous.
- Prepared medium : amber solution, clear, may contain a fine Results
precipitate.
- Typical culture response after 48 hours of anaerobic incubation Growth results in turbidity by microbial
at 37°C : division. The last tube presenting no
bacterial development contains the
Microorganisms Growth antibiotic at the minimal inhibitory
Bacteroides fragilis ATCC® 25285 good to excellent concentration. Multiplying this
concentration by the corresponding
Prevotella melaninogenica ATCC 25611 good to excellent dilution factor indicates the active
Clostridium perfringens ATCC 13124 good to excellent concentration of antibiotic.

Packaging
Dehydrated medium :
Bibliography
(1) Wilkins, T.D., and Chalgren, S. 1976. Medium for use in (3) Lennette, E.H., Balows, A., Hausler, Jr., W.J., and Shadomy,
antibiotic susceptibility testing of anaerobic bacteria. H.J. 1985. Manual of Clinical Microbiology. A.S.M. Washington.
Antimicrob. Agents Chemother., 10 (6): 926. 4th Ed., 988-990.
(2) N.C.C.L.S. 1982. Tentative Standard Reference Agar Dilution
Procedure for Susceptibility Testing of Anaerobic Bacteria,
2, n°3.
258
WL Differential Agar
Intended Use
Product Profile
WL (Wallerstein Laboratory) Agar with Actidione (cycloheximide) is Target : Total microorganisms.
a selective differentiation medium for the culture and enumeration Reconstitution : 80.2 g / L.
of bacteria in fermented beverages, especially beer. Used in the in- Autonomy (500 g) : 415 plates.
process microbiological controls of fermentation, WL Differential
pH (25°C) : 5.5 ± 0.2.
Agar favors bacterial growth while inhibiting the development of
yeasts and limiting that of molds. Supplement required : None.
History
Preparation
WL Differential Agar was described by Green and Gray in their studies
of various fermentation processes. They showed that simple - Suspend 80.2 g of dehydrated medium
microscopic counting was insufficient when carrying out (BK066) in 1 liter of distilled or deionized
microbiological examinations of worts, beer and fermented products. water.
This led to the development of two culture media, one non-selective, - Bring to boiling, stirring until complete
the other containing cycloheximide. dissolution.
- Avoid overheating the medium.
Principles - Dispense in tubes or flasks.
- Casein digest, yeast extract, glucose and a large number of salts in 15 minutes.
the medium assure the growth of microorganisms.
- Cycloheximide inhibits the development of yeasts and molds Instructions for Use
present in samples of fermented beverages. It favors the detection
and enumeration of bacteria which may be present in relatively - Cool and maintain the medium at 47°C.
small numbers. - Transfer 1 mL of the product to analyze
- Bacteria which acidify the medium cause the pH indicator, and its serial tenfold dilutions to sterile
bromcresol green, to turn yellow. Petri dishes.
- Pour in 10 to 15 mL of medium.
Typical Composition - Homogenize by swirling.
(can be adjusted to obtain optimal performance) - Let solidify on a cold surface.
- Incubate at 25 or 30°C from 1 to 15 days,
For 1 liter of medium : using 2 plates per dilution : one
- Tryptone 5.0 g aerobically for the growth of
- Yeast extract 4.0 g Flavobacterium, Proteus and acidophilic
- Glucose 50.0 g bacteria (Acetobacter), the other
- Potassium dihydrogen phosphate 550 mg anaerobically for lactic acid bacteria and
- Potassium chloride 425 mg Pediococcus.
- Calcium chloride 125 mg
- Magnesium sulfate 125 mg Results
- Ferric chloride 2.5 mg
- Manganese sulfate 2.5 mg Count the colonies having grown.
- Bromcresol green 22 mg Only plates containing between 15 and
- Actidione 4 mg 150 colonies are used.
- Bacteriological agar 20.0 g Carry out the required microscopic
verifications.
Quality Control
- Dehydrated medium : slightly greenish powder, free-flowing

and homogeneous.
- Prepared medium : blue-green agar.
Lactobacillus plantarum ATCC® 8014 good to excellent
Proteus mirabilis ATCC 29906 good
259

- Media in tubes or vials : use on the day of preparation.
Packaging
Dehydrated medium :
Bibliography
(1) Green, S.R., and Gray, P.P. 1950. A differential procedure (2) Green, S.R., and Gray, P.P. 1951. A differential procedure for
applicable to bacteriological investigations in brewing. bacteriological studies useful in the fermentation industries.
Wallerstein. Lab. Commun., 13: 357-366. Wallerstein. Lab. Commun., 14: 289-295.
Wort Agar
Intended Use
Product Profile
Wort Agar is used for the growth, isolation and enumeration of Target : Yeasts, Molds.
yeasts and molds. It is particularly well adapted to the enumeration Reconstitution : 48.3 g / L.
of osmophilic yeasts in butter, sugar, syrups, lemonades and more Autonomy (500 g) : 690 plates.
generally in sweet beverages.
pH (25°C) : 4.8.
History Supplement required : Glycerol.
Parfitt successfully developed this medium for the enumeration of
yeasts in butter and syrups.
Preparation
Principles
- Yeasts grow well in media containing maltose, especially at an
water.
acid pH.
- Add 2.35 g of glycerol.
- The formula of the medium simulates the composition of wort,
which favors the growth of yeasts.
- The acidity of the medium inhibits most bacterial species.
Typical Composition of the base media (without glycerol)
15 minutes.
- Pancreatic digest of meat 0.78 g - Cool and maintain the medium at 47°C.
- Malt extract 15.00 g - Adjust the pH to 4.8 by adding sterile 10%
- Maltose 12.75 g lactic or tartaric acid.
- Dextrin 2.75 g - Transfer 1 mL of the product to analyze
- Dipotassium phosphate 1.00 g and its serial tenfold dilutions to sterile
- Ammonium chloride 1.00 g Petri dishes.
- Bacteriological agar 15.00 g - Pour in 10 to 15 mL of medium.
- Incubate at 20-25°C for 3 to 5 days
in the light.
260
Quality Control NOTE
For more selective use, the pH can be adjusted
- Dehydrated medium : beige powder, free-flowing and to 4.5 or 3.5. Never heat the medium after
adding acid to prevent the loss of the gelling
homogeneous.
properties of the agar.
- Prepared medium : amber agar, may present a slight flocculent
after autoclaving.
Results
- Typical culture response after 72 hours of incubation at 25-30°C :
Separately count yeasts and molds. Carry
Microorganisms Growth out a microscopic confirmation test on each
Saccharomyces cerevisiae ATCC® 9763 good to excellent type of colony encountered.
Escherichia coli ATCC 25922 partially to completely inhibited
Dehydrated base medium (without glycerol) : 2-30°C.

Packaging
Dehydrated base medium (without glycerol) :

Bibliography
(1) Parfitt. 1933. J. Dairy Sci., 19: 141.
XLD Agar
Intended Use
XLD (Xylose Lysine Desoxycholate) Agar is used to isolate pathogenic

enterobacteria, especially Shigella and Salmonella from biological and
food samples.
History
The medium was formulated by Taylor in order to increase the

efficiency of recovery of pathogenic enterobacteria, particularly
Shigella and other fastidious species which do not develop in other
formulations containing highly toxic inhibitors.
Principles
- Sodium desoxycholate inhibits contaminating Gram-positive flora.

- Xylose is fermented by practically all enteropathogenic bacteria,
except for Shigella which are thus differentiated from the other
species. After exhausting xylose, Salmonella decarboxylate lysine
(via lysine decarboxylase) to cadaverine, causing the pH to rise.
Colonies of salmonellae resemble those of shigellae in the medium
having become basic.
- The colonies formed are red in the presence of the indicator,
phenol red.
- The addition of lactose and sucrose to the medium enable coliform
bacteria to decarboxylate lysine and thereby produce excess acidity,
making the indicator turn yellow, favoring their differentiation.
261
- In basic medium, pathogenic H2S-producers reduce ferric Product Profile
ammonium citrate and cause a blackening due to the production
of iron sulfide at the center of the colonies. Non-pathogenic Target : Salmonella, Shigella.
bacteria which do not decarboxylate lysine acidify the medium, a Reconstitution : 55.2 g / L.
result of sugar fermentation. The pH decrease prevents the Autonomy (500 g) : 604 plates.
colonies from blackening.
pH (25°C) : 7.4 ± 0.2.
Typical Composition of the complete medium Supplement required : None.
(can be adjusted to obtain optimal performance) Sterilization : Heat to 90°C.
Do not autoclave.
- Yeast extract 3.0 g Preparation
- L-Lysine 5.0 g
- Lactose 7.5 g - Suspend 55.2 g of dehydrated medium
- Sucrose 7.5 g (BK058) in 1 liter of distilled or deionized
- Xylose 3.5 g water.
- Sodium desoxycholate 2.5 g - Heat to about 90°C with frequent
- Sodium chloride 5.0 g agitation.
- Sodium thiosulfate 6.8 g - Stop heating when the medium is
- Ferric ammonium citrate 0.8 g completely dissolved.
- Phenol red 80 mg - Do not autoclave.
NOTE
Excessive heating or prolonged holding at 47°C
may cause precipitation, so the colonies may
furnish less clear-cut reactions. The medium
Quality Control should be clear and orange-red.
- Dehydrated medium : pinkish powder, free-flowing and Instructions for Use

homogeneous.
- Prepared medium : orange red agar. - Cool and maintain the medium at 47°C.
- Typical culture response after 24 hours of incubation at 37°C : - Pour into sterile Petri dishes.
Microorganisms Growth Characteristics - Dry in an incubator with the covers
partially removed.
Salmonella Typhimurium ATCC® 14028 good to excellent red colonies
- Inoculate by streaking on the surface of
with black center
the medium, using enrichment media used
Salmonella Enteritidis ATCC 13076 good to excellent red colonies
for the detection of Salmonella.
with black center - In parallel, transfer the inoculum to
Shigella flexneri ATCC 29903 good to excellent red colonies another selective isolation medium.
Escherichia coli ATCC 25922 partially inhibited yellow colonies - Incubate at 37°C for 24 to 48 hours.
Staphylococcus aureus ATCC 25923 inhibited Results
Storage / Shelf Life Shigella, Providencia, Edwardsiella and

Salmonella Paratyphi ferment xylose slowly
Dehydrated medium : 2-30°C. or not at all, in contrast to other bacteria.
- The expiration date is indicated on the label. Salmonella is distinguished from other
Prepared medium (benchmark value) : genera by its ability to decarboxylate lysine.
- Media in plates : 8 days at 2-8°C. Hydrogen sulfide production in a basic
medium leads to the formation of colonies
Packaging with a black center, while the black color is
inhibited in an acid medium. The colonies
have the following appearance :
Dehydrated medium :
- Yellow colonies
LDC (-), sometimes H2S (+) :
Escherichia coli, Citrobacter, Enterobacter,
Proteus, Serratia, Klebsiella
- Red colonies
LDC (+), H2S (-) :
Shigella, Providencia, Salmonella
- Red colonies with black center
LDC (+), H2S (+) :
Salmonella, Arizona, Edwardsiella
262
Bibliography
(1) Taylor, W.J. 1965. Isolation of Shigellae. I. Xylose lysine agars, (4) NF EN 12824. February 1998. Microbiology of food and animal
new media for isolation of enteric pathogens. Am. J. Clin. feeding stuffs. Horizontal method for the detection of Salmonella.
Path., 44: 471-475. (5) Pharmacopée européenne. Addendum 2000. Contrôle
(2) ISO 6340. December 1995. Water Quality. Detection of microbiologique des produits non stériles (Recherche de
Salmonella. microorganismes spécifiés). Solution et milieux de culture
(3) NF V 08-052. May 1997. Microbiology of food and animal recommandés, 56-61.
feeding stuffs. Detection of Salmonella. Routine method. (6) United States Pharmacopoeia 24. 2000. Microbial Limit Tests,
1814-1818.
XLT4 Agar
Intended Use
XLT4 (Xylose-Lysine-Tergitol 4) Agar is a selective isolation medium for

the detection of Salmonella, except for Salmonella Typhi and
Paratyphi, in poultry and other biological samples.
History
In 1991, Miller and Tate demonstrated that the use of XLT4 Agar
increased the frequency of detection of non-Typhi Salmonella in
poultry samples containing a high secondary microflora, and that
the medium allowed a good differentiation between Salmonella
and Citrobacter. The medium described by these authors
incorporated Tergitol 4 in a modified Xylose-Lysine base, in order to
inhibit a wide spectrum of competitive flora (Proteus, Pseudomonas,
Providencia) which previously had interfered with Salmonella
Salmonella Enteritidis
detection. Further studies performed by Dusch and Altwegg
established that XLT4 Agar could be used for Salmonella detection
in clinical samples, with the exception of Salmonella Typhi and
Product Profile
Salmonella Paratyphi.
- Tergitol 4 inhibits contaminating Gram-positive flora and
numerous Gram-negative strains, notably Proteus. pH (25°C) : 7.4 ± 0.2.
- Xylose is fermented by enteropathogenic bacteria, with the Supplement required : Tergitol 4 Selective
exception of Shigella, which are therefore differentiated from Supplement.
other bacteria. After having exhausted the xylose, Salmonella Sterilization : Heat to boiling.
decarboxylate lysine (via lysine decarboxylase) to cadaverine, which Do not autoclave.
provokes an increase in the pH. In an alkaline (basic) environment
Salmonella forms red colonies in the presence of the pH indicator, Preparation
phenol red.
- Black colonies, due to the appearance of iron sulfide in the colony - Suspend 59.0 g of dehydrated medium
center, are formed through the reduction of ferric ammonium (BK156) in 1 liter of distilled or deionized
citrate by pathogenic hydrogen sulfide producers. water.
- The medium contains two additional sugars, lactose and - Add 4.6 mL of Tergitol 4 Selective
saccharose. Fermentation of either or both sugars results in Supplement (BS039).
acidification of the medium and leads to the formation of yellow - Slowly bring to boiling, stirring until
colonies in the presence of phenol red indicator. complete dissolution.
- Non-pathogenic strains that do not decarboxylase lysine produce - Do not overheat.
- Do not autoclave.
an acidification from the sugar fermentation. The resulting
decrease in pH prevents the blackening of the colonies.
263
Typical Composition of the complete medium Instructions for Use
- Peptone 1.6 g - Let solidify on a cold surface.
- Yeast extract 3.0 g - Dry in an incubator with the covers
- L-lysine 5.0 g partially removed.
- Lactose 7.5 g - To the surface of plates prepared as above,
- Sucrose 7.5 g or to ready-to-use plates (BM036) brought
to room temperature, inoculate by
- Xylose 3.75 g
streaking on the surface of the medium,
using enrichment media used for the
detection of Salmonella.
- Ferric ammonium citrate 0.8 g - In parallel, transfer the inoculum to
- Phenol red 80 mg another selective isolation medium.
- Bacteriological agar 18.0 g - Incubate at 37°C for 24 to 48 hours.
pH of the ready-to-use medium at 25°C : 7.4 ± 0.2. Results
Quality Control Typical (H2S positive) Salmonella colonies

are red with a black center. They may
- Dehydrated medium : pinkish powder, free-flowing and present a yellow halo after 24 hours
homogeneous. incubation. In the event of a prolonged
- Prepared medium : orange red agar. incubation, the colonies become red to pink
- Typical culture response after 24 hours of incubation at 37°C : with a black center or entirely black. H2S-
negative Salmonella appear red to pink
without the black center.
Citrobacter, Klebsiella and Enterobacter
Salmonella Typhimurium ATCC® 14028 good to excellent red colonies cloacae produce yellow colonies. Growth of
with black center Enterobacter aerogenes and Escherichia coli
Salmonella Enteritidis ATCC 13076 good to excellent red colonies is partially inhibited, colonies present on
with black center the medium are yellow.
Citrobacter freundii ATCC 8090 good to excellent yellow colonies Proteus, Pseudomonas and Providencia are
Enterobacter aerogenes ATCC 13048 partially inhibited yellow colonies partially to completely inhibited.
Proteus mirabilis ATCC 29906 inhibited Shigella produces slow growth and pink
Staphylococcus aureus ATCC 25923 inhibited colonies.
Dehydrated base medium (without Tergitol 4) : 2-30°C.

Ready-to-use media in plates :
Tergitol 4 Selective Supplement :
Packaging
- 20 plates : BM03608
Dehydrated base medium (without Tergitol 4) :
Tergitol 4 Selective Supplement :
- 50 mL vial : BS03908
264
Bibliography
(1) Miller, R.G., C.R. Tate, E.T. Mallinson, and J.A. Scherrer. 1991. (5) Wallace, H.A. 1996. Evolution of Methods for the Detection of
Xylose-Lysine-Tergitol 4: an improved selective agar medium for Salmonella in Foods. J. of A.O.A.C. International. 79: 4-12.
the isolation of Salmonella. Poultry Science. 70: 2429-2432. (6) CNEVA Bactériologie animale : Pr 116/00/BA 50/00. Décembre
(2) Miller, R.G., C.R. Tate, E.T. mallinson, and J.A. Scherrer. 1992. 1996. Isolement et identification des salmonelles chez les
Erratum. Xylose-Lysine-Tergitol 4: an improved selective agar mammifères.
medium for the isolation of Salmonella. Poultry Science. 71: 398. (7) CNEVA Bactériologie animale : Pr 116/00/BA 60/00. Décembre
(3) Tate, C.R., R.G. Miller, and E.T. Mallinson. 1992. Evaluation of 1996. Isolement et identification des salmonelles chez les
two isolation and two non-isolation methods for detecting volailles.
naturally occuring salmonellae from broiler flock environmental (8) CNEVA Bactériologie animale : Pr 116/00/BA 70/00. Décembre
drag-swab samples. J. Food Prot. 55: 964-967. 1996. Isolement et identification des salmonelles en élevage
(4) Dusch, H., and M. Altwegg. 1995. Evaluation of five new avicole.
plating media for isolation of Salmonella species. J. Clin.
Microbiol. 33: 802-804.
Yeast Extract Agar
Intended Use
Yeast Extract Agar is used in water microbiology for the enumeration

of culturable microorganisms by colony count at 36 and 22°C.
The method is intended to measure the functional efficiency of
drinking water treatment, and more generally, all types of water.
It is particularly well adapted to the analysis of water destined for
human consumption, including bottled and natural mineral waters.
History
Yeast Extract Agar is derived from Plate Count Agar (PCA). It does
not contain glucose.
Principles
Escherichia coli
- The nutrients supplied by Tryptone and vitamins from yeast extract
favor the growth of most bacteria.
Product Profile
Typical Composition Target : Total microorganisms.
For 1 liter of medium : Autonomy (500 g) : 1755 plates.
- Tryptone 6.0 g pH (25°C) : 7.2 ± 0.2.
Preparation
Quality Control
- Dehydrated medium : cream powder, free-flowing and (BK153) in 1 liter of distilled or deionized
homogeneous. water.
- Prepared medium : light amber agar. - Slowly bring to boiling, stirring until
- Typical culture response after 44 hours of incubation at 36°C : complete dissolution.
15 minutes.
265
Microorganisms Growth Instructions for Use
Escherichia coli WR1 good to excellent - With the ready-to-use media (BM068) or if
Staphylococcus aureus ATCC 25923 good to excellent the media has been prepared in advance
Pseudomonas aeruginosa ATCC 10145 good to excellent from the dehydrated base, melt the
medium for the minimum amount of time
Storage / Shelf Life necessary to achieve total liquefaction.
Petri dishes.
Prepared medium (benchmark value) : - Pour 10 to 15 mL of medium.
- Media in vials : 6 months at 2-8°C. - Homogenize by swirling.
- Media in plates : 1 month at 2-8°C. - Let solidify on a cold surface.
Ready-to-use media in vials : - Incubate :
- Store between 2-8°C, shieldied from light. - One set of inoculated plates at 36°C for
- The expiration date is indicated on the label. 44 hours,
- One set of inoculated plates at 22°C for
Packaging 68 hours.
Ready-to-use media Results

- 10 x 200 mL vials : BM06808
Dehydrated medium : Only plates containing less than 300 colonies
- 500 g bottle : BK153HA are used for enumeration.
Bibliography
(1) NF EN ISO 6222: July 1999. Water quality. Enumeration of

culturable microorganisms. Colony count by inoculation in a
nutrient agar culture medium.
Yersinia Agar (CIN)
Intended Use
Yersinia Agar (CIN) is used for the selective isolation of Yersinia

enterocolitica from biological samples and food products.
History
CIN (Cefsulodin Irgasan Novobiocin) Agar was described by

Schieman in 1979. Compared to results obtained with SS and
MacConkey Agars, CIN was more inhibitory towards normal enteric
flora and its recovery capacity for Yersinia enterocolitica more
satisfactory. The formula was subsequently modified somewhat by
the author in order to refine the performance, so that recovery of
strains from the O:8 serological group could be accomplished.
Principles
- The selectivity of the medium is due to the presence of bile salts, Yersinia enterocolitica
crystal violet and irgasan, which inhibit contaminating Gram-
positive bacteria and most Gram-negative species.
- Supplements added just before use (cefsulodin and novobiocin)
Product Profile
inhibit normal enteric flora. Target : Yersinia enterocolitica.
- The differential properties of the medium are based on the Reconstitution : 59.5 g / L.
fermentation of mannitol. Microorganisms fermenting the sugar
acidify the medium and cause a localized drop in pH around the
colonies. In presence of neutral red, the colonies take on a red color. pH (25°C) : 7.4 ± 0.2.
Mannitol-negative species form colorless and translucent colonies. Supplement required : CIN Selective
- Some strains of Citrobacter freundii, Serratia liquefaciens and Supplement.
Enterobacter agglomerans may grow, but they can be
differentiated easily by the use of specific biochemical tests.
266
Typical Composition of the complete media Preparation
- Polypeptone 20.0 g deionized water.
- Yeast extract 2.0 g - Slowly bring to boiling, stirring until
- Mannitol 20.0 g complete dissolution.
- Sodium desoxycholate 0.5 g - Dispense 100 mL into flasks.
- Sodium cholate 0.5 g - Sterilize in an autoclave at 121°C for
- Sodium chloride 1.0 g 15 minutes.
- Sodium pyruvate 2.0 g - Do not overheat the medium.
- Magnesium sulfate 10 mg
- Neutral red 30 mg Instructions for Use
- Irgasan 4 mg - Aseptically add 1 mL of reconstituted CIN
- Cefsulodin 15 mg Selective Supplement (BS023) to each flask.
- Novobiocin 2.5 mg - Mix well without introducing air bubbles.
partially removed.
Quality Control - Inoculate directly from the sample or after
an appropriate enrichment. Shake the
- Dehydrated medium (base) : slightly pinkish powder, free- inoculating loop to remove excess
flowing and homogeneous. inoculum and streak across the surface
- Prepared plates (complete medium) : pinkish agar. of the plate.
- Typical culture response after 48 hours of incubation at 30°C : - Incubate at 30°C for 24 and 48 hours.
Microorganisms Growth Characteristics Results

Yersinia enterocolitica ATCC ®
27729 good to excellent translucent colonies with
Colonies of Yersinia enterocolitica ferment
a dark pink center
mannitol and are red as a result of the
Escherichia coli ATCC 25922 inhibited acidification of the medium in presence of
Proteus mirabilis ATCC 29906 inhibited neutral red. They have a dark red center
Enterococcus faecalis ATCC 29212 inhibited surrounded by a transparent border. Other
colonies not fermenting mannitol are
Storage / Shelf Life translucent.

CIN Selective Supplement
Packaging
Dehydrated base medium (without supplement) :

CIN Selective Supplement :
Bibliography
(1) Schiemann, D.A. 1979. Synthesis of a selective agar medium (4) Vidon, D.J.M., and Delmas, C.L. 1981. Incidence of Yersinia
for Yersinia enterocolitica. Can. J. Microbiol., 25: 1298-1304. enterocolitica in raw milk in Eastern France. App. Env. Microb.,
(2) Schiemann, D.A. 1980. Yersinia enterocolitica : observations on 41: 355.
some growth characteristics and response to selective agents. (5) Schiemann, D.A. 1982. Development of a two-step enrichment
Can. J. Microbiol., 26: 1232-1240. procedure for recovery of Yersinia enterocolitica from food.
(3) Devenish, J.A., and Schiemann, D.A. 1981. An abbreviated App. Microbiol., 43 (1): 14.
scheme for identification of Yersinia enterocolitica isolated from (6) NF ISO 10273: May 1995. Microbiology. General guidance for the
food enrichments on C I N agar. Can. J. Microbiol., 27: 937-941. detection of presumptive pathogenic Yersinia enterocolitica.
267
Supplements, Reagents,
Ringer
Cefixime-Tellurite Selective Supplement
for CT-SMAC Agar
Intended Use Quality Control
Cefixime-Tellurite Selective Supplement is used with - Appearance : white lyophilisate, giving a clear,
MacConkey Sorbitol Agar (base) for the detection of colorless solution after reconstitution.
Escherichia coli serotype O157:H7. Supplied as a - Bacteriological efficacy in MacConkey Sorbitol Agar
freeze-dried mixture of the antibiotic cefixime and after 24 hours of incubation at 37°C :
the inhibitory compound potassium tellurite, it
inhibits almost all contaminating microflora thereby Microorganisms Growth Characteristics
favoring the growth of Escherichia coli O157:H7. Escherichia coli O157:H7 ATCC® 43895 good to excellent colorless colonies
More information can be found by consulting the technical Escherichia coli ATCC 25922 partially inhibited red colonies
or catalogue sheets for MacConkey Sorbitol Agar. (sorbitol-positive)
Reconstitution Enterococcus faecalis ATCC 29212 inhibited
- Using aseptic techniques, fill the vial of lyophilisate
with 5 mL of sterile distilled water. Storage / Shelf Life
- Turn end-over-end to dissolve. Avoid frothing the
solution. - Store between 2 and 8°C, shielded from light.
- The expiration date is indicated on the vial.
Composition - Once reconstituted, the product can be stored for
a maximum duration of 30 days at 2-8°C, shielded
Per vial : from light.
- Cefixime 0.025 mg
- Potassium tellurite 1.250 mg Packaging
CFC Selective Supplement
CFC Selective Supplement is used with CFC Agar (base) - Appearance : whitish lyophilisate, giving an
for the isolation and enumeration of pigmented and opaque whitish to yellowish solution after
non-pigmented Pseudomonas that contaminate meats reconstitution.
and meat-based products during refrigerated storage. - Bacteriological efficacy in CFC Agar after 72 hours
The supplement contains an inhibitory mixture of incubation at 25°C :
composed of two antibiotics, cephalosporin and
fucidin, and one quaternary ammonium salt (cetrimide, Microorganisms Growth
or cetyltrimethylammonium bromide). Supplied in the Pseudomonas aeruginosa ATCC® 9027 good to excellent
form of a freeze-dried powder, it inhibits practically all Pseudomonas aeruginosa ATCC 27853 good to excellent
contaminating microflora in the samples tested, Pseudomonas putida ATCC 12633 good
favoring the growth of Pseudomonas. More Proteus vulgaris ATCC 13315 partially inhibited
information can be found by consulting the technical Staphylococcus aureus ATCC 25923 inhibited
or catalogue sheets for CFC Agar.
Reconstitution
- Using aseptic techniques, fill the vial of lyophilisate - The expiration date is indicated on the vial.
with 5 mL of sterile distilled water. - Once reconstituted, the product can be stored for a
- Turn end-over-end to dissolve. Avoid frothing the maximum duration of 30 days at 2-8°C, shielded from
solution. light.
Composition Packaging
Per vial : - 10 vial pack : BS02208

- Cetrimide 5.0 mg
- Fucidin 5.0 mg
- Cephalosporin 25.0 mg
270
Chloramphenicol Selective Supplement
Intended Use Composition
Chloramphenicol Selective Supplement is Per vial :

recommended for improving the isolation of yeasts - Chloramphenicol 50.0 mg
and molds in OGA Agar or Sabouraud Agar. The
selective supplement is composed of the antibiotic Quality Control
chloramphenicol. Supplied as a lyophilized
preparation, it inhibits the development of most - Appearance : white lyophilisate, giving a clear
bacterial contaminants, thereby favoring the colorless solution after reconstitution.
isolation of yeasts and molds, particularly - Bacteriological efficacy in OGA Agar base after
cycloheximide-sensitive strains. When incubation 72 hours of incubation at 25°C :
temperature is higher than 25°C, it is preferable to
use chloramphenicol which under these conditions is Microorganisms Growth
more stable than oxytetracycline, which loses Candida albicans ATCC® 10231 good
antimicrobial activity over time. When used in Aspergillus niger ATCC 16404 good
Sabouraud Agar, chloramphenicol can also be Escherichia coli ATCC 25922 inhibited
supplemented with gentamicin, which improves
selectivity overall. Storage / Shelf Life
More information can be found by consulting the
catalogue or technical sheets for Sabouraud and OGA - Store between 2 and 8°C, shielded from light.
Agars. - The expiration date is indicated on the vial.
- Once reconstituted, the product can be stored for a
Reconstitution maximum duration of 30 days at 2-8°C, shielded
from light.
- Using aseptic techniques, fill the vial with 3 mL of
sterile distilled water, then with 2 mL of acetone. Packaging
solution. - 10 vial pack : BS02108
CIN Selective Supplement
The selective supplement for Yersinia Agar (CIN) is used Per vial :
with the base agar for the selective isolation of - Cefsulodin 7.50 mg
Yersinia enterocolitica from biological samples and - Novobiocin 1.25 mg
food products. The supplement contains an inhibitory
selective mixture composed of two antibiotics, Quality Control
cefsulodin and novobiocin. Supplied in the form of a
freeze-dried powder, and combined with irgasan, bile - Appearance : white lyophilisate, giving a colorless,
salts and crystal violet in the base medium, it inhibits very slightly opalescent solution after
the growth of Gram-positive bacteria and most Gram- reconstitution.
negative bacteria contaminating the samples tested, in - Bacteriological efficacy in Yersinia Agar (CIN) after
order to selectively favor cultures of Yersinia 48 hours of incubation at 30°C :
enterocolitica.
More information can be found by consulting the Microorganisms Growth Characteristics
technical or catalogue sheets for Yersinia Agar (CIN). Yersinia enterocolitica ATCC® 27729 good to excellent translucent colonies
with a dark pink center
Reconstitution Pseudomonas aeruginosa ATCC 27853 partially inhibited greenish colonies
- Using aseptic techniques, fill the vial of lyophilisate Proteus mirabilis ATCC 29906 inhibited
with 5 mL of sterile distilled water. Enterococcus faecalis ATCC 29212 inhibited
solution.
271
Storage / Shelf Life Packaging
- Store between 2 and 8°C, shielded from light. - 10 vial pack : BS02308
maximum duration of 30 days at 2-8°C, shielded from
light.
Coagulase Rabbit Plasma
Intended Use Results
Rabbit Plasma recovered over EDTA and freeze-dried is The reaction is considered positive when the clot
used for the detection of staphylocoagulase. Coagulase occupies 3/4 of the initial volume.
is an enzyme that can coagulate blood plasma and was Coagulation in principle occurs within less that 3
demonstrated for the first time by Loeb in 1903 in hours and most often the clot adheres to the tube
some staphylococci. Since that time, a number of walls. Coagulation sometimes occurs more slowly and
authors have tried to link coagulase production in in this case the reaction can be considered positive if
Staphylococcus aureus to its capacity to produce an a clot appears in less than 24 hours.
enterotoxin and thus to its pathogenic power. Today, As a control, add 0.1 mL of sterile Brain Heart Broth
the presence of a coagulase is considered to be the (BK015) to 0.3 mL of reconstituted Coagulase Rabbit
main factor in the determination of the pathogenicity Plasma : this control tube should show no sign of
of staphylococci. Nevertheless, it is not rare to isolate coagulation after 24 hours of incubation.
coagulase-negative strains with proven pathogenicity.
Staphylococcus aureus produces two types of coagulase : Composition
- free extracellular coagulase that reacts with
plasma prothrombin; Per vial :
- bound coagulase, localized in the bacterial wall, - Rabbit Plasma, EDTA, lyophilised.
that reacts with plasma fibrinogen to produce
a clot. Quality Control
In tests done in tubes, free coagulase reacts primarily
by forming a clot in plasma, indicating a positive - Appearance : light beige lyophilisate giving an
reaction. In the case of coagulase-negative amber yellow solution after reconstitution,
staphylococci, it is recommended to screen for the opalescent.
presence of other enzymes such as phosphatase or - Coagulation test in Brain Heart Broth after
deoxyribonuclease, that are also indicators of 24 hours of incubation at 37°C :
pathogenicity.
More information can be found by consulting the Microorganisms Coagulation
catalogue or technical sheets for Baird Parker Agar. Staphylococcus aureus ATCC® 6538P positive
Staphylococcus aureus ATCC 25923 positive
Instruction for Use Staphylococcus epidermidis ATCC 12228 negative
Escherichia coli ATCC 25922 negative
with 6 mL of sterile distilled water. Storage / Shelf Life
solution. - Store between 2 and 8°C, protected from light.
- Add successively into a serological tube : - The expiration date is indicated on the vial.
- 0.3 mL of reconstituted Coagulase Rabbit - Once reconstituted, the product can be stored
Plasma. for a maximum duration of 30 days at 2-8°C,
- 0.1 mL of the 24 hour staphylococcal culture in shielded from light.
Brain Heart Broth (BK015).
- Mix well. Packaging
- Incubate at 37°C in a thermostated water bath.
- 10 vial pack (20 tests per vial) : BR00208
272
D-Cycloserine Selective Supplement
D-Cycloserine Selective Supplement is used with base - Appearance : white lyophilisate, giving a clear
medium for the enumeration of Clostridium colorless solution after reconstitution.
perfringens in samples that may contain it. The - Bacteriological efficacy in TSC Agar after 24 hours
selective supplement contains an antibiotic : of anaerobic incubation at 37°C :
D-cycloserine. Supplied as a freeze-dried preparation,
it inhibits associated contaminating flora in order to Microorganisms Growth Characteristics
favor the enumeration of Clostridium perfringens, Clostridium perfringens ATCC® 13124 good to excellent black colonies
while lowering the dimension of black halos around Bacillus cereus ATCC 11778 inhibited
the colonies. Escherichia coli ATCC 25922 inhibited
technical or catalogue sheets for TSC Agar. Storage / Shelf Life
Reconstitution - Store between 2 and 8°C, shielded from light.

- Using aseptic techniques, fill the vial of lyophilisate - Once reconstituted, the product can be stored for a
with 5 mL of sterile distilled water. maximum duration of 30 days at 2-8°C, shielded
- Turn end-over-end to dissolve. Avoid frothing the from light.
solution.
Packaging
Composition
Per vial :
- D-cycloserine 200 mg
Egg Yolk Enrichment
Egg Yolk Enrichment is used as an additive in the - Egg yolk emulsion at 50% 50 mL
preparation of numerous nutrient bases for the
growth and identification of Clostridium, Bacillus and Quality Control
Staphylococcus as a result of the presence of a
lecithinase in these bacteria. It is used in Mossel's - Appearance : yellowish opaque emulsion
medium for the enumeration of Bacillus cereus, in TSC containing a precipitate that can be resuspended.
medium for Clostridium perfringens or in Baird-Parker - Bacteriological efficacy in Bacillus cereus Agar after
Agar for pathogenic staphylococci. van Netten et al. 24 hours of incubation at 30°C :
also used egg yolk emulsion in PALCAMY medium for
the culture of Listeria. Microorganisms Growth Characteristics
- The presence of lecithinase (in bacteria producing Colony color Lecithinase
the enzyme) is demonstrated through the opacity Bacillus cereus ATCC® 11778 good pinkish positive
which the enrichment imparts on nutrient broths. Bacillus cereus ATCC 14579 good pinkish positive
In agar medium, colonies are surrounded by an
opaque zone. Storage / Shelf Life
- When grown in Baird-Parker Agar, pathogenic
staphylococci form colonies surrounded by a zone - Store between 2 and 8°C, shielded from light.
of opacity which develops inside the lightened - The expiration date is indicated on the bottle.
halo, whose formation is due to egg yolk
proteolysis. Packaging
- The opacity of medium supplemented with egg
yolk results from the breakdown of lecithin into - 50 mL vial : BS00208
insoluble products by lecithinase produced by
certain bacteria.
catalogue or technical sheets for Baird Parker Agar,
Bacillus cereus Agar or TSC Agar.
273
Egg Yolk Tellurite Enrichment
Intended Use Microorganisms Growth Characteristics

Colony color Clear zones
Egg Yolk Tellurite Enrichment is a sterile preparation Staphylococcus aureus ATCC® 6538 good to black present
used in Baird-Parker Agar for the detection and excellent
enumeration of Staphylococcus aureus. Staphylococcus aureus ATCC 25923 good to black present
More information can be found by consulting the excellent
technical or catalogue sheets for Baird Parker Agar. Escherichia coli ATCC 25922 inhibited
Composition Storage / Shelf Life
For 50 mL of enrichment : - Store between 2 and 8°C, shielded from light.

- Egg yolk emulsion at 50% 47 mL - The expiration date is indicated on the bottle.
- Potassium tellurite at 3.5% 3 mL
Packaging
Quality Control
- 50 mL vial : BS00108
- Appearance : yellowish opaque emulsion
containing a resuspendable precipitate.
- Bacteriological efficacy in Baird-Parker Agar after
48 hours of incubation at 37°C :
Fraser Selective Supplement
Fraser Selective Supplement is intended to be used with - Appearance : brown lyophilisate giving a brown
Fraser Broth (base II) as secondary enrichment medium solution after reconstitution, that may contain a
for the detection of Listeria monocytogenes in food precipitate.
products that may contain it. The supplement contains - Bacteriological efficacy of Fraser Broth after
an inhibitory mixture composed of two antimicrobial 24 hours of incubation at 37°C, then subcultured
agents (nalidixic acid and acriflavine) and one reagent, onto PALCAM Agar :
ferric ammonium citrate. Supplied as a freeze-dried
powder, it enables a part of contaminating microflora Microorganisms Growth
to be inhibited, thereby favoring the growth of Listeria. Listeria monocytogenes ATCC® 19115 good to excellent
More information can be found by consulting the (with blackening)
technical or catalogue sheets for Fraser Broth. Listeria monocytogenes ATCC 33152 good to excellent
(with blackening)
Reconstitution Escherichia coli ATCC 25922 inhibited
with 5 mL of a 1:1 solution ethanol / sterile distilled Storage / Shelf Life
water.
- Turn end-over-end to dissolve. Avoid frothing the - Store between 2 and 8°C, shielded from light.
solution. - The expiration date is indicated on the vial.
Composition maximum duration of 30 days at 2-8°C, shielded from
light.
Per vial :
- Nalidixic acid 10.0 mg Packaging
- Acriflavine (hydrochloride) 12.5 mg
- Ferric ammonium citrate 250.0 mg - 10 vial pack : BS03108
274
Gentamicin Selective Supplement
Gentamicin Selective Supplement is recommended to Per vial :

improve the isolation of yeasts and pathogenic fungi - Gentamicin sulfate 25 mg
in clinical samples as well as for the enumeration of
yeasts and molds in food products, particularly in Quality Control
meats and raw seafood. The selective supplement is
composed of the antibiotic gentamicin. Supplied as a - Appearance : white lyophilisate, giving a clear
freeze-dried preparation, it inhibits the development colorless solution after reconstitution.
of most Gram-positive and Gram-negative bacteria - Bacteriological efficacy on Chloramphenicol
(enterobacteria, staphylococci, Pseudomonas), Glucose Agar after 72 hours of incubation at 25°C :
thereby favoring the isolation of yeasts and molds in
Chloramphenicol Glucose Agar or Oxytetracycline Microorganisms Growth
Glucose Agar (for meat and meat products). Candida albicans ATCC® 10231 good
Sabouraud Agar with gentamicin can also be Aspergillus niger ATCC 16404 good
supplemented with chloramphenicol to improve Pseudomonas aeruginosa ATCC 9027 inhibited
selectivity.
More information can be found by consulting the Storage / Shelf Life
technical or catalogue sheets for Sabouraud Agar,
OGA or Chloramphenicol Glucose Agar. - Store between 2 and 8°C, shielded from light.
Reconstitution - Once reconstituted, the product can be stored for a
maximum of 30 days at 2-8°C, shielded from light.
with 5 mL of sterile distilled water. Packaging
Half-Fraser Selective Supplement
Intended Use Freeze-dried supplement for 2.25 liters, BS032 :

Half-Fraser Selective Supplement is intended to be with 20 mL of a 1:1 solution ethanol/sterile distilled
used with Fraser Broth (base II) as primary enrichment water.
medium for the detection of Listeria monocytogenes in - Turn end-over-end to dissolve. Avoid frothing the
food products that may contain it. The supplement solution.
contains an inhibitory mixture composed of two - Aseptically add 2.0 mL of the reconstituted selective
antimicrobial agents (nalidixic acid and acriflavine) and supplement to 225 mL of Fraser Broth (base II,
one reagent : ferric ammonium citrate. Supplied as a BK133), previously autoclaved and cooled to 25°C.
freeze-dried powder, it enables a part of
contaminating microflora to be inhibited, thereby Composition
favoring the growth of Listeria.
More information can be found by consulting the Freeze-dried supplement for 500 mL, BS030 :
technical or catalogue sheets for Half-Fraser Broth. Per vial :
- Nalidixic acid 5.00 mg
Reconstitution - Acriflavine hydrochloride 6.25 mg
- Ferric ammonium citrate 250.00 mg
Freeze-dried supplement for 500 mL, BS030 :
- Using aseptic techniques, fill the vial of lyophilisate Freeze-dried supplement for 2.25 liters, BS032 :
with 5 mL of a 1:1 solution ethanol/sterile distilled Per vial :
water. - Nalidixic acid 22.5 mg
- Turn end-over-end to dissolve. Avoid frothing the - Acriflavine hydrochloride 28.125 mg
solution. - Ferric ammonium citrate 1.125 g
- Aseptically add 2.25 mL of the reconstituted
selective supplement to 225 mL of Fraser Broth
(base II, BK133), previously autoclaved and cooled
to 25°C.
275
Quality Control Storage / Shelf Life
- Appearance : brown lyophilisate giving a brown - Store between 2 and 8°C, shielded from light.
solution after reconstitution, that may contain a - The expiration date is indicated on the vial.
precipitate. - Once reconstituted, the product can be stored for a
- Bacteriological efficacy of Half-Fraser Broth after 24 maximum duration of 30 days at 2-8°C, shielded from
hours of incu-bation at 30°C, then subcultured onto light.
Oxford Agar :
Packaging
Listeria monocytogenes ATCC® 19115 good to excellent - 10 vial pack for 500 mL : BS03008
(with blackening) - 8 vial pack for 2.25 liters : BS03208
Listeria monocytogenes ATCC 33152 good to excellent
(with blackening)
Karmali Selective Supplement
Karmali Selective Supplement is used with Karmali Agar Per vial :

(base) for the selective isolation of Campylobacter in - Vancomycin 10.0 mg
food products and in other samples which may contain - Cefoperazone 16.0 mg
it. The work of Karmali et al. showed that a selective
charcoal medium could favorably replace blood media Quality Control
for the culture of Campylobacter jejuni and
Campylobacter coli. The selective supplement is - Appearance : white lyophilisate, giving a white,
prepared according to the original formula of Karmali slightly opalescent solution after reconstitution.
and is an inhibitory mixture composed of 2 antibiotics, - Bacteriological efficacy in Karmali Agar after
vancomycin and cefoperazone. Supplied as a freeze- 48 hours of incubation at 42°C :
dried preparation, it is used with Karmali Agar base, in
which cycloheximide limits the growth of certain fungi. Microorganisms Growth
Cefoperazone completes the spectrum of action of Campylobacter jejuni ATCC® 33291 good to excellent
vancomycin, leading to a more complete inhibition of Staphylococcus aureus ATCC 25923 inhibited
contaminating bacteria than that obtained with
cephazolin, originally included in selective charcoal Storage / Shelf Life
media.
More information can be found by consulting the - Store between 2 and 8°C, shielded from light.
technical or catalogue sheets for Karmali Agar. - The expiration date is indicated on the vial.
Reconstitution maximum duration of 30 days at 2-8°C, shielded from
light.
- Using aseptic techniques, fill the vial with 5 mL of
sterile distilled water. Packaging
276
MUG Supplement
Intended Use Results / Interpretation
MUG Supplement is used in selective media for the The blue-green fluorescence observed under UV light
detection and enumeration of coliform bacteria in at 366 nm results from glucuronidase activity, thereby
order to simultaneously detect Escherichia coli in constituting a presumptive test for the presence of
samples that may contain it : water, beverages, dairy Escherichia coli in the sample analyzed. Appropriate
products and other food products. It can be used in confirmation tests must be carried out.
both liquid media (Laurylsulfate-Tryptose Broth) and
in solid media (Violet Red Bile Agar). Feng and NOTES :
Hartman used a MUG medium to show that - It is recommended to determine fluorescence in
-glucuronidase activity involved 96% of Escherichia comparison to a reference.
coli, 100% of enterotoxin-producing Escherichia coli, - The presence of an endogenous glucuronidase in the
17% of salmonellas and 40% of shigellas, while the samples may cause false positives.
other bacteria tested were negative. When gas - Verify that the type of glass composing the tubes used for
production by Escherichia coli was inhibited by the liquid media does not affect the results. Alkali-lime
glass is unsuitable.
excessive numbers of Proteus in the samples, the
- In order to avoid false positives, the power of the UV
appearance of fluorescence led to identification in source should not exceed 6 watts.
less than 15 hours. The supplement is a freeze-dried
reagent composed of 4-methylumbelliferyl--D-
Composition
glucuronide (MUG). Escherichia coli possesses a
-D-glucuronidase and hydrolyzes MUG to 4-methyl-
Per vial :
umbelliferone and its corresponding glucuronide.
4-methylumbelliferone is a compound with blue - 4-methylumbelliferyl-ß-D-glucuronide 50 mg
fluorescence, that can be observed by illuminating
with a short-wave UV lamp at 366 nm. Quality Control
technical or catalogue sheets for MacConkey Agar, - Appearance : white lyophilisate, giving a colorless
VRBL Agar, VRBG Agar, Laurylsulfate-Tryptose Broth solution after reconstitution.
or BGBB. - Bacteriological efficacy in Laurylsulfate-Tryptose
Broth after 24 hours of incubation at 37°C :
Microorganisms Growth Gas Fluorescence
- Using aseptic techniques, fill the vial of lyophilisate production
with 5 mL of sterile distilled water. Escherichia coli ATCC® 25922 good to excellent positive positive
- Turn end-over-end to dissolve. Avoid frothing the Enterobacter aerogenes ATCC 13048 good to excellent positive negative
solution. Enterococcus faecalis ATCC 29212 partially inhibited negative negative
- When the appropriate dehydrated medium is
rehydrated, add 1.0 mL of the reconstituted Storage / Shelf Life
supplement to 100 mL of solid media :
- MacConkey Agar - Store between 2 and 8°C, shielded from light.
- VRBL Agar - The expiration date is indicated on the vial.
- VRBG Agar - Once reconstituted, the product can be stored for a
- Alternatively, add 1.0 mL to 200 mL of liquid (broth) maximum duration of 30 days at 2-8°C, shielded
media :
from light.
- Laurylsulfate-Tryptose Broth
- Brilliant Green Bile Broth.
Packaging
- Mix thoroughly to ensure that the preparation is
homogenized. - 10 vial pack : BS02408
- Dispense the media in recipients adapted to the
planned use (after bringing the agar media to
boiling) and follow the instructions in the
corresponding monograph.
Bibliography
(1) Kilian, M., and Bülow, P. 1976. Rapid diagnosis of (4) NF ISO 11866-2. Sept. 1997. Milk and milk products.
enterobacteriaceae I. Detection of Bacterial Glycosidases. Acta. Enumeration of presumptive Escherichia coli. Part 2: Most
path. microbiol. Scand. Sect. B, 84: 245-251. probable number technique using 4-methylumbelliferyl--D-
(2) Feng, C.S., and Hartman, P.A. 1982. Fluorogenic Assays for glucuronide (MUG).
Immediate Confirmation of Escherichia coli. Appl. and Envir. (5) FIL-IDF 170A : 1999. Milk and milk products - Enumeration
Microb., 43 (6): 1320-1329. of presmptive Escherichia coli. Part 2. Most probable number
(3) Trepeta, R.W., and Edberg, S.C. 1984. Methylumbelliferyl-ß-D- techique using using 4-methylumbelliferyl--D-glucuronide
Glucuronide Based Medium for Rapid Isolation and Identification (MUG).
of Escherichia coli. Journ. of Clinical Microb., 19 (2): 172-174.
277
Novobiocin Selective Supplement
The Novobiocin Selective Supplement is used in - Appearance : white lyophilisate, giving a colorless
conjunction with Modified Semi-Solid Rappaport- and limpid solution after reconstitution.
Vassiliadis Medium (MSRV) for the isolation of - Bacteriological efficacy in MSRV Agar after 24
Salmonella. This supplement is also used in mEC and hours of incubation at 42°C :
mTSB Enrichment Broths for the detection of
Escherichia coli serotype O157:H7. The selective Microorganisms Growth Characteristics
supplement contains an antibiotic, novobiocin. Salmonella Typhimurium ATCC 14028 good to
®
Opaque halo of culture
Supplied as a freeze-dried preparation, this excellent of at least 30 mm in
supplement inhibits Gram-positive bacteria and diameter, centered around
the point of inoculation
Proteus.
Proteus vulgaris ATCC 13315 inhibited
technical or catalogue sheets for MSRV, mEC Broth, Proteus mirabilis ATCC 29906 inhibited
or mTSB. Proteus mirabilis ATCC 25933 inhibited
Reconstitution Storage / Shelf Life
- Using aseptic techniques, add 5 mL of sterile - Store between 2 and 8°C, shielded from light.
distilled water to the vial of lyophilisate. - The expiration date is indicated on the vial.
- Turn end-over-end to dissolve. Avoid frothing the - Once reconstituted, the product can be stored for a
solution. maximum duration of 30 days at 2-8°C, shielded
from light.
Composition
Packaging
Per vial :
- Novobiocin 10 mg - 10 vial pack : BS03308
Oxford Selective Supplement (CCCFA)
Oxford Selective Supplement (CCCFA) is used with Per vial :

Oxford Agar (base) for the detection and enumeration - Cycloheximide 200.0 mg
of Listeria. The supplement contains an inhibitory - Colistin sulfate 10.0 mg
mixture composed of 3 antibiotics (colistin, cefotetan - Cefotetan 1.0 mg
and fosfomycin), an antifungal agent (cycloheximide) - Fosfomycin 5.0 mg
and an antiseptic dye (acriflavine). Supplied as a freeze- - Acriflavine 2.5 mg
dried preparation, it inhibits almost all contaminating
microflora in samples tested, to favor the growth of Quality Control
Listeria monocytogenes. More information can be
found by consulting the technical or catalogue sheets - Appearance : yellow lyophilisate, giving a fluorescent
for Oxford Agar. yellow-green solution after reconstitution.
- Bacteriological efficacy in Oxford Agar after
Reconstitution 48 hours of incubation at 37°C :
- Using aseptic techniques, fill the vial of lyophilisate Microorganisms Growth Characteristics
with 5 mL of a 1:1 solution of ethanol/sterile Listeria monocytogenes ATCC® 19115 good to olive-green colonies
distilled water. excellent surrounded by a
- Turn end-over-end to dissolve. Avoid frothing the black halo
solution. Listeria monocytogenes ATCC 35152 good to olive-green colonies
excellent surrounded by a
black halo
Listeria innocua ATCC 33090 good olive-green colonies
surrounded by a
black halo
Staphylococcus aureus ATCC 25293 partially inhibited
278
- Store between 2 and 8°C, shielded from light. - 10 vial pack : BS00308
maximum duration of 7 days at 2-8°C, shielded from
light.
Oxytetracycline Selective Supplement
Oxytetracycline Selective Supplement is used with OGA - Appearance : yellow lyophilisate, giving a yellow,
base for the detection and enumeration of yeasts and limpid solution after reconstitution.
molds. The selective supplement is composed of the - Bacteriological efficacy in Oxytetracycline Glucose
antibiotic oxytetracycline. Supplied as a freeze-dried Agar after 72 hours of incubation at 25°C :
preparation, it inhibits most bacteria in order to favor
the development of yeasts and molds. Microorganisms Growth
More information can be found by consulting the Saccharomyces cerevisiae ATCC® 9763 good to excellent
technical or catalogue sheets for Oxytetracycline Aspergillus niger ATCC 16404 good to excellent
Glucose Agar. Escherichia coli ATCC 25922 inhibited
- Using aseptic techniques, fill the vial of lyophilisate - Store between 2 and 8°C, shielded from light.
with 5 mL of sterile distilled water. - The expiration date is indicated on the vial.
solution. maximum duration of 30 days at 2-8°C, shielded from
light. During this period, the solution may become
Composition opalescent to opaque. This has no influence on the
bacterial activity of the reagent.
Per vial :
- Oxytetracycline hydrochloride 50 mg Packaging
279
PALCAM Selective Supplement
PALCAM Selective Supplement is used with PALCAM Agar - Appearance : yellow lyophilisate, giving a clear
(base) for the detection and enumeration of Listeria. The yellow solution after reconstitution.
supplement contains an inhibitory mixture composed of - Bacteriological efficacy in PALCAM Agar after 48
antibiotics (polymyxin and ceftazidim) and an antiseptic hours of incubation at 37°C :
dye (acriflavine). Supplied as a freeze-dried preparation, it
inhibits almost all contaminating microflora in samples Microorganisms Growth Characteristics
tested, to favor the growth of Listeria monocytogenes. Listeria monocytogenes ATCC® 19115 good to olive-green colonies
More information can be found by consulting the excellent surrounded by
technical or catalogue sheets for PALCAM Agar. a black halo
Listeria monocytogenes ATCC 35152 good to olive-green colonies
Reconstitution excellent surrounded by
a black halo
- Using aseptic techniques, fill the vial of lyophilisate Listeria innocua ATCC 33090 good olive-green colonies
with 5 mL (BS004) or 25 mL (BS049) of sterile
surrounded by
distilled water.
a black halo
solution.
Composition
Freeze-dried supplement for 500 mL, BS004 :
Per vial : - Store between 2 and 8°C, shielded from light.
- Polymyxin B (sulfate) 5.0 mg - The expiration date is indicated on the vial.
- Ceftazidim 10.0 mg - Once reconstituted, the product can be stored for a
- Acriflavine 2.5 mg maximum duration of 30 days at 2-8°C, shielded from
light.
Freeze-dried supplement for 2.5 liters, BS049 :
Per vial : Packaging
- Polymyxin B (sulfate) 25.0 mg
- Ceftazidim 50.0 mg - 10 vial pack for 500 mL : BS00408
- Acriflavine 12.5 mg - 8 vial pack for 2.5 liters : BS04908
Polymyxin B Selective Supplement
Polymyxin B Selective Supplement is used with Mossel's - Appearance : white lyophilisate, giving a clear
medium (base) for the detection and enumeration of colorless solution after reconstitution.
Bacillus cereus. The selective supplement is composed of - Bacteriological efficacy in Bacillus cereus Agar after
polymyxin B. Supplied as a freeze-dried preparation, it 24 hours of incubation at 30°C :
inhibits almost all contaminating microflora in order to
favor the growth of Bacillus cereus.
Colony color Lecithinase
Bacillus cereus ATCC® 11778 good pinkish positive
technical or catalogue sheets for Bacillus cereus Agar
Bacillus cereus ATCC 14579 good pinkish positive
(Mossel).
Pseudomonas aeruginosa ATCC 9027 inhibited
Reconstitution
with 5 mL of sterile distilled water. - Store between 2 and 8°C, shielded from light.
- Turn end-over-end to dissolve. Avoid frothing the - The expiration date is indicated on the vial.
solution. - Once reconstituted, the product can be stored for a
maximum duration of 30 days at 2-8°C, shielded from light.
Composition
Packaging
Per vial :
- Polymyxin B sulfate 5 x 104 IU - 10 vial pack : BS00708
280
Rabbit Plasma Fibrinogen Supplement
Intended Use Rabbit Plasma Fibrinogen Supplement, BS038

(for 450 mL base) :
Rabbit Plasma Fibrinogen Supplement is a freeze-dried Per vial :
preparation intended to be incorporated into Baird- - Rabbit plasma, EDTA 12.5 mL
Parker Agar base. The resulting medium allows the - Bovine fibrinogen 2.5 g
simultaneous enumeration and confirmation of - Trypsin inhibitor 12.5 mg
coagulase positive staphylococci in a single operation. - Potassium tellurite 12.5 mg
Consequently, significant reductions in the time and
labor needed for the analysis of foods, dairy products Quality Control
or other samples may be achieved.
More information can be found by consulting the - Appearance : whitish lyophilisate, giving an amber
technical or catalogue sheets for Baird Parker RPF Agar. and opalescent solution after reconstitution.
- Bacteriological efficacy on Baird-Parker Agar after
Reconstitution 48 hours of incubation at 37°C :
Rabbit Plasma Fibrinogen Supplement, BS034 Microorganisms Growth Characteristics

(for 90 mL base) Colony color Fibrin halo
- Using aseptic techniques, fill the vial of lyophilisate Staphylococcus aureus ATCC® 6538 good to excellent black presence
with 10 mL of sterile distilled water, preheated to Staphylococcus aureus ATCC 25923 good to excellent black presence
37°C. Staphylococcus epidermidis ATCC 12228 slowed grey absence
- Turn end-over-end to dissolve. Avoid frothing the Bacillus subtilis ATCC 6633 inhibited
solution. Immediate dissolution is not always Escherichia coli ATCC 25922 inhibited
obtained. To accelerate the process, a mechanical
agitator (vortex) for tubes can be used to fully Storage / Shelf Life
dissolve the lyophilisate. The supplement should be
completely dissolved before adding to Baird Parker - Store between 2 and 8°C, shielded from light.
Agar base. - The expiration date is indicated on the vial.
- Once reconstituted, the product must not be stored
Rabbit Plasma Fibrinogen Supplement, BS038 for more than 3 hours at room temperature. Do not
(for 450 mL base) refrigerate or freeze.
with 50 mL of sterile distilled water, preheated to Packaging
37°C.
solution. The supplement should be completely
dissolved before adding to Baird Parker Agar base.
Composition
Rabbit Plasma Fibrinogen Supplement, BS034

(for 90 mL base) :
Per vial :
- Rabbit plasma, EDTA 2.5 mL
- Bovine fibrinogen 0.5 g
- Trypsin inhibitor 2.5 mg
- Potassium tellurite 2.5 mg
281
Ringer’s Solution (1/4 strength)
Intended Use Typical Composition

Ringer’s Solution 1/4 strength is a diluent used to
prepare dispersion solutions for the microbiological For 1 liter of medium :
control of water, milk, dairy and other food products. - Sodium chloride 2.250 g
It is also widely used to prepare serial tenfold - Potassium chloride 0.105 g
dilutions. - Calcium chloride 0.120 g
- Sodium hydrogen carbonate 0.050 g
Principles
Quality Control
Ringer’s Solution 1/4 strength is an isotonic solution
that prevents osmotic shock from occurring, when
- Dehydrated medium : white tablets.
bacteria are removed from their usual environment.
- Prepared medium : colorless solution, limpid.
- Typical cultures response after maintaining diluted
Preparation
for 30 minutes at 20°C, followed by inoculation in
Tryptone Soy Agar :
- Put one tablet (BR001) in 500 mL of distilled or
deionized water.
Escherichia coli ATCC® 25922 good
- Dispense in tubes (9 mL) or in vials (90 or 225 mL).
- Sterilize in an autoclave at 121°C for 15 minutes.
Instructions for Use Storage / Shelf Life
Preparation of dispersion solutions Tablets : 2-30°C.

- Cool to 25°C. - The expiration date is indicated on the label.
- Aseptically add 10 or 25 g of the product to analyze Prepared solution (benchmark value) :
to a tared flask containing 90 or 225 mL of the - Solutions in tubes or vials : 6 months at 2-8°C.
medium in order to prepare a 1/10 suspension
(or a solution). Packaging
Tablets :
Preparation of tenfold dilutions - Bottle of 100 pieces : BR00108
- Cool to 25°C.
- Add 1 mL of homogenized sample or stock
suspension to a tube containing 9 mL of diluent.
- Repeat this operation until the desired dilution
is obtained.
Bibliography
(1) NF T 90-413. October 1985. Testing water. Detection and (3) FIL-IDF 122 C: 1996. Milk and milk products. Preparation
enumeration of coliforms and thermotolerant coliforms. General of samples and dilutions for microbiological examination.
method by inoculation in liquid medium (MPN).
(2) ISO 8199:1988. Water quality. General guide to the
enumeration of micro-organisms by culture.
282
Rosolic Acid Selective Supplement
Rosolic Acid Selective Supplement is designed for use - Appearance : crystalline, red-brown powder, giving
with mFC Agar for the detection and enumeration of a dark red, limpid solution.
thermotolerant (or fecal) coliforms in water using the - Bacteriological efficacy in mFC Agar after 24 hours
membrane filtration technique. The selective of incubation at 44°C :
supplement is comprised of rosolic acid in powdered
form and functions as a part of the pH indicator system Microorganisms Growth Characteristics
present in the mFC formulation. Associated with aniline Escherichia coli ATCC® 25922 good to excellent blue colonies
blue, it imparts a blue coloration on thermotolerant Enterobacter cloacae ATCC 13047 good to excellent blue colonies
coliforms growing on the agar medium. Salmonella Typhimurium ATCC 14028 good colorless colonies
More information can be obtained by consulting the Staphylococcus aureus ATCC 25923 inhibited
technical or catalogue sheets for mFC Agar. Enterococcus faecalis ATCC 29212 inhibited
Dissolve 0.1 g of the selective supplement in 10 mL - Store between 2 and 30°C, shielded from light.
of a 0.2N NaOH solution. - The expiration date is indicated on the label.
Composition maximum duration of 15 days at 2-8°C, shielded from
light.
Per vial :
- Rosolic acid crystalline powder. Packaging
- 1 g vial : BS04008
Sulfamethazine Selective Supplement
Because of its inhibition of the growth of Proteus, - Appearance : whitish lyophilisate, giving a clear
Sulfamethazine Selective Supplement is used in Baird- and colorless solution after reconstitution.
Parker Agar to favor the detection and enumeration of - Bacteriological efficacy in Baird-Parker Agar after
Staphylococcus aureus in food products. The 24 hours of incubation at 37°C :
supplement is composed of the antibiotic
sulfamethazine. Supplied in the form of a freeze-dried Microorganisms Growth Characteristics
powder, it assures the total or partial inhibition of Staphylococcus aureus ATCC® 25923 good black colonies surrounded
Proteus in Baird Parker Agar and thereby prevents or by a clear halo
limits the invasion of the medium, in order to facilitate Proteus vulgaris ATCC 13315 limited black colonies without halo,
reading. without swarming
More information can be found by consulting the Proteus mirabilis ATCC 29906 limited black colonies without halo,
technical or catalogue sheets for Baird Parker Agar. without swarming
- Using aseptic techniques, fill the vial of lyophilisate - Store between 2 and 8°C, shielded from light.
with 5 mL of sterile distilled water. - The expiration date is indicated on the vial.
solution. maximum duration of 30 days at 2-8°C, shielded from
light.
Composition
Packaging
Per vial :
- Sulfamethazine 25 mg - 10 vial pack : BS02808
283
Tergitol 4 Selective Supplement
Intended Use
- Bacteriological efficacy in XLT4 Agar after 24 hours
Tergitol 4 Selective Supplement is used in XLT4 Agar of incubation at 37°C :
base for the selective isolation and differentiation of
Salmonella , with the exception of Salmonella Typhi Microorganisms Growth Characteristics
and Paratyphi. The supplement is a 26-28% solution of Salmonella Enteritidis ATCC® 13076 good to excellent red colonies with black center
an anionic detergent, 7-ethyl 2-methyl 4-undecyl Enterobacter aerogenes ATCC 13048 partially inhibited yellow colonies
sulfate, in a sodium salt form, of which the commercial Proteus mirabilis ATCC 29906 inhibited
name is Tergitol 4. Staphylococcus aureus ATCC 25923 inhibited
technical or catalogue sheets for XLT4 Agar. Storage / Shelf Life
Composition - Store between 2 and 25°C, shielded from light.

Per vial :
- Tergitol 4 50 mL Packaging
Quality Control - 50 mL vial : BS03908
- Appearance : white-yellowish solution, limpid.
Thayer-Martin Growth Supplement
Intended Use Reconstitution
Thayer-Martin Growth Supplement is used as a vitamin - Using aseptic techniques, fill the vial with all the
supplement for the culture of particularly fastidious rehydrating diluent.
microorganisms. It is recommended for use in Thayer- - Turn end-over-end to dissolve. Avoid frothing the
Martin Medium for the isolation of Neisseria and solution.
Haemophilus. The composition of Thayer-Martin
Growth Supplement is chemically defined. Supplied as a Composition
freeze-dried preparation, it is composed of a sterile
mixture of growth factors (vitamins, coenzymes, amino Per reconstituted vial :
acids, mineral salts and glucose). They cannot be - Cyanocobalamin 0.050 mg
autoclaved and so are added to the medium aseptically - Thiamine hydrochloride 0.015 mg
at the moment of use. - Cocarboxylase 0.500 mg
- Vitamins (thiamine, cyanocobalamin,
- Para-aminobenzoic acid 0.065 mg
cocarboxylase, para-aminobenzoic acid) are
- Adenine 5.000 mg
essential for the growth of bacteria with high
- Guanine hydrochloride 0.150 mg
requirements in terms of coenzyme synthesis.
- L-glutamine 50.000 mg
- Purine bases (adenine and guanine) are the
building blocks for nucleic acid synthesis. - Nicotinamide adenine dinucleotide 1.250 mg
- Glutamine is a growth factor specifically utilized by - L-cysteine hydrochloride 129.500 mg
certain strains of Neisseria gonorrhoeae. - L-cystine, 2HCl 5.500 mg
- NAD (nicotinamide adenine dinucleotide) is - Ferric nitrate, 9H20 0.100 mg
required by certain strains of Haemophilus. It - Glucose 1000.000 mg
participates principally in respiratory chains in most
microorganisms. Quality Control
- Cysteine and cystine are sources of organic sulfur
that can act on the redox potential of the medium, - Appearance : pink-white lyophilisate, giving a clear
have a protective effect towards certain toxic pinkish solution after reconstitution.
compounds, and also chelate certain metals. - Bacteriological efficacy in GC Agar after 48 hours
- Soluble iron, required by most bacteria, is supplied of incubation at 37°C :
in the form of ferric nitrate.
- Glucose is an energy source. Added after Microorganisms Growth
autoclaving, it retains all its properties since it does Neisseria meningitidis ATCC® 13090 good to excellent
not participate in Maillard reactions which would Streptococcus pneumoniae ATCC 6303 good to excellent
occur if it was sterilized. Streptococcus pyogenes ATCC 19615 good to excellent
technical or catalogue sheets for GC Agar.
284
- Store between 2 and 8°C, shielded from light. - 10 vial pack

- The expiration date is indicated on the vials (5 vials of lyophilisate + 5 vials of diluent) : BS01008
(lyophilisate and diluent).
maximum duration of 24 hours at 2-8°C, shielded
from light.
TTC Supplement 12.5 mg
TTC Supplement 12.5 mg is used with Tergitol 7 Agar Per vial :

for the identification and enumeration of coliform and - Triphenyltetrazolium chloride 12.5 mg
Escherichia coli in water samples, using the membrane
filtration method. The supplement is composed of Quality Control
2,3,5-triphenyltetrazolium chloride. Supplied in the
form of a freeze-dried powder, it is used to - Appearance : white lyophilisate, giving a colorless
differentiate between Escherichia coli and other solution after reconstitution.
bacteria that may grow and whose red color of the - Bacteriological efficacy in Tergitol 7 Agar
colonies is due to the reduction of TTC to insoluble (base acc. to NF EN ISO) after 24-48 hours
formazan. of incubation at 37°C :
technical sheets for Tergitol 7 Agar (acc. to NF EN ISO) Microorganisms Growth Characteristics
or Tergitol 7 Agar. Escherichia coli ATCC® 25922 good yellow-orange colonies
Salmonella Typhimurium ATCC 14028 good dark red colonies
Instruction for Use Shigella flexneri ATCC 29903 good red colonies
- Using aseptic techniques, fill the vial of lyophilisate Storage / Shelf Life
with 5 mL of sterile distilled water.
- Turn end-over-end to dissolve. Avoid frothing the - Store between 2 and 8°C, shielded from light.
solution. - The expiration date is indicated on the vial.
- Aseptically add 1.6 mL of the reconstituted - Once reconstituted, the product can be stored for a
supplement to 100 mL of Tergitol 7 Agar (base maximum duration of 30 days at 2-8°C, shielded from
BK038) autoclaved and cooled to 47°C or 1.0 mL of light.
reconstituted supplement to 100 mL of molten
Tergitol 7 Agar (base acc. to NF EN ISO ; BK 123), Packaging
autoclaved and cooled to 47°C.
- Mix well. - 10 vial pack : BS02608
- Pour into sterile Petri dishes (the agar layer should
be 5 mm thick).
285
TTC Supplement 50 mg
TTC supplement 50 mg is used with Slanetz and Bartley - Appearance : white lyophilisate, giving a colorless
Agar (base) or KF Streptococcus Agar (base) for the solution after reconstitution.
enumeration of enterococci in tap water, beverages, - Bacteriological efficacy in Slanetz and Bartley Agar
sewage and various biological products, using both the after 48 hours of incubation at 37°C :
membrane filtration technique and the classical
technique of enumeration in Petri dishes. The Microorganisms Growth Characteristics
supplement is composed of 2,3,5-triphenyltetrazolium Enterococcus faecalis ATCC® 29212 good to excellent pink to red-brown
chloride. Supplied in the form of a freeze-dried colonies
powder, it is used to indicate bacterial growth. Enterococcus faecalis ATCC 33186 good to excellent pink to red-brown
Enterococci reduce it to an insoluble formazan inside colonies
the cell. This reaction is manifested by the appearance
of red to maroon colonies. Storage / Shelf Life
technical or catalogue sheets for Slanetz and Bartley - Store between 2 and 8°C, shielded from light.
Agar or KF Streptococcus Agar. - The expiration date is indicated on the vial.
Reconstitution maximum duration of 30 days at 2-8°C, shielded from
light.
with 5 mL of sterile distilled water. Packaging
Composition
Per vial :
- Triphenyltetrazolium chloride 50 mg
286
Microplates
Microplate MUD/SF
Intended Use Principles
Microplate MUD/SF allows the practical identification - Each microtiter plate contains 96 wells (12 rows of 8
and enumeration of enterococci in water, according wells).
to standard NF EN ISO 7899-1. The use of microtiter - The substrate demonstrating the bacterial
plates, with wells containing a specifically developed enzymatic activity in question is MUD
medium, was conceived for use in the analysis of (4-methylumbelliferyl--D-glucoside). This component
several types of water, notably swimming and beach is incorporated in a culture media specifically
water in salt and fresh water, surface (still) water and designed for the detection of enterococci. The
post-treatment water. The method is applicable to culture medium is dehydrated and fixed to the
all water samples, including those rich in suspended bottom of the microtiter wells. Actual rehydration
matter. of the medium is achieved when the water sample
For the enumeration of enterococci, the microtiter itself is introduced into the wells. Enterococci
technique with MUD has been recognized as the eventually present in the sample inoculum,
most specific, precise, and rapid of all previously used hydrolyze the MUD into 4-methylumbelliferone and
methods. glucose. The production of 4-methylumbelliferone,
This technique represents an important evolution as indicated by a blue fluorescence, can be observed
regards to existing technologies for enterococci, with the aid of a UV lamp at 366 nm. Once reading
which among the indicator microorganisms of fecal of the wells has been performed, the number of
contamination, present a particular interest in water fluorescent wells is counted for each dilution. From
quality controls. an obtained characteristic number, a statistical
analysis, based on Poisson's law allows the
History calculation of enterococci in the analyzed sample.
- The media composition, with its high level of
Among the numerous methods used for the isolation peptone and galactose, allows excellent
and enumeration of Group D streptococci, GTC recuperation.
media, originally developed by Donnelly & Hartman - Polysorbate, monopotassium phosphate and sodium
in 1978 was very selective, but did not allow the hydrogenocarbonate increase the performance of
separation of fecal streptococci from non-fecal the medium.
streptococci. - Nalidixic acid blocks replication of DNA in bacteria
The use of fluorogenic substrates has attracted sensitive to it and thallium acetate inhibits nearly all
numerous studies for the identification of other contaminating microflora.
microorganisms via simple and rapid procedures. - Incubation at 44°C was studied in order to inhibit
In 1982, Littel & Hartman selected, from amongst the growth of the majority of contaminating
numerous fluorogenic substrates, the one that allowed microorganisms.
the characterization and differentiation of fecal
streptococci from other streptococci. They Instructions for Use
demonstrated that 4-methylumbelliferyl--D-glucoside
(MUD) was hydrolyzed by nearly all fecal streptococci In order to proceed with the analysis of samples from
with the exception of Streptococcus mitis. chlorinated, brominated or ozonized waters, it is
The microtiter technique, as well as the statistical necessary to add, by sterile methods, excess sodium
method of interpreting the results, was used and thiosulfate in the collecting container to neutralize
validated by Hernandez in 1988, with the aid of 5 the oxidents. In this way, total recuperation of the
partner laboratories for a comparative study microorganisms to be detected can be obtained.
between currently used methods and the new
miniaturized fluorogenic method, on sea water off Preparation and dilutions
the French coasts. The level of recovery was found to
be equal or superior to other methods in tubes or by - Mix the sample well in order to obtain a
membrane filtration. In particular, the miniaturized homogeneous repartition of the microorganisms.
method has shown a higher specificity than - Proceed with an initial 1 : 2 dilution by transferring
membrane filtration. This method can therefore be 9 mL of the sample in a tube containing 9 mL of diluent.
considered as a reliable means to detect enterococci - For fresh water, bathing water and other surface
in water. water, (salinity less than 30 g/Kg), use 9 mL
of the special microplate diluent (Synthetic
Sea Salt - BR003).
- For sea water with a salinity greater than
30 g/Kg (measured with the aid of a
refractometer), use sterile distilled water as a
diluent.
- Perform successive serial dilutions with the diluent
BR003. The number of dilutions to be inoculated is
a function of the level of contamination in the
288
tested sample. MPN (Most Probable Number) and CONFIDENCE
- The detection ranges for the number of INTERVAL CALCULATION
microorganisms present in each sample category are
listed in the following table : - The most probable number represents a statistical
estimation of the concentration of microorganisms in
Sample Number of Number of Detection range for a given sample. This estimation follows Poisson's Law.
type dilutions wells per dilution microorganisms The upper and lower limits correspond to a 95%
present (per 100 mL) confidence level.
Bathing / swimming 2 64 wells at 1:2 1.5.101 to 3.5.104
water 32 wells at 1:20 - In the case of bathing water where 2 successive
Standing fresh water 4 24 wells at 1:2 4.0.101 to 3.2.106 dilutions at 1:2 and 1:20 are inoculated into 64 and
24 wells at 1:20 32 wells, respectively, the following example
24 wells at 1:200 demonstrates the determination of the characteristic
24 wells at 1:2000 number, the MPN and its upper and lower limits.
Effluent and post- 6 16 wells at 1:2 6.0.101 to 6.7.108
treatment waters up to 16 wells 1 : 2 dilution : 25 positive wells out of 64
at 1.200000 1 : 20 dilution : 3 positive wells out of 32
The characteristic number is 25/3
Sample Inoculation
In referring to the statistical table furnished in
- Transfer the initial dilution into an appropriate Annex 1, the example 25/3 indicates :
sterile container.
- By using a multi-channel pipette (8 sterile tips), MPN : 524 enterococci / 100 mL
inoculate 200 L into each microtiter plate well. Lower limit : 360 enterococci / 100 mL
- In the same fashion, inoculate the subsequent Upper limit : 763 enterococci / 100 mL
dilutions (1:20, 1:200, 1:2000, etc.) by using a new
recipient and new sterile pipette tips. ➔ For all other cases, refer to the annexes A and B of
- Well inoculation should be performed with care in standard NF EN ISO 7899-1: March 1999. For standing
order to avoid cross-contamination. fresh water and effluent water, the statistical tables
- Cover each microtiter plate with a sterile adhesive can be furnished on demand.
cover furnished in the kit. This measure limits
dehydration of the media in the wells and protects Typical Composition
the plate from external contamination during the
incubation period. - MUD/SF medium
Each microtiter plate well is filled with 100 L of
Incubation medium, of which the formula can be adjusted to
obtain optimal performances :
Incubate the microtiter plates at 44°C for at least
36 hours, not exceeding a maximum of 72 hours. For 1 liter of medium :
- Tryptose 40.0 g
Reading - Galactose 2.0 g
- Polysorbate (Tween 80) 1.5 mL
- The wells showing a blue fluorescence under 366
nm UV light are considered positive.
- Sodium hydrogenocarbonate 4.0 g
- Reading may be performed after the minimal
- Nalidixic acid 0.25 g
period of incubation as the fluorescence does not
- Thallium acetate 2.0 g
diminish over time.
- 2,3,5 triphenyltetrazolium chloride (TTC) 0.1 g
- The transparent microtiter plates (BT004) are
specifically adapted to automated reading by - MUD 0.15 g
various appropriate systems.
- The opaque microtiter plates (BT003) were pH : 7.5 ± 0.2
developed for a visual reading and manual
counting. Depending on the microtiter plate reader, - Synthetic Sea Salt
it may also be possible to use these plates with The diluent is composed of Synthetic Sea Salt
those systems. (BR003), presented in dehydrated form.
The reconstitution of this reagent is as follows :
Results
- Dissolve 22.5 g of synthetic sea salt in 1 liter of
- Determine the characteristic number from the distilled or deionized water.
number of positive wells for each of the chosen - Mix slowly until complete dissolution.
dilutions. - Distribute into 20 x 200 mm tubes ; 9 mL per tube.
- In the event that more than 3 dilutions are - Sterilize in an autoclave at 121°C for 15 minutes.
inoculated, a characteristic number comprised of 3
numbers (if possible ending in 0) should be retained.
289
Quality Control (following NF EN ISO 7899-1: Presentation
March 1999, annex E) :
- Kit containing 25 opaque microtiter plates
- Background less than 25% of the positive threshold. (white) + packet of 25 sterile transparent adhesive
- Fluorescence more than twice the positive covers : BT00308
threshold. - Kit containing 25 transparent microtiter plates
- Fertility: 0.66 to 1.5 times the target value. + packet of 25 sterile transparent adhesive
- Interfering microorganisms : fluorescence less than covers : BT00408
25% of the positive threshold; absence of turbidity - Synthetic Sea Salt, dehydrated (100 g) : BR00308
or formazan precipitate.
Microtiter plates : 2-8°C.

Synthetic Sea Salt : 2-30°C.
Bibliography
(1) Cochran, W.G. 1950. "Estimation of bacterial densities by (6) Littel, K.J.,and Hartman P.A. 1983. "Fluorogenic Selective and
means of the Most Probable Number." Biometrics, 6: 105-116. Differential Medium for Isolation of Fecal Streptococci." Appl.
(2) Fung, D.Y.C., and Kraft, A.A. 1969. "Rapid evaluation of viable and Environ. Microb., 45 (2): 622-627.
cell counts by using the microtiter system and MPN (7) Hernandez J.F., Guibert J.M., Delattre J.M., Oger C., Charrière
technique." J. of Milk and Food Techn., 32 (10): 408-409. C., Hughes B., Serceau R., and Sinègre F. 1991. "Miniaturized
(3) Hawksworth, G., Drasar, B.S., and Hill, M.J. 1971. "Intestinal fluorogenic assays for enumeration of E.coli and enterococci in
bacteria and the hydrolysis of glycosidic bonds." J. of Med. marine water." Wat. Sci. Tech., 24 (2): 137-141.
Microb., 4: 451-459. (8) NF EN ISO 7899-1. March 1999. Water quality - Detection and
(4) de Man, J.C. 1975. "The Probability of Most Probable enumeration of intestinal enterococci in surface and waste
Numbers." Europ J. Appl. Microbiol., 1: 67-78. water - Part 1: Miniaturized method (Most Probable Number)
(5) Hurley, M.A., and Roscoe, M.E. 1983. "Automated statistical by inoculation in liquid medium.
analysis of microbial enumeration by dilution series." J. of
Appl. Bact., 55: 159-164.
Microplate MUG/EC
Intended Use History
Microplate MUG/EC allows the practical identification Buehler et al, in 1949, were the first to reveal the
and enumeration of Escherichia coli in water, according presence of a -D-glucuronidase activity in
to ISO standard NF EN ISO 9308-3. The use of microtiter Escherichia coli. In 1976, Kilian and Bülow
plates, with wells containing a specifically developed demonstrated this enzymatic activity characterized
medium, was conceived for use in the analysis of only the genera Escherichia, Shigella and Salmonella.
several types of water, notably swimming and beach From these observations, Feng and Hartman, in 1982,
water in salt and fresh water, surface (still) water and developed procedures for the specific detection of
post-treatment water. The method is applicable to all Escherichia coli in water and food products.
water samples, including those rich in suspended Following this, Trepeta and Edberg incorporated
matter. For the enumeration of Escherichia coli, the MUG in a culture medium in order to detect the
microtiter technique with MUG has been recognized as presence of -glucuronidase. The majority of studies
the most specific, precise, and rapid of all previously have shown that from 94 to 97 % of Escherichia coli
used methods. of human or environmental origin possess this
This technique represents an important evolution as activity. Recent data also shows that this enzyme can
regards to existing technologies for Escherichia coli be detected in certain species of Citrobacter,
testing. By virtue of its role as an indicator Enterobacter, Klebsiella, Salmonella, Shigella and
microorganism of fecal contamination, this bacteria is Yersinia, but its presence only concerns a limited
of special interest due to its exclusively intestinal number of strains in each of the aforementioned
origin, which confers on it a more particular status as genera.
a priority bioindicator in water quality controls.
290
The microtiter technique, as well as the statistical Preparation and dilutions
method of interpreting the results, was used and
validated by Hernandez in 1988, with the aid of - Mix the sample well in order to obtain a
5 partner laboratories for a comparative study homogeneous repartition of the microorganisms.
between currently used methods and the new - Proceed with an initial 1:2 dilution by transferring
miniaturized fluorogenic method, on sea water off 9 mL of the sample in a tube containing 9 mL of
the French coasts. The level of recovery was found to diluent.
be equal or superior to other methods in tubes or by - For fresh water, bathing water and other surface
membrane filtration. In particular, the miniaturized water (salinity less than 30 g/Kg), use 9 mL of the
method has shown a higher specificity than special microplate diluent (Synthetic Sea Salt -
membrane filtration. BR003).
-D-glucuronidase can therefore be considered as a - For sea water with a salinity greater than 30g/Kg
reliable indicator for the detection of Escherichia coli (measured with the aid of a refractometer), use
in water. sterile distilled water as a diluent.
- Perform successive serial dilutions with the diluent
Principles BR003. The number of dilutions to be inoculated is
a function of the level of contamination in the
- Each microplate contains 96 wells (12 rows of 8 tested sample.
wells). - The detection ranges for the number of
- The substrate demonstrating the bacterial microorganisms present in each sample category are
enzymatic activity in question is MUG listed in the following table:
(4-methylumbelliferyl--D-glucuronide). This
component is incorporated in a culture medium Sample Number of Number of Detection range for
derived from A1 Medium cited by the American type dilutions wells per dilution microorganisms
Public Health Association for the determination of present (per 100 mL)
the presence of fecal coliforms in water. The culture Bathing / swimming 2 64 wells at 1:2 1.5.101 to 3.5.104
medium is dehydrated and fixed to the bottom of water 32 wells at 1:20
the microtiter wells. Actual rehydration of the Standing fresh water 4 24 wells at 1:2 4.0.101 to 3.2.106
medium is achieved when the water sample itself is 24 wells at 1:20
introduced into the wells. Escherichia coli, 24 wells at 1:200
eventually present in the sample inoculum 24 wells at 1:2000
hydrolyses the MUG into 4-methylumbelliferone Effluent and post- 6 16 wells at 1:2 6.0.101 to 6.7.108
and its glucuronide constituent. The production of treatment waters up to 16 wells
4-methylumbelliferone, indicated by a blue at 1.200000
fluorescence, can be observed with the aid of a UV
lamp at 366 nm. Once reading of the wells has been Sample Inoculation
performed, the number of fluorescent wells is
counted for each dilution. From an obtained - Transfer the initial dilution into an appropriate
characteristic number, a statistical analysis, based on sterile container.
Poisson's law allows the calculation of Escherichia - By using a multi-channel pipette (8 sterile tips),
coli in the analyzed sample. It is important to note inoculate 200 L into each microtiter plate well.
that certain strains of Escherichia coli do not possess - In the same fashion, inoculate the subsequent
-D-glucuronidase, in particular the dilutions (1:20, 1:200, 1:2000, etc.) by using a new
enterohemorrhagic strain O157:H7. recipient and new sterile pipette tips.
- The media composition, with its high level of - Well inoculation should be performed with care in
peptone and salicin, allows excellent recuperation. order to avoid cross-contamination.
- Triton X favors microorganism and fluorogen - Cover each microtiter plate with a sterile adhesive
dispersion in the wells of the microtiter plate. cover furnished in the kit. This measure limits
- Incubation at 44°C was studied in order to inhibit dehydration of the media in the wells and protects
the growth of the majority of contaminating the plate from external contamination during the
microorganisms. incubation period.
Instructions for Use Incubation
In order to proceed with the analysis of samples from Incubate the microtiter plates at 44°C for at least 36
chlorinated, brominated or ozonized waters, it is hours, not exceeding a maximum of 72 hours.
necessary to add, by sterile methods, excess sodium
thiosulfate in the collecting container to neutralize Reading
the oxidents. In this way, total recuperation of the
microorganisms to be detected can be obtained. - The wells showing a blue fluorescence under
366 nm UV light are considered positive.
- Reading may be performed after the minimal
period of incubation as the fluorescence does not
diminish over time.
291
- The transparent microtiter plates (BT002) are Typical Composition
specifically adapted to automated reading by
various appropriate systems. - MUG/EC medium
- The opaque microtiter plates (BT001) were Each microtiter plate well is filled with 100 L
developed for a visual reading and manual of medium, of which the formula can be adjusted
counting. Depending on the microtiter plate reader, to obtain optimal performances :
it may also be possible to use these plates with
those systems. For 1 liter of medium :
- Tryptone 40.0 g
Results - Salicine 1.0 g
- Triton X 100 1.0 g
- Determine the characteristic number from the - MUG 0.1 g
number of positive wells for each of the chosen
dilutions. pH : 6.9 ± 0.2.
- In the event that more than 3 dilutions are
inoculated, a characteristic number comprised of 3 - Synthetic Sea Salt
numbers (if possible ending in 0) should be retained. The diluent is composed of Synthetic Sea Salt
(BR003), presented in dehydrated form.
MPN (Most Probable Number) and CONFIDENCE The reconstitution of this reagent is as follows :
INTERVAL CALCULATION - Dissolve 22.5 g of synthetic sea salt in 1 liter of
distilled or demineralized water.
- The most probable number represents a statistical - Mix slowly until complete dissolution.
estimation of the concentration of microorganisms in - Distribute into 20 x 200 mm tubes ; 9 mL per tube.
a given sample. This estimation follows Poisson's Law. - Sterilize in an autoclave at 121°C for 15 minutes.
The upper and lower limits correspond to a 95%
confidence level. Quality Control (following NF EN ISO 9308-3 :
- In the case of bathing water where 2 successive March 1999, annex E) :
dilutions at 1:2 and 1:20 are inoculated into 64 and
32 wells, respectively, the following example - Background less than 25% of the positive threshold.
demonstrates the determination of the characteristic - Fluorescence more than twice the positive
number, the MPN and its upper and lower limits. threshold.
- Fertility: 0.66 to 1.5 times the target value.
1 : 2 dilution : 25 positive wells out of 64
1 : 20 dilution : 3 positive wells out of 32 Storage / Shelf Life
The characteristic number is 25/3
Microtiter plates : 2-8°C.
In referring to the statistical table furnished in - The expiration date is indicated on the label.
Annex 1, the example 25/3 indicates : Synthetic Sea Salt : 2-30°C.
MPN : 524 Escherichia coli / 100 mL - The expiration date is indicated on the label.
Lower limit : 360 Escherichia coli / 100 mL
Upper limit : 763 Escherichia coli / 100 mL Presentation
➔ For all other cases, refer to the annexes A and B of - Kit containing 25 opaque microtiter plates
standard NF EN ISO 9308-3: March 1999. For standing (white) + packet of 25 sterile transparent
fresh water and effluent water, the statistical tables adhesive covers : BT00108
can be furnished on demand. - Kit containing 25 transparent microtiter plates
+ packet of 25 sterile transparent
adhesive covers : BT00208
- Synthetic Sea Salt, dehydrated (100 g) : BR00308
Bibliography
(1) Buehler, H.J., Katzman, P.A., and Doisey, E.A. 1949. "Bacterial (6) Hurley, M.A., and Roscoe, M.E. 1983. "Automated statistical
glucuronidase." Federation Proceedings, 8: 189. analysis of microbial enumeration by dilution series.
(2) Cochran, W.G. 1950. "Estimation of bacterial densities by means " J. of Appl. Bact., 55: 159-164.
of the Most Probable Number." Biometrics, 6: 105-116. (7) Trepeta R.W., and Edberg S.C. 1984. "Methylumbelliferyl--D-
(3) De Man, J.C. 1975. "The Probability of Most Probable Numbers." Glucuronide-Based Medium for Rapid Isolation and Identification
European J. Appl. Microbiol., 1: 67-78. of Escherichia coli." J. of Clin. l Microb., 19 (2): 172-174.
(4) Kilian, M., and Bülow, P. 1976. "Rapid diagnosis of Entero- (8) Hernandez J.F., Guibert J.M., Delattre J.M., Oger C., Charrière C.,
bacteriaceae." Acta. Path. Microbiol. Scand. Sect. B, 84: 245-251. Hughes B., Serceau R., and Sinègre F. 1991. "Miniaturized
(5) Feng, P.C.S. and Hartman P.A. 1982. "Fluorogenic assays for fluorogenic assays for enumeration of E. coli and enterococci in
immediate confirmation of Escherichia coli." App. Environ. marine water." Wat. Sci. Tech., 24 (2): 137-141.
Microbiol., 43(6): 1320-1329. (9) NF EN ISO 9308-3. March 1999. Water quality - Detection and
enumeration of Escherichia coli and coliform bacteria in surface
and waste water - Part 3: Miniaturized method (Most Probable
Number) by inoculation in liquid medium.
292
ANNEX 1
Enumeration : Escherichia coli (NF EN ISO 9308-3)

: Enterococci (NF EN ISO 7899-1)
Bathing water MPN : the microplate method
Microplate MUG/EC BT00108 (opaque) : Escherichia coli

Microplate MUG/EC BT00208 (transparent) : Escherichia coli
Microplate MUD/SF BT00308 (opaque) : Enterococci

Microplate MUD/SF BT00408 (transparent) : Enterococci
Example calculation
Characteristic number (CN) : 34 / 6
MPN = 841 / 100 mL
[ LL = 609 / 100mL UL = 1162 / 100mL ]
Confidence interval = 95 %
293
Bathing water :
MPN calculation in microplates
CN MPN LL UL CN MPN LL UL CN MPN LL UL CN MPN LL UL

0 1 15 2 106 8 0 127 63 253 14 4 302 189 482 20 2 390 256 594
0 2 30 7 120 8 1 143 74 275 14 5 319 202 504 20 3 408 270 617
0 3 45 14 140 8 2 159 85 296 14 6 336 215 526 20 4 426 284 640
0 4 60 22 161 8 3 175 96 317 14 7 353 228 548 20 5 445 298 664
1 0 15 2 106 8 4 191 108 339 15 0 253 152 419 20 6 463 312 687
1 1 30 7 121 8 5 207 119 360 15 1 270 165 441 20 7 482 327 710
1 2 45 14 141 8 6 223 131 381 15 2 287 178 463 20 8 500 340 734
1 3 60 22 162 9 0 144 75 276 15 3 304 191 485 21 0 375 244 575
1 4 75 31 183 9 1 160 86 298 15 4 322 204 507 21 1 393 258 598
1 5 90 40 204 9 2 176 97 319 15 5 339 217 529 21 2 412 272 622
2 0 30 8 121 9 3 192 109 340 15 6 356 230 552 21 3 430 287 645
2 1 45 15 141 9 4 209 120 362 15 7 374 243 574 21 4 449 301 669
2 2 61 23 162 9 5 225 132 383 16 0 272 167 444 21 5 467 316 692
2 3 76 31 183 9 6 241 144 404 16 1 289 180 466 21 6 486 330 716
2 4 91 40 204 10 0 161 87 299 16 2 307 193 488 21 7 505 345 739
2 5 106 50 225 10 1 177 98 321 16 3 324 206 511 21 8 524 359 763
3 0 46 15 142 10 2 194 110 342 16 4 342 219 533 22 0 397 261 603
3 1 61 23 163 10 3 210 121 364 16 5 359 232 555 22 1 415 275 626
3 2 76 32 184 10 4 226 133 385 16 6 377 246 578 22 2 434 290 650
3 3 92 41 205 10 5 243 145 406 16 7 394 259 600 22 3 453 304 674
3 4 107 50 226 10 6 259 157 428 17 0 292 181 469 22 4 472 319 697
3 5 122 60 247 11 0 179 99 323 17 1 309 194 492 22 5 490 334 721
4 0 61 23 163 11 1 195 111 344 17 2 327 208 514 22 6 509 348 745
4 1 77 32 185 11 2 212 123 366 17 3 344 221 537 22 7 528 363 769
4 2 92 41 206 11 3 228 134 387 17 4 362 235 559 22 8 547 378 793
4 3 108 51 227 11 4 245 147 409 17 5 380 248 582 23 0 419 278 631
4 4 123 61 248 11 5 261 159 430 17 6 398 262 604 23 1 438 293 655
4 5 139 71 269 11 6 278 171 452 17 7 416 276 627 23 2 457 307 679
5 0 77 32 186 11 7 295 184 473 17 8 433 289 649 23 3 476 322 703
5 1 93 42 207 12 0 197 112 346 18 0 312 196 495 23 4 495 337 727
5 2 108 51 228 12 1 213 124 368 18 1 330 210 518 23 5 514 352 751
5 3 124 62 250 12 2 230 136 390 18 2 347 223 540 23 6 533 367 775
5 4 140 72 271 12 3 247 148 411 18 3 365 237 563 23 7 553 382 799
5 5 155 83 292 12 4 263 160 433 18 4 383 251 586 23 8 572 397 823
5 6 171 94 312 12 5 280 173 454 18 5 401 264 603 23 9 591 412 848
6 0 94 42 208 12 6 297 185 476 18 6 419 278 631 24 0 442 296 660
6 1 109 52 230 12 7 314 198 498 18 7 437 292 654 24 1 461 311 684
6 2 125 62 251 13 0 215 125 370 18 8 455 306 677 24 2 480 326 708
6 3 141 73 272 13 1 232 137 392 19 0 332 212 521 24 3 500 341 733
6 4 156 84 293 13 2 249 149 414 19 1 350 226 544 24 4 519 356 757
6 5 172 94 314 13 3 266 162 436 19 2 368 239 567 24 5 538 371 781
6 6 188 106 335 13 4 282 174 457 19 3 386 253 590 24 6 558 386 806
7 0 110 52 231 13 5 299 187 479 19 4 405 267 613 24 7 577 401 830
7 1 126 63 252 13 6 316 200 501 19 5 423 281 636 24 8 597 417 855
7 2 142 73 273 13 7 333 213 523 19 6 441 295 659 24 9 617 432 880
7 3 158 84 295 14 0 234 138 394 19 7 459 309 682 25 0 465 314 689
7 4 174 95 316 14 1 251 151 416 19 8 478 323 705 25 1 485 329 714
7 5 189 107 337 14 2 268 163 438 20 0 353 228 548 25 2 504 344 738
7 6 205 118 358 14 3 285 176 460 20 1 371 242 571 25 3 524 360 763
64 and 32 wells - Dilutions at 1:2 and 1:20.
294
25 4 543 375 783 30 1 612 429 874 34 6 841 609 1162 38 7 1006 739 1370
25 5 563 390 812 30 2 633 445 901 34 7 865 628 1191 38 8 1032 759 1402
25 6 583 406 837 30 3 654 462 927 34 8 888 646 1220 38 9 1057 779 1434
25 7 603 421 862 30 4 676 479 954 34 9 911 664 1249 38 10 1083 799 1467
25 8 623 437 887 30 5 697 495 980 34 10 935 683 1279 38 11 1109 820 1500
25 9 643 453 912 30 6 718 512 1007 34 11 958 701 1309 38 12 1135 840 1533
26 0 489 333 720 30 7 740 529 1034 35 0 736 526 1029 39 0 868 631 1196
26 1 509 348 744 30 8 761 546 1061 35 1 759 544 1058 39 1 893 650 1227
26 2 529 363 769 30 9 783 563 1088 35 2 782 562 1086 39 2 918 670 1258
26 3 549 379 794 30 10 805 581 1116 35 3 805 580 1116 39 3 943 690 1290
26 4 568 395 819 31 0 619 434 882 35 4 828 599 1145 39 4 969 710 1322
26 5 588 410 844 31 1 640 451 909 35 5 851 617 1174 39 5 994 730 1354
26 6 609 426 869 31 2 661 467 936 35 6 875 636 1204 39 6 1020 750 1387
26 7 629 442 895 31 3 683 484 963 35 7 898 654 1233 39 7 1045 770 1419
26 8 649 458 920 31 4 704 501 990 35 8 922 673 1263 39 8 1071 790 1452
26 9 669 474 945 31 5 726 518 1017 35 9 946 692 1293 39 9 1097 811 1485
27 0 514 352 751 31 6 748 536 1044 35 10 970 710 1323 39 10 1123 831 1519
27 1 534 367 776 31 7 770 553 1071 35 11 994 729 1354 39 11 1150 852 1552
27 2 554 383 801 31 8 792 570 1099 36 0 767 551 1069 39 12 1176 873 1586
27 3 574 399 826 31 9 814 587 1127 36 1 791 569 1098 40 0 904 659 1241
27 4 594 415 851 31 10 836 605 1155 36 2 814 588 1127 40 1 930 679 1273
27 5 615 431 877 32 0 647 456 917 36 3 838 606 1157 40 2 955 699 1305
27 6 635 447 902 32 1 668 473 944 36 4 861 625 1187 40 3 981 719 1338
27 7 655 463 928 32 2 690 490 972 36 5 885 644 1217 40 4 1007 740 1371
27 8 676 479 954 32 3 712 507 999 36 6 909 663 1247 40 5 1033 760 1404
27 9 696 495 980 32 4 734 525 1027 36 7 933 682 1277 40 6 1059 781 1437
28 0 539 371 782 32 5 756 542 1054 36 8 957 701 1308 40 7 1086 802 1470
28 1 559 387 808 32 6 778 559 1082 36 9 982 720 1338 40 8 1112 822 1504
28 2 580 403 833 32 7 800 577 1110 36 10 1006 739 1369 40 9 1139 843 1538
28 3 600 419 859 32 8 823 595 1138 36 11 1031 758 1401 40 10 1166 864 1572
28 4 621 435 885 32 9 845 612 1166 37 0 800 577 1109 40 11 1193 885 1607
28 5 641 452 910 32 10 868 630 1195 37 1 824 595 1139 40 12 1220 907 1642
28 6 662 468 936 33 0 675 479 953 37 2 848 614 1169 41 0 942 689 1288
28 7 683 484 962 33 1 697 496 981 37 3 872 633 1200 41 1 968 709 1321
28 8 704 501 989 33 2 720 513 1009 37 4 896 652 1230 41 2 994 730 1354
28 9 725 517 1015 33 3 742 531 1037 37 5 920 671 1261 41 3 1020 750 1388
2810 746 534 1041 33 4 764 549 1065 37 6 945 691 1292 41 4 1047 771 1421
29 0 565 392 815 33 5 787 566 1093 37 7 969 710 1323 41 5 1074 792 1455
29 1 585 408 840 33 6 809 584 1121 37 8 994 730 1354 41 6 1100 813 1489
29 2 606 424 866 33 7 832 602 1150 37 9 1019 749 1385 41 7 1128 834 1524
29 3 627 440 892 33 8 855 620 1178 37 10 1044 769 1417 41 8 1155 856 1558
29 4 648 451 919 33 9 878 638 1207 37 11 1069 788 1449 41 9 1182 877 1593
29 5 669 473 945 33 10 901 656 1236 38 0 834 603 1152 41 10 1210 899 1629
29 6 690 490 971 34 0 705 502 991 38 1 858 622 1182 41 11 1238 920 1664
29 7 711 506 998 34 1 728 520 1019 38 2 882 642 1213 41 12 1266 942 1700
29 8 732 553 1024 34 2 750 537 1047 38 3 907 661 1244 42 0 981 719 1338
29 9 753 540 1051 34 3 773 555 1075 38 4 931 680 1275 42 1 1007 740 1371
2910 775 557 1078 34 4 796 573 1104 38 5 956 700 1307 42 2 1034 761 1405
30 0 591 412 848 34 5 818 591 1133 38 6 981 720 1338 42 3 1061 782 1439
295
55 0 1706 1279 2276 57 12 2544 1883 3437 60 1 2296 1711 3083 62 5 2994 2183 4107
55 1 1749 1312 2333 57 13 2608 1927 3529 60 2 2361 1756 3174 62 6 3093 2247 4260
55 2 1793 1344 2392 57 14 2672 1971 3624 60 3 2427 1803 3269 62 7 3197 2312 4421
55 3 1838 1378 2451 57 15 2739 2015 3723 60 4 2496 1850 3367 62 8 3306 2380 4593
55 4 1883 1411 2512 57 16 2807 2061 3824 60 5 2567 1899 3470 62 9 3421 2450 4777
55 5 1929 1445 2575 57 17 2877 2107 3929 60 6 2639 1948 3576 62 10 3543 2523 4973
55 6 1976 1480 2638 57 18 2949 2154 4038 60 7 2715 1999 3687 62 11 3671 2599 5184
55 7 2023 1514 2703 58 0 1988 1489 2656 60 8 2792 2051 3802 62 12 3806 2678 5410
55 8 2072 1550 2770 58 1 2041 1527 2728 60 9 2873 2104 3923 62 13 3951 2761 5653
55 9 2121 1585 2838 58 2 2095 1566 2802 60 10 2956 2158 4049 62 14 4104 2847 5916
5510 2172 1622 2908 58 3 2150 1606 2878 60 11 3042 2214 4181 62 15 4267 2937 6200
5511 2223 1658 2979 58 4 2206 1646 2956 60 12 3132 2271 4319 62 16 4442 3032 6508
5512 2275 1695 3053 58 5 2263 1687 3036 60 13 3225 2329 4465 62 17 4628 3132 6841
5513 2328 1733 3128 58 6 2322 1729 3119 60 14 3322 2389 4618 62 18 4828 3238 7201
5514 2382 1771 3204 58 7 2383 1771 3205 60 15 3422 2451 4779 62 19 5043 3351 7590
5515 2438 1810 3283 58 8 2444 1814 3293 60 16 3527 2514 4948 62 20 5273 3472 8009
5516 2494 1849 3364 58 9 2508 1858 3384 60 17 3636 2579 5127 62 21 5520 3602 8459
5517 2551 1888 3448 58 10 2573 1903 3479 60 18 3750 2646 5316 62 22 5784 3743 8939
56 0 1792 1343 2390 58 11 2640 1948 3576 60 19 3870 2715 5516 62 23 6068 3897 9448
56 1 1838 1378 2451 58 12 2708 1995 3677 60 20 3994 2785 5728 62 24 6371 4065 9985
56 2 1884 1412 2514 58 13 2779 2042 3782 60 21 4124 2858 5952 62 25 6694 4249 10545
56 3 1931 1447 2578 58 14 2851 2090 3890 61 0 2383 1772 3206 63 0 2773 2038 3773
56 4 1980 1483 2644 58 15 2926 2138 4003 61 1 2453 1821 3306 63 1 2867 2100 3914
56 5 2029 1518 2711 58 16 3002 2188 4120 61 2 2526 1871 3410 63 2 2966 2165 4064
56 6 2079 1555 2780 58 17 3082 2239 4241 61 3 2601 1922 3519 63 3 3071 2232 4224
56 7 2130 1592 2850 58 18 3163 2291 4368 61 4 2678 1975 3633 63 4 3181 2302 4395
56 8 2182 1629 2923 58 19 3247 2343 4499 61 5 2759 2029 3752 63 5 3297 2374 4579
56 9 2235 1667 2997 59 0 2104 1573 2814 61 6 2843 2084 3877 63 6 3421 2450 4776
5610 2290 1706 3073 59 1 2161 1614 2893 61 7 2929 2141 4008 63 7 3552 2529 4989
5611 2345 1745 3151 59 2 2219 1656 2974 61 8 3020 2199 4146 63 8 3693 2612 5220
5612 2401 1784 3232 59 3 2279 1698 3059 61 9 3114 2259 4291 63 9 3843 2699 5472
5613 2459 1825 3314 59 4 2341 1742 3146 61 10 3212 2321 4444 63 10 4005 2791 5746
5614 2518 1865 3399 59 5 2404 1786 3235 61 11 3315 2385 4606 63 11 4179 2888 6047
5615 2578 1907 3487 59 6 2469 1831 3329 61 12 3422 2451 4778 63 12 4368 2992 6377
5616 2640 1949 3577 59 7 2536 1878 3425 61 13 3534 2518 4960 63 13 4573 3102 6741
5617 2703 1991 3669 59 8 2604 1924 3525 61 14 3652 2588 5153 63 14 4796 3220 7142
5618 2767 2034 3765 59 9 2675 1972 3628 61 15 3776 2661 5359 63 15 5039 3349 7584
57 0 1885 1413 2516 59 10 2748 2021 3736 61 16 3906 2736 5578 63 16 5306 3489 8070
57 1 1934 1449 2582 59 11 2823 2071 3848 61 17 4044 2813 5812 63 17 5598 3643 8601
57 2 1984 1486 2650 59 12 2900 2122 3964 61 18 4188 2894 6063 63 18 5918 3815 9179
57 3 2035 1523 2719 59 13 2980 2174 4086 61 19 4341 2977 6330 63 19 6267 4007 9802
57 4 2087 1560 2790 59 14 3063 2227 4213 61 20 4503 3064 6616 63 20 6648 4223 10467
57 5 2140 1599 2864 59 15 3148 2281 4345 61 21 4674 3156 6921 63 21 7063 4466 11169
57 6 2194 1637 2939 59 16 3237 2337 4483 61 22 4854 3251 7247 63 22 7511 4738 11905
57 7 2249 1677 3016 59 17 3328 2393 4628 62 0 2559 1893 3459 63 23 7994 5042 12675
57 8 2305 1717 3095 59 18 3423 2451 4779 62 1 2639 1948 3575 63 24 8513 5378 13478
57 9 2363 1758 3177 59 19 3521 2510 4938 62 2 2722 2004 3698 63 25 9070 5746 14318
5710 2422 1799 3261 59 20 3623 2571 5105 62 3 2809 2062 3827 63 26 9666 6146 15203
5711 2482 1841 3348 60 0 2234 1666 2995 62 4 2900 2122 3963 63 27 10305 6579 16141
296
42 4 1089 804 1474 45 12 1471 1100 1965 49 2 1372 1025 1838 52 3 1599 1198 2134
42 5 1116 825 1509 45 13 1503 1125 2007 49 3 1406 1051 1881 52 4 1638 1228 2185
42 6 1143 847 1544 46 0 1154 855 1557 49 4 1440 1077 1925 52 5 1677 1257 2236
42 7 1171 869 1579 46 1 1183 878 1595 49 5 1474 1103 1970 52 6 1716 1287 2289
42 8 1199 890 1615 46 2 1213 901 1633 49 6 1509 1130 2015 52 7 1756 1317 2342
42 9 1227 912 1651 46 3 1244 925 1672 49 7 1544 1156 2061 52 8 1797 1347 2397
4210 1256 935 1688 46 4 1274 949 1711 49 8 1579 1183 2108 52 9 1838 1378 2452
4211 1284 957 1725 46 5 1305 973 1751 49 9 1615 1210 2155 52 10 1880 1409 2508
4212 1313 979 1762 46 6 1336 997 1791 49 10 1651 1238 2203 52 11 1922 1440 2566
43 0 1021 751 1389 46 7 1368 1021 1832 49 11 1688 1265 2251 52 12 1965 1472 2624
43 1 1049 773 1423 46 8 1399 1045 1873 49 12 1725 1293 2300 52 13 2009 1504 2683
43 2 1076 794 1458 46 9 1431 1070 1914 49 13 1762 1321 2350 52 14 2053 1536 2744
43 3 1104 816 1494 46 10 1463 1095 1956 49 14 1800 1350 2401 52 15 2098 1569 2806
43 4 1132 838 1529 46 11 1496 1120 1998 49 15 1838 1378 2452 52 16 2143 1601 2869
43 5 1160 860 1565 46 12 1529 1145 2041 50 0 1363 1017 1825 53 0 1554 1164 2075
43 6 1188 882 1601 46 13 1562 1170 2085 50 1 1397 1044 1870 53 1 1593 1194 2126
43 7 1217 904 1638 46 14 1595 1195 2129 50 2 1431 1070 1914 53 2 1633 1224 2178
43 8 1246 927 1675 47 0 1202 893 1619 50 3 1466 1097 1960 53 3 1673 1254 2231
43 9 1275 949 1712 47 1 1233 916 1658 50 4 1502 1124 2006 53 4 1713 1284 2285
4310 1304 972 1750 47 2 1264 941 1698 50 5 1537 1151 2053 53 5 1754 1315 2339
4311 1333 995 1788 47 3 1295 965 1738 50 6 1573 1179 2100 53 6 1796 1346 2395
4312 1363 1018 1826 47 4 1327 989 1779 50 7 1610 1207 2148 53 7 1838 1378 2452
4313 1393 1041 1865 47 5 1358 1014 1820 50 8 1647 1235 2197 53 8 1881 1410 2510
44 0 1064 784 1442 47 6 1391 1039 1861 50 9 1684 1263 2246 53 9 1924 1442 2568
44 1 1092 806 1478 47 7 1423 1064 1904 50 10 1722 1291 2297 53 10 1969 1474 2628
44 2 1120 828 1514 47 8 1456 1089 1946 50 11 1760 1320 2348 53 11 2013 1507 2690
44 3 1148 851 1550 47 9 1489 1114 1989 50 12 1799 1349 2400 53 12 2059 1540 2752
44 4 1177 873 1587 47 10 1522 1140 2033 50 13 1838 1378 2452 53 13 2105 1574 2816
44 5 1206 896 1624 47 11 1556 1166 2077 50 14 1878 1408 2506 53 14 2152 1608 2881
44 6 1235 919 1661 47 12 1590 1192 2122 50 15 1918 1437 2560 53 15 2200 1642 2948
44 7 1265 941 1699 47 13 1625 1218 2167 51 0 1423 1064 1903 53 16 2249 1677 3016
44 8 1294 964 1737 47 14 1659 1244 2213 51 1 1458 1091 1949 54 0 1627 1220 2171
44 9 1324 988 1776 48 0 1253 932 1684 51 2 1494 1118 1996 54 1 1668 1251 2225
4410 1354 1011 1815 48 1 1285 957 1725 51 3 1531 1146 2044 54 2 1710 1282 2280
4411 1385 1034 1854 48 2 1317 982 1766 51 4 1567 1174 2092 54 3 1752 1314 2336
4412 1416 1058 1894 48 3 1349 1007 1808 51 5 1605 1203 2141 54 4 1794 1345 2393
4413 1446 1082 1934 48 4 1382 1032 1850 51 6 1642 1231 2191 54 5 1838 1378 2452
45 0 1108 819 1498 48 5 1415 1057 1893 51 7 1681 1260 2241 54 6 1882 1410 2511
45 1 1136 841 1535 48 6 1448 1083 1936 51 8 1719 1289 2293 54 7 1927 1443 2571
45 2 1166 864 1572 48 7 1482 1109 1980 51 9 1758 1318 2345 54 8 1972 1477 2633
45 3 1195 887 1610 48 8 1516 1135 2024 51 10 1798 1348 2398 54 9 2018 1511 2696
45 4 1225 910 1648 48 9 1550 1161 2069 51 11 1838 1378 2452 54 10 2065 1545 2761
45 5 1254 933 1686 48 10 1585 1188 2115 51 12 1879 1408 2507 54 11 2113 1579 2827
45 6 1285 957 1725 48 11 1620 1214 2161 51 13 1920 1439 2563 54 12 2162 1614 2894
45 7 1315 980 1764 48 12 1655 1241 2208 51 14 1962 1470 2620 54 13 2211 1650 2963
45 8 1345 1004 1803 48 13 1691 1268 2256 51 15 2004 1501 2677 54 14 2261 1686 3033
45 9 1376 1028 1843 48 14 1727 1295 2304 52 0 1486 1112 1986 54 15 2312 1722 3105
4510 1407 1052 1883 49 0 1306 974 1753 52 1 1523 1141 2035 54 16 2365 1759 3179
4511 1439 1076 1924 49 1 1339 999 1795 52 2 1561 1169 2084 54 17 2418 1796 3255
297
CN MPN LL UL
63 28 10991 7047 17143
63 29 11730 7551 18222
64 0 3045 2215 4184
64 1 3162 2290 4365
64 2 3286 2368 4562
64 3 3420 2450 4775
64 4 3564 2536 5008
64 5 3720 2628 5265
64 6 3889 2725 5548
64 7 4074 2830 5864
64 8 4277 2942 6217
64 9 4502 3064 6615
64 10 4753 3198 7065
64 11 5035 3346 7575
64 12 5352 3513 8154
64 13 5712 3704 8807
64 14 6119 3925 9540
64 15 6581 4184 10350
64 16 7101 4489 11233
64 17 7683 4845 12182
64 18 8329 5258 13195
64 19 9043 5727 14277
64 20 9826 6254 15439
64 21 10687 6840 16699
64 22 11636 7487 18084
64 23 12687 8201 19626
64 24 13864 8993 21371
64 25 15199 9879 23383
64 26 16740 10880 25756
64 27 18563 12030 28643
64 28 20795 13381 32315
64 29 23671 15017 37313
64 30 27726 17088 44987
64 31 34659 19864 60473
298
Agars, Peptones,
Extracts
Acid Hydrolysate of Casein
Intended Use Total amino acids

(expressed as g per 100 g of product)
Acid Hydrolysate of Casein is included in the Aspartic acid................................................................4.4
composition of culture media intended for Threonine ....................................................................2.2
microbiological assays of vitamins and of tryptophan, Serine...........................................................................2.7
as well as for the study of the resistance of bacteria Glutamic acid ............................................................12.5
to antibiotics, to sulfamides in particular. It is Proline .........................................................................6.1
included in Mueller-Hinton Agar. Glycine .........................................................................1.2
Alanine ........................................................................2.0
Description Valine...........................................................................3.9
Cystine...........................................................not assayed
Acid Hydrolysate of Casein is obtained by the Methionine..................................................................1.2
hydrochloric acid hydrolysis of a high quality casein. Isoleucine.....................................................................2.4
As a result of the relatively consistant quality of the Leucine ........................................................................3.4
starting material used, Acid Hydrolysate of Casein Tyrosine........................................................................0.6
furnishes constant results. Hydrochloric acid is Phenylalanine..............................................................2.5
neutralized after hydrolysis, explaining the high Lysine ...........................................................................5.6
levels of inorganic salts. The hydrolysate is composed Histidine ......................................................................1.8
primarily of free amino acids, except for tryptophan Arginine.......................................................................2.2
and cystine. It contains no vitamins. Tryptophan ..................................................................0.0
Typical Analysis Quality control
Physical characteristics - Total aerobic

Appearance, color ........................cream-white powder mesophilic count ................................... < 5000 cfu / g
Solubility in water at 5% ........................................total - Heat resistant spores in 1 g............................ absence
pH of an aqueous 5% solution ........................5.2 ± 0.5
Stability at pH 7.0 after autoclaving Packaging
for 15 min. at 121°C ..............................................stable
- 500 g bottle : A1404HA
Chemical characteristics - 5 kg drum : A1404GC
Biuret reaction...................................................negative
Total nitrogen..........................................................7.5%
-amino nitrogen ....................................................5.4%
-amino/total nitrogen ratio ...................................0.72
Total carbohydrates.................................less than 0.5%
Indole ....................................................................absent
Nitrites...................................................................absent
Chlorides (expressed as NaCl)...............................40.0%
Calcium ..................................................................0.05%
Loss on drying..........................................less than 5.0%
300
Bacteriological Agar Type A
Intended Use Chemical characteristics

Foreign substances ..................................less than 1.0%
Bacteriological agars are used as gelling agents in the Starch.....................................................................absent
preparation of solid culture media, at concentrations Gelatin...................................................................absent
between 12 and 20 g/L. Sulfuric ash...............................................less than 6.0%
Heavy metals........................................less than 0.004%
Description Lead......................................................less than 0.001%
Arsenic................................................less than 0.0003%
Agars are extracted from purified and dried marine Loss on drying........................................less than 10.0%
algae. Extraction in acid solution is followed by
successive purification to yield clear agars with no Quality control
precipitate after autoclaving and which are free of
inhibitory substances. Agar is composed primarily of - Total aerobic
agarose (70%) and agaropectin (30%) which mesophilic count ................................... < 5000 cfu / g
together form a gel after heating and cooling. - Heat resistant spores in 1 g ...............................absent
Agarose is a linear polymer composed uniquely of
D-galactose and of 3,6-anhydro-L-galactose bound Packaging
1-3 and 1-4. The structure of agaropectin is close
to that of agarose but also contains anionic groups - 500 g bottle : A1010HA
bound to calcium or magnesium, as well as organic - 5 kg drum : A1010GC
acids (glucuronic and pyruvic). The 3,6-anhydro--L-
galactoside bond is very sensitive to acid pH values so
that prolonged heating of acid media will lead to
depolymerization of the agar and thus a decreased
gel strength. Bacteriological Agar type A is an
American type which has a lower gel strength than
the European type E.
Typical Analysis
Physical characteristics
Appearance, color ........................cream-white powder
Melting point ......................................................85-95°C
Gelling point .......................................................36-34°C
Precipitation after autoclaving ........................negative
pH of a 1.5% gel after autoclaving ....................6.5-7.5
Gel strength of 1.5% agar
after autoclaving ......................................500-700 g/cm2
301
Bacteriological Agar Type E
Intended Use Typical Analysis
Bacteriological agars are used as gelling agents in the Physical characteristics

preparation of solid culture media, at concentrations Appearance, color ........................cream-white powder
between 12 and 20 g/L. Melting point ......................................................82-86°C
Gelling point .......................................................36-34°C
Description Precipitation after autoclaving ........................negative
pH of a 1.5% gel after autoclaving ....................6.5-7.5
Agars are extracted from purified and dried marine Gel strength of 1.5% agar
algae. Extraction in acid solution is followed by after autoclaving ......................................700-900 g/cm2
successive purification to yield clear agars with no
precipitate after autoclaving and which are free of Chemical characteristics
inhibitory substances. Agar is composed primarily of Foreign substances ..................................less than 1.0%
agarose (70%) and agaropectin (30%) which Starch.....................................................................absent
together form a gel after heating and cooling. Gelatin...................................................................absent
Agarose is a linear polymer composed uniquely of Sulfuric ash...............................................less than 6.0%
D-galactose and of 3,6-anhydro-L-galactose bound Heavy metals........................................less than 0.004%
1-3 and 1-4. The structure of agaropectin is close Lead......................................................less than 0.001%
to that of agarose but also contains anionic groups Arsenic................................................less than 0.0003%
bound to calcium or magnesium, as well as organic Loss on drying........................................less than 10.0%
acids (glucuronic and pyruvic). The 3,6-anhydro--L-
galactoside bond is very sensitive to acid pH values so Quality control
that prolonged heating of acid media will lead to
depolymerization of the agar and thus a decreased - Total aerobic
gel strength. Bacteriological Agar type E is a mesophilic count ................................< à 5000 cfu / g
European type which has a higher gel strength than - Heat resistant spores in 1 g ...............................absent
the American type A.
Packaging

- 5 kg drum : A1012GC
Bacteriological Malt Extract
Malt extract is intended primarily for the culture and Physical characteristics
enumeration of yeasts and molds in malt extract Appearance, color ..............................yellowish powder
media. It favors the sporulation of molds such as Solubility in water at 3% ........................................total
Aspergillus and Penicillium. Because of it high pH of an aqueous 3% solution ........................5.5 ± 0.5
carbohydrate content, malt extract should not be Stability at pH 7.0 after autoclaving
subjected to excessive heating, since the culture for 15 min. at 121°C ..............................................stable
medium will turn brown.
Chemical characteristics
Description Maltose ..................................................................80.0%
Dextrins..................................................................10.0%
Bacteriological Malt Extract is obtained by successive Protein substances...................................................5.0%
purifications such that all residual enzymatic activity Inorganic substances ...............................................1.5%
is removed. It is rich in carbohydrates (maltose, Detection of glycerol ........................................negative
glucose, fructose, sucrose, trisaccharides, dextrins) Loss on drying..........................................less than 5.0%
and vitamins (thiamine, biotin, riboflavine, nicotinic
acid, folic acid, inositol). These compounds ensure the
rapid growth of yeasts and molds.
302
Total amino acids Quality control
Aspartic acid................................................................0.9 - Total aerobic
Threonine ....................................................................0.4 mesophilic count .................................. < 5000 cfu / g
Serine...........................................................................0.4 - Heat resistant spores in 1 g............................negative
Glutamic acid ..............................................................1.6
Proline .........................................................................0.6 Packaging
Glycine .........................................................................0.4
Alanine ........................................................................0.4 - 500 g bottle : A1101HA
Valine...........................................................................0.6 - 5 kg drum : A1101GC
Methionine..................................................................0.2
Isoleucine.....................................................................0.5
Leucine ........................................................................0.6
Tyrosine........................................................................0.3
Phenylalanine..............................................................0.7
Lysine ...........................................................................0.6
Histidine ......................................................................0.6
Arginine.......................................................................0.5
Bacteriological Meat Extract

Combined with peptones of different origins, Aspartic acid................................................................8.2
Bacteriological Meat Extract is an excellent Threonine ....................................................................3.0
nutritional complement destined for the preparation Serine...........................................................................2.9
of a variety of culture media. It is normally used in Glutamic acid ..............................................................9.5
the concentration range of 0.2 to 1.0%. Proline .........................................................................4.1
Glycine .........................................................................7.2
Description Alanine ........................................................................5.3
Valine...........................................................................4.9
Meat Extract is prepared from selected animal tissues Cystine...........................................................not assayed
which confer on it excellent nutritive properties. Methionine..................................................................0.7
Isoleucine.....................................................................3.0
Typical Analysis Leucine ........................................................................6.0
Tyrosine........................................................................1.2
Physical characteristics Phenylalanine..............................................................3.7
Appearance, color..........................light brown powder Lysine ...........................................................................6.5
Solubility in water at 5% ........................................total Histidine ......................................................................1.9
pH of an aqueous 5% solution ........................7.0 ± 0.5 Arginine.......................................................................3.7
Stability at pH 7.0 after autoclaving Tryptophan ..................................................................0.4
Quality control
Total nitrogen........................................................12.0% - Total aerobic
-amino nitrogen ....................................................5.0% mesophilic count.....................................< 5000 cfu / g
Creatinine ................................................................2.5% - Heat resistant spores in 1 g............................negative
Indole ....................................................................absent
Nitrates..................................................................absent Packaging
Nitrites...................................................................absent
Chlorides (expressed as NaCl).................................2.0% - 500 g bottle : A1710HA
Calcium ....................................................................0.1% - 5 kg drum : A1710GC
Iron ......................................................................0.007%
Sulfuric ash.............................................less than 15.0%
303
Casein-Meat Peptone

Casein-Meat Peptone is suitable for a number of uses Aspartic acid................................................................7.2
in general microbiology. It contains a broad spectrum Threonine ....................................................................3.2
of nitrogen compounds, from free amino acids to Serine...........................................................................2.4
high molecular weight proteins. It is thus suitable for Glutamic acid ............................................................11.3
the preparation of media for the culture of Proline .........................................................................6.6
enterobacteria, staphylococci, streptococci and a Glycine .........................................................................4.6
wide variety of other microorganisms. Alanine ........................................................................4.5
Valine...........................................................................5.5
Description Cystine...........................................................not assayed
Methionine..................................................................1.9
Casein-Meat Peptone is composed of a mixture of Isoleucine.....................................................................3.5
Tryptone and Peptic Digest of Meat which confer on Leucine ........................................................................6.6
it a perfect protein and vitamin balance as a result of Tyrosine........................................................................1.9
the complementarities of its constituents. Phenylalanine..............................................................3.1
Lysine ...........................................................................5.1
Typical Analysis Histidine ......................................................................3.1
Arginine.......................................................................3.1
Physical characteristics Tryptophan ..................................................................0.9
Appearance, color ....................................beige powder
Solubility in water at 5% ........................................total Microbiological characteristics
pH of an aqueous 5% solution ........................7.0 ± 0.5 Indole production ...............................................positive
Stability at pH 7.0 after autoclaving Hydrogen sulfide production.............................positive
for 15 min. at 121°C ..............................................stable Acetylmethylcarbinol production ......................positive
Detection of fermentable sugars .........slightly positive
Biuret reaction ....................................................positive Quality control
Total nitrogen........................................................12.5%
-amino nitrogen ....................................................5.0% - Total aerobic
-amino/total nitrogen ratio ...................................0.40 mesophilic count.....................................< 5000 cfu / g
Indole ....................................................................absent - Heat resistant spores in 1 g............................negative
Nitrites...................................................................absent
Chlorides (expressed as NaCl).................................2.0%
Calcium ....................................................................0.2% Packaging
Loss on drying..........................................less than 5.0% - 500 g bottle : A1408HA
Mycopeptone
Mycopeptone is used for the culture of Physical characteristics

dermatophytes, as well as for saprophytic yeasts Appearance, color ....................................beige powder
and molds. It was formulated especially to be Solubility in water at 5% ........................................total
incorporated in solid media destined for the isolation pH of an aqueous 5% solution ........................6.0 ± 0.5
and diagnosis of pathogenic and non-pathogenic Stability at pH 6.0 after autoclaving
fungi. for 15 min. at 121°C ..............................................stable
Description
Mycopeptone is composed of a mixture of peptones

of various origins in order to optimize the growth of
yeasts and molds and to obtain characteristic
morphology and pigmentation.
304
Chemical characteristics Quality control
Biuret reaction ....................................................positive
Total nitrogen..........................................................8.0% - Total aerobic
-amino nitrogen ....................................................3.0% mesophilic count.....................................< 5000 cfu / g
-amino/total nitrogen ratio ...................................0.38 - Heat resistant spores in 1 g............................negative
Indole ....................................................................absent
Nitrites...................................................................absent
Chlorides (expressed as NaCl).................................8.0% Packaging
Calcium ....................................................................0.2%
Sulfuric ash.............................................less than 20.0% - 500 g bottle : A1801HA
Total amino acids

Aspartic acid................................................................7.5
Threonine ....................................................................3.0
Serine...........................................................................2.7
Glutamic acid ..............................................................9.9
Proline .........................................................................5.2
Glycine .........................................................................6.2
Alanine ........................................................................6.0
Valine...........................................................................5.0
Methionine..................................................................1.4
Isoleucine.....................................................................3.1
Leucine ........................................................................6.3
Tyrosine........................................................................1.8
Phenylalanine..............................................................2.7
Lysine ...........................................................................4.7
Histidine ......................................................................2.8
Arginine.......................................................................3.8
Tryptophan ..................................................................0.8
Pancreatic Digest of Casein Codex
Pancreatic Digest of Casein Codex is suitable for the Physical characteristics

preparation of culture media destined for usual Appearance, color ....................................white powder
bacteriological controls. Solubility in water at 5% ........................................total
Combined with meat peptones and as a result of its pH of an aqueous 5% solution ........................5.5 ± 0.5
appropriate nutritional qualities and pH, it is used in Stability at pH 7.0 after autoclaving
media destined for the growth of yeasts and molds. for 15 min. at 121°C ..............................................stable
It is also used to grow lactic acid bacteria and for the
development of sporulated aerobic bacteria such as Chemical characteristics
Bacillus species. Biuret reaction ....................................................positive
Total nitrogen........................................................14.5%
Description -amino nitrogen ....................................................5.5%
Pancreatic digest of casein is obtained by the Indole ....................................................................absent
pancreatic digest of a high grade casein. As a result Nitrites...................................................................absent
of the relatively constant quality of the starting Chlorides (expressed as NaCl) .................less than 1.0%
material used, this peptone furnishes consistent and Calcium ....................................................................0.2%
efficient results. The method of preparation was Sulfuric ash...............................................less than 5.0%
specially designed to reduce the ash level to a Loss on drying..........................................less than 5.0%
minimum and increase the protein content
proportionally so as to favor the development of a
number of microorganisms.
305
Total amino acids Quality control
Aspartic acid................................................................6.4 - Total aerobic
Threonine ....................................................................3.4 mesophilic count.....................................< 5000 cfu / g
Serine...........................................................................4.2 - Heat resistant spores in 1 g............................negative
Glutamic acid ............................................................17.4
Proline .........................................................................8.4 Packaging
Glycine .........................................................................1.9
Alanine ........................................................................2.6 - 500 g bottle : A1402HA
Valine...........................................................................5.1 - 5 kg drum : A1402GC
Methionine..................................................................2.4
Isoleucine.....................................................................4.3
Leucine ........................................................................7.2
Tyrosine........................................................................2.0
Phenylalanine..............................................................4.1
Lysine ...........................................................................6.5
Histidine ......................................................................2.7
Arginine.......................................................................3.1
Tryptophan ..................................................................1.2
Pancreatic Digest of Gelatin
Intended Use Chemical characteristics

Pancreatic Digest of Gelatin has a relatively low Total nitrogen........................................................14.5%
nutritive potential. It is used in media destined for -amino nitrogen ....................................................3.5%
non-fastidious bacteria. It is used primarily in -amino/total nitrogen ratio ...................................0.24
formulations in which the medium is required to be Indole ....................................................................absent
as colorless as possible. Compatible with phosphates Nitrites...................................................................absent
and Brain Heart Infusion, it is included in the Chlorides (expressed as NaCl).................................1.0%
formulation of Brain Heart Broth which is required to Calcium ....................................................................0.2%
be very clear and have perfect stability. Sulfuric ash.............................................less than 15.0%
Description
Total amino acids
Gelatin digest is obtained by the pancreatic digestion (expressed as g per 100 g of product)
of a selected starting material. It is characterized by a Aspartic acid................................................................6.9
reduced content of sulfur amino acids (cystine, Threonine ....................................................................1.7
methionine) and contains no tryptophan. The proline Serine...........................................................................2.5
and hydroxyproline contents are higher than in most Glutamic acid ..............................................................8.8
other peptones. Proline .......................................................................10.9
As a result of its special composition, it is most often Glycine .......................................................................19.2
included in media in combination with other Alanine ........................................................................8.2
nitrogen substrates such as Tryptone, meat extract or Valine...........................................................................3.3
yeast extract, with which it is perfectly compatible. Cystine...........................................................not assayed
Methionine..................................................................0.8
Typical Analysis Isoleucine.....................................................................1.6
Leucine ........................................................................2.8
Physical characteristics Tyrosine........................................................................0.5
Appearance, color ....................................white powder Phenylalanine..............................................................2.1
Solubility in water at 5% ........................................total Lysine ...........................................................................4.2
pH of an aqueous 5% solution ........................7.0 ± 0.5 Histidine ......................................................................0.9
Stability at pH 7.0 after autoclaving Arginine.......................................................................6.9
for 15 min. at 121°C ..............................................stable Tryptophan ..................................................................0.0
Hydroxyproline .........................................................10.0
306
Microbiological characteristics Packaging
Indole production .............................................negative
Hydrogen sulfide production.............................positive - 500 g bottle : A1001HA
Acetylmethylcarbinol production ......................positive
Detection of fermentable sugars .....................negative
Quality control
- Total aerobic
mesophilic count.....................................< 5000 cfu / g
- Heat resistant spores in 1 g............................negative
Pancreatic Digest of Meat Type 2

Pancreatic Digest of Meat Type 2 is suitable for the Aspartic acid................................................................6.8
culture of a large number of aerobic and anaerobic Threonine ....................................................................2.8
bacteria. Serine...........................................................................3.1
It is recommended for spore production in anaerobic Glutamic acid ............................................................10.2
bacteria such as Clostridium perfringens. Proline .........................................................................4.7
Glycine .........................................................................8.9
Description Alanine ........................................................................6.0
Valine...........................................................................4.6
Pancreatic Digest of Meat Type 2 is obtained by the Cystine...........................................................not assayed
pancreatic digestion of certain highly selected animal Methionine..................................................................1.2
tissues. Isoleucine.....................................................................2.8
Leucine ........................................................................5.4
Typical Analysis Tyrosine........................................................................1.0
Phenylalanine..............................................................3.2
Physical characteristics Lysine ...........................................................................5.4
Appearance, color ....................................beige powder Histidine ......................................................................1.8
Solubility in water at 5% ........................................total Arginine.......................................................................4.1
pH of an aqueous 5% solution ........................7.0 ± 0.5 Tryptophan ..................................................................0.7
Stability at pH 7.0 after autoclaving
for 15 min. at 121°C ..............................................stable Quality control
Chemical characteristics - Total aerobic

Biuret reaction ....................................................positive mesophilic count.....................................< 5000 cfu / g
Total nitrogen........................................................12.5% - Heat resistant spores in 1 g............................negative
-amino nitrogen ....................................................5.0%
-amino/total nitrogen ratio ...................................0.40 Packaging
Indole ....................................................................absent
Nitrites...................................................................absent - 500 g bottle : A1702HA
Chlorides (expressed as NaCl).................................2.0% - 5 kg drum : A1702GC
Calcium ....................................................................0.3%
307
Papaic Digest of Soybean Meal USP

As a result of its balanced content of essential amino Aspartic acid................................................................7.2
acids, carbohydrates and vitamins, Papaic Digest of Threonine ....................................................................2.5
Soybean Meal USP leads to the very rapid and Serine......................................................................... 3.4
abundant growth of a wide variety of Glutamic acid .......................................................... 13.2
microorganisms, including yeasts and molds. It is Proline ........................................................................ 4.2
often combined with Tryptone in Tryptone-Soy Broth Glycine....................................................................... 2.9
and Tryptone-Soy Agar for the growth of fastidious Alanine ........................................................................2.9
microorganisms. Valine...........................................................................4.6
Cystine ......................................................... not assayed
Description Methionine..................................................................0.8
Isoleucine.....................................................................3.0
Papaic Digest of Soybean Meal is obtained by the Leucine ........................................................................5.2
action of papain on dilipidated soybean meal. It Tyrosine........................................................................1.3
contains a high proportion of carbohydrates which Phenylalanine..............................................................3.4
explains why it is not suitable for studying sugar Lysine ...........................................................................4.6
fermentations. Histidine ......................................................................1.9
Arginine.......................................................................4.2
Typical Analysis Tryptophan................................................................. 1.1
Physical characteristics Microbiological characteristics

Appearance, color ....................................beige powder Indole production ............................................negative
Solubility in water at 5% ........................................total Hydrogen sulfide production.............................positive
pH of an aqueous 5% solution ........................7.0 ± 0.5 Acetylmethylcarbinol productio ....................... positive
Stability at pH 7.0 after autoclaving Detection of fermentable sugars .................... positive
Quality control
Biuret reaction ....................................................positive - Total aerobic
Total nitrogen........................................................10.0% mesophilic count ................................... < 5000 cfu / g
-amino nitrogen ....................................................2.5% - Heat resistant spores in 1 g............................negative
Indole ....................................................................absent Packaging
Nitrites...................................................................absent
Chlorides (expressed as NaCl).................................0.5% - 500 g bottle : A1601HA
Calcium ................................................................... 0.5% - 5 kg drum : A1601GC
Sulfuric ash ........................................... less than 15.0%
Loss on drying......................................... less than 5.0%
308
Peptic Digest of Meat USP

Peptic Digest of Meat USP is used for the growth of Aspartic acid................................................................7.9
yeasts and molds, enterobacteria, staphylococci, and Threonine ....................................................................2.8
numerous other microorganisms. Serine...........................................................................2.6
After addition to blood agar, it favors the growth of Glutamic acid ..............................................................9.3
pneumococci, as well as the production of Proline .........................................................................5.6
characteristic hemolysis. Because of its high content Glycine .........................................................................6.8
of sulfur compounds, it is used for the detection of Alanine ........................................................................6.5
hydrogen sulfide producers. Valine...........................................................................5.1
Description Methionine..................................................................1.5
Isoleucine.....................................................................3.2
Obtained by the enzymatic digestion of selected Leucine ........................................................................6.6
animal tissues, Peptic Digest of Meat USP is Tyrosine........................................................................2.0
characterized by a predominance of high molecular Phenylalanine..............................................................2.5
weight polypeptides. Lysine ...........................................................................4.8
Histidine ......................................................................2.9
Typical Analysis Arginine.......................................................................3.5
Tryptophan ..................................................................0.8
Appearance, color ...........................light beige powder Microbiological characteristics
Solubility in water at 5% ........................................total Indole production ...............................................positive
pH of an aqueous 5% solution ........................7.0 ± 0.5 Hydrogen sulfide production.............................positive
Stability at pH 7.0 after autoclaving Acetylmethylcarbinol production ......................positive
for 15 min. at 121°C ..............................................stable Detection of fermentable sugars .........slightly positive
Biuret reaction ....................................................positive Quality control
Total nitrogen........................................................12.5%
-amino nitrogen ....................................................5.0% - Total aerobic
-amino/total nitrogen ratio ...................................0.40 mesophilic count.....................................< 5000 cfu / g
Indole ....................................................................absent - Heat resistant spores in 1 g............................negative
Nitrites...................................................................absent
Chlorides (expressed as NaCl).................................2.0% Packaging
Calcium ..................................................................0.05%
Sulfuric ash.............................................less than 15.0% - 500 g bottle : A1708HA
Loss on drying..........................................less than 5.0% - 5 kg drum : A1708GC
309
Tryptone USP

Tryptone USP is suitable for a wide variety of uses in Aspartic acid................................................................6.9
general bacteriology for the preparation of culture Threonine ....................................................................3.3
media. As a result of its high tryptophan Serine...........................................................................4.1
concentration, it is used for the detection of indole Glutamic acid ............................................................18.5
production. Lacking fermentable carbohydrates, it is Proline .........................................................................9.0
included in media used to study sugar fermentations. Glycine .........................................................................3.2
Because of its compatibility with other ingredients, Alanine ........................................................................3.1
peptones or extracts, Tryptone USP is included in the Valine...........................................................................6.1
composition of media intended for the growth and Cystine...........................................................not assayed
enumeration of bacteria in water, milk, other food Methionine..................................................................2.4
products, pharmaceuticals and cosmetics. Isoleucine.....................................................................4.9
Combined with papaic digest of soybean meal, it is Leucine ........................................................................8.1
used for the preparation of Tryptone-Soy Broth and Tyrosine........................................................................1.5
Tryptone-Soy Agar. Phenylalanine..............................................................4.9
Lysine ...........................................................................7.6
Description Histidine ......................................................................2.9
Arginine.......................................................................3.2
Tryptone USP is obtained by the pancreatic digestion Tryptophan ..................................................................1.2
of a high quality casein. As a result of the relatively
constant purity of the starting material used, Microbiological characteristics
Tryptone USP furnishes efficient and consistent Indole production ...............................................positive
results, in particular in studies on the metabolism and Hydrogen sulfide production.............................positive
growth of various microorganisms. Acetylmethylcarbinol production ......................positive
The method of preparation has been designed to Detection of fermentable sugars .....................negative
maximally reduce the level of calcium in the finished
product. This is why Tryptone USP can be used to Quality control
prepare phosphate-containing media characterized
by their clear, light nature, as well as by the absence - Total aerobic
of significant precipitates after autoclaving. mesophilic count.....................................< 5000 cfu / g
- Heat resistant spores in 1 g............................negative
Typical Analysis
Packaging
Appearance, color ........................cream-white powder - 500 g bottle : A1401HA
Solubility in water at 5% ........................................total - 5 kg drum : A1401GC
pH of an aqueous 5% solution ........................7.0 ± 0.5
Stability at pH 7.0 after autoclaving
Total nitrogen........................................................12.5%
-amino nitrogen ....................................................4.0%
Indole ....................................................................absent
Nitrites...................................................................absent
Chlorides (expressed as NaCl) .................less than 1.0%
Calcium.....................................................less than 0.1%
Sulfuric ash .......................................... less than 15.0%
310
Yeast Extract

Yeast Extract is considered as a principal enrichment Aspartic acid................................................................6.5
factor in culture media. It accelerates the growth of a Threonine ....................................................................2.6
wide variety of microorganisms, including yeasts and Serine...........................................................................2.4
molds. Because of its carbohydrate content, it is not Glutamic acid ........................................................... 11.5
suitable for media intended for the study of sugar Proline ........................................................................ 2.3
fermentation. Glycine ........................................................................ 3.0
Alanine ........................................................................4.8
Description Valine...........................................................................4.1
Controlled enzymatic digestion of the cellular Methionine..................................................................0.9
constituents of yeast (Saccharomyces cerevisiae) by its Isoleucine.................................................................... 3.1
own enzymes (autolysis) furnishes an extract rich in Leucine ....................................................................... 4.2
amino acids, oligopeptides, vitamins (especially the B Tyrosine ...................................................................... 1.1
group), growth factors, carbohydrates, and purine Phenylalanine..............................................................2.6
and pyrimidine bases from nucleic acids. Lysine .......................................................................... 4.6
Histidine ......................................................................1.4
Typical Analysis Arginine.......................................................................3.2
Tryptophan ..................................................................0.9
Appearance, color ..................................yellow powder Vitamins
Solubility in water at 5% ........................................total (expressed as mg per 100 g of product)
pH of an aqueous 5% solution ........................7.0 ± 0.5 Thiamine......................................................................2.1
Stability at pH 7.0 after autoclaving Riboflavine ................................................................12.5
for 15 min. at 121°C ..............................................stable Pyridoxine....................................................................2.4
Nicotinic acid.............................................................60.0
Chemical characteristics Pantothenic acid .......................................................10.5
Heat-coagulated proteins ....................................absent Folic acid......................................................................0.6
Total nitrogen........................................................10.6% Choline ....................................................................150.0
-amino nitrogen ....................................................5.0% Biotin ...........................................................................0.4
Indole ....................................................................absent
Nitrites...................................................................absent Quality control
Chlorides (expressed as NaCl) .................less than 1.0%
Calcium ....................................................................0.1% - Total aerobic
Sulfuric ash.............................................less than 15.0% mesophilic count.....................................< 5000 cfu / g
Loss on drying..........................................less than 5.0% - Heat resistant spores in 1 g............................negative
Packaging

- 5 kg drum : A1202GC
311

Biokar Catalogus

Diunggah oleh

Informasi Dokumen

Judul Asli

Hak Cipta

Format Tersedia

Bagikan dokumen Ini

Bagikan atau Tanam Dokumen

Opsi Berbagi

Apakah menurut Anda dokumen ini bermanfaat?

Apakah konten ini tidak pantas?

Hak Cipta:

Format Tersedia

Biokar Catalogus

Diunggah oleh

Hak Cipta:

Format Tersedia

BIOKAR

Biokar Diagnostics Quality 25

Supplements, Reagents, Ringer 269

Agars, Peptones, Extracts 299

Supplements, Reagents, Ringer

A1010 Bacteriological Agar Type A 301

A1012 Bacteriological Agar Type E 302

A1101 Bacteriological Malt Extract 302

A1202 Yeast Extract 311

A1401 Tryptone USP 310

A1402 Pancreatic Digest of Casein Codex 305

A1404 Acid Hydrolysate of Casein 300

A1408 Casein-Meat Peptone 304

A1601 Papaic Digest of Soybean Meal USP 308

A1702 Pancreatic Digest of Meat Type 2 307

A1708 Peptic Digest of Meat USP 309

A1710 Bacteriological Meat Extract 303

A1801 Mycopeptone 304

BK001 TSN Agar 252

BK002 Brilliant Green Bile Broth (BGBB) 85

BK003 Nutrient Broth 188

BK004 Bismuth Sulfite Agar 78

BK005 Minerals Glutamate Medium (base) 172

BK006 Meat-Yeast Agar 167

BK007 Chloramphenicol Glucose Agar 101

BK009 Selenite-Cystine Broth 223

BK010 Laurylsulfate-Tryptose Broth 146

BK011 Violet Red Bile Glucose Agar (VRBG) 255

BK012 M17 Broth 156

BK013 Wort Agar (base) 260

BK014 Tryptone-Salt Broth 242

BK015 Brain-Heart Broth 81

BK017 Thioglycollate Medium with Resazurin 239

BK018 Buffered Peptone Water (25.5 g/L) 95

BK019 Columbia Agar (base) 104

BK020 CLED Agar 102

BK021 Nutrient Agar 187

BK022 SS Agar 226

BK023 Bromcresol Purple Lactose Agar 87

BK024 Meat-Liver Glucose - 0.6% Agar 166

BK025 Sabouraud Dextrose Agar 215

BK026 Sabouraud Dextrose Broth 216

BK027 Sabouraud Chloramphenicol Agar 214

BK028 Tryptone-Soy Agar (Blood Agar base) 245

BK029 Brain-Heart Agar 80

BK030 Mannitol Salt Agar 163

BK031 TSC Agar (base) 248

BK032 Önöz Agar 189

BK033 Rogosa Agar 208

BK034 Kligler Iron Agar 140

BK036 Drigalski Lactose Agar 117

BK037 Slanetz and Bartley Agar 225

BK038 Tergitol 7 Agar (base) 233

BK039 Yersinia Agar (CIN base) 266

BK040 TCBS Agar 231

BK042 Dextrose Tryptone Agar 116

BK044 Desoxycholate Citrate Agar (DCA) 111

BK045 Malt Agar 162

BK046 Tryptone-Soy Broth 247

BK047 Tryptone-Soy Agar 244

BK048 Mueller Hinton Agar 182

BK049 Cetrimide Agar (base) 98

BK050 MacConkey Agar 157