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KIRBY-BAUER DISK DIFFUSION TEST (Susceptibility/Sensitivity Test) Mueller Hinton Agar allows several microorganisms to grow pH 7.2 7.

.4 culture media with thickness of 4mm *4mm = decreased zone of inhibition *4mm = increase of zone of inhibition uses antimicrobial disks which will act on organisms (saturated by antimicrobial drug) Sensitive growth of organism is prevented - drug gives a very large zone of inhibition Intermediate Resistant Example: Antimicrobial Drug Code 1. Ampicillin Am10

Zone of Inhibition (sensitive) (intermediate) (resistant)

18mm reading in zone of inhibition Result: Sensitive

Intermediate Sensitive (zone of inhibition)

Resistant (no zone of inhibition)

Overlapping Streaking Incubate for 18-24 hours *Intermediate can be given to patients but with an increase in dosage *Confluent even distribution of microorganisms

McFarland Standard 0.5% concentration composed of BaCl2 and H2SO4 used in adjusting the inoculums turbidity before serial dilution, colony count, and minimal inhibitory concentration Code C30 SXT P10 NIT300 A25 INH S25 NA30 Antibiotic Chloramphenicol Sulfamethoxazole Trimethoprim Penicillin G Nitrofurantoin Ampicillin Isoniazid Streptomycin Nalidixic Acid Code CN10 / G30 B10 NN10 CL10 VA30 NV30 CH30 K10 Antibiotic Gentamycin Bacitracin Tobramycin Colistin Vancomycin Novobiocin Cephalothin Kanamycin

Pharmaceutical Microbiology and Parasitology | Suzette Pamela G. Santos | 2F-Pharmacy | 1

SERIAL DILUTION inoculums based on 0.5 McFarland Standard

diluent: 4.5 mL sterile distilled water the last test tube has the least no. of colonies (1:10, 1:100, 1:1000, 1:10000, 1:100,000) Nutrient Agar ten-fold of microorganisms Nutrient Agar / Trypticase Soy Agar / Mueller Hinton Agar can be used as substitute Hockey Stick bended glass tube, to spread inoculums from inside to outside

COLONY COUNT Spread Method o incubate plates at 37 C for 18-24 hours Count the isolated colonies using the Quebec counter consider counting the plates with 30 300 isolated colonies* average = Colony Forming Unit (CFU/mL) Pour Method uses melted agar (MHA or NA) MINIMUM INHIBITORY CONCENTRATION 1 mL of antibiotic + 1 mL of sterile water (100 mg/mL) dilution: 1:2, 1:4, 1:8, 1:16, (50mg) (25mg) (12.5mg) (6.25mg) 1:8 least concentration of antibiotic that will inhibit growth o Incubate at 37 for 18-24 hours Two-fold serial dilution 0.01 mL inoculum (standardized to 0.5mL McFarland) ISOLATION OF GRAM POSITIVE BACTERIA ENRICHED MEDIA has additives (e.g. Blood) Blood Agar Plate (BAP) unheated blood ( -

1:32 3.125mg

)
Uninoculated

Chocolate Agar Plate (CAP) heated blood Two special growth factors: 1. X Factor = hemin, a heat stable derivative of hemoglobin 2. V Factor = heat-labile coenzyme (Nicotinamide Adenine Dinucleotide) - presence/absence of hemolysis

Uninoculated

Pharmaceutical Microbiology and Parasitology | Suzette Pamela G. Santos | 2F-Pharmacy | 2

Phenyl Ethyl Agar (PEA) - selective media for gram positive (+) - Enterococcus, Staphylococcus, Streptococcus

Uninoculated G(-)
E. coli

G(+)
E. faecalis

Mannitol Salt Agar (MSA) differential media 7.5% NaCl (selective media for gram positive) Mannitol carbohydrate present Non-Mannitol Fermenter Pink Non-mannitol fermenter S. epidermidis Yellow Mannitol Fermenter organism : Staphylococcus (it can resist high salt concentration) Biochemical Tests: Coagulase Test - medium : Plasma (+) coagulation/clumping (production of white granules) aureus produces coagulation use glass slide + 1-2 drops plasma + organism (inoculating loop) Catalase Test medium : H2O2 (+) bubbling/effervescence (release of oxygen) Staph (+) Strep (-)

| Mannitol Fermenter

S. aureus

1.

2.

ISOLATION OF GRAM NEGATIVE ENTEROBACTERIACEAE MacConkey Agar differential media isolation of gram-negative bacilli differentiates LF & NLF
Uninoculated Lactose Fermenter Non-Lactose Fermenter

Eosin Methylene Blue (EMB) differential media isolation of gram-negative bacilli differentiates LF from NLF ,,

coli
E. coli, Citrobacter

aerogenes
Klebsiella Enterobacter

Non Lactose Fermenter Pseudomonas

two "classical" types of coliform colonies: Coli - very dark, almost black when observed directly against the light. - a green sheen can be seen which is due to the precipitation of methylene blue - most strains of E. coli and some strains Citrobacter. - methyl red positive (MR+) lactose fermenters Aerogenes- a dark center is seen surrounded by a wide, light-colored, mucoid rim resulting in a "fish-eye" type of colony. - most strains of Klebsiella and Enterobacter. - methyl red negative (MR-) lactose fermenters

Pharmaceutical Microbiology and Parasitology | Suzette Pamela G. Santos | 2F-Pharmacy | 3

Hektoen Enteric Agar (HEA) selection of enteric pathogens and inhibition of normal flora coliforms
Uninoculated Lactose Fermenter Non Lactose Fermenter

sodium thiosulfate and ferric ammonium citrate for H2S production

Salmonella Shigella Agar (SSA) isolation and differentiation of Salmonella and Shigella inhibition of normal flora coliforms NLF without black center = Shigella NLF with black center = Salmonella Xylose Lysine Desoxycholate (XLD) selective for gram negative enteric pathogens xylose as carbohydrate for fermentation lysine for decarboxylation sodium thiosulfate and ferric ammonium citrate for hydrogen sulfide production Bismuth Sulfite Agar (BSA) suitable for the isolation of Salmonella typhi produces metallic colonies with a black ring
Salmonella typhi

Klebsiella pneumoniae

Salmonella typhimurium

Thiosulfate Citrate Bile Salts Sucrose (TCBS) alkaline, 8.6 inhibits gram positive bacteria and somelactose fermenting gram negative bacilli differentiate V. cholerae(+) and V. parahaemolyticus(-) in sucrose fermentation ferric ammonium citrate on H2S production
Medium

Inhibitor
Eosin Methylene Blue Crystal violet Bile salt Bile Salt

pH Indicator
Eosin Methylene Blue Neutral Red Phenol Red

EMB MacConkey XLD

Carbohydrate present Lactose Lactose Lactose Xylose Sucrose Lactose Sucrose Salicin Lactose Sucrose Glucose Glucose Lactose Sucrose Glucose

Lactose Fermenter
Purple Green metallic sheen Pink -> Red *

Non Lactose Fermenter


Colorless

H2S Indicators

Colorless

HEA

Bile Salt

Bromthymol Blue

Carrot Orange

Green

Sodium Thiosulfate Ferric ammonium citrate Sodium Thiosulfate Ferric ammonium citrate Ferric ammonium citrate

SSA TCBS BSA TSI

Brilliant green

Neutral Red

Red

White

Oxgall Sodium cholate Brilliant green

Bromthymol Blue Bismuth Sulfite

Yellow No Growth

Green Metallic with black ring

Phenol Red

Ferric ammonium citrate Ferrous sulfate Ferric ammonium citrate

LIA

Bromcresol Purple

Pharmaceutical Microbiology and Parasitology | Suzette Pamela G. Santos | 2F-Pharmacy | 4

Triple Sugar Iron (TSI) screening medium for the identification of Enterobacteriaceae #2 Yellow acid fermentation (A) #1 Red no acid fermentation (K) Gas production cracks or displacement of medium (G/encircle) H2S production blackening on the butt (+) deamination of amino acids (all +) aerobic alkaline rxn.
Symbol Bacteria Glucose Fermentation with Acid Gas Production Lactose / Sucrose Fermented H2S Production

A/AG A/AG K/A


+

K/A K/AG

K/AG

Escherichia Klebsiella Enterobacter Citrobacter Shigella Serratia Providencia Anaerogenic E. coli Salmonella typhi Morganella Salmonella Proteus Arizona Citrobacter Edwardsiella

Lysine Iron Agar (LIA) for identification of Salmonella, Proteus, Providencia and Morganella Lysine amino acid present #3 Purple butt lysine decarboxylation #1 & 2 Yellow butt no lysine decarboxylation #2 Red slant lysine deamination #4 Black butt H2S production
Symbol Bacteria Serratia Enterobacter Escherichia Klebsiella Salmonella Edwardsiella Citrobacter Enterobacter Shigella Proteus Providencia Morganella Lysine Decarboxylation
(alkaline cadaverine production)

Lysine deamination

Glucose Fermentation

H2S Production

K/K

K/K

K/A

R/A

x x x x x x

Pharmaceutical Microbiology and Parasitology | Suzette Pamela G. Santos | 2F-Pharmacy | 5

Other Biochemical Tests: TRYPTONE BROTH (TB) indole production test Indicator: Kovacs or Erlichs reagent ( p-aminobenzaldehyde ) Red ring (+) Yellow ring (-) METHYL RED VOGES PROSKAUER ( MRVP ) Methyl Red Test mixed acid fermentation ( lactic, acetic, formic, succinic ) pH 4.4 Red solution (+) Yellow solution (-) Voges Proskauer Test Acetoin KOH+ air Pink-red complex (+) Yellow brown (-)

Diacetyl

Naphthol + creatine

SIMMON CITRATE AGAR ( SCA ) citrate utilization test determine if the organism utilizes citrate as its sole source of carbon Sodium citrate ammonia NH4OH Indicator: Bromthymol blue Prussian blue (+) Green (-) UREA BROTH (UB) urease test Indicator: Phenol Red Red (+) Pale Yellow (-) SULFITE INDOLE AGAR (SIM) for motility test, indole production

Pharmaceutical Microbiology and Parasitology | Suzette Pamela G. Santos | 2F-Pharmacy | 6