Method GS2/3-41 (2011)
The Determination of the Total
Mesophilic Bacterial Count in Refined
Sugar Products by the Pour Plate
Method or the Membrane Filtration
Method - Official
1 Scope and Field of Application
This method is use¢ for the determination of the total mesophilic
bacteria count in refined sugar products (General Subjects 2 and 3)
[1,2,3,4,5,6,7]. With higher colony counts and/or for dark-coloured
products use the pour plate method. With low levels of
microorganisms use the membrane fiter method [12]. This method
isa combination of the previous GS2/3-41 (1998) and GS2/3-43
(1998) methods.
2 Definitions
2.1 Colony counts ~ are the enumeration of colony forming units
(CFU). The results are reported as CFU per 10 g of sugar.
2.2 Mesophilic bacteria. Mesophilic bacteria have their growth
optima between 20°C and 45°C. The Incubation temperature
selected is an average for optimum growth. Conditions favouring the
growth of a broad spectrum of bacterial species are established by
using a general nutrient medium.
2.3 Pour plate. These are agar plates which have been inoculated
by mixing known amounts of diluted sample with liquid agar medium
(47 21°C).
2.4 Membrane filter. These are sterile filters of knowin pore size
which retain microorganisms on their surface for further culture.3 Principle
+ Pour plate method. Diluted aliquots of the sample are mixed with
‘small vclumes of cooled molten agar in Petri dishes and incubated
when the agar medium has solidified. The microorganisms ara
detected as colony forming units.
+ Membrane filter method. Test solutions of sugars are fitered
through sterile membranes of known pore size. The microorganisms
are retained on the surface. The membrane is transferred onto a
nutrient medium or pad and incubated. The microorganisms are
detected as colony forming units.
Sterile equipment and an aseptic technique are used throughout the
method.
4 Diluents, Culture Media, Reagents and
other Products
4,1 Sterile distilled water - autoclaved at 121°C for 15 min.
4.2 Culture media [3,4,8].
Use either nutrient agar or plate count medium,
Nutrient Agar (1 L):
Beef extract 19 Sodium chloride 5 9
Yeast extract, 20 Agar 150
Peptone Sa pH 7.4 £0.2
(This is commercial available as Oxoid CM0003)..
Plate Count medium (1 L):
Peptone 3a Agar 109
Yeast extract, 259 pH 7040.2
Glucose 1g
(This is commercial available as Merck 5463, Oxoid CM0325, BBL
11638).
Alternatively use:
Nutrient pad sets in Petri dishes with membrane filters (0.45 um
pore-size) commercially available from Sartorius Stedim, Standard SM
140 55 ACN.
4.3 Disinfectant - for the working area.
4.4 Alcohol - for flaming the filter funnel assembly (5.13).5 Apparatus and Glassware [8,9,11]
ition at 180°C - for glassware
5.1 Apparatus for dry ste
sterilisation.
5.2 Autoclave, 121 = 1°C - for water and culture media
sterilisation.
5.3 Incubator ~ operating at 30 + 1 °C.
5.4 Bottles - for culture medium.
5.5 Erlenmeyer flasks - 200 mL with 2 100 mL mark and plugs for
autodaving.
5.6 Test tubes - or preparation of dilutions.
5.7 Graduated Pipettes - 1 mL to 5 ml for sample distribution; 10
ML for dilutions, glass or sterile disposable plastic.
5.8 Petri dishes - diameter 90 to 100 mm, sterile disposable plastic
oF glass; alternatively nutrient pad sets.
5.9 Water bath or similar apparatus - operating at 47 + 1 °C.
5.10 pH meter - accurate to + 0.1 pH units at 25°C.
5.11 Colony counting equipment.
5.12 Bunsen burner.
The following additional apparatus is required for the membrane
fitration technique.
5.13 Filter funnel, base with support and clamp. These can be
stainless steel, autoclavable plastic, class or sterile disposable plastic,
50 mm diameter with a capacity of 100 mL.
5.14 Flask for mounting filter apparatus ~ 2 with side arm.
5.15 Vacuum pump or vacuum supply.
5.16 Forceps ~ fer handling membranes.
5.17 Membranes - sterile 0.45 um pore size, 45 mm diameter.
6 Samples
Collect representative samples in sterile containers, 200 g minimum.
size for sugar or 200 mL for liquid sugar. Retain the samples until the
analysis has been completed (see Method GS2/3-42)..7 Procedure [2,8,9]
7.1 Preparation. Clean and disinfect the working area before
starting the analysis.
Mark the sterile Petri dishes with type of medium, sample number
and date.
7.2 Medium preparation
+ Agar medium. Following the manufacturer's instructions
rehydrate the culture medium with distilled or de-ionised water.
Dispense the medium into bottles and sterilise at 121 °C for 15
min. Check the pH value of the medium.
Medium for immediate use should be cooled to 47°C + 1 °Cina
water bath. Alternatively the medum could be allowed to solidify
and be stored in the dark at 0 to 5 °C for up to 1 month. Media i
bottles can be remelted, but only once.
+ Nutrient pads. Wet the nutrient pads with 3 to 3.5 mL of sterile
distilled or de-ionised water. Ideally a slight access of liquid should
be visible at the edge of the pad [11].
7.3 Glassware preparation. Sterile the Erlenmeyer flasks with
the plugs and the pipettes in the apparatus for dry sterilisation
(180°C) for 2h [10].
7.4 Sample preparation. aseptically place 10 9 of crystaline sugar
or, in the case of liquid sugar, the amount corresponding to 109 of
dry matter, in the 200 mL Erlenmeyer flask which is marked to
indicate 100 mL. Add sterilised water (distilled or de-ionised) up to
the 100 mL mark. Shake thoroughly to dissolve and mix the sugar
solution.
NOTE - 20 g/100 mL must be taken according to the NCA Standard
for “Canners Sugars’.7.5 Inoculation and incubation. For the pour plate method take
two sterile Petri dishes. Transfer to each dish 1 mL of th q
sample solution (7.4).
If necessary prepare serial dilutions from the initial solution (10-*)
adding 1 mL to mL of sterle water In a test tube to give a serles
107, 10? etc and plate out similarly.
NOTE - 5 plates must be taken according to the NCA Standard for
“Canners Sugars”
Add approximately 15 mL of the medium, previously melted and
maintained at 47 + 1°C in a water bath, into each Petri dish. Mix the
Inoculum carefully with the medium by swirling the plate and allow
the mixture to solicify. Prepare a control plate just containing culture
medium to check its sterility.
Invert the Petridishes and inaubate at 30°C for 48-72 h.
NOTE ~ 72 h incubation is specified in the NCA Standard for “Canners
Sugars’.
7.6 Filtration and incubation. Using the membrane filter method
connect the filter apparatus to the vacuum pump or vacuum supply.
If not already sterile, flame the fiter funnel and base with alcohol.
After cooling place the membrane centrally on the base with sterile
forceps. Place the sterile funnel on the top and clamp in place.
Pour the sample (7.4) into the funnel, apoly the vacuum and fiter
the sample. Turn off the vacuum. Rinse the funnel with sterile water
and apply the vacuum once again. Remove the funnel and use
flamed forceps to place the membrane on an agar plate or moistened
nutrient pad. When posttioning the membrane on the surface ensure
that no air bubbles are trapped between them.
Invert the dishes an
tubate at 30°C for 48-72 h.
NOTE - 72 h incubation is specified in the NCA Standard for “Canners
Sugars’8 Expression of Results
Count the colonies in each dish usingthe colony counting equipment.
For samples showing more than 100 cfu/plate examine the dilutions.
The pour plate method is not suitable when colony counts are below
10 cfu/plate. The membrane filtration method should be used
instead.
Calculate the number of CFU per 10 g sugar or 10 a dry matter by
the following calculations.
8.1 Pour plates.
Ye
(em 404-1) -d
CFU/10 9 of sugar or dry matter
where:
Ze — cum of the colonies counted on all the plates,
ny = number of plates counted in the first Dilution,
m2 = number of plates counted in the second Dilution,
¢ = dilution from which the first counts were obtained (10-!).
Example:
Two Petri dishes from two dilutions were incubated and the observed
colony counts were:
Dilution 10-2 tstplate = 97 CFU
2né plate = 110CFU
Dilution 10-2 tstplete = 35 CFU
2nd plate = 46 CFU
97+ 110435446 288
(1-240.1-2)-107 0.22
1309 CFU /g sugar
For 10 g of sugar or dry matter, the result is multiplied by 10 giving
the final result:
1.3 x 104 CFU/10 9 of sugar or dry matter.8.2 Membrane filter.
Ne
n
CFU/10 9 of sugar or dry matter
where:
Ec = sumof the colonies counted on all the plates,
n= number of plates.
Example:
‘Two Petri cishes were incubated and the observed colony counts
were:
istplte = 98 CFU
2nd plate = 110 CFU
98 +110
104 CFU / 10g
2
WARNING AND SAFETY PRECAUTIONS
PLACE THE USED DISPOSABLE PLASTIC PETRI DISHES IN
AUTOCLAVABLE PLASTIC BAGS AND STERILISE PRIOR TO
DISPOSAL ACCORDING TO LOCAL REGULATIONS. GLASS
PETRI DISHES REQUIRE IMMEDIATE STERILISATION.
1
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