ISO 21528-2:2017

Microbiology of the food chain –Horizontal method for the detection and enumeration of Enterobacteriaceae
PART 2: COLONY - COUNT TECHNIQUE
Sample Types
✓ Human Food.
✓ Animal Feed.
Materials (Equipment, reagents, chemicals, and media)
✓ Culture medium: VRBG (violet, red bile glucose).
✓ Sub culturing medium: Nutrient agar.
✓ Confirmation test: oxidase discs and glucose of medium.
✓ Apparatus and glassware:
1. Apparatus for wet sterilization (autoclave).
2. Incubator, maintained at 37⁰C ± 1 ⁰C.
3. Water bath, maintained at 47 ⁰C to 50 ⁰C.
4. Test tubes, flasks, bottles of suitable capacity.
5. Pipettes, having a nominal capacity of 1 ml and 10ml.
6. Petri dishes, of approximately 90 mm diameter.
7. Sterile disposable Loops.
8. PH - meter, capable of measuring to an accuracy of ± 0.1 PH unit.
Media preparation
VRBG
✓ Dissolve the dehydrated complete medium in the water by boiling.
✓ Do not sterilize the medium in autoclave.
✓ Cool the medium in a water bath set at 47⁰C to 50⁰C.
✓ Adjust the PH, if necessary, so that after boiling it is 7.4 ± 0.2 at 25⁰C.
✓ Prepare the medium just before use. Use the molten medium within 4h of its
preparation.
Non-selective agar medium
✓ Dissolve the dehydrated complete medium in the water by heating with frequent
agitation. 
✓ Sterilize for 15 min in an autoclave at 121⁰C.
✓ Cool the medium in a water bath set at 47⁰C to 50⁰C.
✓ Adjust the PH, if necessary, so that the PH after sterilization, is 7.0 ± 0.2 at 25⁰C.
Glucose of medium
✓ Dissolve the dehydrated complete medium in the water by heating if necessary.
✓ Dispense the culture medium into sterile flasks of appropriate capacity (10 ml).
✓ Sterilize for 15 min in an autoclave at 121⁰C.
✓ Adjust the PH, if necessary, so that after boiling it is 6.8 ± 0.2 at25⁰C.
✓ The medium may be stored for up to 4 weeks at 5⁰C ± 3 ⁰C.
Sample Preparation

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 ISO 21528-2:2017
Microbiology of the food chain –Horizontal method for the detection and enumeration of Enterobacteriaceae
✓ Prepare sample in a 1:9 ratio in sample as diluent (weigh 10g of sample and add 90ml of
diluent) in stomacher bag.
✓ Mix sample with diluent in the stomacher.
Inoculation
✓ Using a sterile pipette or a micropipette, transfer to a sterile petri dish 1ml of the test
sample (if liquid) or 1ml of the initial dilution (10^-1) in the case of other product.
✓ Repeat the procedure described with the further dilutions, using a fresh sterile pipette
for each dilution.
✓ Pour into each petri dish approximately 15ml of the VRBG medium, previously cooled at
47⁰c - 50⁰c in the water bath.
✓ Carefully mix the inoculum with the medium by horizontal movements and allow the
medium to solidify.
✓ After complete solidification of mixture, add a covering layer of approximately 5ml to 10
ml of VRBG to achieve semi-anaerobic conditions. Allow to solidify.
✓ Invert inoculated dishes so that the bottom is upper most and place them in an
incubator set at 37⁰C for 24h ± 2h.
Results Interpretation
✓ Characteristic colonies are pink to red or purple (with or without precipitation)

Characteristic colonies of Enterobacteriaceae on VRBG Agar

Counting and selection of colonies for confirmation
✓ Select the dishes containing less than 150 characteristic colonies, count these colonies.
✓ Then choose at random five such colonies from each dish for sub culturing for the
biochemical confirmation tests.
✓ If there are less than five colonies on the plate, take all presumptive colonies present.
✓ Spreading colonies shall be considered as single colonies.
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 ISO 21528-2:2017
Microbiology of the food chain –Horizontal method for the detection and enumeration of Enterobacteriaceae
✓ If less than one-quarter of the dish is overgrown by spreading, count the colonies on the
unaffected part of the dish and calculate by extrapolation the theoretical number of
colonies for the entire plate.
✓ If more than one-quarter is overgrown by spreading colonies, discard the count.
Sub culturing selected colonies
✓ Streak the selected colonies onto the surface of pre-dried non-selective agar medium, in
a manner which well allow well-isolated colonies to develop.
✓ Incubate these plates at 37⁰C for 24h ± 2h.
✓ Select a well-isolated colony from each plate for the biochemical confirmation tests.
Biochemical confirmation test
✓ Oxidase reaction:
Using a sterile loop, take a portion of each well-isolated colony and streak onto a filter
paper moistened with the oxidase reagent or onto a commercially available disc.
Consider the test to be negative when the color of the disc or filter paper does not turn
dark purple within 10s. Enterobacteriaceae are oxidase negative.

Oxidase test
Biochemical confirmation test (cont.)
✓ Fermentation test:
Using a wire, stab the same colonies selected from the subculture medium that gave a
negative oxidase test into tubes containing Glucose of medium.
Overly the surface of the medium with minimal 1 cm of sterile mineral oil.
Incubate these tubes at 37⁰C for 24h ± 2h.
If a yellow color develops throughout the content of the tube, regard the reaction as
being positive.
Colonies that are oxidase-negative and glucose-positive are confirmed as
Enterobacteriaceae.

Prepared by Eng. Mohamed Badr

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