Enterobacteriaceae

Revision: 11-Jul-2023
Standard Method

Purpose
This method is intended to detect Enterobacteriaceae in ingredients and environmental
samples. The Family Enterobacteriaceae includes those microorganisms which are
facultatively anaerobic, oxidase negative, gram-negative straight rods which ferment
glucose to acid. They are used as hygiene indicators in the manufacturing process.

Safety
The safety section doesn’t supersede any safety procedures indicated by your local
Chemical Hygiene Plan.

NOTE: Safe lab practices must be followed at all times. Become familiar with all
potential hazards and wear appropriate personal protection equipment before using
this method.)

Equipment
Incubator, 35ºC ± 2°C or 37ºC ± 1°C

Reagents and Materials

• Media
• Diluent
• 0.45µm membrane filter with grid
• Swabs, sponges for environmental samples
• Sterile mineral oil

Samples Preparation and Storage

Refer to Microbiological Sample Preparation, SM-PR-640.

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Standard Method

Table 1. Method and Media and Incubation

Method Media(See Notes 1, 2 and 3) Incubation

• Violet Red Bile Glucose

Agar (VRBGA) or Violet
Red Bile Dextrose Agar
For filterable samples: (VRBDA).
• RAPID’Enterobacteriacea
Membrane Filtration (SM-PR-625) (BIO-RAD)

35°C ± 2°C for 18-

24 hours (VRBGA
• Violet Red Bile Glucose or VRBDA)
Agar (VRBGA) or Violet
Red Bile Dextrose Agar
For non-filterable samples:
(VRBDA).
Pour Plate (SM-PR-655) • RAPID’Enterobacteriacea 37°C ± 1°C for 18-
(BIO-RAD) 24 hours ( RAPID’
Enterobacteriaceae
Medium)
• Violet Red Bile Glucose
Agar (VRBGA) or Violet
For environmental samples: Red Bile Dextrose Agar
(VRBDA).
Environmental Sampling
• RAPID’Enterobacteriacea
Technique (SM-PR-694)
(BIO-RAD)

• 3M™ Petrifilm™ Follow the

3M™ Petrifilm™ Methodology
Enterobacteriaceae Count manufacturers’
(SM-PR-661)
Plates instructions.

Note 1: Refer to Media Preparation and Handling (SM-PR-641) for preparing media and
diluents.

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Standard Method

Note 2: When using pour plate method with VRBG Agar it is necessary to overlay the plate
with 5-8 ml VRBGA. The aim of the second layer is to limit invasion of the surface,
which can interfere with reading.

Note 3: When using pour plate method with RAPID’Enterobacteriacea an overlay is

recommended with 5-8ml agar in case abundant mesophilic flora is expected in the
matrix.

Procedure
Refer to the appropriate procedure in Table 1 (see above) to proceed and start by diluting
samples if necessary, using Decimal Dilution SM-PR-648
1. Incubate plates in an inverted position at 35°C ± 2°C for 18-24 hours or 37°C ± 1°C for 18-
24 hours depending on medium used.
2. Count all characteristics colonies (see below under Interpretation) of Enterobacteriaceae
present on the Petri dish at the end of the incubation period (18-24 hours). If dilutions have
been plated, multiply the number of typical Enterobacteriaceae colonies by the reciprocal of
the dilution used.
3. Retain the plate(s) in the event further analysis is required.
4. Report results as the number of Enterobacteriaceae colony-forming units (CFU) per sample
size.

Interpretation

Violet Red Bile Count all purple-red • Crystal violet and bile salts inhibit the
Glucose Agar colonies, 0.5 mm in Gram positive flora.
(VRBGA) - See diameter or larger,
Note 4 • Degradation of glucose is accompanied
surrounded by a by production of acid which results in a
purple halo which is decreased pH, which is indicated by a
a zone of color change to purple/red and by zones
precipitated bile of precipitated bile acids
surrounding the colonies
acids.

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Standard Method

Count all red • Crystal violet and bile salts inhibit the Gram
colonies with a positive flora.
RAPID’Enterobacteri diameter equal to
acea Medium – See or exceeding 0.5 • Degradation of glucose allows a high level of
Note 5 mm with or without contrast of Enterobacteriaceae colonies which
a zone of appear red on a clear gray medium
precipitation.

Follow the • https://multimedia.3m.com/mws/media/156674

interpretation guide 8O/enterobacteriaceae-count-plate-
3M™ Petrifilm™ from the interpretation-guide-en-hr.pdf
Methodology - See manufacturer.
Note 5

Note 4: In case VRBG medium is used, confirmatory tests are necessary, in particular when the
specification is to have zero or not detected organisms. Also certain Enterobacteriaceae may
cause decolouration of their colonies or of the medium. Therefore, choose at random five purple-
red colonies (if there are less than 5 typical colonies on the plate, take all presumptive colonies
present) or when no characteristic colonies are present, choose five whitish colonies for
confirmation. Oxidase test and glucose fermentation are the biochemical tests to be performed.

If desired an identification, it can be performed using PCR, Vitex, API Strips or other identification
method.

Note 5: There is no need to proceed confirmatory tests using the RAPID’Enterobacteriacea

Medium and 3M™ Petrifilm™ Methodology.

Confirmatory Tests: (colonies from VRBG medium)

Oxidase test :

1. To ensure the oxidase test is carried out with pure cultures, subculture presumptive
Enterobacteriaceae colonies on a non-selective agar e.g. PCA, TSA, NA, and
incubate at 37 °C for 24 h ± 2 h.
2. Use a commercial oxidase test following manufactures’ instructions or add 2 to 3
drops of fresh oxidase reagent onto a filter paper in a Petri-dish. The colonies which
must be confirmed are transferred onto the pretreated filter paper using a plastic or
platinum inoculating loop. A positive oxidase reaction is shown by the appearance of
a dark-blue color within 30 s. This shall not be observed for Enterobacteriaceae
bacteria since they are oxidase negative

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Standard Method

Glucose Fermentation test

1. Using a wire transfer the same colonies selected that gave a negative oxidase test into tubes
containing Glucose OF medium . Overlay the surface of the medium with minimal 1 cm of
sterile mineral oil.
2. Incubate these tubes at 37 °C for 24 h ± 2 h.
3. If a yellow colour develops throughout the content of the tube, regard the reaction as being
positive.
Interpretation of results:

Colonies that are oxidase-negative and glucose-positive are confirmed as Enterobacteriaceae.

References
Media Preparation and Handling SM-PR-641
Decimal Dilutions SM-PR-648

Pour Plate Method SM-PR-655

Membrane Filtration SM-PR-625

Enviromental Monitoring Samples SM-PR-694

3M™Petrifilm™ Methodology SM-PR-661
ISO 21528-2 - Microbiology of the food chain — Horizontal method for the
detection and enumeration of Enterobacteriaceae — Part 2: Colony-count
technique

Compendium of Methods for the Microbiological Examination of

Foods, fifth edition – Chapter 9

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Standard Method

Revision History

Revision Date Summary of Change

11-Jul-2023 • Additon RAPID’Enterobacteriacea Medium (Biorads)
• Additon clarification related overlay in the media
• Included environmental samples in the scope
• Formatted in the new template
• Included 3M Petrifilm methodology
• Included confirmatory tests (oxidase and glucose fermentation) when
using VRBG medium

13-Dec-2013 New document.

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