SOP: Determination of Coliforms in Foods Using Violet Red Bile Agar

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This method enumerates coliforms in foods and food ingredients to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. Where an Official Method is specified for certain products, that method is followed. The method is not applicable to the enumeration of coliforms in fish, fish products or other “seafood”.

The presence of coliforms in a food may indicate manufacture under unsanitary conditions. This procedure estimates the number of viable coliforms per gram or millilitre of product: a portion of the product is mixed and incubated with a selective medium by the pour-plate technique, a selection of typical red-purple colonies is subjected to confirmatory tests, and the number of confirmed coliforms is calculated from the ratio of colonies confirmed to colonies tested.

2. Apparatus required

• Stomacher, blender or equivalent
• pH meter or pH paper capable of distinguishing 0.3 to 0.5 pH units within a range of 5.0 to 8.0
• Light microscope
• Incubator capable of maintaining 35°C
• Colony counting device (optional)

3. Media and reagents

• Peptone (0.1%) water diluent (PW)
• Basal medium: peptone — 1.0 g; distilled water — 1.0 L. Final pH 7.0 ± 0.2.
• To prepare: suspend 1.0 g of peptone in 1.0 L of distilled water ↓ mix thoroughly, and dispense into dilution bottles or tubes in amounts that will allow for volume lost due to autoclaving ↓ autoclave at 121 °C for 15 min.
• 2% aqueous sodium citrate, tempered to 45°C (the source prints “450C”) — for cheese samples only
• To prepare: dissolve 20.0 g of trisodium citrate dihydrate (Na3C6H5O7·2H2O) in 1.0 L of distilled water by heating to 45–50 °C; adjust the pH to 7.5 ± 0.2; dispense; autoclave at 121 °C for 15 min.
• Nutrient agar (NA)
• Basal medium: peptone — 5.0 g; beef extract — 3.0 g; agar — 15.0 g; distilled water — 1.0 L. pH 6.8 ± 0.2.
• To prepare: suspend the ingredients in 1.0 L of distilled water and heat to boiling with frequent swirling ↓ dispense into tubes or plates and autoclave at 121 °C for 15 min. Cool tubes in a slanted position. Plates can be stored for up to 8 weeks at 4 °C.
• Tryptic soy agar (TSA)
• Basal medium: tryptone — 15.0 g; soya peptone — 5.0 g; sodium chloride (NaCl) — 5.0 g; agar — 15.0 g; distilled water — 1.0 L. pH 7.3 ± 0.2.
• To prepare: suspend the basal medium ingredients in 1.0 L of distilled water ↓ heat to boiling with constant stirring to dissolve all ingredients; do not overheat ↓ autoclave at 121 °C for 15 min.
• Levine’s eosin methylene blue (L-EMB) agar or Endo agar
• L-EMB agar: peptone — 10.0 g; lactose — 10.0 g; dipotassium phosphate (K2HPO4) — 2.0 g; agar — 15.0 g; eosin Y — 0.4 g; methylene blue — 0.065 g; distilled water — 1.0 L. Final pH 7.1 ± 0.2.
• To prepare (L-EMB): add all the ingredients to distilled water ↓ heat to boiling to obtain a complete solution ↓ mix well ↓ dispense as required (usually in 100–200 mL volumes) ↓ sterilize at 121 °C for 15 min. Avoid overheating, because the agar is likely to become soft and unsuitable for streaking. When the agar is to be used as a streaking medium, melt it in flowing steam or boiling water, swirl gently to distribute the flocculant precipitate, and pour a thick layer into Petri plates. Dry the surface completely. Plates can be stored at 2 °C to 7 °C in the dark for 1 month.
• Brilliant green lactose 2% bile (BGLB) broth
• Basal medium: peptone — 10.0 g; lactose — 10.0 g; ox-gall — 20.0 g; brilliant green — 0.0133 g. Final pH 7.2 ± 0.2.
• To prepare: dissolve the peptone and lactose in 500 mL of water ↓ add the ox-gall dissolved in 200 mL of water ↓ bring the volume to approximately 975 mL with water and adjust the pH ↓ add 13.3 mL of a 1% aqueous solution of brilliant green ↓ bring the total volume to 1.0 L, stir, and filter through cotton if necessary to clarify ↓ dispense in 10.0 mL volumes into tubes containing gas vials ↓ sterilize at 121 °C for 12 min.
• Lauryl sulfate tryptose (LST) broth
• Basal medium: tryptose, tryptone or trypticase — 20.0 g; lactose — 5.0 g; dipotassium phosphate (K2HPO4) — 2.75 g; monopotassium phosphate (KH2PO4) — 2.75 g; sodium chloride (NaCl) — 5.0 g; sodium lauryl sulphate — 0.1 g; distilled water — 1.0 L. Final pH 6.8 ± 0.2.
• To prepare: dissolve the ingredients in 1.0 L of distilled water ↓ dispense in 10.0 mL volumes into tubes containing gas vials ↓ sterilize at 121 °C for 15 min.
• Violet red bile (VRBA) agar
• Basal medium: yeast extract — 3.0 g; peptone — 7.0 g; sodium chloride (NaCl) — 5.0 g; bile salts no. 3 — 1.5 g; lactose — 10.0 g; neutral red — 0.03 g; crystal violet — 0.002 g; agar — 15.0 g; distilled water — 1.0 L. Final pH 7.4 ± 0.2.
• To prepare: suspend the basal medium ingredients in 1.0 L of distilled water ↓ heat to boiling to dissolve completely (boil for no more than 2 min) ↓ do not autoclave ↓ mix well before pouring. Prepare the medium on the day of use. Do not incubate prepared plates longer than 24 hours, as the selectivity diminishes.
• Double strength violet red bile agar
• Basal medium: yeast extract — 3.0 g; peptone — 7.0 g; sodium chloride (NaCl) — 5.0 g; bile salts no. 3 — 3.0 g; lactose — 10.0 g; neutral red — 0.06 g; crystal violet — 0.004 g; agar — 15.0 g; distilled water — 1.0 L. (Double the usual concentrations of bile salts no. 3, neutral red and crystal violet.)
• To prepare: suspend the basal medium ingredients in 1.0 L of distilled water ↓ heat to boiling to dissolve completely (boil for no more than 2 min) ↓ do not autoclave ↓ mix well before pouring. Prepare the medium on the day of use. Do not incubate prepared plates longer than 24 hours, as the selectivity diminishes.
• 1 N HCl and 1 N NaOH
• 1 N HCl — To prepare: dilute concentrated hydrochloric acid with distilled water to 1.0 L at a final concentration of 1 mol/L (1 N).
• 1 N NaOH — To prepare: dissolve 40.0 g of sodium hydroxide in distilled water and make up to 1.0 L.
• Gram stain reagents
• Positive and negative control cultures, ATCC or equivalent (see the quality-control table below)

Media 1 and 3 to 8 are commercially available; prepare and sterilize them according to the manufacturer’s instructions.

• Quality-control organisms:

Use the strains listed or ones giving the same reaction: Escherichia coli ATCC 25922; Enterobacter aerogenes ATCC 13048; Staphylococcus aureus ATCC 25923; Salmonella enteritidis ATCC 13076.

4. Sample preparation

Each sample unit may be analysed individually, or the analytical units may be combined.

• In the laboratory before analysis, except for shelf-stable foods, keep sample units refrigerated (0–5°C) or frozen, depending on the nature of the product; thaw frozen samples in a refrigerator, or under time and temperature conditions that prevent microbial growth or death
• Analyse sample units as soon as possible after receipt in the laboratory
• Have ready sterile peptone water
• Clean the surface of the working area with a suitable disinfectant
• To obtain a truly representative analytical unit, agitate liquids or free-flowing materials until the contents are homogeneous; if the sample unit is a solid, take the analytical unit from several locations within the sample unit; to reduce the workload, the analytical units may be combined for analysis — a composite should contain not more than 500 g
• Prepare a 1:10 dilution of the food by aseptically blending 25 g or mL (the analytical unit) into 225 mL of the required diluent, as indicated in the initial-dilution table below; if a sample size other than 25 g or mL is used, maintain the 1:10 sample-to-dilution ratio, such as 11 (10) g or mL into 99 (90) mL (volume in brackets indicates the alternate procedure for marking dilutions)
• With products that require blending, blend or stomach for the minimum time required to produce a homogeneous suspension and to avoid overheating; blending time should not exceed 2.5 min; shake dilutions 25 times through a 1-foot (30 cm) arc in approximately 7 seconds
• Allow the food homogenate (1:10 dilution) of dry foods to stand at room temperature for 15 min; in all other instances, continue the analysis without this delay
• Check the pH of the food suspension; if the pH is outside 5.5–7.6, adjust the pH to 7.0 by adding sterile 1 N NaOH or 1 N HCl
• While blending foods that tend to foam, use the blender at low speed and remove the aliquot from below the liquid/foam interface
• Prepare succeeding decimal dilutions as required, using a separate sterile pipette for each transfer

• Shake all dilutions as described above, immediately prior to making transfers, to ensure uniform distribution of the microorganisms present

• Preparation of the initial dilution:

5. Procedure

Pipette 1 mL of the required dilutions into each of duplicate sterile Petri plates

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For unprocessed foods: pour into each plate approximately 10 mL of VRBA agar, mix by gentle rotation and allow to solidify on a level surface

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Overlay with approximately 5 mL of VRBA and allow to solidify (this helps prevent surface growth and spreading of the colonies)

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Invert and incubate the plates at 35°C for 24 ± 2 h

For refrigerated, frozen and processed foods:

Pipette 1 mL of the required dilutions into each of duplicate sterile Petri plates

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Pour into each plate approximately 10 mL of TSA, mix by gentle rotation and allow to solidify on a level surface

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Incubate the plates at room temperature for 2 ± 0.5 hours

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Overlay with 5 mL, or an amount to give a thin, even layer, of double strength VRBA and allow to solidify

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Invert and incubate the plates at 35°C for 24 ± 2 h

Confirmed coliform count:

Count plates containing 20–200 purple-red colonies, about 0.5 mm in diameter or larger, surrounded by a reddish zone of precipitated bile

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Pick representative numbers of colonies of each morphological type and transfer each to a tube of BGLB broth; purify the colonies as required on VRBA

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Incubate the inoculated BGLB broth tubes at 35°C for 24 ± 2 h; examine for gas formation and record results

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Incubate gas-negative tubes for an additional 24 ± 2 h, re-examine, record the numbers of additional gas-positive tubes and add to the result obtained above

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Formation of gas during 48 ± 4 h incubation constitutes a positive confirmed test; perform Gram stains on any tube with a pellicle, to exclude Gram-positive, lactose-fermenting bacilli

Supplementary analysis for completed test (optional):

If it is necessary to do a completed test for the presence of coliforms, submit all positive BGLB tubes to the completed test as soon as they are recorded as positive

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Gently shake positive BGLB broth tubes, and from each tube streak L-EMB or Endo agar plates to obtain well-isolated colonies; incubate the plates at 35°C for 24 ± 2 h

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Examine the plates: typical coliform colonies on these media may be nucleated with or without a metallic sheen, or they may be opaque, mucoid and pink; all other types should not be considered as belonging to the coliform group

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Select, preferably with the assistance of a stereoscopic microscope or other magnifying device, two typical coliform colonies from each plate; if no typical coliform colonies are present, select two colonies considered most likely to belong to the coliform group; pick isolated colonies; where no clearly isolated colonies are visible, purify the colonies by restreaking on fresh selective agar

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From the selected colonies, inoculate NA and fresh LST broth tubes

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Incubate the inoculated NA at 35°C for 24 ± 2 h or 48 ± 4 h; incubate the inoculated LST broth at 35°C for 24 ± 2 h; examine for gas formation and record results

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Incubate gas-negative tubes for an additional 24 ± 2 h, re-examine, record the number of additional gas-positive tubes and add to the results obtained above

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Where an LST tube shows gas after 24 ± 2 h or 48 ± 4 h, prepare a Gram stain from the matching NA slope culture; examine it microscopically for cell shape and Gram reaction, and record the results

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Gas in an LST tube, together with Gram-negative, non-sporeforming rod-shaped bacteria in the agar cultures, counts as a positive completed test for coliform organisms; if both isolates fail to produce gas in LST and/or are not Gram-negative, non-sporeforming rods, then coliforms are considered absent from the BGLB tube from which they originated

6. Calculation

Confirmed coliform count per g or mL = (P ÷ 100) × C × D

Where,

• P = percent of BGLB tubes confirmed as positive
• C = VRBA presumptive count (the 20–200 purple-red colony count)
• D = dilution factor

Report the average of the duplicate plates. The same formula is used after the optional completed test.

• Where the plate with the fewest colonies exceeds 200, count or estimate the colonies and mark the result with the letter E (estimate), e.g. 1.8 × 10⁶ E, showing a lower degree of accuracy; if the number is too high to be estimated, record the smallest number that can be estimated with a > sign, e.g. >2.0 × 10⁶
• Where the plate with the most colonies stays below 20, mark the result with the letter E, e.g. 1.2 × 10³ E
• If no presumptive colonies are found, record the count as <0.5 × the dilution factor

7. Precautions

• It is the responsibility of each laboratory to ensure that the temperatures of the incubators or water baths are maintained at the recommended temperatures: where 35°C is recommended, the incubator may be 35 ± 1.0°C; similarly, lower temperatures of 30 or 25°C may be ± 1.0°C; however, where higher temperatures are recommended, such as 43 or 45.5°C, it is imperative that the incubators or water baths be maintained within 0.5°C, due to the potential lethality of higher temperatures on the microorganism being isolated
• Use ATCC or equivalent control strains; the strains suggested in the quality-control table above, or ones giving the same reaction
• Where an Official Method for certain products is specified, that method shall be followed
• This method is not applicable to fish, fish products or other “seafood”

References

Health Canada, Compendium of Analytical Methods, Volume 2 — HPB Method MFHPB-31, March 2001: “Determination of Coliforms in Foods Using Violet Red Bile Agar.” (Replaces MFHPB-31, dated April 1997.)

The method’s own bibliography: Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition; American Public Health Association (APHA). 1992. Compendium of Methods for the Microbiological Examination of Foods. Third edition; International Commission on Microbiological Specifications for Foods (ICMSF). 1978. Microorganisms in Foods; Their Significance and Method of Enumeration. Second edition; ICMSF. 1980. Microbial Ecology of Foods; Factors Affecting Life and Death of Microorganisms; Food and Drug Administration. 1995. Bacteriological Analytical Manual. Eighth edition.