SOP: Enumeration of Clostridium perfringens in Foods

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This procedure estimates the number of viable Clostridium perfringens per g or mL of food. A portion of the product is mixed with a selective sulfite cycloserine agar by the pour-plate technique and incubated anaerobically. Typical black colonies are counted as presumptive C. perfringens; a minimum of five of them are put through confirmatory tests (motility, nitrate reduction, lactose fermentation and gelatin liquefaction). The confirmed count is calculated from the ratio of confirmed to tested presumptive colonies.

2. Apparatus required

• Stomacher, blender or equivalent
• Anaerobic system, such as anaerobic jars, the AnaeroGen anaerobic atmosphere generation system (Oxoid) or an anaerobic incubator capable of maintaining 35°C
• Aerobic incubator capable of maintaining 35°C
• Water bath capable of maintaining 45°C (where sodium citrate is used)
• Colony counting device (optional)

Note on temperatures: the laboratory is responsible for holding its incubators and water baths at the stated temperatures. Where 35°C is recommended, 35 ± 1.0°C is acceptable; lower temperatures of 30 or 25°C may vary by ± 1.0°C. Where higher temperatures are recommended (such as 43 or 45.5°C), the equipment must be held within 0.5°C, since higher temperatures may be lethal to the organism being isolated.

3. Media and reagents

• Sulfite cycloserine agar (SC)
• Nitrate-motility (NM) agar
• Basal medium: peptone — 5.0 g; beef extract — 3.0 g; potassium nitrate — 1.0 g; disodium phosphate — 2.5 g; galactose — 5.0 g; glycerol — 5.0 g; agar — 3.0 g; distilled water — 1.0 L. pH 7.3 ± 0.2.
• To prepare: add the components, except the agar, to the distilled water ↓ mix thoroughly and adjust the pH ↓ add the agar and gently heat to boiling to dissolve it ↓ dispense into tubes and autoclave at 121 °C for 15 min. If the medium is not used within 4 h, deaerate it by heating for 10 min in boiling water or flowing steam, then cool.
• Nitrate reagents
• 2% sodium citrate (pre-warmed to 45°C, for cheese samples)
• 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.
• Peptone water diluent (0.1%)
• 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.
• Lactose gelatin (LG)
• Zinc dust (for the nitrate confirmation)
• Gas mixture: 5% CO₂, 10% H₂ and 85% N₂ (where an anaerobic incubator or jars with a venting system are used)
• Anaerobic indicator

The media and reagents above are commercially available and are used, prepared and sterilized according to the manufacturer’s instructions; media formulas follow the Compendium’s Media Glossary. Where a laboratory varies a listed formulation, it must demonstrate equivalency; equivalency data is forwarded to the Editor of the Compendium of Analytical Methods for consideration of modification of the method.

4. Sample preparation

• Analyze each sample unit individually; keep refrigerated or frozen (except shelf-stable foods) and analyze as soon as possible after receipt. Thaw frozen samples in a refrigerator, or under time and temperature conditions that prevent microbial growth or death.
• Prepare the diluent: sterile 0.1% peptone water (or the diluent indicated in Table I below).
• Clean the surface of the working area with a suitable disinfectant.
• For a representative analytical unit, agitate liquids or free-flowing materials until homogeneous; for solids, take portions from several locations within the sample unit.
• Prepare a 1 in 10 (1:9) dilution by aseptically shaking, stomaching or blending 25 g or mL of food into 225 mL of diluent, as Table I indicates. If another sample size is used, keep the 1 in 10 ratio (e.g. 11 g in 99 mL, or 10 g in 90 mL).
• Blend for the minimum time needed for a homogeneous suspension (not longer than 2.5 min, to avoid overheating); with foaming foods, blend at low speed and draw the aliquot from below the foam surface. When shaking in a dilution bottle, shake 25 times through a 30 cm arc in about 7 s. With a stomacher, macerate for 1 min.
• Let the 1 in 10 homogenate of dry foods stand at room temperature for 15 min; in all other cases continue the analysis as soon as possible.
• Prepare further decimal dilutions as required, using a separate sterile pipette for each transfer; shake every dilution immediately before transfer.

Table I — Preparation of the initial dilution

• Liquids (milk, water, etc.): pipette directly into Petri plates and/or peptone water diluent; swirl to mix or shake as appropriate
• Viscous and non-miscible liquids: weigh into peptone water diluent; shake
• Solids — water-soluble solids, spices: weigh into peptone water diluent; shake
• Solids — powder, meats, shellfish: weigh into peptone water diluent; stomach or blend
• Cheese: weigh into 2% aqueous sodium citrate pre-warmed at 45°C; stomach or blend

The sample may go into an empty sterile stomacher bag, blender jar or dilution bottle with the diluent added before mixing.

5. Procedure

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

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Pour about 20 mL of sulfite cycloserine agar into each plate and mix by gentle rotation

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Incubate the plates anaerobically in an upright position at 35°C for 20 h

• For a small number of plates, anaerobic jars or the AnaeroGen system may be used according to the manufacturer’s instructions; for a large number of plates an anaerobic incubator is preferable
• Anaerobic incubators and jars need three evacuations and replacements with the 5% CO₂ / 10% H₂ / 85% N₂ mixture; each jar and incubator must contain an anaerobic indicator
• Longer incubation may cause excess blackening along the bottom rim of the plates; do not invert the plates, since inversion may displace the agar by gas

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After 20 h, check the indicators to confirm anaerobiosis; without anaerobiosis, discontinue the analysis

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Select plates with 20–200 black colonies about 1–2.5 mm in diameter; do not count pinpoint black colonies. If no plates fall within that range, plates containing fewer than 20 or more than 200 colonies can be used

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Count the presumptive colonies and average the duplicate plates

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Randomly select a minimum of five presumptive colonies from plates in the acceptable range (or all of them if fewer than five were found); stab-inoculate nitrate-motility (NM) agar with each, and in parallel inoculate the same colonies deep into lactose gelatin (LG)

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Close the tubes and incubate anaerobically at 35–37°C for 24 h; incubate uninoculated NM and LG tubes as controls

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To each NM tube showing a distinct line of non-motile growth along the stab, add 0.2–0.4 mL of nitrate reagent: a red colour at the top means nitrate was reduced to nitrite (positive). Faint reactions only slightly stronger than the blanks count as negative

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To a negative culture, add a small quantity of zinc dust: a developing red colour is a negative test; no colour change is positive (no nitrate remains — the culture has reduced it beyond the nitrite stage). Where growth is limited to the lower part of the tube with little or no colour, aspirate and discard the upper part of the medium and add nitrate reagent to the remainder

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Examine the LG tubes for gas production and for a red-to-yellow colour change (lactose fermentation)

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Place the LG tubes in ice water for 10 min or in a refrigerator for 1 h; examine for liquefaction. If no liquefaction has occurred but the organism is non-motile, reduced nitrate to nitrite and is lactose positive, re-incubate the LG tube for another 24 h at 35–37°C. Isolates that are non-motile, reduce nitrate, are lactose positive and liquefy gelatin within 48 h are confirmed C. perfringens

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Optional: in addition to the tests mentioned, rapid identification kits may be used, such as the API 20A, API An-ident or Vitek 2 Anaerobic cards

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Calculate the confirmed count (section 6 below)

6. Calculation

Presumptive count per g (mL):

N = A × D

• N = presumptive count (colonies per g or mL)
• A = average presumptive count from duplicate plates
• D = dilution factor

Reporting rules:

• If the lowest number of colonies per plate exceeds 200, count or estimate and record with the letter E to show lower accuracy, e.g. 1.8 × 10⁶ E
• If too high to estimate, record the minimum number estimable with a > sign, e.g. >2.0 × 10⁶
• If the highest number of colonies per plate is below 20, record with the letter E, e.g. 1.2 × 10³ E
• If no presumptive colonies are found, record the count as <0.5 × D

Confirmed count per g (mL):

Confirmed count = Presumptive count × (No. of colonies confirmed ÷ No. of colonies tested)

• If no colonies are confirmed, record the count as <0.5 × D, where D is the dilution factor

7. Precautions

• The laboratory supervisor must see that the analysis is carried out in accordance with ISO/IEC 17025:2005 (or the latest version), “General Requirements for the Competence of Testing and Calibration Laboratories”
• Anaerobic work is essential: without confirmed anaerobiosis after incubation, the analysis must be stopped
• Do not prolong incubation beyond 20 h — excess blackening along the plate rims can obscure the count
• Pinpoint black colonies are not counted

References

Health Canada, Compendium of Analytical Methods, Volume 2 — HPB Method MFHPB-23, June 2015: “Enumeration of Clostridium perfringens in foods” (replaces MFHPB-23, November 2001).

• Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.
• Hauschild, A.H.W. and R. Hilsheimer. 1974. Enumeration of food-borne Clostridium perfringens in egg yolk-free tryptose-sulfite-cycloserine agar. Appl. Microbiol. 27:521-526.
• Hauschild, A.H.W. 1975. Criteria and procedures for implicating Clostridium perfringens in food-borne outbreaks. Can. J. Public Health 66:388-392.
• Hauschild, A.H.W., R.J. Gilbert, S.M. Harmon, M.F. O’Keeffe and R. Vahlefield. 1977. ICMSF methods studies. VIII. Comparative study for the enumeration of Clostridium perfringens in foods. Can. J. Microbiol. 23:884-892.