SOP: Determination of Commercial Sterility and the Presence of Viable Microorganisms in Canned Foods

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

The contents of hermetically sealed foods are sampled aseptically and inoculated into suitable media to determine whether any viable microorganisms are present. A portion of the product is mixed with specified media and incubated under defined conditions of time and temperature. Under those conditions every viable microorganism is expected to multiply and produce either a visible colony on solid media or turbidity in liquid media. The method also allows the isolates to be characterized: mixed or pure population; anaerobic, aerobic or facultative; mesophilic or thermophilic; and coccal, bacillal or spiral cell morphology.

Which procedure to follow depends on the food’s acidity and water activity:

• Low-acid foods — any component of the food has a pH above 4.6 and a water activity above 0.85.
• Acid and acidified low-acid foods — the food has a pH of 4.6 or below.

2. Apparatus required

• Laminar air-flow cabinet or biological safety cabinet, located in a clean environment (a laboratory, or a portion of a laboratory, with no open windows and designed so that there are no direct drafts across the work area). The particle count of the laminar flow must not exceed a total of 100 particles of size 0.5 micron or greater per cubic foot (Class 100, U.S. Federal Standard 209B). A Class 10,000 clean room is acceptable under the additional quality-assurance provisions, and a Class 100,000 clean room has also proved satisfactory where those additional quality-assurance measures are taken.
• Sterile can openers (e.g., Bacti-Disc Cutters) or other sterile opening devices
• Sterile screw-cap culture tubes (20 × 150 mm)
• Sterile cotton-plugged transfer pipettes (liquids and semi-liquids)
• Sterile spoons, spatulas, probes, pliers, cork borers (viscous, semi-solid and solid products)
• Sterile loops
• Anaerobe jars
• Sanitizing solution (e.g., chlorine); 2–10 L soaking container; chlorine-indicating tape
• 70% alcohol (optional)
• pH meter or paper capable of distinguishing 0.3–0.5 pH over the range 6.0–7.5
• Sterile, sealable containers for sample storage
• Gram stain solutions; light microscope
• Diamond-point pen or other marking device
• Sterile examination trays
• Sterile plastic bags (for swollen containers)
• Protective clothing: clean laboratory smock with protective sleeves and tight-fitting cuffs; surgical gloves; cap to cover hair; mask covering mouth, nose and facial hair (face masks are optional, or an equivalent). Disinfect gloved hands as required.
• Incubators at 30, 35 and 55°C (temperature tolerances: ±1.0°C at 30–35°C; ±0.5°C at 43–55°C)
• Water baths at 80 and 100°C

3. Media and reagents

Most of the media below are commercially available and are prepared and sterilized per the manufacturer’s instructions. Preparation formulae for the in-house media are given in section 8.

Low-acid foods — enrichment: Cooked Meat Medium (CMM); Modified PE-2 medium.
Low-acid foods — plating: Plate Count Agar (PCA); Tryptic Soy Agar (TSA); Blood Agar (BA); Reinforced Clostridial Medium (RCM); Liver Veal Agar (LVA).
Low-acid foods — optional: Baird-Parker agar; MacConkey’s agar.
Acid and acidified low-acid foods — enrichment: Acid Broth (AB). Adjust the pH of the broths and agars to the pH of the food under analysis. Prior knowledge of the food’s pH may be sufficient, but when the food’s pH is unknown, determine the pH of the contents of at least one can.
Acid and acidified low-acid foods — plating: Potato Dextrose Agar (PDA); Sabouraud Dextrose Agar (SDA); Thermoacidurans Agar (TA); Orange Serum Agar (OSA; filter aid is needed).

• Control strains (or equivalents): Clostridium sporogenes ATCC 7955 (NFPA 3679) or C. histolyticum — anaerobic mesophilic sporeformer; Bacillus coagulans ATCC 8038 (NFPA 43P) — aerobic growth at 30°C; Bacillus stearothermophilus ATCC 12016 (NFPA 2184) — obligate aerobic thermophile; Clostridium thermosaccharolyticum ATCC 7956 — obligate anaerobic thermophile.
• Chlorine disinfecting solution (see section 8.3).
• For shelf-stable milk and milk products: add 1 mL of re-sterilized product to each litre of tempered medium before pouring the plates, to aid recovery.

4. Procedure

For aseptically processed and packaged products collected less than 30 days after packaging: pre-incubate each container for 7 days at 30–35°C before taking the analytical unit. This does not apply to illness investigations or to cases of evident lack of commercial sterility.

Analyse each sample unit individually

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Identify each sample unit legibly, directly on the container, in a way that survives subsequent handling; record the complete code

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Put coincident marks on the label and container, then remove the label as intact as possible and mark it with the sample and sample-unit numbers (not applicable to lithographed cans and printed flexible packages)

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Clean the external container surface; immerse completely in chlorine solution (or flood the end to be opened) for at least 20 min; verify periodically that available chlorine stays at or above 100 ppm (re-make the solution when it falls below this level) — alternatively decontaminate with 2% peracetic acid in a wetting agent (e.g., 0.1% polysorbate 80) for 5 min

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Place the container with the end to be opened facing up (usually the non-coded end) under a properly operating laminar air-flow hood; let the surface dry completely (air-dry under laminar flow or use sterile disposable tissues)

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Open the container with a sterile opener, with the container’s coded end down. For swollen containers: place the container in a sterile plastic bag and, holding the open end of the bag firmly about the opener shaft, pierce the container; do not remove the container until it has completely vented (see Precautions). This bag method has in practice proven the best for swollen flexible pouch products.

To disinfect an opener for immediate re-use: remove any adhering product; immerse the complete tip in 70% aqueous ethanol and flame the entire end, both the piercing and the cutting edges. Let it cool under the laminar-flow hood before re-use. Alternatively, immerse the opener in the chlorine disinfecting solution for at least 20 minutes, then air-dry under laminar flow or dry by flaming.

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Cut the opening to ¼ inch (6.35 mm) from the double seam

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Immediately transfer at least 50 g of the food aseptically to a sealable sterile container; label and refrigerate as the retained portion

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Withdraw product for the media inoculations: 1–2 mL (or g) per tube of liquid medium; for direct plating, streak each plate with at least one loopful (approx. 0.01 mL), or use pour plates

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Sample the product according to its texture:

• Liquids and semi-liquids: shake the unopened container end over end at least 20–30 times before opening; sample with sterile pipettes
• Solids and semi-solids: take a core with a sterilized cork borer for the centre; remove the entire contents to a sterile tray for the external surface and scrape it, especially near the seams, with a sterile spatula (remove the can end only with a sterile conventional opener; finish any vacuum-leak test first). If both double seams are suspect and their integrity is to be tested, open the can by another aseptic means — for example, a scored can can be opened with its key or a similar device, after checking that the score area is not damaged

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If the product’s normal storage or handling temperature is above 40°C, analyse for THERMOPHILES; otherwise analyse for MESOPHILES only (swollen cans, underprocessed lots and food-poisoning samples are analysed for BOTH)

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MESOPHILES — LOW-ACID FOODS. Enrichment: inoculate 2 tubes of sterile PE-2 and 2 tubes of sterile CMM (include positive and negative media controls); incubate aerobically at 30–35°C for at least 14 days; examine daily. From all positive and suspected-positive tubes, prepare duplicate streak plates of PCA, TSA or BA (RCM or LVA for anaerobes); incubate one plate aerobically and one anaerobically at 35°C for 2–5 days; examine daily; determine Gram reaction and cell morphology for each colony type. Direct plating: streak or pour two PCA plates (RCM or LVA for anaerobes); incubate one aerobically and one anaerobically at 35°C for 2–5 days; record colony types, Gram reaction and cell morphology. When specific microorganisms are suspected, use the corresponding media — for example, Baird-Parker agar, MacConkey’s agar and similar.

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MESOPHILES — ACID AND ACIDIFIED LOW-ACID FOODS (adjust medium pH to the food’s pH). Enrichment: inoculate 2 tubes of sterile Acid Broth — or, when the product is fluid or semi-fluid, 2 tubes of the re-sterilized food product itself (add recently boiled distilled water to solid foods for consistency); incubate one set aerobically and one anaerobically at 30–35°C for at least 14 days; examine after 3, 7 and 14 days — because growth is often hard to see in food products used as the growth medium, use direct smears and streak plates to determine whether growth has occurred; prepare duplicate streak PDA plates and duplicate pour plates of TA, or duplicate plates of SDA and OSA; incubate aerobically and anaerobically at 30°C for 2–5 days; examine daily; determine Gram reaction and cell morphology for each colony. Direct plating: streak or pour two PDA plates and two TA plates — or two SDA plates and two OSA plates; incubate one aerobically and one anaerobically at 35°C for 2–5 days; record colony types, Gram reaction and cell morphology. Inoculate other appropriate media when specific organisms are suspected.

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THERMOPHILES — LOW-ACID FOODS. Enrichment: inoculate 2 tubes of sterile PE-2 and 2 tubes of sterile CMM; heat-shock one tube of each medium for 10 min at 80°C (start timing only after the medium reaches 80°C); incubate all tubes at 55°C for up to 7 days; examine daily; treat positive and suspected-positive tubes as in the mesophile enrichment, but incubate plates at 55°C. Direct plating: streak or pour two PCA plates (two RCM or LVA for anaerobes); incubate one aerobically and one anaerobically at 55°C for 2–5 days; record colony types, Gram reaction and cell morphology.

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THERMOPHILES — ACID AND ACIDIFIED LOW-ACID FOODS. Enrichment: inoculate 2 tubes of sterile Acid Broth or 2 tubes of re-sterilized food product; heat-shock and incubate as for thermophilic low-acid enrichment. Direct plating: streak or pour two PDA plates and two TA plates — or two SDA plates and two OSA plates; incubate one plate aerobically and one anaerobically at 55°C for 2–5 days; record colony types, Gram reaction and cell morphology. Inoculate other appropriate media when specific organisms are suspected.

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Examine the product organoleptically (off-odours, frothing, curdling, discolouration — record; never taste)

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Measure the pH of the sampled food as soon as possible after sampling (growth often shifts pH)

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Identify isolates when required (compliance actions, recalls): Gram reaction and cell morphology for each colony type; send cultures to a Reference Laboratory if needed

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Examine the product directly under the microscope: direct microscopic examination of the product often reveals evidence of microbial growth before or after processing. This test does not require aseptic conditions, but prepare the smears for examination after sampling the container contents.

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Dispose safely: autoclave the container and contents after sampling if commercial sterility is in doubt — this is MANDATORY before disposal whenever C. botulinum is suspected

5. Interpretation of results

• No visible growth in any enrichment tube and no colonies on any direct plate: the sample unit meets the commercial-sterility expectation.
• Growth (turbidity in liquid media or colonies on solid media) indicates viable microorganisms are present; record their aerobic/anaerobic requirements, temperature range (mesophilic/thermophilic) and morphology.
• Note that the retained sample unit must be interpreted with care, since refrigeration can change bacterial densities.
• A retained sample unit may be used for toxin analysis (C. botulinum: see MFHPB-16; S. aureus: see MFLP-47), chemical analysis (e.g., pH) or repeat microbiological analysis.

6. Quality control

• Negative food control: if needed, autoclave an aliquot of the product at 121°C for 15 min and inoculate 2 tubes of PE-2 and 2 tubes of CMM with it. Record all results.
• Negative media control: pre-incubate media for at least two days (or one unit of medium per sample per batch used). Record all results.
• Positive media controls, one unit per batch: C. sporogenes or C. histolyticum on PE2, CMM or LVA at 35°C; B. coagulans on Thermoacidurans Agar, Acid Broth or PDA at 30°C; B. stearothermophilus on PE2, CMM or PCA at 55°C; C. thermosaccharolyticum on PE2, CMM or LVA at 55°C. Record all results.
• Laminar-flow cabinets: carry out a DiOctyl Phthalate (DOP) challenge test or an equivalent test, and measure the air velocity, at least annually and more often as required; record the results of the tests. Biosafety cabinets: certify at least annually and record the results of the certification tests.
• Clean rooms Class 10,000 and 100,000: check the microbiological quality of the air with a Lusella slit-type air sampler (or another type of air sampler) and/or open plates exposed for one hour — at least once per operation (Class 10,000) or continuously during every test operation (Class 100,000). Record the results of all tests.

7. Precautions

• During storage and transport, keep swollen or leaking containers and suspect food-poisoning samples properly contained — in sealable plastic bags placed in water-tight metal or plastic bins — and refrigerated. Wherever possible, transfer the contents of open containers aseptically to sterile containers.
• HANDLE ALL SWOLLEN CONTAINERS AS IF THEY CONTAINED CLOSTRIDIUM BOTULINUM AND/OR ITS TOXINS UNTIL PROVEN OTHERWISE. NEVER TASTE FOOD FROM A CONTAINER UNDER INVESTIGATION. ALWAYS USE MECHANICAL PIPETTING DEVICES.
• Carry out all microbiological analyses under the laminar air-flow cabinet or biological safety cabinet located in a clean environment.
• Swollen containers (especially hard swells) can spray their contents when punctured — handle in a Class II biosafety cabinet, which protects both operator and product.
• Follow all C. botulinum precautions and methodology in MFHPB-16.

8. Media preparation (per the source method)

For steam sterilization, the load must be sufficiently pre-heated before the sterilization period starts. This varies considerably with the nature and size of the load, so follow proper exposure times to ensure sterilization of flask solutions and heat-stable culture media, particularly when prepared in large volumes.

8.1 Acid Broth (AB): proteose peptone 5 g; yeast extract 5 g; dextrose 5 g; K₂HPO₄ 4 g; distilled water 1000 mL. Adjust to the pH of the food under analysis with 1 N HCl; sterilize at 121°C for 20 min.
8.2 Cooked Meat Medium (CMM) — from scratch: beef heart 454 g; proteose peptone 20 g; dextrose 2 g; sodium chloride 5 g. Or use dehydrated medium: distribute 2.5 g of prepared dehydrated medium per 20 × 150 mm screw-cap tube; add 20 mL of cold distilled water; mix and let stand to wet all particles; sterilize 15 min at 121°C. Final pH 7.2. Keeps up to 4 weeks at 0–4°C; before use, de-aerate at 100°C for 10 min and cool.
8.3 Chlorine disinfecting solution: stock buffer — KH₂PO₄ 136 g in 1000 mL distilled water, adjusted to pH 6.2 with 1 N NaOH. Dilute the stock 20-fold (e.g., 5 mL to 100 mL with distilled water). If the diluted buffer is held longer than one working day before use, sterilize it at 121°C for 15 minutes in suitable bottles first. Add 10 mL of commercial chlorine solution (e.g., Javex at 5.25% available chlorine) per litre of diluted buffer: final available chlorine 500–525 ppm at pH 6.5; the solution remains effective until available chlorine falls below 100 ppm.
8.4 Modified PE-2 medium: peptone 20 g; yeast extract 3 g; 2 mL of 2% alcoholic bromocresol purple solution; distilled water 1000 mL. Dispense 19 mL per 20 × 150 mm screw-cap tube containing 8–10 untreated Alaska seed peas; sterilize 30 min at 121°C. Keeps up to 4 weeks at 0–4°C; de-aerate at 100°C for 10 min before use and cool.
8.5 Plate Count Agar (PCA): tryptone 5 g; glucose 1 g; yeast extract 2.5 g; agar 15 g per litre of distilled water; boil to dissolve; cool to 45–60°C; adjust so that the post-autoclave pH is 7.0 ± 0.1; sterilize 15 min at 121°C.
8.6 Potato Dextrose Agar, acidified (PDA): infusion from white potatoes 200 mL; dextrose 20.0 g; agar 15.0 g; distilled water to 1000 mL; autoclave 15 min at 121°C; cool to 45–50°C; add 16 mL of sterile 10% tartaric acid to reach pH 3.5 ± 0.1. Do not reheat after acidification. Alternative — from dehydrated medium: suspend 39 g of commercial dehydrated PDA ingredients in distilled water; boil to dissolve; autoclave 15 min at 121°C; cool to 45–50°C; lower the pH to 3.5 ± 0.1 with 16 mL of sterile 10% tartaric acid; pour into Petri plates. Do not reheat the acidified medium — reheating hydrolyses the agar.
8.7 Reinforced Clostridial Medium (RCM): yeast extract 3 g; beef extract 10 g; peptone 10 g; soluble starch 1 g; dextrose 5 g; cysteine hydrochloride 0.5 g; sodium acetate (anhydrous) 3 g; sodium chloride 5 g; agar 15 g per litre of distilled water; boil to dissolve; adjust pH to 7.4; sterilize 15 min at 121°C.
8.8 Sabouraud Dextrose Agar (SDA): dextrose 40.0 g; peptone 10.0 g; agar 15.0 g per litre of distilled water; boil to dissolve; sterilize 15 min at 121°C. pH 5.6 ± 0.2. Do not over autoclave.
8.9 Thermoacidurans Agar (TA): yeast extract 5.0 g; proteose peptone 5.0 g; dextrose 5.0 g; dipotassium phosphate 4.0 g; agar 20.0 g per litre of distilled water; boil to dissolve; autoclave 15 min at 121°C. Final pH should be 5.0.
8.10 Liver Veal Agar (LVA): liver infusion from 50 g; veal infusion from 500 g; proteose peptone 20 g; neopeptone 1.3 g; tryptone 1.3 g; dextrose 5.0 g; soluble starch 10.0 g; isoelectric casein 2.0 g; sodium chloride 5.0 g; sodium nitrate 2.0 g; agar 15.0 g per litre of distilled water; sterilize 15 min at 121°C. Final pH 7.3 ± 0.3.
8.11 Orange Serum Agar (OSA): tryptone 10.0 g; yeast extract 3.0 g; dextrose 4.0 g; dipotassium phosphate 2.5 g; agar 17.0 g; cysteine 0.001 g; orange serum 200 mL; distilled water 800 mL. Prepare the orange serum by heating 1 L of freshly extracted orange juice (or reconstituted concentrate) to about 93°C; add 30 g of filter aid; filter under suction through a Buchner funnel with coarse filter paper pre-coated with filter aid; discard the first few mL. Sterilize 15 min at 121°C; post-autoclave pH about 5.5; do not over-autoclave.

8.12 Tryptic Soy Agar (TSA): tryptone 15.0 g; soya peptone 5.0 g; sodium chloride 5.0 g; agar 15.0 g per 1.0 L of distilled water; pH 7.3 ± 0.2. Suspend the 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.

8.13 Blood Agar (Trypticase Blood Agar Base): trypticase 10.0 g; beef extract 3.0 g; sodium chloride 5.0 g; agar 15.0 g per 1.0 L of distilled water; final pH 7.2 ± 0.1. Suspend the basal ingredients in 1.0 L of distilled water; heat to boiling to dissolve; sterilize 15 min at 121°C; cool to 45–50°C and aseptically add 50.0 mL of sterile defibrinated blood; mix thoroughly without incorporating air bubbles; dispense.

8.14 Baird-Parker (BP) agar: tryptone 10.0 g; beef extract 5.0 g; yeast extract 1.0 g; glycine 12.0 g; lithium chloride 5.0 g; sodium pyruvate 10.0 g; agar 20.0 g per 1.0 L of distilled water; final pH 7.0 ± 0.2. Add the basal components to the water; heat gently and bring to the boil; autoclave at 121°C for 15 min; cool to 45–50°C; add the supplements (filter-sterilized 1% potassium tellurite solution and 50% egg yolk emulsion — for example, aseptically add 50.0 mL of pre-warmed (45–50°C) commercial egg yolk/tellurite emulsion, mix thoroughly) and dispense. Dry the agar surface before inoculation; freshly prepared plates may be toxic to injured cells — store the plates at refrigeration temperature overnight before inoculation.

8.15 MacConkey agar: peptone 20.0 g; lactose 10.0 g; bile salts No. 3 1.5 g; sodium chloride 5.0 g; neutral red 0.03 g; crystal violet 0.001 g; agar 13.5 g per 1.0 L of distilled water; final pH 7.1 ± 0.2. Suspend the ingredients in 1.0 L of distilled water and heat to boiling with frequent swirling; autoclave at 121°C for 15 min and cool to 45–50°C; dispense the medium into plates and allow to dry with the lids partially removed.

References

Health Canada, Compendium of Analytical Methods, Volume 2 — HPB Method MFHPB-01, March 2001: “Determination of commercial sterility and the presence of viable microorganisms in canned foods.”

Health Canada, Compendium of Analytical Methods, Volume 2 — Supplement to HPB Method MFHPB-01, October 2001.

Association of Official Analytical Chemists (AOAC). 1995. Chapter 21. Bacteriological Analytical Manual. Arlington, Virginia, USA.

Association of Official Analytical Chemists (AOAC). 1995, Supplement March 1997. Official Methods of Analysis of AOAC International, 16th Edition. Gaithersburg, Maryland, USA.

American Public Health Association (APHA). 1992. Compendium of Methods for the Microbiological Examination of Foods, 3rd edition. Washington, D.C., USA.

Bergey’s Manual of Determinative Bacteriology. 9th edition. 1994. W.R. Hensyl (editor). Williams and Wilkins Co., Baltimore, USA.

Federal Standard (U.S.) 209B. 1973. Clean Room and Work Station Requirements, Controlled Environment.

Atlas, R.M. 1997. Handbook of Microbiological Media, 2nd edition. L.C. Parks (editor). CRC Press Inc.

Austin, P.R. 1970. Design and Operation of Clean Rooms. Revised edition. Business News Publishing Company, Birmingham, Michigan, USA.

Health Canada. 1996. Laboratory Biosafety Guidelines, 2nd edition. Laboratory Centre for Disease Control, Health Canada.