SOP: Enumeration of Coagulase-Positive Staphylococci (Staphylococcus aureus) in a Variety of Foods using the TEMPO STA (Staphylococcus aureus) method

GIFSQ — Global Institute for Food Safety & Quality

Principle

This method is applicable to the enumeration of coagulase-positive staphylococci (Staphylococcus aureus) to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act and/or other relevant federal regulations. This method has been validated for use in all foods excluding strongly coloured foods (see the note under Preparation of sample).

The TEMPO STA (Staphylococcus aureus) test is intended for use exclusively with the TEMPO system for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus) in 24–27 hours.

The TEMPO STA test consists of a vial of culture medium and a card, which are specific to this method. The culture medium is inoculated with the sample to be tested. The inoculated medium is transferred by the TEMPO Filler instrument into the card containing 48 wells of three different volumes. The card contains 3 sets of 16 wells (small, medium and large wells) with a one log difference in volume for each set of wells. The card is designed to simulate the Most Probable Number (MPN) method. The card is then hermetically sealed in order to avoid any risk of contamination during subsequent handling.

The culture medium contains a fluorescent pH indicator which, when its pH is neutral, emits a signal detected by the TEMPO Reader instrument. The staphylococci present in the card assimilate the nutrients in the culture medium during incubation, resulting in a decrease in pH and the extinction of the fluorescent signal. Depending on the number and type of the positive wells, the TEMPO system calculates the number of staphylococci present in the original sample according to a calculation based on the MPN method.

Apparatus

• TEMPO system, which includes a TEMPO Filler, a TEMPO Reader, and all necessary components for its intended use
• TEMPO STA (reference 80002) assay kit, which contains the necessary reagents including cards and culture medium in vials for the analysis of 48 test samples
• TEMPO Filter Bags (reference 80015)
• Pipette to dispense 0.10 mL or 1.0 mL, and disposable tips
• Vortex type mixer
• Stomacher, blender, or equivalent
• Incubator capable of maintaining 35 °C
• TEMPO Control Kit (reference 80000)

Note: it is the responsibility of each laboratory to ensure that the temperatures of the incubators or water baths are maintained at the recommended temperatures. The following applies to steps of the method which apply to growth only. Where a temperature of ≤ 37 °C is recommended in the text of the method, the temperature may be ± 1.0 °C, e.g., 35 ± 1.0 °C. However, where higher temperatures are recommended, it is imperative that the incubators or water baths be maintained within 0.5 °C due to potential lethality of the higher temperatures on the microorganism(s) being isolated.

Reagents

The method formulates no media or reagents itself; the following are used as named:

• 0.1% Peptone water
• To prepare: dissolve 1.0 g of peptone in distilled water and make up to 1.0 L.
• Sterile deionized or distilled water

The media listed above are commercially available and are to be prepared and sterilized according to the manufacturer’s instructions.

Sample preparation

Handling of sample units:

• In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated or frozen, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.
• Analyze sample units as soon as possible after their receipt in the laboratory.

Preparation for analysis:

• Have primary and secondary sterile diluent prepared, pre-warmed to room temperature before use.
• Clean the surface of the working area with a suitable disinfectant.

Preparation of sample:

• To ensure a representative analytical unit, agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit.
• Prepare a 1 in 10 primary dilution of the food in a TEMPO Bag by aseptically adding 25 g or mL (the analytical unit) to 225 mL of 0.1% Peptone water. Shake, stomach or blend according to the type of food. (The source prints “stomach” as a verb.) If a smaller sample size other than 25 g or mL is used, maintain the 1 in 10 dilution (e.g., 11 g in 99 mL or 10 g in 90 mL).
• Note: the fluorescent signal may be affected if the primary dilution is strongly coloured (e.g., fruit purées and cocoa) or if it becomes coloured during incubation in the card due to oxidation reaction (e.g., raw mushrooms): for the TEMPO STA test, a dilution of at least 1 in 400 is recommended for these matrices.
• Blend/stomach for the minimum time required to produce a homogeneous suspension; to avoid overheating, blending time should not exceed 2 minutes. (The source prints “Blend/stomach”.) With foods that tend to foam, use a blender at low speed and remove aliquot from below liquid/foam interface.
• Note: the interval between the homogenization of the primary dilution and its transfer into the TEMPO card must not exceed 45 minutes.

Procedure

Card set-up:

• Set the dispenser(s) containing the secondary diluent to the appropriate volumes and prime the pump by eliminating the first two (2) volumes dispensed.
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• Log on to the TEMPO preparation station.
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• Reconstitute the culture medium (TEMPO vial) by dispensing the appropriate volume of the secondary diluent using the dispenser.
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• Note: for aromatic herbs, spices, teas and herbal teas, which may have an inhibitory effect, a minimum dilution of 1 in 400 should be used.
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• Note: for butter and margarine, separate the aqueous and lipid phase by melting the sample in a water bath at 45°C in a volume of diluent. To calculate the volume of diluent necessary, consider the fat level of the sample. For example, for margarines containing 84% fat and an analytical unit of 25 g, add 25 x 0.84 = 21 mL of diluent. Once melted, shake and allow the phases to separate. The analysis should be done on the aqueous phase; 1 mL corresponds to 1 g of the sample.
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• Choose the enumeration range: a 1 in 40 dilution will enable enumeration between 10 and 4.9 x 10⁴ CFU/g — aseptically transfer 1 mL of the primary dilution into one TEMPO STA vial containing 3.0 mL of sterile deionized or distilled water to create a 1 in 40 dilution; a 1 in 400 dilution will enable enumeration between 100 and 4.9 x 10⁵ CFU/g — aseptically transfer 0.1 mL of the primary dilution into one TEMPO STA vial containing 3.9 mL of sterile deionized or distilled water to create a 1 in 400 dilution; if higher enumeration ranges are required, prepare serial dilutions of the original sample to achieve dilutions of 1 in 4 000, 1 in 40 000, 1 in 400 000, etc. according to the manufacturer’s instructions.
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• Agitate each dilution to resuspend the material that may have settled during the preparation.
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• Using a sterile pipette, transfer the appropriate volume from the TEMPO Bag containing the sample to its corresponding vial with the reconstituted medium. Vortex or agitate manually for approximately 5 seconds.
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• Following the instructions of the preparation station user interface, identify the sample to be tested.
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• Remove one TEMPO STA card and scan the card information into the TEMPO system by following the prompts.
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• Scan the TEMPO STA vial containing the sample then insert the TEMPO STA fill tube into its corresponding TEMPO STA vial. Place the combination TEMPO STA card and vial into a filler rack.
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• Repeat the card and vial scan steps for each vial prepared.
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• Fill the TEMPO cards using the TEMPO Filler. After the cycle, visually inspect that the vials are empty. Dispose of the used vials in an appropriate receptacle.
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• Remove the filled TEMPO STA cards from the filler rack and insert them into the TEMPO incubation rack.

Incubation:

• Incubate the cards at 35 ± 1 °C for 24–27 hours.
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• Note: the incubation time for the test is managed by the TEMPO Read software which integrates a theoretical interval of 15 minutes between the reading of the card barcode and the start of incubation. If the real interval is greater than 15 minutes (without exceeding 2 hours), this extra time must be added to the remaining incubation time displayed by the TEMPO Read software. Reading must always be performed within the time limit authorized by the software.

Reading results:

• Remove the TEMPO incubation rack from the incubator.
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• Log on to the TEMPO reading station.
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• Introduce the TEMPO incubation rack containing the cards to be read into the TEMPO Reader. The reader scans the barcode of each card and interprets the results of fluorescence in the wells. It automatically associates the sample identifier with the type of test, the dilution and the enumeration results as colony forming units (CFU) per gram or milliliter of initial sample.
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• At the end of the analysis, remove the cards from the rack and dispose of them into an appropriate receptacle.

Precautions

• The laboratory supervisor must ensure that completion of the analysis described in this method is done in accordance with the International Standards reference “ISO/IEC 17025 (latest version): General Requirements for the Competence of Testing and Calibration Laboratories”.
• It is the responsibility of the laboratory to ensure equivalency if any variations of the media formulations listed here are used (either product that is commercially available or made from scratch).

References

• Health Canada. 2020. MFLP-109: Enumeration of Coagulase-Positive Staphylococci (Staphylococcus aureus) in a Variety of Foods using the TEMPO STA (Staphylococcus aureus) method. Compendium of Analytical Methods, Volume 3. Health Products and Food Branch. No “replaces” line exists in this method.
• Health Products and Food Branch, Health Products and Food Branch, MFHPB-21 Enumeration of Staphylococcus aureus in Foods, Compendium of Analytical Methods, Volume 2, September 2005. (The source prints the branch name twice in this reference; the link points to the GIFSQ SOP for MFHPB-21.)
• Cochran, W.G. (1950) Estimation of bacterial densities by means of the “Most Probable Number”, Biometrics 6, 105-116.
• International Standard EN ISO 6887-4 (2003) – Microbiology of food and animal feeding stuffs – Preparation of test samples, initial suspension and decimal dilutions for microbiological examination. Part 4: Specific rules for the preparation of products other than milk and milk products, meat and meat products, and fish and fishery products.
• International Standard EN ISO 6887-5 (2010) – Microbiology of food and animal feeding stuffs – Preparation of test samples, initial suspension and decimal dilutions for microbiological examination. Part 5: Specific rules for the preparation of milk and milk products.
• Woodward R.L. (1957) How probable is the most probable number? J. Am. Water Works Assoc. 49, 1060, 1068.