SOP: Determination of Staphylococcus aureus Thermostable Nuclease in Foods

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

Staphylococcus aureus makes a thermostable nuclease (TNase) that survives the heating steps used in food processing, much like staphylococcal enterotoxins. Viable cells may die off during processing and storage, so counting S. aureus in a finished food is not always a reliable guide to whether enterotoxin could be present. Because TNase output tracks closely with enterotoxin output, finding TNase in a food shows that the food once held a large population of S. aureus — at least 10⁶ cells per g — and may therefore contain staphylococcal enterotoxin. The method therefore detects TNase on toluidine blue DNA agar, where nuclease activity turns the dye blue to pink zones, and confirms specificity with anti-staphylococcal TNase. The method has shown satisfactory results with naturally contaminated meats, fish, poultry, vegetables, cereals and dairy products.

2. Apparatus required

• Stomacher, blender or equivalent
• Centrifuge capable of 23,000 × g (14,000 RPM with a rotor of 4.25 in. radius, or 12,000 RPM with a rotor of 5.75 in. radius)
• Incubator capable of maintaining 35°C
• pH meter or pH paper able to distinguish 0.3 to 0.5 pH units over pH 3.5 to 9.5
• Single narrow combination electrode for measuring the pH of the small-volume extract
• Boiling water bath or steam source
• Microscope slides, plastic immunoplate, or flat-bottom Petri dishes (internal diameter 85–90 mm)
• Cork borer or suitably cut Pasteur pipette (for 2 mm wells)
• Moist chamber
• Cork borer template (Figure 1 of the method is used as the alignment example)

3. Media and reagents

• 3N hydrochloric acid (HCl)
• To prepare: dilute concentrated hydrochloric acid with distilled water to 1.0 L at a final concentration of 3 mol/L (3 N).
• 3M trichloroacetic acid (TCA), used cold
• 0.05M Tris buffer, pH 9.0
• 1N and 0.1N sodium hydroxide (NaOH)
• 1 N NaOH — To prepare: dissolve 40.0 g of sodium hydroxide in distilled water and make up to 1.0 L.
• 0.1 N NaOH — To prepare: dissolve 4.0 g of sodium hydroxide in distilled water and make up to 1.0 L.
• Toluidine blue DNA agar (TDA), for use on microscope slides or in Petri dishes
• Non-fat dry milk (NFDM), must be free from TNase
• Kimwipes (Kimberley-Clark Ltd.) or Miracloth (Calbiochem Ltd.)
• Positive control: coagulase-positive S. aureus, for example ATCC 27154 or ATCC 25923
• Anti-staphylococcal TNase

4. Handling of sample units

• Except for shelf-stable foods, keep sample units refrigerated or frozen in the laboratory before analysis, according to the nature of the product
• Thaw frozen samples in a refrigerator or under time and temperature conditions that prevent microbial growth or death
• Each sample unit may be analyzed on its own, or two sample units may be combined into one composite

5. Procedure

Take the analytical unit from the surface of solid packed food, meat, cheese and especially canned products, preferably along the seam — do not take a core portion

↓

Extract the TNase according to the food type:

• Dry foods: add 5 g of NFDM and 75 ml of water to each 30 g of analytical unit; for dried foods such as pasta, blend 2 min before and 2 min after adding the NFDM and water, refrigerate 1–2 h, then blend a third 2 min to get a homogeneous suspension
• Liquid foods: shake 80 ml of product with 5 g of NFDM
• All other foods: blend 30 g of food with 5 g of NFDM and 60 ml of water — no NFDM is needed for dairy products
• Composite option: combine 30 g from each of two sample units into a 60 g composite; for dry foods add 7.5 g of NFDM and 150 ml of water; for liquid foods combine 100 ml from each of the two sample units into a 200 ml composite and shake with 7.5 g of NFDM; for other foods blend with 7.5 g of NFDM and 120 ml of water — no NFDM is needed for dairy products

↓

With constant stirring, adjust the food homogenate to pH 3.8 ± 0.2 with 3N HCl

↓

Centrifuge the homogenate for 30 min at 23,000 × g — lower g-forces may be adequate for some foods; for high-fat foods a refrigerated centrifuge may be needed to form a solid compact fat layer at the surface, which is pushed aside to reach the supernatant

↓

Filter the supernatant through two layers of Kimwipes or a single pre-wetted layer of Miracloth

↓

To the entire filtrate, add cold 3M TCA to a final ratio of 1 part TCA to 20 parts filtrate; mix thoroughly

↓

Centrifuge the TCA-treated filtrate at 23,000 × g for 15 min

↓

Discard the supernatant; dissolve the sediment in 2 ml of 0.05M Tris buffer (4 ml for extracts from composited products); with constant agitation adjust the pH to 7.5 ± 0.5 with 1N or 0.1N NaOH, using the single narrow combination electrode (narrow-range pH paper may also be used); cap the centrifuge tube and place it in a boiling water bath or steam for 15 min, then cool; if the TNase test cannot be completed at once, the extract may be refrigerated overnight at this point before proceeding

↓

With constant stirring, carefully adjust the pH to 9.0 ± 0.2 with 0.1N or 1N NaOH so that the final volume does not exceed 2.5 ml (5.0 ml for composited-product extracts) — the pH must not go above 9.2, to avoid possible inactivation of TNase; if the extract is viscous or very cloudy, centrifuge at 23,000 × g for 10 min; keep the fluid part of the extract

↓

Run the thermonuclease test on TDA:

• Pipette 3 ml of molten TDA mixture onto a microscope slide or plastic immunoplate, or pipette sufficient TDA onto a flat-bottom Petri dish to give a 1.8 mm thickness (12–13 ml for dishes of 85–90 mm internal diameter)
• With the cork borer or cut pipette, cut into the agar one pair of wells of 2 mm internal diameter with their centres 8–10 mm apart for each sample; remove the agar plugs by aspiration
• Add 5 µl of the heated fluid extract into each well
• Incubate the slide, immunoplate or Petri dish for 4 h at 35–37°C, then measure and record the diameter of the pink zone that indicates TNase activity; leave the slide 16–18 h at room temperature and measure and record the pink-zone diameter again; keep plates in an environment that will not let the agar dry; set up a reagent control by adding known staphylococcal TNase to sample wells
• If the pink-zone diameter, inclusive of the well, is equal to or greater than 4 mm after 4 h at 35–37°C, record as positive; if it is less than 4 mm after 4 h at 35–37°C and increases in size after overnight incubation at room temperature, record as positive; if either of the above occurs without inhibition, record as negative; if the pink-zone diameter is less than 4 mm after 4 h at 35–37°C and it does not increase in size, or disappears after overnight incubation at room temperature, record as negative

↓

Run the seroinhibition test on the same TDA:

• When preparing the agar, make two additional anti-TNase wells between the two sample wells, placed so that at the closest point the outer edge of an anti-TNase well is 2–3 mm from the sample wells
• In each anti-TNase well add 5 µl of the heated fluid extract
• In one anti-TNase well add 5 µl of a suitable dilution of anti-TNase; in the other anti-TNase well add 5 µl of 0.05M Tris buffer — the buffer-containing well serves two purposes: (a) if non-specific flattening of the pink zones occurs, it appears in the zones separated by the buffer well too, so the reaction is recorded as negative for staphylococcal TNase (such reactions are rare but have been observed); (b) genuine inhibition by the anti-TNase is easier to detect, even the smallest zone, by comparison with the uninhibited zone in the two adjacent sample wells separated by the buffer-containing well
• Incubate the slide, immunoplate or Petri dish in a moist chamber for 4 h at 35–37°C, then look for seroinhibition shown by flattening of the pink-zone segment near the well containing the anti-TNase; leave the slide 16–18 h at room temperature and note any seroinhibition; keep plates in an environment that will not let the agar dry
• If the pink-zone diameter, inclusive of the well, is equal to or greater than 4 mm after 4 h at 35–37°C and is inhibited by the anti-TNase, record as positive; if it is less than 4 mm after 4 h at 35–37°C, increases in size after overnight incubation at room temperature and is inhibited, record as positive; if either of the above occurs without inhibition, record as negative; if the pink-zone diameter is less than 4 mm after 4 h at 35–37°C and it does not increase in size and is not inhibited, or disappears after overnight incubation at room temperature, record as negative

↓

Interpret the result:

• A positive TNase test indicates the food is a potential hazard: at some point in its history it was likely contaminated with at least 10⁶ cells of S. aureus per g, and the food may therefore contain staphylococcal enterotoxin
• The method also serves as a screening tool and an indicator of staphylococcal enterotoxin contamination, and it confirms that S. aureus had at some time been present in the food

6. Precautions

• The laboratory supervisor must ensure the analysis is completed in accordance with ISO/IEC 17025:2005 (or the latest version), General Requirements for the Competence of Testing and Calibration Laboratories
• The laboratory is responsible for ensuring equivalency if any variations of the media formulations are used (commercially available product or made from scratch); equivalency data are to be forwarded to the Editor of the Compendium of Analytical Methods for consideration of modification of this method
• Where 35°C is recommended, the incubator may be at 35 ± 1°C; lower temperatures of 30 or 25°C may be ± 1°C; where higher temperatures are recommended (43 or 45.5°C), the incubators or water baths must be maintained within 0.5°C because of the potential lethality of higher temperatures on the microorganism being isolated
• Do not use an Ag/AgCl electrode when adjusting the pH of a solution containing Tris buffer — Tris complexes with Ag, giving inaccurate pH readings; a calomel combination electrode is recommended

References

Health Canada, Compendium of Analytical Methods, Volume 2 — HPB Method MFHPB-28, March 2016: “Determination of Staphylococcus aureus Thermostable Nuclease in Foods.”

• Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.
• Chesbro, W.R. and K. Auborn. 1967. Enzymatic detection of the growth of Staphylococcus aureus in foods. Appl. Microbiol. 15:1150-1159.
• Lachica, R.V.F., P.D. Hoeprich and C. Genigeorgis. 1971. Metachromatic agar-diffusion microslide technique for detecting staphylococcal nuclease in foods. Appl. Microbiol. 21:585-587.
• Lachica, R. V. F. 1976. Simplified thermonuclease test for rapid indentification of Staphylococcus aureus recovered on agar media. Appl. Environ. Microbiol. 32: 633-634.
• Rayman, M.K., C.E. Park, J. Philpott and E.C.D. Todd. 1975. Reassessment of the coagulase and thermostable nuclease tests as means of identifying Staphylococcus aureus. Appl. Microbiol. 29:451-454.