BAM Chapter 12: Staphylococcus aureus (2023)

Bacteriological Analytical Manual (BAM) main page

Authors: Sandra Talent, Jennifer Hait, Reginald W. Bennett (retired) and Gayle A. Lancette (retired)

revision history:

• March 2016: The incubation temperature ofS aureushas been changed from 35°C to 35-37°C.

staphylococci goldenIt is very susceptible to destruction from heat treatment and almost all disinfectants. Therefore, the presence of this bacterium or its enterotoxins in processed foods or on food processing equipment is usually an indication of poor hygiene.S.goldencan cause severe food poisoning. It has been identified as a causative agent in many foodborne illness outbreaks and is likely responsible for even more cases in individuals and family groups than records show. Food is examined for the presence ofS.goldenand/or its enterotoxins to confirm thisS.goldenthe causative agent of foodborne illness, to determine if a food is a potential source of "staph" foodborne illness and to detect post-processing contamination, which often occurs through human contact or contaminated food contact surfaces. Conclusions about the meaning ofS.goldenin food should be consumed with caution. The presence of a large number ofS.goldenOrganisms in a food can indicate poor handling or hygiene; However, it is not enough to blame a food as the cause of food poisoning. the isolatedS.goldenmust be shown to produce enterotoxins. On the other hand, small Staph populations at the time of testing may be remnants of large populations that have produced enterotoxins in sufficient quantities to cause foodborne illness. Therefore, when analyzing a food, the analyst must consider all possibilitiesS.golden.

Methods of recognizing and enumeratingS.goldendepend on the reasons for testing the food and the history of the test material. Processed foods can contain relatively low numbers of impaired viable cells, the presence of which must be detected by appropriate means. Food analysis forS.goldenmay result in legal action against the party or parties responsible for a contaminated food. The methods of analysis forS.goldenthat have been collectively researched and found appropriate to provide the type of information required by FDA requirements are presented in this chapter.

There has been considerable controversy over the meaning and correct method of reading the clotting test. Research results indicated that weak coagulase activity, represented by 1+, 2+, and 3+ responses, rarely coincides with other associated criteriaS.golden(4). A peer consensus concluded that a coagulase 4+ reaction is required for the unequivocal identification ofS.golden. The tribes are suspectedS.goldenbased on coagulase reactions below 4+ should be confirmed by other tests such as anaerobic glucose fermentation, lysostaphin sensitivity and thermonuclease production. Colony morphological studies on Baird-Parker agar, lysostaphin sensitivity, coagulase and thermonuclease production, and glucose and mannitol fermentation were performed on 100 enterotoxigenic and 51 non-enterotoxigenic strains ofS.golden(3). In all cases, the responses of enterotoxigenic and non-enterotoxigenic strains varied by 12% or less. This research shows that none of these tests can be used to distinguish between toxic and non-toxic staph.

Direct plate counting method

This method is suitable for the analysis of food containing more than 100S.goldencells/g expected. It corresponds to the method in Ref. 1.

1. equipment and materials

   1. Same basic equipment as conventional plate counting (chapter 3).
   2. Drying cabinet or incubator for drying the surface of agar plates
   3. Sterile curved glass rods, shaped like a hockey stick or pickaxe, with fire-polished ends, 3-4 mm in diameter, 15-20 cm in length, with an angled opening face 45-55 mm in length

2. mediaereagents

   1. Half Baird-Parker (M17)
   2. Trypticase (Tryptic) Soja-Agar (TSA) (M152)
   3. Brain Heart Infusion Broth (BHI) (M24)
   4. Plasmakoagulase (Coelho) mit EDTA
   5. Toluidinblau-DNA-Agar (M148)
   6. Lysostaphin (Schwartz-Mann, Mountain View Ave., Orangeburg, NY 10962)
   7. Trypton-Hefeextrakt-Agar (M165)
   8. Paraffin oil, sterile
   9. 0,02 M Salzphosphatpuffer (R61), with 1% NaCl
   10. Tests for catalase (R12)
3. Sample preparation (see BAMChapter 1).

4. Isolation and enumeration ofS.golden

   (Video) ISO 21702 - Test Method Introduction
   1. For each dilution to be inoculated, aseptically transfer 1 mL of sample suspension to 3 Baird-Parker agar plates and distribute 1 mL of inoculum evenly among 3 plates (e.g. 0.4 mL, 0.3 mL and 0.3 mL). Spread the inoculum over the surface of the agar plate using a sterile curved glass rod. Keep the plates upright until the inoculum is absorbed by the agar (approximately 10 min on properly dried plates). If the inoculum is not readily adsorbed, place the plates upright in the incubator for approximately 1 h. Invert plates and incubate at 35-37°C for 45-48 h. Select plates with 20-200 colonies unless only lower dilution plates (>200 colonies) have colonies with the typical appearance ofS.golden. colonies ofS.goldenthey are circular, smooth, convex, moist, 2-3 mm in diameter in unclumped plaques, gray to jet black, often with a pale (whitish) border, surrounded by an opaque zone and often with a pale outer zone; Colonies have a buttery to rubbery consistency when touched with the piercing needle. Occasionally, non-lipolytic strains of similar appearance can be found in various foods and dairy products, except that the surrounding opaque and clear zones are absent. Strains isolated from frozen or dried foods that have been stored for a long time often develop a less black coloration than typical colonies and can have a rough appearance and a dry texture.
   2. Count and record colonies. If several types of colonies are observed that appear to beS.goldenOn selected plates, count the number of colonies of each type and record the counts separately. If the lowest dilution plates contain <20 colonies, they can be used. When plates with >200 colonies have colonies with the typical appearance ofS.goldenand typical colonies do not appear at higher dilutions, use these plates for enumerationS.golden, but do not count outlier colonies. Pick > 1 colony of each type counted and assay for coagulase production. Add the number of colonies in triplicate plates represented by positive coagulase test colonies and multiply by the sample dilution factor. Report this number asS.golden/g food tested.

5. Coagulase-Test

   transfer suspectS.goldenPlace colonies in small tubes containing 0.2–0.3 mL of BHI broth and emulsify thoroughly. Agar slant with suitable maintenance medium, e.g. B. TSA, with the loop filled with BHI suspension. Incubate BHI culture suspension and invert for 18-24 h at 35-37°C. Keep cultures tilted at room temperature for supplemental testing, or repeat if coagulase test results are questionable. Add 0.5 mL of EDTA-reconstituted coagulase plasma (B-4, above) to the BHI culture and mix well. Incubate at 35-37°C and examine periodically for 6 h for clot formation. Only a firm, complete clot that stays in place when the tube is tipped or inverted is considered positiveS.golden. Partial coagulation, formerly coagulase 2+ and 3+ reactions, should be tested later (4). Test known positive and negative cultures concurrently with cultures suspected of unknown coagulase activity. Stain all suspect cultures with Gram's reagent and observe microscopically. A latex agglutination test (AUREUS TEST^MT, Trisum Corp., Taipei, Taiwan) can replace the coagulation test when a faster procedure is desired.

6. auxiliary exams

   1. catalase test. Use the TSA slant growth for catalase testing on a glass slide or dot plate and illuminate properly to observe gas bubble formation.
   2. Anaerobic utilization of glucose. Inoculate tubes with carbohydrate fermentation medium containing glucose (0.5%). Immediately inoculate each tube vigorously with wire loop. Make sure the inoculum reaches the bottom of the tube. Cover the surface of the agar with a layer of sterile paraffin oil at least 25 mm thick. Incubate for 5 days at 35-37°C. Acid is being produced anaerobically when the indicator turns yellow throughout the tube, indicating the presence of acidS.golden. Run controls simultaneously (positive and negative cultures and medium controls).
   3. Anaerobic utilization of mannitol. Repeat 2 above, using mannitol as a carb in between.S.goldenit is usually positive, but some strains are negative. Run the controls at the same time.
   4. Lysostaphin sensitivity. Transfer the isolated colony from the loophole agar plate to 0.2 ml phosphate buffered saline and emulsify. Transfer half of the suspended cells to another tube (13 × 100 mm) and mix with 0.1 mL of phosphate buffered saline as a control. Add 0.1 mL of lysostaphin (dissolved in 0.02 M phosphate buffered saline with 1% NaCl) to the original tube to achieve a concentration of 25 µg lysostaphin/mL. Incubate both tubes at 35-37°C for no longer than 2 h. If the turbidity in the test mixture decreases, the test is considered positive. If there is no clarification within 2 hours, the test is negative.S.goldenit is usually positive.
   5. Production of thermostable nuclease. This test is considered to be as specific as the coagulase test but less subjective as it involves a color change from blue to light pink. It is not a replacement for the coagulase test, but a supportive test, especially for coagulase 2+ reactions. Prepare microslides by spreading 3 mL of toluidine blue deoxyribonucleic acid agar on the surface of each slide. When the agar has solidified, cut 2 mm diameter wells (10-12 per slide) in the agar and remove the agar plug by aspiration. Place approximately 0.01 mL of a warmed (15 min in boiling water bath) sample of the broth cultures used for the coagulase test well onto the prepared slide. Incubate the slides in a humid chamber for 4 h at 35-37 °C. The development of a light pink halo extending at least 1mm from the periphery of the well indicates a positive reaction.
7. Some typical features of 2 species of staphylococci and micrococci that may be useful in their identification are listed in Table 1.

Table 1. Typical properties ofS.golden,S.Epidermis, and Mikrokokos^(A)

Most likely number method forstaphylococcispp.

The most probable number (MPN) method (2) is recommended for routine monitoring of products with low numbers ofS.goldenexpected and in foods containing large populations of competing species.

1. Equipment and Materials - Same as the direct plate counting method above.
2. Media and Reagents - Same as direct plate counting method above. Additionally: Trypticase (tryptic) soy broth (TSB) with 10% NaCl and 1% sodium pyruvate (M154a).
3. Sample Preparation - Same as the direct plate counting method above.

4. Determination of the MPN

   Inoculate 3 TSB tubes containing 10% NaCl and 1% sodium pyruvate (B, above) with 1 mL portions of decimal dilutions of each sample. The highest dilution should give a negative endpoint. Incubate the tubes at 35-37°C for 48 ± 2 h. Using a 3 mm loop, transfer 1 loop from each tube showing growth (turbidity) to a properly surface dried Baird-Parker medium plate. Mix the tubes before spreading if growth is only visible on the bottom or sides of the tubes. Seed the inoculum to obtain isolated colonies. Incubate plates at 35-37°C for 48 h. Transfer at least 1 suspected colony from each plate showing growthS.goldento BHI broth (see Direct Plate Count procedures D and E above). Proceed with the identification and verification process ofS.golden(E and F, Direct Plate Count, above). reportS.golden/g as MPN/g, according to tables inSchedule 2, MPN Provision.

references

1. AOAC INTERNATIONAL. 1995. Official Methods of Analysis, 16ª ed., sek. 975,55. AOAC INTERNACIONAL, Arlington, VA.
2. AOAC INTERNATIONAL. 1995. Official Methods of Analysis, 16ª ed., sek. 987.09. AOAC INTERNACIONAL, Arlington, VA.
3. Bennett, R.W., M. Yeterian, W. Smith, C.M. Coles, M. Sassaman und F.D. McClure. 1986.staphylococci goldenIdentifiers and enterotoxinity.J. FoodSci. 51:1337-1339.
4. Sperber, W.H. and S.R. Tatini. 1975. Interpretation of the coagulase tube test for the identification ofstaphylococci golden.appl. Microbiol. 29:502-505.

Hypertext source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter 12.