Chapter 19: Staphylococcus aureus

• Potential Food Safety Hazard
• Control Measures
• FDA Guidelines
• State Guidelines
• Recommended Microbiological Limits
• ICMSF Recommended Microbial Limits
• Caniadian Bacteriological Guidelines
• Growth
• Toxin Production
• Heat Resistance
• Analytical Procedures
• Food Sampling and Preparation of Sample Homogenate
• S. aureus
• Most Probable Number Method for Staphylococcus spp.
• Staphylococcal Enterotoxins: Micro-slide Double Diffusion and ELISA-based Methods
• Chromatographic Separation of Toxin from Foods for Micro-Slide Double Diffusion
• Microslide Gel Double Diffusion Test
• Extraction of Enterotoxins from Foods for ELISA Assays
• Visual ELISA: Polyvalent (Types A-E) Screening for Determining Enterotoxigenicity and Identifying Staphylococcal Enterotoxins in Foods
• Automated Multiparametric Immunoanalyzer, VIDAS™, VIDAS Staph (SET) for the Identification of the Staphylococcal Enterotoxins
• Transia™ Immunoenzymatic Test for the Identification of Staphylococcal Enterotoxin
• Electrophoretic and Immunoblot Analysis of Staphylococcal Enterotoxins in Food
• Other analytical procedures
• Commercial Test Products
• References

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Potential Food Safety Hazard

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Control Measures

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FDA Guidelines

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State Guidelines

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Recommended Microbiological Limits

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ICMSF Recommended Microbial Limits

Plate counts below "m" are considered good quality. Plate counts between "m" and "M" are considered marginally acceptable quality, but can be accepted if the number of samples does not exceed "c." Plate counts at or above "M" are considered unacceptable quality (ICMSF, 1986).

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Growth

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Toxin Production

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Heat Resistance

Table 19-7. Heat resistance of S. aureus toxin.

Temp.		D-Value	Medium	Reference
(ºC)	(ºF)	(min.)
98.9	210	68.5	Milk	Read and Bradshaw, 1966
104.4	220	46.2	Milk	Read and Bradshaw, 1966
110	230	26.1	Milk	Read and Bradshaw, 1966
115.6	240	16.6	Milk	Read and Bradshaw, 1966
121.1	250	9.4	Milk	Read and Bradshaw, 1966
126.7	260	6.2	Milk	Read and Bradshaw, 1966

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Analytical Procedures

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Food Sampling and Preparation of Sample Homogenate (Andrews and June, 1998)

The adequacy and condition of the sample or specimen received for examination are of primary importance. If samples are improperly collected and mishandled or are not representative of the sampled lot, the laboratory results will be meaningless. Because interpretations about a large consignment of food are based on a relatively small sample of the lot, established sampling procedures must be applied uniformly. A representative sample is essential when pathogens or toxins are sparsely distributed within the food or when disposal of a food shipment depends on the demonstrated bacterial content in relation to a legal standard.

The number of units that comprise a representative sample from a designated lot of a food product must be statistically significant. The composition and nature of each lot affects the homogeneity and uniformity of the total sample mass. The proper statistical sampling procedure, according to whether the food is solid, semisolid, viscous, or liquid, must be determined by the collector at the time of sampling by using the Investigations Operation Manual (FDA, 1993). Sampling and sample plans are discussed in detail in ICMSF (1986).

Whenever possible, submit samples to the laboratory in the original unopened containers. If products are in bulk or in containers too large for submission to the laboratory, transfer representative portions to sterile containers under aseptic conditions. There can be no compromise in the use of sterile sampling equipment and the use of aseptic technique. Sterilize one-piece stainless steel spoons, forceps, spatulas, and scissors in an autoclave or dry-heat oven. Use of a propane torch or dipping the instrument in alcohol and igniting is dangerous and may be inadequate for sterilizing equipment.

Use containers that are clean, dry, leak-proof, wide-mouthed, sterile, and of a size suitable for samples of the product. Containers such as plastic jars or metal cans that are leak-proof may be hermetically sealed. Whenever possible, avoid glass containers, which may break and contaminate the food product. For dry materials, use sterile metal boxes, cans, bags, or packets with suitable closures. Sterile plastic bags (for dry, unfrozen materials only) or plastic bottles are useful containers for line samples. Take care not to overfill bags or permit puncture by wire closure. Identify each sample unit (defined later) with a properly marked strip of masking tape. Do not use a felt pen on plastic because the ink might penetrate the container. Whenever possible, obtain at least 100 g for each sample unit. Submit open and closed controls of sterile containers with the sample.

Deliver samples to the laboratory promptly with the original storage conditions maintained as nearly as possible. When collecting liquid samples, take an additional sample as a temperature control. Check the temperature of the control sample at the time of collection and on receipt at the laboratory. Make a record for all samples of the times and dates of collection and of arrival at the laboratory. Dry or canned foods that are not perishable and are collected at ambient temperatures need not be refrigerated. Transport frozen or refrigerated products in approved insulated containers of rigid construction so that they will arrive at the laboratory unchanged. Collect frozen samples in pre-chilled containers.

Place containers in a freezer long enough to chill them thoroughly. Keep frozen samples solidly frozen at all times. Cool refrigerated samples, except shellfish and shell stock, in ice at 0-4ºC and transport them in a sample chest with suitable refrigerant capable of maintaining the sample at 0-4ºC until arrival at the laboratory. Do not freeze refrigerated products. Unless otherwise specified, refrigerated samples should not be analyzed more than 36 h after collection. Special conditions apply to the collection and storage of shucked, unfrozen shellfish and shell stock (APHA, 1985). Pack samples of shucked shellfish immediately in crushed ice (no temperature specified) until analyzed; keep shell stock above freezing but below 10ºC. Examine refrigerated shellfish and shell stock within 6 h of collection but in no case more than 24 h after collection. Further details on sample handling and shipment may be found in the Investigations Operation Manual (FDA, 1993) and the Laboratory Procedures Manual (FDA, 1989). The Investigations Operation Manual (FDA, 1993) contains sampling plans for various microorganisms. Some of those commonly used are presented here.

1. Sampling plans
1. Aerobic plate counts, total coliforms, fecal coliforms, Escherichia coli (including enteropathogenic strains), Staphylococcus spp., Vibrio spp., Shigella spp., Campylobacter spp., Yersinia spp., Bacillus cereus, and Clostridium perfringens
1. Sample collection. From any lot of food, collect ten 8 ounce (227 g) subsamples (or retail packages) at random. Do not break or cut larger retail packages to obtain an 8 ounce (227 g) subsample. Collect the intact retail unit as the subsample even if it is larger than 8 ounce (227 g).
2. Sample analysis. Analyze samples as indicated in current compliance programs.
2. Equipment and materials
1. Mechanical blender. Several types are available. Use blender that has several operating speeds or rheostat. The term "high-speed blender" designates mixer with 4 canted, sharp-edge, stainless steel blades rotating at bottom of 4 lobe jar at 10,000-12,000 rpm or with equivalent shearing action. Suspended solids are reduced to fine pulp by action of blades and by lobular container, which swirls suspended solids into blades. Waring blender, or equivalent, meets these requirements.
2. Sterile glass or metal high-speed blender jar. 1000 ml, with cover, resistant to autoclaving for 60 min at 121ºC.
3. Balance, with weights. 2000 g capacity, sensitivity of 0.1 g.
4. Sterile beakers. 250 ml, low-form, covered with aluminum foil.
5. Sterile graduated pipets. 1.0 and 10.0 ml.
6. Butterfield's phosphate-buffered dilution water (R11). Sterilized in bottles to yield final volume of 90 ± 1 ml.
7. Sterile knives, forks, spatulas, forceps, scissors, tablespoons, and tongue depressors. For sample handling.
3. Receipt of samples
1. The official food sample is collected by the FDA inspector or investigator. As soon as the sample arrives at the laboratory, the analyst should note its general physical condition. If the sample cannot be analyzed immediately, it should be stored as described later. Whether the sample is to be analyzed for regulatory purposes, for investigation of a foodborne illness outbreak, or for a bacteriological survey, strict adherence to the recommendations described here is essential.
2. Condition of sampling container. Check sampling containers for gross physical defects. Carefully inspect plastic bags and bottles for tears, pinholes, and puncture marks. If sample units were collected in plastic bottles, check bottles for fractures and loose lids. If plastic bags were used for sampling, be certain that twist wires did not puncture surrounding bags. Any cross-contamination resulting from one or more of above defects would invalidate the sample, and the collecting district should be notified (see 3-e, below).
3. Labeling and records. Be certain that each sample is accompanied by a completed copy of the Collection Report (Form FD-464) and officially sealed with tape (FD-415a) bearing the sample number, collecting official's name, and date. Assign each sample unit an individual unit number and analyze as a discrete unit unless the sample is composited as described previously in this chapter.
4. Adherence to sampling plan. Most foods are collected under a specifically designed sampling plan in one of several ongoing compliance programs. Foods to be examined for Salmonella, however, are sampled according to a statistically based sampling plan designed exclusively for use with this pathogen. Depending on the food and the type of analysis to be performed, determine whether the food has been sampled according to the most appropriate sampling plan.
5. Storage. If possible, examine samples immediately upon receipt. If analysis must be postponed, however, store frozen samples at -20°C until examination. Refrigerate unfrozen perishable samples at 0-4°C not longer than 36 h. Store nonperishable, canned, or low-moisture foods at room temperature until analysis.
6. Notification of collecting district. If a sample fails to meet the above criteria and is therefore not analyzed, notify the collecting district so that a valid sample can be obtained and the possibility of a recurrence reduced.
4. Thawing

Use aseptic technique when handling product. Before handling or analysis of sample, clean immediate and surrounding work areas. In addition, swab immediate work area with commercial germicidal agent. Preferably, do not thaw frozen samples before analysis. If necessary to temper a frozen sample to obtain an analytical portion, thaw it in the original container or in the container in which it was received in the laboratory. Whenever possible, avoid transferring the sample to a second container for thawing. Normally, a sample can be thawed at 2-5ºC within 18 h. If rapid thawing is desired, thaw the sample at less than 45ºC for not more than 15 min. When thawing a sample at elevated temperatures, agitate the sample continuously in thermostatically controlled water bath.

5. Mixing

Various degrees of non-uniform distribution of microorganisms are to be expected in any food sample. To ensure more even distribution, shake liquid samples thoroughly and, if practical, mix dried samples with sterile spoons or other utensils before withdrawing the analytical unit from a sample of 100 g or greater. Use a 50 g analytical unit of liquid or dry food to determine aerobic plate count value and most probable number of coliforms. Other analytical unit sizes (e.g., 25 g for Salmonella) may be recommended, depending on specific analysis to be performed. Use analytical unit size and diluent volume recommended for appropriate Bacteriological Analytical Manual method being used. If contents of package are obviously not homogeneous (e.g., a frozen dinner), macerate entire contents of package and withdraw the analytical unit, or, preferably, analyze each different food portion separately, depending on purpose of test.

6. Weighing

Tare high-speed blender jar; then aseptically and accurately (± 0.1 g) weigh unthawed food (if frozen) into jar. If entire sample weighs less than the required amount, weigh portion equivalent to one-half of sample and adjust amount of diluent or broth accordingly. Total volume in blender must completely cover blades.

7. Blending and diluting of samples requiring enumeration of microorganisms
1. All foods other than nut meat halves and larger pieces, and nut meal. Add 450 ml Butterfield's phosphate-buffered dilution water to blender jar containing 50 g analytical unit and blend 2 min. This results in a dilution of 10^-1. Make dilutions of original homogenate promptly, using pipets that deliver required volume accurately. Do not deliver less than 10% of total volume of pipet. For example, do not use pipet with capacity greater than 10 ml to deliver 1 ml volumes; for delivering 0.1 ml volumes, do not use pipet with capacity greater than 1.0 ml. Prepare all decimal dilutions with 90 ml of sterile diluent plus 10 ml of previous dilution, unless otherwise specified. Shake all dilutions vigorously 25 times in 30 cm (1 foot) arc in 7 s. Not more than 15 min should elapse from the time sample is blended until all dilutions are in appropriate media.
2. Nut meat halves and larger pieces. Aseptically weigh 50 g analytical unit into sterile screw-cap jar. Add 50 ml diluent (7-a, above) and shake vigorously 50 times through 30 cm arc to obtain 10⁰ dilution. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations.
3. Nut meal. Aseptically weigh 10 g analytical unit into sterile screw-cap jar. Add 90 ml of diluent (7-a, above) and shake vigorously 50 times through 30 cm arc to obtain 10^-1 dilution. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations.

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S. aureus (Bennett and Lancette, 1998)

Foods are examined for the presence of S. aureus and/or its enterotoxins to confirm that S. aureus is the causative agent of food-borne illness, to determine whether a food is a potential source of "staph" food poisoning, and to demonstrate post-processing contamination, which is generally due to human contact or contaminated food-contact surfaces. Conclusions regarding the significance of S. aureus in foods should be made with circumspection. The presence of a large number of S. aureus organisms in a food may indicate poor handling or sanitation; however, it is not sufficient evidence to incriminate a food as the cause of food poisoning. The isolated S. aureus must be shown to produce enterotoxins. Conversely, small staphylococcal populations at the time of testing may be remnants of large populations that produced enterotoxins in sufficient quantity to cause food poisoning. Therefore, the analyst should consider all possibilities when analyzing a food for S. aureus.

Methods used to detect and enumerate S. aureus depend on the reasons for testing the food and on the past history of the test material. Processed foods may contain relatively small numbers of debilitated viable cells, whose presence must be demonstrated by appropriate means. Analysis of food for S. aureus may lead to legal action against the party or parties responsible for a contaminated food. The methods of analysis for S. aureus that have been studied collaboratively and found suitable for use in providing the type of information necessary for FDA requirements are presented in this chapter.

There has been considerable controversy about the significance and correct method of reading the coagulase test. Research results have indicated that the weak coagulase activity represented by 1+, 2+, and 3+ reactions seldom corresponds with other criteria associated with S. aureus (Sperber and Tatini, 1975). A consensus of peers has established that a 4+ coagulase reaction is necessary for unquestioned identification of S. aureus. Those strains suspected of being S. aureus on the basis of coagulase reactions of less than 4+ should be confirmed by other tests, such as anaerobic glucose fermentation, lysostaphin sensitivity, and thermonuclease production. Studies of colonial morphology on Baird-Parker agar, lysostaphin sensitivity, coagulase and thermonuclease production, and glucose and mannitol fermentation were conducted on 100 enterotoxigenic and 51 nonenterotoxigenic strains of S. aureus (Bennett et al., 1986). In all cases, the reactions of enterotoxigenic and nonenterotoxigenic strains varied by 12% or less. This research indicates that none of these tests can be relied upon to differentiate toxic and nontoxic staphylococci.

Direct Plate Count Method

This method is suitable for the analysis of foods in which more than 100 S. aureus cells/g may be expected. It conforms to the method in AOAC (1995).

1. Equipment and materials
1. Same basic equipment as for conventional plate count (Chapter 9)
2. Drying cabinet or incubator for drying surface of agar plates
3. Sterile bent glass streaking rods, hockey stick or hoe-shaped, with fire-polished ends, 3-4 mm diameter, 15-20 cm long, with an angled spreading surface 45-55 mm long
2. Media and reagents

Codes, e.g., "M27" refer to media recipes in the FDA Bacteriological Analytical Manual (Merker, 1998).

1. Baird-Parker medium (M17)
2. Trypticase (tryptic) soy agar (TSA) (M152)
3. Brain heart infusion (BHI) broth (M24)
4. Coagulase plasma (rabbit) with EDTA
5. Toluidine blue-DNA agar (M148)
6. Lysostaphin (Schwartz-Mann, Mountain View Ave., Orangeburg, NY 10962)
7. Tryptone yeast extract agar (M165)
8. Paraffin oil, sterile
9. 0.02 M phosphate-saline buffer (R61), containing 1% NaCl
10. Catalase test (R12)
3. Preparation of sample (see Chapter 9)
4. Isolation and enumeration of S. aureus
1. For each dilution to be plated, aseptically transfer 1 ml sample suspension to 3 plates of Baird-Parker agar, distributing 1 ml of inoculum equitably to 3 plates (e.g., 0.4 ml, 0.3 ml, and 0.3 ml). Spread inoculum over surface of agar plate, using sterile bent glass streaking rod. Retain plates in upright position until inoculum is absorbed by agar (about 10 min on properly dried plates). If inoculum is not readily adsorbed, place plates upright in incubator for about 1 h. Invert plates and incubate 45-48 h at 35ºC. Select plates containing 20-200 colonies, unless only plates at lower dilutions (>200 colonies) have colonies with typical appearance of S. aureus. Colonies of S. aureus are circular, smooth, convex, moist, 2-3 mm in diameter on uncrowded plates, gray to jet-black, frequently with light-colored (off-white) margin, surrounded by opaque zone and frequently with an outer clear zone; colonies have buttery to gummy consistency when touched with inoculating needle. Occasionally from various foods and dairy products, nonlipolytic strains of similar appearance may be encountered, except that surrounding opaque and clear zones are absent. Strains isolated from frozen or desiccated foods that have been stored for extended periods frequently develop less black coloration than typical colonies and may have rough appearance and dry texture.
2. Count and record colonies. If several types of colonies are observed which appear to be S. aureus on selected plates, count number of colonies of each type and record counts separately. When plates of the lowest dilution contain <20 colonies, these may be used. If plates containing >200 colonies have colonies with the typical appearance of S. aureus and typical colonies do not appear at higher dilutions, use these plates for the enumeration of S. aureus, but do not count nontypical colonies. Select > 1 colony of each type counted and test for coagulase production. Add number of colonies on triplicate plates represented by colonies giving positive coagulase test and multiply by the sample dilution factor. Report this number as number of S. aureus/g of food tested.
5. Coagulase test

Transfer suspect S. aureus colonies into small tubes containing 0.2-0.3 ml BHI broth and emulsify thoroughly. Inoculate agar slant of suitable maintenance medium, e.g., TSA, with loopful of BHI suspension. Incubate BHI culture suspension and slants 18-24 h at 35ºC. Retain slant cultures at room temperature for ancillary or repeat tests in case coagulase test results are questionable. Add 0.5 ml reconstituted coagulase plasma with EDTA (B-4, above) to the BHI culture and mix thoroughly. Incubate at 35ºC and examine periodically over 6 h period for clot formation. Only firm and complete clot that stays in place when tube is tilted or inverted is considered positive for S. aureus. Partial clotting, formerly 2+ and 3+ coagulase reactions, must be tested further (Sperber and Tatini, 1975). Test known positive and negative cultures simultaneously with suspect cultures of unknown coagulase activity. Stain all suspect cultures with Gram reagent and observe microscopically. A latex agglutination test (AUREUS TEST™ , Trisum Corp., Taipei, Taiwan) may be substituted for the coagulase test if a more rapid procedure is desired.

6. Ancillary tests
1. Catalase test. Use growth from TSA slant for catalase test on glass slide or spot plate, and illuminate properly to observe production of gas bubbles.
2. Anaerobic utilization of glucose. Inoculate tube of carbohydrate fermentation medium containing glucose (0.5%). Immediately inoculate each tube heavily with wire loop. Make certain inoculum reaches bottom of tube. Cover surface of agar with layer of sterile paraffin oil at least 25 mm thick. Incubate 5 d at 37ºC. Acid is produced anaerobically if indicator changes to yellow throughout tube, indicating presence of S. aureus. Run controls simultaneously (positive and negative cultures and medium controls).
3. Anaerobic utilization of mannitol. Repeat 2, above, using mannitol as carbohydrate in medium. S. aureus is usually positive but some strains are negative. Run controls simultaneously.
4. Lysostaphin sensitivity. Transfer isolated colony from agar plate with inoculating loop to 0.2 ml phosphate-saline buffer, and emulsify. Transfer half of suspended cells to another tube (13 x 100 mm) and mix with 0.1 ml phosphate-saline buffer as control. Add 0.1 ml lysostaphin (dissolved in 0.02 M phosphate-saline buffer containing 1% NaCl) to original tube for concentration of 25 µg lysostaphin/ml. Incubate both tubes at 35ºC for not more than 2 h. If turbidity clears in test mixture, test is considered positive. If clearing has not occurred in 2 h, test is negative. S. aureus is generally positive.
5. Thermostable nuclease production. This test is claimed to be as specific as the coagulase test but less subjective, because it involves a color change from blue to bright pink. It is not a substitute for the coagulase test but rather is a supportive test, particularly for 2+ coagulase reactions. Prepare microslides by spreading 3 ml toluidine blue-deoxyribonucleic acid agar on the surface of each microscope slide. When agar has solidified, cut 2 mm diameter wells (10-12 per slide) in agar and remove agar plug by aspiration. Add about 0.01 ml of heated sample (15 min in boiling water bath) of broth cultures used for coagulase test to well on prepared slide. Incubate slides in moist chamber 4 h at 35ºC. Development of bright pink halo extending at least 1 mm from periphery of well indicates a positive reaction.

7. Some typical characteristics of 2 species of staphylococci and the micrococci, which may be helpful in their identification, are shown in Table 19-8.

Table 19-8. Typical characteristics of S. aureus, S. epidermidis, and micrococci^a

Characteristic	S. aureus	S. epidermidis	Micrococci
Catalase activity	+	+	+
Coagulase production	+	-	-
Thermonuclease production	+	-	-
Lysostaphin sensitivity	+	+	-
Anaerobic utilization of glucose	+	+	-
Anaerobic utilization of mannitol	+	-	-

^a+, Most (90% or more) strains are positive; -, most (90% or more) strains are negative.

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Most Probable Number Method for Staphylococcus spp. (Bennett and Lancette, 1998)

The most probable number (MPN) method (AOAC, 1995b) is recommended for routine surveillance of products in which small numbers of S. aureus are expected and in foods expected to contain a large population of competing species.

1. Equipment and materials--Same as for Direct Plate Count Method, above.
2. Media and reagents--Same as for Direct Plate Count Method, above. In addition: Trypticase (tryptic) soy broth (TSB) containing 10% NaCl and 1% sodium pyruvate (M154a).
3. Preparation of sample--Same as for Direct Plate Count Method, above.
4. Determination of MPN

Inoculate 3 tubes of TSB containing 10% NaCl and 1% sodium pyruvate (B, above) with 1 ml portions of decimal dilutions of each sample. Highest dilution must give negative endpoint. Incubate tubes 48 ± 2 h at 35ºC. Using 3 mm loop, transfer 1 loopful from each tube showing growth (turbidity) to plate of Baird-Parker medium with properly dried surface. Vortex-mix tubes before streaking if growth is visible only on bottom or sides of tubes. Streak inoculum to obtain isolated colonies. Incubate plates 48 h at 35ºC. From each plate showing growth, transfer at least 1 colony suspected to be S. aureus to BHI broth (see D and E of Direct Plate Count Method, above). Continue procedure for identification and confirmation of S. aureus (E and F, Direct Plate Count, above). Report S. aureus/g as MPN/g, according to tables in Chapter 10, MPN Determination.

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Staphylococcal Enterotoxins: Micro-slide Double Diffusion and ELISA-based Methods (Bennett, 1998)

Among the metabolites produced by S. aureus, and other staphylococcal spp., enterotoxins (Baird-Parker, 1990; Bergdoll, 1990; Genigeorgis, 1989) present the greatest food-borne risk to the health of consumers. Staphylococcal enterotoxins are basic proteins produced by certain Staphylococcus strains in a variety of environments, including food substrates. These structurally-related, toxicologically similar proteins are produced primarily by S. aureus, although S. intermedius and S. hyicus also have been shown to be enterotoxigenic (Adesiun et al., 1984). Normally considered a veterinary pathogen (Raus and Love, 1983; Talan et al., 1989), S. intermedius was isolated from butter blend and margarine in a food poisoning outbreak (Bennett, 1996; Khambaty et al., 1994). A coagulase negative S. epidermidis was reported to have caused least one outbreak (Breckinridge and Bergdoll, 1971). These incidents support testing staphylococci other than S. aureus for enterotoxigenicity, if they are present in large numbers in a food suspected of causing a food poisoning outbreak.

When large numbers of enterotoxigenic staphylococci grow in foods, they may elaborate enough toxin to cause food poisoning after the foods are ingested. The most common symptoms of staphylococcal food poisoning, which usually begin 2-6 h after contaminated food is consumed, are nausea, vomiting, acute prostration, and abdominal cramps. To date, 8 enterotoxins (types A, B, C1, C2, C3, D, E, and H) have been identified as distinct serological entities. Current methods to detect enterotoxins use specific polyclonal or monoclonal antibodies (Notermans et al., 1987; Thompson et al., 1985; Thompson et al., 1986).

The threshold amount of enterotoxin for causing illness in humans is not known. However, information from food poisoning outbreaks (Bergdoll, 1990; Evenson et al., 1988) and human challenge studies (Dangerfield, 1973) indicates that individuals experiencing illness probably consumed at least 100 ng of enterotoxin A, the serotype most frequently involved in food-borne staphylococcal illness (Casman et al., 1967). The microslide gel double diffusion technique requires at least 30-60 ng enterotoxin/g of food. Chromatographic purification and concentration are used to achieve this toxin concentration so that the serological assay can be performed (AOAC, 1990).

The microslide method is approved by AOAC International (AOAC, 1990) and is the current standard for evaluating new methods. Other methods used for food extracts should be at least as sensitive as the microslide method, which requires concentrating extracts from 100 g food in as much as 600 ml to about 0.2 ml. Less sensitive methods are inadequate.

Techniques such as radioimmunoassay (RIA), agglutination, and enzyme-linked immunosorbent assay (ELISA), require less concentration of the food extracts; thus, they save time and are more sensitive. Latex agglutination (Bergdoll, 1990) appears promising as a serological tool for identifying staphylococcal enterotoxins. Several ELISA methods (Freed et al., 1982; Kauffman, 1980; Kuo and Silverman, 1980; Notremans et al., 1978;Saunders and Bartlett, 1977; Simon and Terplan, 1977; Stiffler-Rosenberg and Fey, 1978) have been proposed for the identification of enterotoxins in foods, but, except for a polyvalent ELISA (Bennett and Atrache, 1989; Bennett et al., 1989), their specificity has not been studied extensively. Among ELISA methods, the "double antibody sandwich" ELISA is the method of choice, because reagents are commercially available in polyvalent and monovalent formats for both toxin screening and serotype specific identification(Chen and Wong, 1997). An automated enzyme-linked fluorescent immunoassay (ELFA) has been developed and is commercially available. This method has undergone specificity and sensitivity evaluations and has proven to be an effective serological system for the identification of staphylococcal enterotoxin in a wide variety of foods (Bennett and Matthews, 1995). Other methods, which have been used in the identification of the staphylococcal enterotoxins and may have application in foods, are the T-cell proliferation assay (Rasooly et al., 1997), and polyacrylamide gel electrophoresis (PAGE) combined with Western blotting (Anderson et al., 1996).

Examining staphylococci isolated from foods for enterotoxin production helps establish potential sources of enterotoxin in foods. Of the methods developed for laboratory testing of enterotoxin production, the semisolid agar procedure (Casman et al., 1963) is approved by AOAC International. It is simple to perform and requires minimal, routine laboratory equipment. Another simple approach is the use of pH 5.5 brain heart infusion (BHI) broth (Bennett and Matthews, 1995). The major problem with identifying enterotoxins in foods is that minute concentrations are sufficient to cause food poisoning. Pasteurization and thermal processing may render most toxin serologically unreactive. Consequently, false negatives may result, if detection methods lack sufficient sensitivity to detect active toxin (Bennett and Berry, 1987).

This chapter presents a technique for the routine culturing of suspect staphylococci, procedures for the extraction of enterotoxin from foods and selected serological methods (Microslide gel double diffusion precipitation test, two manual ELISAs [Tecra™, Transia™], an automated qualitative "enzyme-linked fluorescent immunoassay" [ELFA™, Vidas™], and sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]-immunoblotting) for the identification of staphylococcal enterotoxin from isolates and from foods.

Recommended for routine analysis of foods for staphylococcal enterotoxin is the use, initially, of two different polyvalent ELISA kits. If results from different polyvalent ELISA methods yield conflicting results, retest using another method (e.g., another polyvalent ELISA method or the SDS-polyacrylamide gel electrophoresis-immunoblot assay for S. aureus enterotoxin A). Methods were developed to restore serological activity to heat-altered toxin in extracts of heat-processed foods (Anderson, 1996; Bennett, 1992; Bennett et al., 1993; Bennett, 1994; Brunner and Wong, 1992; Tatini, 1976; Van der Zee and Nagel, 1993). However, current toxin detection assays (described above) are sensitive enough to detect unaltered toxin that may persist after heat without such treatment (Anderson et al., 1996).

These procedures are to be performed with extreme caution. Staphylococcal enterotoxins are highly toxic and procedures that may create aerosols should be performed in appropriate containment facilities, such as a biosafety hood.

Contents

Chromatographic Separation of Toxin from Foods for Micro-Slide Double Diffusion (Bennett, 1998)

1. Special equipment and materials
1. Refrigerated cabinet or cold room. The carboxymethyl cellulose (CMC) column extraction is performed at about 5ºC, primarily because the column is allowed to run overnight. Storing food materials and extracts in a cold room or cabinet eliminates the need for a refrigerator.
2. Waring blender or Omnimixer. Grind foods into slurry for adequate extraction of enterotoxin. An Omnimixer (DuPont) is convenient for grinding food directly into stainless steel centrifuge tubes.
3. pH meter. The pH during extraction and the pH of buffers used in the extraction are important. Make adjustments within ± 0.1 pH unit.
4. Refrigerated centrifuge. Food extracts are centrifuged at relatively high speeds at 5ºC in a refrigerated centrifuge, such as a Sorvall RC-2B, which can reach 20,000 rpm. The lower the centrifuge speed, the more difficult is the clarification of extracts.
5. Carboxymethyl cellulose (CMC). The extract is partially purified by absorption onto CMC, Whatman CM 22, 0.6 meq/g (H. Reeve Angel, Inc., 9 Bridewell Place, Clifton, NJ). Soluble extractants are removed by this step.
6. Centrifuge tubes. Use 285 ml stainless steel centrifuge bottles (Sorvall No. 530).
7. Magnetic stirrer. A magnetic stirrer keeps test samples agitated during pH adjustments, dialysis, etc.
8. Filter cloth. At various stages in the procedures, food is filtered through several layers of coarse material such as cheesecloth placed in a funnel. Wetting cheesecloth before placing it in the funnel reduces adherence of food to cloth. The coarse material allows rapid flow with efficient removal of food particles, the chloroform layer, etc.
9. Chromatographic tube (with stopcock or rubber tube attachment with finger clamp). Enterotoxin in food is partially purified by using CMC, with elution in a chromatographic tube. For this purpose, a 19 mm id column, e.g., chromaflex, plain with stopcock, size 234 (Kontes Glass Co., Vineland, NJ) is recommended.
10. Polyethylene glycol (PEG). Food extracts are concentrated with PEG (Carbowax 20,000; Union Carbide Corp., Chemical Division, 230 North Michigan Ave., Chicago, IL 60638).
11. Lyophilizer. The extract is finally concentrated by freeze-drying, which conveniently reduces the volume to 0.2 ml and completely recovers the extract.
12. Dialysis tubing. Cellulose casing of 1-1/8 inches (2.9 cm) flat width and an average pore diameter of 48 Angstrom units is used (12,000-14,000 mol wt exclusion).
13. Separatory funnels. Separatory funnels of various sizes are needed for CHCl₃ extractions and with the chromatographic column.
14. Glass wool. Glass wool makes ideal plugs for chromatographic columns.
15. Chloroform. Food extract is treated with CHCl₃ (several times in some instances) to remove lipids and other substances that interfere with concentration of extract to small volumes. NOTE: Chloroform is hazardous. Wear gloves, avoid contact with skin, and perform extraction in a chemical fume hood.

2. Reagents
1. 0.2 M NaH₂PO₄·H₂O
2. 0.2 M Na₂HPO₄
3. H₃PO₄ (0.005 M, 0.05 M)
4. Na₂HPO₄ (0.005 M, 0.05 M)
5. NaCl (crystal)
6. 1 N (or 0.1 N) NaOH
7. 1 N (or 0.1 N) HCl

3. Preparation of materials and reagents
1. Polyethylene glycol (PEG). Prepare 30% (w/v) PEG 20,000 mol wt solution by adding 30 g PEG for each 70 ml distilled water. Cut dialysis tubing (1/8 inch [2.9 cm] flat width) long enough to accommodate food extract to be concentrated. Soak tubing in 2 changes of distilled water to remove glycerol coating. Tie one end of tubing with 2 knots close together. Fill tube with distilled water and test for leaks by squeezing filled sac while holding untied end tightly closed. Empty sac and place it in distilled water until use.
2. Sodium phosphate buffer solutions
1. Phosphate buffer, pH 5.7, 0.2 M (stock). Prepare solution by adding 0.2 M NaH₂PO₄·H₂O (27.60 g in 1 L water) to 0.2 M Na₂HPO₄ (28.39 g in 1 L water) to final pH of 5.7.
2. 0.005 M Phosphate buffer. Dilute 0.2 M, pH 5.7 buffer (stock) with water (1 + 39). Adjust pH to 5.7 with 0.005 M H₃PO₄ .
3. 0.2 M Phosphate buffer, pH 6.4 (stock). Add 0.2 M Na₂HPO₄ to 0.2 M NaH₂PO₄ to pH 6.4.
4. 0.05 M Sodium phosphate-NaCl buffer, pH 6.5. Add NaCl (11.69 g/L) to pH 6.4, 0.2 M solution (stock) to give 0.2 M NaCl, pH about 6.3. Dilute with water (1 + 3), and adjust to pH 6.5 with 0.05 M H₃PO₄ or 0.05 M Na₂HPO₄.

3. Reservoir (separatory funnel). Attach about 60 cm latex tubing to stem of separatory funnel of appropriate size and attach other end of tube to glass tubing inserted through No. 3 rubber stopper to fit chromatographic column. Suspend separatory funnel from ring stand above chromatographic tube.
4. 4. Carboxymethy1 cellulose (CMC) column. Suspend 1 g CMC in 100 ml 0.005 M sodium phosphate buffer, pH 5.7, in 250 ml beaker. Adjust pH of CMC suspension with 0.005 M H₃PO₄ . Stir suspension intermittently for 15 min, recheck pH, and readjust to 5.7 if necessary. Pour suspension into 1.9 cm chromatographic tube, and let CMC particles settle. Withdraw liquid from column through stopcock to within 1 inch (2.5 cm) of surface of settled CMC. Place loosely packed plug of glass wool on top of CMC. Pass 0.005 M sodium phosphate buffer, pH 5.7, through column until washing is clear (150-200 ml). Check pH of last wash; if not 5.7, continue washing until pH is 5.7. Leave enough buffer in column to cover CMC and glass wool to prevent column from drying out.

4. Extraction and chromatographic separation of enterotoxin from food (see Figure 19-1 --Scheme).

Note: This procedure and other procedures that may generate aerosols of pathogenic microorganisms should be performed in an approved biohazard hood.

Grind 100 g food in Waring blender at high speed for 3 min with 500 ml 0.2 M NaCl. Use Omnimixer for smaller quantities. Adjust pH to 7.5 with 1 N NaOH or HCl if food is highly buffered, and 0.1 N NaOH or HCl if food is weakly buffered (e.g., custards). Let slurry stand for 10 to 15 min, recheck pH, and readjust if necessary.

Transfer slurry to two 285 ml stainless steel centrifuge bottles. Centrifuge at 16,300 x g for 20 min at 5ºC. Lower speeds with longer centrifuge time can be used, but clearing of some foods is not as effective. Separation of fatty materials is ineffective unless food is centrifuged at refrigeration temperature. Decant supernatant fluid into 800 ml beaker through cheesecloth or other suitable filtering material placed in a funnel. Re-extract residue with 125 ml of 0.2 M NaCl by blending for 3 min. Adjust pH to 7.5 if necessary. Centrifuge at 27,300 x g for 20 min at 5ºC. Filter supernatant through cheesecloth, and pool filtrate with original extract.

Place pooled extracts in dialysis sac. Immerse sac in 30% (w/v) PEG at 5ºC until volume is reduced to 15-20 ml or less (usually overnight). Remove sac from PEG and wash outside thoroughly with cold tap water to remove any PEG adhering to sac. Soak in distilled water for 1-2 min and in 0.2 M NaCl for a few min. Pour contents into small beaker.

Rinse inside of sac with 2-3 ml 0.2 M NaCl by running fingers up and down outside of sac to remove material adhering to sides of tubing. Repeat rinsing until rinse is clear. Keep volume as small as possible.

Adjust pH of extract to 7.5. Centrifuge at 32,800 x g for l0 min. Decant supernatant fluid into graduated cylinder to measure volume. Add extract with ¼ to ½ volume of CHCl₃ to separatory funnel. Shake vigorously 10 times through 90 degree arc. Centrifuge CHCl₃ extract mixture at 16,300 x g for 10 min at 5ºC. Return fluid layers to separatory funnel. Draw off CHCl₃ layer from bottom of separatory funnel, and discard. Measure volume of water layer and dilute with 40 volumes of 0.005 M sodium phosphate buffer, pH 5.7. Adjust pH to 5.7 with 0.005 M H₃PO₄ or 0.005 M Na₂HPO₄. Place diluted solution in 2 L separatory funnel.

Place stopper (attached to bottom of separatory funnel) loosely into top with liquid from separatory funnel. Tighten stopper in top of tube and open stopcock of separatory funnel. Let fluid percolate through CMC column at 5ºC at 1-2 ml/min by adjusting flow rate with stopcock at bottom of column so that percolation can be completed overnight. If all liquid has not passed through column overnight, stop flow when liquid level reaches glass wool layer. If all liquid has passed through overnight, rehydrate column with 25 ml distilled water.

After percolation is complete, wash CMC column with 100 ml 0.005 M sodium phosphate buffer (1-2 ml/min); stop flow when liquid level reaches glass wool layer. Discard wash. Elute enterotoxin from CMC column with 200 ml 0.05 M sodium phosphate buffer, pH 6.5 (0.05 M phosphate-0.05 M NaCl buffer, pH 6.5), at flow rate of 1-2 ml/min at room temperature. Force last of liquid from CMC by applying air pressure to top of chromatographic tube.

Place eluate in dialysis sac. Place sac in 30% (w/v) PEG at 5ºC and concentrate almost to dryness. Remove sac from PEG and wash. Soak sac in 0.2 M phosphate buffer, pH 7.4. Remove concentrated material from sac by rinsing 5 times with 2-3 ml 0.01 M sodium phosphate buffer, pH 7.4-7.5. Extract concentrated solution with CHCl₃. Repeat CHCl₃ extractions until precipitate is so lacy that it falls apart in CHCl₃ layer in cheesecloth.

Place extract in short dialysis sac (about 15 cm). Place sac in 30% (w/v) PEG, and let it remain until all liquid is removed from inside sac (usually overnight). Remove sac from PEG and wash outside with tap water. Place sac in distilled water for 1-2 min. Remove contents by rinsing inside of sac with 1 ml portions of distilled water. Keep volume below 5 ml. Place rinsings in test tube (18 x 100 mm) or other suitable container and freeze-dry. Dissolve freeze-dried test sample in as small an amount of saline as possible (0.1-0.15 ml). Check for enterotoxins by microslide method.

1. General precautions

For raw or fermented foods and culture fluids from staphylococcal growth in laboratory media, check after extraction or collection of the culture fluid to determine if the test preparation contains peroxidase, which could interfere with the proper interpretation of results. To determine peroxidase presence, add 50 µl of sample to 50 µl of ELISA kit substrate reagent in an untreated microtiter plate (no antibody to staphylococcal enterotoxin) and let stand 10 min. If color changes to blue (or bluish-green), the sample contains intrinsic peroxidase, which must be inactivated. If sample remains colorless (or original color), analyze it for enterotoxin by ELISA. For inactivation of intrinsic peroxidase, prepare a 30% (w/v) solution of sodium azide and add 1 ml of this solution (30% w/v sodium azide) to 4 ml of test sample (final sodium azide concentration 6% (w/v)). Mix sample with azide solution, add extra sample additive, and let stand 1-2 min at room temperature. Retest sample for peroxidase presence (50 µl sodium azide-treated sample with 50 µl ELISA kit substrate reagent), as described above. If reaction is colorless (or original color), proceed with ELISA to identify enterotoxin in the peroxidase-inactivated sample. CAUTION: Use appropriate safety waste containers for disposal of preparations containing sodium azide, a hazardous material.

When examining processed foods with obvious can defects which might result in the growth of organisms that produce peroxidase, test the extract for peroxidase production and inactivate as described above before testing for staphylococcal enterotoxin.

2. Procedures

NOTE: Raw food (e.g., vegetables), see General Precautions, above. Follow directions under 4, Other Foods, below.

1. Milk and milk powder. Reconstitute milk powder (25 g) by mixing with 125 ml 0.25 M Tris, pH 8.0. Treat reconstituted milk powder in same way as fluid milk. For milk samples (5.0 ml), ensure that pH is in range 7-8; then add 50 µl sample additive (in TECRA™ kit). For clearer extract, adjust pH to 4.0 with concentrated HCl. For milk samples (50 ml), ensure that pH is in range 7-8; then add 50 µl sample additive (in kit). Centrifuge sample for at least 10 min at 1000-3000 x g. Decant extract and pump about 5.0 ml through syringe containing wetted absorbent cotton into polypropylene tube. Readjust pH to 7.0-8.0 (use pH paper), add 50 µl additive (in kit), and mix thoroughly.
2. Dehydrated food ingredients. Add 125 ml 0.25 Tris, pH 8, to 25 g of food, and homogenize in blender for about 3 min at high speed. Centrifuge sample for about 10 min at 1000-3000 x g and collect extract. Remove plunger from plastic syringe containing prewetted absorbent cotton and carefully pump solution through, collecting eluate. Take 5 ml of eluate; adjust pH to 7.0-8.0; then add 50 µl of sample additive, and mix thoroughly.
3. Cheeses. Add 50 ml water to 25 g of cheese and homogenize for about 3 min at high speed in blender. Adjust pH to 4 (pH paper) with concentrated HCl. Centrifuge sample for about 10 min at 1000-3000 x g. Remove plunger of plastic syringe containing prewetted cotton, and place 5.0 ml of extract into syringe; insert plunger and carefully pump solution through, collecting eluate. Take 5 ml of eluate, and add NaOH to adjust pH to 7.0-8.0; add 50 µl of sample additive, and mix thoroughly.
4. Other foods. Prepare foods other than those described above as follows: Add 50 ml 0.25 M Tris, pH 8, to 25 g of food and homogenize for about 3 min at high speed in blender. Centrifuge sample for about 10 min in bench centrifuge at 1000-3000 x g. Remove plunger from plastic syringe containing prewetted absorbent cotton and place 5 ml of extract into syringe; insert plunger and carefully pump solution through, collecting eluate in polypropylene tube. Take 5 ml of eluate; adjust pH, if necessary, to 7.0-8.0; add 50 µl of sample additive, and mix thoroughly.

1. Special equipment

Materials and reagents supplied in kit:

1. 30 SET Reagent strips

The SET Reagent Strip (refer to the table below) is a polypropylene strip of 10 wells covered with a foil seal and paper label. The first well of the strip is for the sample. The last well of the strip, an optically clear cuvette, is for the fluorometric determination. The eight wells in the center of the strip contain the various reagents for the assay. (See description of reagent strip below).

DESCRIPTION OF THE STAPH ENTEROTOXIN REAGENT STRIP

Wells	Reagents
1	Sample Well: 0.5 ml of food extract is placed into the well
2	Pre-Wash Solution (0.4 ml): TBS - Tween with 0.1% (w/v) sodium azide
3-4-5-7-8-9	Wash Solution (0.6 ml): TBS - Tween with 0.1% (w/v) sodium azide
6	Conjugate (0.4 ml): alkaline phosphatase labeled polyclonal antibodies with 0.1% (w/v) sodium azide
10	Cuvette with substrate (0.3 ml): 4-methyl-umbelliferyl phosphate with 0.1% (w/v) sodium azide

The name of the test, the lot number, and the expiration date of the kit are included on a bar code which is printed on the SET Reagent Strip. The test identification, lot number and calibration parameters are both clearly indicated in the kit's specification sheet and printed with a bar code.

2. 30 SET SPRs

The interior of the SET SPR is coated at the time of manufacture with anti-enterotoxin antibodies.

3. 1 Bottle standard (3 ml)

Purified staphylococcal enterotoxin B (5 ng/ml) with 0.1% (w/v) sodium azide and protein stabilizers.

CAUTION: Handle with care!

4. 1 Bottle positive control (6 ml)

Purified staphylococcal enterotoxin B (5 ng/ml) with 0.1% (w/v) sodium azide and protein stabilizers. Control range indicated on the vial label. CAUTION: Handle with care!

5. 1 Bottle negative control (6 ml)

TRIS buffered saline (TBS) - Tween with 0.1% (w/v) sodium azide.

6. 1 Bottle concentrated extraction buffer (55 ml)

2.5 mol/1 TRIS - 1% (w/v) Tween with 1% (w/v) sodium azide.

Materials required by user but not provided in kit:

1. Pipette which will dispense a minimum of 0.5 ml
2. Tips, plastic, to deliver 500 µl
3. Centrifugation/filtration tubes (bioMerieux Product Number: 30550) or plastic syringes (20 ml optional
4. Omni mixer, Waring blender (or equivalent) for preparation of food extracts
5. pH paper (range 0-14)
6. Centrifuge
7. Centrifuge cups
8. Polyethylene glycol (15,000-20,000 mol wt)
9. Sodium hydroxide solution (1.0 N Na0H)
10. Hydrochloric acid
11. Sodium hypochlorite
12. Dialysis tubing, flat width 32 mm or comparable

Contents

Electrophoretic and Immunoblot Analysis of Staphylococcal Enterotoxins in Food (Rasooly, 1998)

Immunoblotting can detect S. aureus enterotoxin A in food. The method may also be adapted to other toxins in foods.

Staphylococcal enterotoxins (SE), a family of five major serological types of heat stable (Anderson et al., 1986; Denny et al., 1971; Fung et al., 1973; Lee et al., 1977; Read and Bradshaw, 1966a; Read and Bradshaw, 1996b; Schwabe et al., 1990; Tibana et al., 1987), emetic enterotoxins (SEA through SEE), are encoded by five genes, which share 50 to 85% homology at the predicted amino acid level (Bergdoll, 1972; Marrack and Kappler, 1990). Enterotoxin A (SEA), a 27 kDa monomeric protein, is an extremely potent gastrointestinal toxin (Archer and Young, 1988; Evenson et al., 1988)and requires very sensitive methods to detect the low levels in foods (ng/g food) that can cause illness.

After antibodies to SEA were produced, immunological testing became the method of choice for SEA detection (Bergdoll et al., 1959). Radioimmunoassay (Miller et al., 1976), microslide double diffusion and enzyme-linked immunosorbent assay (ELISA), have been used for testing food samples. ELISA is especially useful, because it is simple, sensitive (0.5 ng/ml), rapid, and available in commercial kits that use distinct antibodies, either polyclonal or monoclonal.

Cross-reaction with unrelated antigens (Park et al., 1992; Park et al., 1993) or endogenous peroxides in particular foods that react with colorigenic reagents may not be distinguishable from positive results by some methods without extensive controls (Park et al., 1994). In addition, heat-treated SEA (in heat processed foods) may give negative results, because heat-treated enterotoxin may aggregate, reducing its reactivity with antibodies. However, it may retain toxicity after heat treatment (Anderson et al., 1986; Bennett, 1992).

Methods for analysis of regulatory samples of foods must resolve or avoid "false positive" and "false negative" reactions. Before antibody is applied, the SDS-PAGE immunoblot method, described below, solubilizes and separates proteins, to discriminate cross reactions to heterologous proteins that may occur.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a common method for protein separation (Laemmli, 1970; Orden et al., 1992). An electrical field is applied so that charged molecules migrate through a polyacrylamide matrix to the electrode bearing the opposite charge. The negatively-charged detergent, SDS, denatures and strongly binds proteins. Then, SDS-bound proteins migrate to the positive pole at rates inversely proportional to their molecular weights.

In general, two-part discontinuous gels are used (Laemmli, 1970). The sample is loaded onto the upper portion (stacking gel), which has a low acrylamide concentration, low pH, and low resolving ability. When a sample runs through the stacking gel, all proteins are concentrated into a narrow band. That narrow band then enters the lower portion (resolving gel) that separates proteins by size. The acrylamide concentration chosen for the resolving gel depends on the sizes of proteins to be separated. Smaller proteins are resolved at higher acrylamide concentrations and vice versa. SEs are 25-30 kDa; 12.5% acrylamide is useful for separating proteins in that range.

Immunoblotting (also known as "Western" blotting) is widely used for analyzing proteins separated by SDS-PAGE. The proteins are transferred from the gel to a membrane. Then, the membrane is probed with an antibody ("primary antibody") against the specific antigen. To detect the antibody-antigen complex, a secondary antibody is used. Usually, this is a polyclonal antibody (e.g., anti-mouse if the primary antibody is a mouse monoclonal) tagged with a biochemically detectable marker. Some common secondary antibody tags are fluorescent molecules (e.g., FITC, rhodamine), horseradish peroxidase, alkaline phosphatase, or biotin. Then, simple colorimetric reactions are carried out to reveal the location of the complex in a band on the membrane at a position corresponding to the molecular weight of the antigen.

Immunoblots have two important advantages for food testing. First, even though heat and other treatments during food processing can cause proteins to aggregate, the aggregates are solubilized and unfolded in SDS gels. Other antibodybased methods of food analysis, such as ELISA, do not have an SDS solubilization step. Instead, the sample is applied directly to the antibody, because SDS in the sample would denature the detecting antibody. Second, cross-reacting antigens usually can be distinguished from the desired antigen on the basis of molecular weight in a Western blot. In ELISA, and other assays in which samples are evaluated without separation or purification, cross-reacting antigens increase the background.

1. Equipment and materials
1. Equipment: Electrophoretic apparatus: Vertical mini gel unit with 8 x 10 cm or 10 x 12 cm plates (Bio-Rad Mini-Protean II or Hoefer SE-260 or equivalent), 1.5 mm spacers, and 1.5 mm 10 well comb. (Wider combs and spacers for larger volumes may be custom-made.)
2. Transfer unit, Mini electroblotting unit (Bio-Rad Mini-Trans-Blot or equivalent).
3. Power supply: Constant voltage of at least 200V and constant amperage of at least 400 mA (Bio-Rad PowerPac 300 or equivalent). A power supply with timer is recommended.
4. Microcentrifuge: A centrifuge that accommodates 1.5 ml (microcentrifuge) tubes and attains speeds of at least 10,000 rpm.
5. Homogenizer: An Omni µH or other homogenizer for grinding small amounts of food.
6. Membrane - Nitrocellulose membranes (similar to Sigma N8142) the same size as gel.
7. Miscellaneous small equipment: Rotator or rocking platform for mixing samples, 100 x 15 mm square culture dishes (Falcon 1012 or equivalent), Pyrex baking dish (at least 10 inches [25.4 cm] square) and 1.5 ml plastic centrifuge (microfuge) tubes.
8. Scanner (recommended): A flatbed scanner with resolution of at least 600 DPI (similar to Hewlett Packard 4C).
2. Reagents
1. 30% Acrylamide/bis-acrylamide (0.8% bis) pre-mixed solution (Sigma A-3699 or equivalent).

WARNING! ACRYLAMIDE IS NEUROTOXIC. ALWAYS WEAR GLOVES AND OTHER APPROPRIATE PROTECTION WHEN USING.

2. BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium), alkaline phosphatase substrate solution (Sigma B-6404 or equivalent).
3. Protein molecular weight color markers (6-200 kDa) (Sigma C-3437 or equivalent).
4. Purified Staphylococcal enterotoxin A (similar to Sigma S9399 or equivalent).
5. Molecular biology grade reagents:
1. Tris base
2. SDS
3. EDTA
4. Tween 20 (Polyoxyethylenesorbitan Monolaurate)
5. NaCl
6. 6N HCl
7. Glycine
8. Ammonium Persulfate
9. TEMED
10. Methanol
11. Non-fat dry milk (similar to Carnation)
12. ß-mercaptoethanol
13. Bromphenol blue
6. Antibodies:
1. Mouse monoclonal anti-SEA (IGEN Mab 3A #506-022-01)
2. Rabbit polyclonal anti-SEA (Sigma S-7656 or equivalent)
3. Goat anti-rabbit alkaline phosphatase conjugate (Sigma A2556 or equivalent)
4. Goat anti-mouse alkaline phosphatase conjugate (Sigma A4937 or equivalent)
3. Preparation of materials
1. 1M Tris, pH 7. Dissolve 121.1 g Tris base in 750 ml H O. Add 6N HCl to pH 7.02 (approximately 160 ml). Add H₂O to final volume of 1 liters.
2. 1M Tris, pH 8. Dissolve 121.1 g Tris base in 750 ml H O. Add 6N HCl to pH 8.02 (approximately 90 ml). Add H₂O to final volume of 1 liters.
3. 20% (w/v) SDS. (Wear a mask when weighing SDS.)
4. Gel Buffer A (3M Tris, pH 8.8). Dissolve 181.6 g Tris base in 250 ml H₂O. Add 40 ml 6N HCl. Add H₂O to final volume of 500 ml.
5. Gel Buffer D (0.25M Tris, pH 6.8). Dissolve 15.1 g Tris base in 50 ml H₂O. Add 18 ml 6N HCl. Add H₂O to final volume of 100 ml.
6. 10X Running buffer (250 mM Tris; 1.92M glycine; 1.0% SDS). Dissolve 121 g Tris base and 576 g glycine in 4 l H₂O. Stir until completely dissolved. Add 40 g SDS (wear a mask when weighing SDS).
7. 10X Western blot transfer buffer (250 mM Tris; 1.92M glycine). Add 121 g Tris base and 576 g glycine to 4 l H₂O; stir until completely dissolved.
8. 1X Western blot transfer buffer. Mix 400 ml 10X Western blot transfer buffer with 2800 ml H₂O. Then add 800 ml methanol.
9. Tris-Tween blocking buffer (10 mM Tris, pH 8; 500 mM NaCl; 0.5% Tween-20). Mix 116 g NaCl with 3 L H₂O. Add 40 ml 1.0 M Tris pH 8 and stir until dissolved. Add 20 ml Tween-20 and stir gently. Add water to 4 liters.
10. Loading buffer (250 mM Tris, pH 7; 4% SDS; 20% glycerol; 10% b -mercaptoethanol; 0.05% bromphenol blue). Mix 12.5 ml 1.0 M Tris pH 7 with 10 ml glycerol and 5 ml b -mercaptoethanol. Add H₂O to 40 ml. Add 10 ml 20% SDS and 25 mg bromphenol blue.
11. 10% (w/v) APS (ammonium persulfate). Prepare fresh weekly; cover in aluminum foil.

4. Casting gels

Commercially prepared gels are available through suppliers (depending on apparatus).

1. Assemble the casting tray. The gel is cast between two glass plates. Follow the manufacturer's instructions and assemble the plates for casting two gels, using the 1.5 mm spacers. Always cast two gels; the second can be stored, used as a duplicate or as a backup for the first.
2. Prepare lower gel. Always wear gloves when handling acrylamide solutions Prepare the resolving (lower) gel (Table 19-9) in a 50 ml tube. Add acrylamide last. The amount of APS and TEMED is higher than normally recommended, so the solution polymerizes rapidly. If you find it hard to work fast enough, half the amount of APS and TEMED.
3. Pour lower gel. Quickly pour the gel solution between the glass plates to 2/3 of the height. Immediately overlay (GENTLY) with 300 µl H₂O or isobutanol. Note, however, that isobutanol may react with the plastic of the apparatus
4. Prepare gel to add stacking gel. The gel will polymerize within 15 min, forming a clearly visible interface between the water and the gel. The gel can now be used immediately or covered with Saran Wrap and stored (at 4°C) for later use. Right before using the gel, pour off the water (or isobutanol).
5. Prepare and pour stacking (upper) gel. Prepare the stacking gel solution (Table 19-10), pour to top of plates and insert 1.5 mm comb. After the gel polymerizes, GENTLY remove the comb and wash the wells with H₂O. Drain the wells carefully.

Table 19-9. Lower (resolving) gel (for two 0.75 mm gels)

% Gel	12.5%	15%	16%	17%
Buffer A	2.8m l	2.8m l	2.8m l	2.8m l
H2O	1.5 ml	0.83 ml	0.58 ml	0.33 ml
20% SDS	50m l	50m l	50m l	50m l
10% APS	30m l	30m l	30m l	30m l
TEMED	30m l	30m l	30m l	30m l
Acrylamide solution	3.1 ml	3.75 ml	4 ml	4.25 ml
Total	7.5 ml

Table 19-10. Stacking gel (for two 0.75 mm gels)

% Gel	4.5%
Buffer D	0.625 ml
H2O	1.5 ml
20% SDS	30 m l
10% APS	20m l
TEMED	20m l
Acrylamide solution	0.375 ml
Total	2.57 ml

6. Checking the gels. Assemble the electrode unit (follow the manufacturer's instructions) and fill the upper buffer with H₂O to test for leaks.

5. Sample preparation
1. Mushroom samples. Homogenize at least 1 g mushrooms (follow sampling procedure) with the homogenizer. Add an equal amount (w/v) of the can brine and homogenize again. Transfer 300 µl (approximately) to a 1.5 ml tube. Volume measurements may be inaccurate because these samples are very viscous. It may help to cut the micropipette tip to a larger aperture, or add approx. 300 mg to a preweighed tube. Add an equal volume of Loading Buffer. Heat at 90ºC for 2 min and centrifuge for 1 min.
2. Purified SEA (positive control). Make a 1 µg/ml stock solution in H O. For 1 ng add 1 µl to 192 µl H O, and add an equal volume of Loading Buffer. Heat at 90ºC for 2 min and centrifuge for 12 min.

6. Running the gel
1. Loading. Load gels with 40 µl (sample size depends on combs and spacer use; wider combs and spacers for larger volumes are available through the FDA upon request) of the sample per well. Whenever possible, skip one lane between samples, to minimize cross-contamination. Be sure to apply the positive control several lanes from any test samples. Place 5 µl (25 µg protein) of prestained (color) MW marker in a nearby well to monitor the progress of the run, the effectiveness of transfer and the size of the bands.
2. Running the gel. Pour the running buffer into the outer chamber and then GENTLY add buffer to the upper chamber, using a pipette so as not to disturb the samples. When attaching the leads, VERIFY correct electrical polarity. Wrong electrical orientation is the most common mistake in SDS-PAGE. Run at 150 V for 1.5 h (one gel) or 100 V for 2.2 h (two gels), or until the bromphenol blue reaches the bottom of the resolving gel. When running two gels, make sure not to overheat the apparatus (lower the voltage if necessary).

7. Immunoblotting
1. Stop the gel, dissemble the electrophoresis unit, and remove the top glass plate of the gel.
2. Assembling the transfer apparatus. Use gloves when handling nitrocellulose filters. Assemble the transfer unit according to manufacturer's instructions, in a baking dish filled with cold transfer buffer to avoid air bubbles.
3. Transferring proteins. Add ice to the unit's cooling reservoir to keep it cool while running. Connect the electric lead and VERIFY correct electrical polarity. Electroblot at 400 mA for 1½ h (you may need to change the ice). (Consult your manual.)
4. Dissemble the unit. Confirm successful transfer of colored MW markers. Discard gel and put membrane into a baking dish.
5. Block the membrane. Incubate in Tris-Tween Blocking Buffer for 20 min with gentle shaking in a square culture dish; use at least 20 ml solution.
6. Primary antibody. Add anti-SEA in Tris-Tween Blocking Buffer. If using the monoclonal anti-SEA from IGEN, dilute 1:300. Use at least 10 ml of solution (20 ml for two membranes). For SIGMA anti SEA dilute 1:1000. Shake gently for 2 h.
7. Washing. Wash for 20 min in 20 ml or more of Tris-Tween Blocking Buffer with gentle shaking.
8. Secondary antibody. Incubate 1-2 h with the secondary antibody. If a mouse monoclonal was used as the primary antibody, use an anti-mouse alkaline phosphatase conjugate diluted 1:1000 in 10 ml Tris-Tween Blocking Buffer.
9. Washing and color development. Wash vigorously three times for 20 min with at least 20 ml Tris-Tween Blocking Buffer. Add 10 ml BCIP/ NBT color reagent for detection. Watch for signal development and for background to determine the correct incubation time empirically for each sample and membrane (approximately 10 min).

8. Densitometry (recommended)
1. Dry the membrane and then scan the blot to quantitate the signal. Set the scan mode to 256 gray scale black and white photograph scanning. The approximate size of the file (in TIFF format) of a mini gel blot is 1.4 Mb. While contrast and brightness can be adjusted to improve the data for presentation, this will affect the quantitation of the image. Quantitation should always be performed with the raw data compared to a standard curve of known amounts of toxin. While there is no established method for immunoblot quantitation, the bands can be quantitated using NIH Image software (public domain software for MacIntosh).

9. Data presentation

Problems and troubleshooting:

Problem	Cause
Slow or no polymerization of the gel	APS is old, OR APS, TEMED or acrylamide were left out
No tracking dye observed	Wrong polarity
Smile effect or gel overheats	High voltage leads to excessive heat.
Sample floats in the well or diffuses out of well	Wrong concentration of glycerol in the loading dye
No transfer	Wrong polarity or problem with transfer buffer.
Transfer OK but no signal in the positive control, membrane turns purple	problem with antibodies.
Transfer OK but no signal in the positive control, membrane is colorless	problem with detection reagents
High background	problems with blocking. increase time and/or add 0.5%-1% non-fat dry milk to the Blocking Buffer
Diffuse /distorted marker bands	Too little SDS
Distorted SEA band in samples	Too much protein in sample (overloading). Dilute sample or use chromatography or immunoprecipitation to remove major proteins

Limitations of Western blotting:

Western blotting has some limitations, which are important to recognize when applying the method to food analysis.

1. First, inactive and active SE are nearly indistinguishable by Western blotting (or any other antibodybased method).
2. Second, only small sample volumes (30-50 µl) can be loaded onto a gel (Wider combs and spacers for larger volumes are available through the FDA upon request)., which may limit the sensitivity of the method. Preparative methodology (tube gels and preparative electrophoresis), which is under development, may overcome this limitation. The present technology with small samples is nevertheless extremely sensitive. When compared directly with ELISA using contaminated mushroom samples, Western blotting was as sensitive as ELISA with native samples and much more sensitive with heat-denatured samples.
3. A third limitation of Western blots is that cross-reactive bands potentially could co-migrate with the antigen. Cross-reactivity is an inherent problem with all immunological methodology, because antibodies recognize small regions of proteins and similar epitopes may occur in other proteins. This is a major concern in ELISA and other methods in which the proteins are not separated. It is a smaller concern with Western blotting because the proteins are separated, but false positives are still a potential problem.

There are several ways to minimize this problem. One is to increase the specificity of the reaction by using monoclonal antibodies. Alternatively, several independently isolated antibodies and control samples of uncontaminated similar food can be used to determine whether the bands represent toxin or unrelated antigens.

4. Finally, it is important to note that Western blots have a linear response over a broad range of toxin concentrations. However, at very low levels, the signal is not linear.

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Other analytical procedures

• Staphylococcus aureus in foods: Most probable number method for isolation (AOAC, 1995c).
• Staphylococcus aureus in foods: Surface plating method for isolation and enumeration (AOAC, 1995d).
• Staphylococcus aureus isolated from foods: Latex agglutination test method (AOAC, 1995e).
• Staphylococcal enterotoxin in foods: Extraction and separation methods (AOAC, 1995f).
• Staphylococcal enterotoxin in foods: Microslide gel double diffusion test (AOAC, 1995g).
• Staphylococcal enterotoxin in selected foods: Polyvalent enzyme immunoassay method (AOAC, 1995h).

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Commercial Test Products

Table 19-11. Commercial test products for S. aureus. 

Test Kit	Analytical Technique	Approx. Total Test Time1	Supplier
3M Petrifilm ™  Rapid S. aureus Count Plate	An indicator of the presence of a thermostable nuclease. Dry rehydratable film method.	26-30 h	3M Microbiology Products  3M Center, Building 275-5W-05  St. Paul, MN  55144-1000  Phone: 800/228-3957; 651/737-6501  E-mail: innovation@mmm.com Web: www.3m.com/microbiology/
AccuPROBE® Culture Staphylococcus aureus Culture Identification Test	Nucleic acid hybridization	Up to 72 h	Gen-Probe  Contact: Customer Service  10210 Genetic Center Dr.  San Diego, CA  92121  Phone: 858/410-8000 Web: www.gen-probe.com
API RAPIDEC Staph  [Used to identify Staphylococcus aureus]	Fluorescent test for S. aureus.  Biochemical identification of Staphylococcus	2 h	bioMérieux Inc. Contact: bioMérieux Industry 595 Anglum Rd.  Hazelwood, MO  63042  Phone: 800/638-4835; 314/731-8500  E-mail: usa@na.biomerieux.com Web: www.biomerieux.com
API Staph  [Used to identify Staphylococcus aureus]	Biochemical reaction	24 h	bioMérieux Inc. Contact: bioMérieux Industry 595 Anglum Rd.  Hazelwood, MO  63042  Phone: 800/638-4835; 314/731-8500  E-mail: usa@na.biomerieux.com Web: www.biomerieux.com
BACTiStaph [Used to identify Staphylococcus aureus]	Latex agglutination	After a 24 h plate culture, 60 s	Remel Contact: Customer Service 12076 Santa Fe Dr.  Lenexa, KS  66206  Phone: 800/255-6730; 913/888-0939   E-mail: remel@remelinc.com Web: www.remelinc.com
CHECK 3 Staph aureus	Chemical, visual detection	4-18 h	Contamination Sciences LLC  Contact: Robert Steinhauser  4230 East Towne Blvd., Suite 191  Madison, WI  53704  Phone: 608/825-6125  E-mail: bsteinha@contam-sci.com Web: www.contam-sci.com
GENE-TRAK Staphylococcus aureus Assay [Used to detect Staphylococcus aureus]	Nucleic acid hybridization	28 h	GENE-TRAK Systems  Contact: Linda Dragone  94 South St.  Hopkinton, MA  01748  Phone: 508/435-7400 E-mail: MCyr@vysis.com
ISO-GRID Method for Staphylococcus aureus Count using Baird-Parker Agar	Membrane filtration with selective and differential culture medium	48-54 h	QA Life Sciences, Inc.  6645 Nancy Ridge Dr.  San Diego, CA  92121  Phone: 800/788-4446; 858/622-0560  E-mail: bugsy@qalife.com
RIDASCREEN SET (R4101) (R-Biopharm GmbH)  [Used to identify S. aureus enterotoxins A, B, C, D, or E]	ELISA	3 h	R-Biopharm, Inc. Contact: Thomas Grace 7950 US 27 South Marshall, MN 49068 Phone: 616/789-3033 E-mail: RbioST@voyager.net
RIDASCREEN Staphylococcus aureus Thermonuclease (R4001) (R-Biopharm GmbH)  [Used to identify S. aureus at levels to identify intoxication]	Immunodiffusion inhibition assay	4 h	R-Biopharm, Inc. Contact: Thomas Grace 7950 US 27 South Marshall, MN 49068 Phone: 616/789-3033 E-mail: RbioST@voyager.net
SET-RPLA (Oxoid)   [Used to identify staphylococcal enterotoxin A, B, C, D and E]	Reversed passive latex agglutination	18 h	Oxoid, Inc.  Contact: Jim Bell  217 Colonnade Rd.  Nepean, Ontario K2E 7K3  Canada  Phone: 613/226-1318  E-mail: jbell@oxoid.ca
Slidex Staph Kit  [Used to identify Staphylococcus aureus]	Latex agglutination	Once organism grown (24 h), 20 s test	bioMérieux Inc. Contact: bioMérieux Industry 595 Anglum Rd.  Hazelwood, MO  63042  Phone: 800/638-4835; 314/731-8500  E-mail: usa@na.biomerieux.com Web: www.biomerieux.com
Staphytect Plus (Oxoid)  [Used to confirm the presence of coagulase positive or coagulase negative staphylococci.  Confirms the presence of Staphylococcus aureus]	Latex agglutination	After a 24 to 48 h plate culture, approx. 60 s	Oxoid, Inc.  Contact: Jim Bell  217 Colonnade Rd.  Nepean, Ontario K2E 7K3  Canada  Phone: 613/226-1318  E-mail: jbell@oxoid.ca
TECRA Staphylococcus aureus Visual Immunoassay   [Used to identify Staphylococcus aureus]	ELISA	26 h	InternationalBioProducts  Contact: Mike Yeager 14780 NE 95th St.  Redmond, WA  98052  Phone: 800/729-7611; 425/883-1349  E-mail: myeager@intlbioproducts.com Web: intlbioproducts.com
TECRA Staphylococcal Enterotoxin (SET) Visual Immunoassay2  [Used to identify Staphylococcal enterotoxins A, B, C1, C2, C3, D, E and enterotoxin producing staphylococci]	ELISA	Thermally processed foods: 21 h  All other foods: 4 hours  Analysis of Staphylococcal cultures for toxin production: 4 h	InternationalBioProducts  Contact: Mike Yeager 14780 NE 95th St.  Redmond, WA  98052  Phone: 800/729-7611; 425/883-1349  E-mail: myeager@intlbioproducts.com Web: intlbioproducts.com
Transia Tube SET	ELISA	1½ h	GENE-TRAK Systems  Contact: Linda Dragone  94 South St.  Hopkinton, MA  01748  Phone: 508/435-7400 E-mail: MCyr@vysis.com
Vidas SET  [Used to identify Staphylococcus aureus]	Enzyme linked fluorescent assay	2 h	bioMérieux Inc. Contact: bioMérieux Industry 595 Anglum Rd.  Hazelwood, MO  63042  Phone: 800/638-4835; 314/731-8500  E-mail: usa@na.biomerieux.com Web: www.biomerieux.com

¹Includes enrichment

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References