Chapter 14: Coliforms, Fecal Coliforms and Escherichia coli

• Potential Food Safety Hazard
• Coliforms
• Fecal coliforms
• E. coli
• Control Measures
• FDA Guidelines
• State Guidelines
• Recommended Microbiological Limits
• ICMSF Recommended Microbiological Limits
• Canadian Bacteriological Guidelines
• Growth
• Heat Resistance
• Analytical Procedures
• Food Sampling and Preparation of Sample Homogenate
• Coliforms and E. coli
• Conventional method for determining coliforms and E. coli
• Rapid Method (RM-2) Using A-1 Medium for Recovery of Fecal Coliforms from Shellfish-Growing Waters
• Bottled water
• Examination of shellfish and shellfish meats
• LST-MUG Method for Detecting E. coli in Chilled or Frozen Foods Exclusive of Bivalve Molluscan Shellfish
• EC-MUG Method for Determining E. coli MPNs in Shellfish Meats
• Hydrophobic Grid Membrane Filter Methods for Rapid Enumeration of Total Coliforms, Fecal Coliforms, and E. coli
• Enterovirulent E. coli
• Isolation and identification of enterovirulent E. coli (EEC)
• Serological characterization
• Tests for Enteroinvasive E. coli (EIEC)
• Tests for Enterotoxigenic E. coli (ETEC)
• Isolation Methods for Enterohemorrhagic (EHEC) E. coli O157:H7
• Tests for Toxins of Enterohemorrhagic E. coli (EHEC)
• Tests for Enteropathogenic E. coli (EPEC)
• Other Analytical Procedures
• Commercial Test Products
• References

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Potential Food Safety Hazard

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Coliforms

Coliforms are Gram-negative, rod-shaped facultatively anaerobic bacteria. Identification criteria used are production of gas from glucose (and other sugars) and fermentation of lactose to acid and gas within 48 h at 35ºC (Hitchins et al., 1998).

The coliform group includes species from the genera Escherichia, Klebsiella, Enterobacter, and Citrobacter, and includes E. coli. Coliforms were historically used as indicator microorganisms to serve as a measure of fecal contamination, and thus potentially, of the presence of enteric pathogens in fresh water. Although some coliforms are found in the intestinal tract of man, most are found throughout the environment and have little sanitary significance (Greenberg and Hunt, 1985).

The presence of large numbers of coliforms in foods is highly undesirable, but it would be almost impossible to eliminate all forms (Jay, 1978). Because they are easily killed by heat, coliform counts can be useful when testing for post-processing contamination of cooked fish and fishery products.

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Fecal coliforms

Because coliform counts are inadequate to differentiate between fecal and nonfecal contamination, a fecal coliform test was developed. Fecal coliforms are coliforms that ferment lactose in EC medium with gas production within 48 h at 45.5ºC. With all shellfish isolates, an incubation temperature of 44.5ºC (rather than 45.5ºC) is used. Fecal coliforms are considered to be more directly associated with fecal contamination from warm-blooded vertebrates than are other members of the coliforms. E. coli usually makes up 75-95% of the fecal coliform count in shellfish growing areas, but at times can represent less than 1% of the coliform count (APHA, 1970; Greenberg and Hunt, 1985; Paille et al., 1987).

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E. coli

E. coli are naturally found in the intestinal tracts of all warm-blooded animals, including humans. Most forms of the bacteria are not pathogenic and serve useful functions in the intestine. Pathogenic strains of E. coli are transferred to seafood through sewage pollution of the coastal environment or by contamination after harvest. E. coli food infection causes abdominal cramping, water or bloody diarrhea, fever, nausea and vomiting (Ward et al., 1997).

Some E. coli strains may be only weakly lactose-positive (delayed lactose fermentation) or even lactose-negative (APHA, 1970; Ewing, 1986).

Enterovirulent E. coli (EEC) strains include several major subgroups:

Enterohemorrhagic E. coli (EHEC) causes hemorrhagic colitis and hemolytic uremic syndrome. Six verotoxins have been identified within this group, but only stx-1 and stx-2 seem to be important in human infections. E. coli O157:H7 is the principle serotype of this group (Reed, 1994).

Enteroinvasive E. coli (EIEC) causes a diarrheal illness similar to shigellosis (Reed, 1994).

Enterotoxigenic E. coli (ETEC) is a major cause of travelers' diarrhea and infant diarrhea in developing countries. These strains product a heat-labile toxin (LT) and/or a heat-stable toxin (ST) (Reed, 1994).

Enteropathogenic E. coli (EPEC) is an important cause of infant diarrhea (Reed, 1994).

Enteroadherent E. coli (EAEC) is a newly added category and not fully characterized (Hitchins et al., 1998).

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Control Measures

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FDA Guidelines

Table 14-3. FDA guidelines for E. coli in fish and fishery products.

Product	Guideline	Reference
Crabmeat, fresh and frozen	MPN of at least 3.6 per gram (IMVIC confirmed) in one or more of a minimum of 6 subsamples and inspectional evidence indicating the most probable source of the E. coli.	FDA, 1996b
Shrimp, raw breaded	Mean log of 16 units of finished product breaded shrimp collected prior to freezing is greater than 0.56 (i.e., geometric mean greater than 3.6/g) and exceeds the mean log of 16 units of stock shrimp by more than twice the standard error of their difference (2 SED).	FDA, 1996a
Oysters and clams, fresh or frozen - imports	MPN of 230/100 g (average of subs or 3 or more of 5 subs)	FDA, 1998a
Oysters, clams, mussels, fresh or frozen - domestic	1 or more of 5 subs exceeding MPN of 330/100 g or 2 or more exceeding 230/100 g	FDA, 1998a

Table 14-4. FDA guidelines for enterotoxigenic E. coli in fish.

Product	Guideline	Reference
Ready-to-eat fishery products (minimum cooking by consumer)	1 x 103 ETEC/g, LT1 or ST2 positive	FDA, 1998a

¹Heat-labile toxin; ²Heat-stable toxin

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State Guidelines

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Recommended Microbiological Limits

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ICMSF Recommended Microbial Limits

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Heat Resistance

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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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Coliforms and E. coli

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Conventional method for determining coliforms and E. coli (Hitchins et al., 1998)

The standard weight of analytical portions of food samples examined for the presence of sanitary and enteropathogenic E. coli is 25 g. If desired, 50 g portions may be used with appropriate scale-up of the suspending medium. See Chapter 9 and current FDA field instructions on sampling and compositing before proceeding with E. coli and coliform analyses (Hitchins et al., 1998).

1. Equipment and materials
1. Covered water bath, with circulating system to maintain temperature of 45.5 ± 0.2ºC. Water level should be above the medium in immersed tubes.
2. Immersion-type thermometer, 1-55ºC, about 55 cm long, with 0.1º subdivisions, certified by National Institute of Standards and Technology (NIST), or equivalent
3. Incubator, 35 ± 1ºC
4. Balance with capacity of ³ 2 kg and sensitivity of 0.1 g
5. Blender and blender jar (see Chapter 9)
6. Sterile graduated pipets, 1.0 and 10.0 ml
7. Sterile utensils for sample handling (see Chapter 9)
8. Dilution bottles made of borosilicate glass, with stopper or polyethylene screw caps equipped with Teflon liners
9. Quebec colony counter, or equivalent, with magnifying lens
10. Longwave UV light
11. pH meter
2. Media and reagents
1. Brilliant green lactose bile (BGLB) broth, 2% (M25)
2. Lauryl tryptose (LST) broth (M76)
3. EC broth (M49)
4. Levine's eosin-methylene blue (L-EMB) agar (M80)
5. Tryptone (tryptophane) broth (M164)
6. MR-VP broth (M104)
7. Koser's citrate broth (M72)
8. Plate count agar (PCA) (standard methods) (M124)
9. Butterfield's phosphate-buffered dilution water (R11) or equivalent diluent (except for shellfish)
10. Kovacs' reagent (R38)
11. Voges-Proskauer (VP) reagents (R89)
12. Gram stain reagents (R32)
13. Methyl red indicator (R44)
14. Violet red bile agar (VRBA) (M174)
15. VRBA-MUG agar (M175)
16. EC-MUG medium (M50)
17. Lauryl tryptose MUG (LST-MUG) broth (M77)
18. Peptone diluent, 0.1% (R56)
3. Presumptive test for coliform bacteria
1. Weigh 50 g (see comment on sample size on first page of this chapter) food (unthawed if frozen) into sterile high-speed blender jar. Add 450 ml Butterfield's phosphate-buffered dilution water and blend 2 min. Frozen sample can be softened by refrigerating 25 g portion for 18 h at 2-5ºC. If necessary, the analytical sample may differ from 50 g in the range of 25-50 g -- depending on availability of the sample -- as long as the diluent is adjusted accordingly.
2. Prepare decimal dilutions with 90 ml sterile dilution water plus 10 ml from previous dilution. Number of dilutions to be prepared depends on anticipated coliform density. Shake all suspensions 25 times in 30 cm arc for 7 s. Do not use pipets to deliver <10% of their total volume. Transfer 1 ml portions to 3 LST tubes for each dilution for 3 consecutive dilutions. Hold pipet at angle so that its lower edge rests against tube. Let pipet drain 2-3 s. Not more than 15 min should elapse from time sample is blended until all dilutions are in appropriate media.
3. Incubate tubes 48 ± 2 h at 35ºC. Examine tubes at 24 ± 2 h for gas, i.e., displacement of medium in fermentation vial or effervescence when tubes are gently agitated. Reincubate negative tubes for additional 24 h. Examine a second time for gas. Perform a confirmed test on all presumptive positive (gassing) tubes.
3. Confirmed test for coliforms

Gently agitate each gassing LST tube and transfer loopful of suspension to tube of BGLB broth. Hold LST tube at angle and insert loop to avoid transfer of pellicle (if present). Incubate BGLB tubes 48 ± 2 h at 35ºC.

Examine for gas production and record. Calculate most probable number (MPN) (see Chapter 10) of coliforms based on proportion of confirmed gassing LST tubes for 3 consecutive dilutions.

4. Coliform group: solid medium method
1. Prepare violet red bile agar (VRBA) and pasteurize it by boiling for 2 min on day of use. NOTE: Overheating may result in decreased productivity (Hartman and Hartman, 1976). If autoclave is used to sterilize VRBA, heat small aliquots of about 100 ml no longer than 5 min at 121ºC. Store sterile medium in the dark no longer than 2 weeks before use, and remelt agar in flowing steam, boiling water, or in a microwave oven. Cool to 48ºC before use; pH, 7.0-7.2. Homogenize 25 g sample at high speed for 1 min in 225 ml Butterfield's phosphate-buffered dilution water or 0.1% peptone water. Prepare serial tenfold dilutions in Butterfield's diluent or 0.1% peptone water in accordance with anticipated level of coliforms. Transfer two 1 ml aliquots of each dilution to petri dishes.
2. Use either of two plating methods (APHA, 1970). For conventional method, pour 10 ml VRBA tempered to 48ºC into plates. Swirl plates to mix, and let solidify. To prevent surface growth and spreading of colonies, overlay with 5 ml VRBA, and let solidify. If resuscitation is necessary, pour basal layer of 8-10 ml of tryptic soy agar tempered to 48ºC. Swirl plates to mix, and incubate at room temperature for 2 ± 0.5 h. Then overlay with 8-10 ml of melted, cooled VRBA and let solidify. To find E. coli among coliforms, use 100 mg 4 methyl-umbelliferyl-a -D-glucuronide (MUG) per ml in the VRBA overlay and observe for fluorescent colonies under longwave UV light. (See LST-MUG section for theory and applicability.) Use aliquots of up to 4 ml of dilution when deeper plates are used and 15 ml VRBA is added.
3. Invert solidified plates and incubate 18-24 h at 35ºC. Incubate dairy products at 32ºC (APHA, 1985b). Examine plates with illumination under magnifying lens. Count purple-red colonies that are 0.5 mm or larger in diameter and surrounded by zone of precipitated bile acids. Plates should have 25-250 colonies. For confirmation, select colonies representing different types in accordance with their relative numbers, and transfer each to tube of BGLB broth. Incubate tubes at 35ºC. Examine at 24 and 48 h for gas production.
4. Confirm colonies producing gas as coliform organisms. Perform Gram stain on sample from any tube showing a pellicle to exclude Gram-positive, lactose-fermenting bacilli. Determine number of coliforms/g by multiplying percentage of tubes confirmed as positive by original VRBA count, i.e., number of suspicious coliform colonies multiplied by dilution factor. A modification of this method has been commercialized (Marshall et al., 1987); see later section on rehydratable dry-film method.
3. EC broth method for fecal coliforms and confirmed test for E. coli

The EC broth MPN method may be used for seawater and shellfish since it conforms to recommended procedures (APHA, 1970).

1. Gently agitate each gassing LST tube (or, less preferably, use gassing BGLB tubes) and transfer loopful of each suspension to tube of EC broth. Incubate EC tubes 48 ± 2 h at 45.5 ± 0.2ºC. Examine for gas production at 24 ± 2 h; if negative, examine again at 48 ± 2 h. Use results of this test to calculate fecal coliform MPN. Streak loopful of suspension from each gassing tube to L-EMB agar. One portion of plate must exhibit well-separated colonies. Incubate 18-24 h at 35ºC. Examine plates for suspicious E. coli colonies, i.e., dark centered and flat, with or without metallic sheen. Transfer 2 suspicious colonies from each L-EMB plate to PCA slants for morphological and biochemical tests. Incubate PCA slants 18-24 h at 35ºC. If typical colonies are not present, transfer one or more colonies most likely to be E. coli. Pick one colony from every plate.
2. Perform Gram stain. Examine all cultures appearing as Gram-negative short rods or cocci for the following biochemical activities:
1. Indole production. Inoculate tube of tryptone broth and incubate 24 ± 2 h at 35ºC. Test for indole by adding 0.2-0.3 ml of Kovacs' reagent. Appearance of distinct red color in upper layer is positive test. NOTE: Studies (Ewing, 1986) indicate that indole-negative strains of E. coli are infrequent and probably belong to other species of Enterobacteriaceae. Examine these strains by using additional reactions suggested in Ewing (1986).
2. Voges-Proskauer (VP)-reactive compounds. Inoculate tube of MR-VP broth and incubate 48 ± 2 h at 35ºC. Transfer 1 ml to 13 x 100 mm tube. Add 0.6 ml a-naphthol solution and 0.2 ml 40% KOH, and shake. Add a few crystals of creatine. Shake and let stand 2 h. Test is positive if eosin pink color develops.
3. Methyl red-reactive compounds. Incubate MR-VP tube additional 48 ± 2 h at 35ºC after VP test. Add 5 drops to methyl red solution to each tube. Distinct red color is positive test. Yellow is negative reaction.
4. Citrate. Lightly inoculate tube of Koser's citrate broth; avoid detectable turbidity. Incubate 96 h at 35ºC. Development of distinct turbidity is positive reaction.
5. Gas from lactose. Inoculate tube of LST broth and incubate 48 ± 2 h at 35ºC. Displacement of medium from inner vial or effervescence after gentle agitation is positive reaction.
6. Interpretation. All cultures that (a) ferment lactose with production of gas within 48 h at 35ºC, (b) appear as Gram-negative nonsporeforming rods or cocci, and (c) give IMViC patterns ++-- (biotype 1) or -+-- (biotype 2) are considered to be E. coli. Calculate MPN (see Chapter 10) of E. coli based on proportion of EC tubes in 3 successive dilutions that contain E. coli.

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Rapid Method (RM-2) Using A-1 Medium for Recovery of Fecal Coliforms from Shellfish-Growing Waters

This rapid test may be used to enumerate fecal coliforms and as a presumptive test for E. coli in shellfish-growing waters but not, however, in shellfish tissues. Because geographical differences in these waters may affect the efficiency of this test, analysts should compare its results with those of the conventional method before using the A-1 medium method exclusively.

1. Equipment, materials, and media
1. Covered water bath, 44.5 ± 0.2ºC
2. A-1 medium (M1)
3. Levine's eosin-methylene blue (L-EMB) agar (M80)
4. Tryptone (tryptophane) broth (M164)
5. MR-VP broth (M104)
6. Koser's citrate broth (M72)
7. Butterfield's phosphate-buffered dilution water (R11)
8. Air incubator, 35 ± 0.5ºC
2. Procedure for recovery of fecal coliforms from shellfish-growing waters

Prepare samples as in presumptive test for coliform bacteria (see C, above). Incubate A-1 medium tubes 3 h at 35 ± 0.5ºC in air incubator. Transfer to water bath and incubate 21 ± 2 h at 44.5 ± 0.2ºC. Maintain water level in bath above level of liquid in inoculated tubes.

Presence of gas in inverted vial or of dissolved gas which can be removed by slight agitation is positive test. Report results as fecal coliform MPN/100 ml sample. NOTE: Fecal coliform counts tend to be greater than E. coli counts since no effort is made to obtain pure cultures and to identify them. Interpretation of data requires understanding of the microflorae of the specimen.

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Bottled Water

For microbial quality of bottled water, FDA traditionally follows the guidelines of the Environmental Protection Agency (EPA) for E. coli and coliforms in drinking water. EPA's current methodology (EPA, 1989) is based partly on the APHA methodology (APHA, 1989) and partly, for coliforms only, on the Edberg et al. (1989) method. For information on the methodology used by FDA for water, contact A.D. Hitchins, FDA, Division of Microbiological Studies (HFS-516), Washington, DC 20204.

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Examination of Shellfish and Shellfish Meats

The official bivalve molluscan FDA procedure for bacteriological analysis of domestic and imported shellfish is fully and properly described only in the 1970 edition of APHA's Recommended Procedures of the Examination of Sea Water and Shellfish (APHA, 1970). Methods, including the conventional 5-tube fecal coliform MPN test, are described for examining shell stock, fresh-shucked meats, fresh-shucked frozen shellfish, and shellfish frozen on the half shell. This procedure does not apply to the examination of crustaceans (crabs, lobsters, and shrimp) or to processed shellfish meats such as breaded, shucked, pre-cooked, and heat-processed products.

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LST-MUG Method for Detecting E. coli in Chilled or Frozen Foods Exclusive of Bivalve Molluscan Shellfish

About 94% of E. coli, including many anaerogenic (non-gas-producing) strains, produce the enzyme b -glucuronidase (GUD). Although some shigellae (44%) and salmonellae (29%) also produce GUD, its production by other Enterobacteriaceae is infrequent (Feng and Hartman, 1982). EHEC strains, which are present in about 2% of beef, pork, lamb, and poultry samples, do not produce GUD (Doyle and Schoeni, 1987). The lack of GUD activity in enterohemorrhagic E. coli O157:H7 has been used as a selection criterion for this pathogen. Reently, about 34% of human fecal isolates of E. coli were reported to be GUD-negative (Chang et al., 1989). There is evidence, however, that GUD enzyme activity may be under catabolite repression control (Chang et al., 1989) and that the genetic sequences for the GUD enzyme (uidA gene) are present in most GUD-negative E. coli isolates (Bej et al., 1991; Feng et al., 1991).

The LST-MUG assay can presumptively identify E. coli within 24 h (Feng and Hartman, 1982; Moberg et al., 1988). The assay is based on the presence of GUD in E. coli, which cleaves the MUG substrate to release 4-methylumbelliferone (MU). When exposed to longwave (365 nm) UV light, MU exhibits a bluish fluorescence which is easily visualized. When MUG is incorporated into LST medium, coliforms can be enumerated on the basis of gas production from lactose.

E. coli are presumptively identified by fluorescence in the medium under longwave UV light. The LST-MUG method described has been adopted as final action by the AOAC (Moberg et al., 1988).

CAUTION: To observe for fluorescence, examine inoculated LST-MUG tubes under longwave (365 nm) UV light in the dark. A 6-watt hand-held UV lamp is satisfactory and safe. When using a more powerful UV source, such as a 15-watt fluorescent tube lamp, wear protective glasses or goggles (available from laboratory supply houses) if personal exposure exceeds a few minutes a day. Also, examine all glass tubes for fluorescence before use. Cerium oxide, which is sometimes added to glass as a quality control measure, will fluoresce under UV light and interfere with the MUG assay (Hartman, 1989).

NOTE: Some foods, such as shellfish, contain natural GUD activity (Rippey, 1987). In these instances the EC-MUG confirmatory test may be used. Recently, a hydrophobic grid membrane filter/MUG (HGMF/MUG) method for enumeration of total coliform and E. coli in foods was also adopted official first action by the AOAC (Entis, 1989).

1. Equipment and materials
1. Incubator, 35 ± 1ºC
2. Balance with capacity of ³ 2 kg and sensitivity of 0.1 g
3. Blender and blender jar
4. Sterile graduated pipets, 1.0 and 10.0 ml
5. Sterile utensils for sample handling (see Chapter 9)
6. Dilution bottles made of borosilicate glass, with stopper or polyethylene screw caps equipped with Teflon liners
7. UV lamp and protective eyewear
2. Media and reagents
1. Lauryl tryptose MUG (LST-MUG) broth (M77)
2. Lauryl tryptose (LST) broth (M76)
3. Levine's eosin-methylene blue (L-EMB) agar (M80)
4. MR-VP broth (M104)
5. Koser's citrate broth (M72)
6. Plate count agar (PCA) (standard methods) (M124)
7. Butterfield's phosphate-buffered dilution water (R11) or equivalent diluent
8. Kovacs' reagent (R38)
9. Voges-Proskauer (VP) reagents (R89)
10. Gram stain reagents (R32)
11. Methyl red indicator (R44)
3. Presumptive LST-MUG test for E. coli

1. Prepare food samples as described for Conventional Method for Determining Coliforms, C-1 above.
2. Prepare decimal dilutions as described for Conventional Method for Determining Coliforms, C-2 above, and inoculate 1 ml portions to 3 LST-MUG tubes for each dilution for 3 consecutive dilutions. Also inoculate one tube of LST-MUG with a known positive E. coli isolate as positive control.
3. Incubate tubes for 24 ± 2 h at 35ºC and examine each tube for growth (turbidity, gas, or fluorescence). To observe fluorescence, examine tubes in the dark under longwave UV lamp (365 nm). A bluish fluorescence is positive presumptive test for E. coli. Studies by Moberg et al. (1988) show that a 24 h fluorescence reading is an accurate predictor of E. coli and can identify 83-95% of the E. coli-positive tubes. After 48 h of incubation, 96-100% of E. coli-positive tubes can be identified (33). Perform a confirmed test on all presumptive positive tubes.
4. Confirmed LST-MUG test for E. coli
1. Streak loopful of suspension from each fluorescent tube to L-EMB agar and incubate 24 ± 2 h at 35ºC. Follow protocols outlined in F, above, for Gram stains, IMViC tests, and production of gas from lactose to confirm E. coli.
2. Interpretation. All cultures that (a) fluoresce, (b) ferment lactose with production of gas within 48 h at 35ºC, (c) appear as Gram-negative nonsporeforming rods or cocci, and (d) give IMViC patterns of ++-- (biotype 1) or -+--- (biotype 2) are considered to be E. coli. Calculate MPN of E. coli based on proportion of fluorescent tubes in 3 successive dilutions that contain E. coli.

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EC-MUG Method for Determining E. coli MPNs in Shellfish Meats

The EC-MUG method (Rippey et al., 1987), uses MUG hydrolysis to detect E. coli (see LST-MUG section for theory and applicability). The very few other bacterial species that can hydrolyze MUG are seldom encountered in shellfish meats. Thus, by including MUG in EC broth at 44.5ºC and examining the incubated EC-MUG tubes for fluorescence under UV irradiation, an E. coli MPN can be readily obtained from a conventional 5-tube fecal coliform MPN determination for shellfish meats. The EC-MUG medium is inoculated from regular LST cultures of shellfish meats.

1. Equipment and materials
1. All those required for performing the conventional 5-tube MPN method for determining fecal coliforms
2. New, disposable borosilicate glass tubs (100 x 16 mm) for EC-MUG broths (5 ml)
3. New, disposable borosilicate glass Durham vials (50 x 9 mm) for inverted gas collection tubes in EC-MUG broths
4. Longwave UV lamp, 4 watt or equivalent
5. Positive control species: E. coli
6. Negative control species: Klebsiella pneumoniae
2. Media and reagents
1. All those required to determine fecal coliforms by the conventional MPN method
2. EC-MUG broth (M50)
3. Determining fluorescence of EC-MUG broth cultures
1. Use commercially prepared dehydrated EC-MUG, or prepare medium by adding MUG to EC broth (0.05 g/L). Several sources of MUG compound are suitable: Marcor Development Corp., Hackensack, NJ; Biosynth International, Skokie, IL; and Sigma Chemical Co., St. Louis, MO. Sterilize EC-MUG broth at 121ºC for 15 min; store up to 1 week at room temperature or refrigerate up to 1 month. Inoculate medium from LST cultures (24 h at 35ºC) of shellfish meat homogenate. Determination of fluorescence in EC-MUG broth requires control cultures, which are examined with MPN EC-MUG tubes tested for fluorescence. Use 3 control tubes, 2 of which are inoculated and grown with bacterial species: E. coli, positive culture control; K. pneumoniae, negative culture control; and uninoculated, EC medium batch control. Inoculate the 2 culture control strains when EC-MUG broths are being inoculated from positive LST broths. Incubate all 3 control tubes at 44.5 ± 0.2ºC for 24 h with the other EC-MUG broths.
2. Determine fluorescence in darkened or partially darkened room. Ordinarily, turning off all room lights is sufficient. In some instances it may also be necessary to decrease light from windows by closing blinds or using shades.
3. The UV light source can affect the determination of fluorescence. Hold a 4 watt, longwave UV lamp 5-10 cm (2-4 inches [5.1-10.2 cm]) from EC-MUG cultures and shine UV light directly on sides of tubes. Use control EC-MUG broth tubes as references for judging whether tube is positive or negative for fluorescence.
4. A few isolates (<10%) of E. coli are MUG-positive, yet are anaerogenic (gas-negative). Include all tubes determined positive for fluorescence in E. coli MPN calculations. Obtain E. coli MPN from tables by determining tube code for EC-MUG broth cultures that fluoresce under UV irradiation.

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Hydrophobic Grid Membrane Filter Methods for Rapid Enumeration of Total Coliforms, Fecal Coliforms, and E. coli

These methods are described in the APHA Compendium of Methods for the Microbiological Examination of Foods (APHA, 1992) and the AOAC Official Methods of Analysis (AOAC, 1990). Dry Rehydratable Film Method for the Enumeration of Total Coliforms and E. coli. The Petrifilm^TM Coliform Count Plate and the PetrifilmTM E. coli Count Plate Methods (AOAC^® Official Method^SM 986.33, Bacterial and Coliform Counts in Milk—Dry Rehydratable Film Methods, AOAC® Official Method^SM 989.10, Bacterial and Coliform Counts in Diary Products—Dry Rehydratable Film Methods, and AOAC® Official Method^SM 991.14, Coliform and Escherichia coli Counts in Foods—Dry Rehydratable Film Methods) are described in the APHA Standard Methods for the Examination of Dairy Products and in the Official Methods of Analysis of AOAC International.

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Enterovirulent E. coli

Contents

Isolation and Identification of Enterovirulent E. coli (EEC)

1. Equipment and materials
1. Balance, 50 g with 0.1 g sensitivity
2. Blender, Waring or equivalent, 2-speed standard model with low speed operation at 8000 rpm, with 1 liter glass or metal jar
3. Incubators, 22 ± 2ºC and 35 ± 2ºC
4. Water bath, 44.0 ± 0.2ºC
5. Petri dishes, glass, clean, unscratched, 15 x 150 mm or 20 x 150 mm
6. Pipets, Pasteur
7. Pipet filler is recommended for distributing viable cultures
8. pH test paper, range 6.0-8.0
2. Media
1. Tryptone phosphate (TP) broth (M162)
2. Brain heart infusion (BHI) broth (M24)
3. Levine's eosin-methylene blue (L-EMB) agar (M80)
4. MacConkey agar (M91)
5. Triple sugar iron (TSI) agar (M149)
6. Blood agar base (BAB) (M21)
7. Tryptone (tryptophane) broth (M164)
8. Bromcresol purple both (M26) supplemented individually with the following carbohydrates, each at 0.5% (w/v): glucose, adonitol, cellobiose, sorbitol, arabinose, mannitol, and lactose
9. 9. Urea broth (M171)
10. 10. Decarboxylase basal medium (lysine, Falkow) (M44)
11. Potassium cyanide (KCN) broth (M126)
12. MR-VP broth (M104)
13. Indole nitrite medium (tryptic nitrate) (M66)
14. Acetate agar (M3)
15. Mucate broth (M105)
16. Mucate control broth (M106)
17. Malonate broth (M92)
18. Koser's citrate broth (M72)
3. Reagents, inorganic, organic, and biological
1. Sodium bicarbonate solution, 10%, aqueous (sterile) (R70)
2. ONPG (o-nitrophenyl-b-D-galactopyranoside) disks (R53)
3. Physiological saline solution, 0.85% (sterile) (R63)
4. Kovacs' reagent (R38)
5. VP reagents (R89)
6. Oxidase test reagent (R54)
7. Nitrite detection reagents (R48)
8. Mineral oil, heavy sterile (R46)
9. Gram stain reagents (R32)
4. Enrichment of EEC

Analyze samples promptly after they arrive. Do not freeze. If product is subject to microbiological alteration, refrigerate it before examination. CAUTION: Most pathogenic biotypes lose viability at 6ºC. The approach recommended here permits qualitative determination of the presence of EEC. If enumeration is essential, consider either the dilution endpoint or MPN technique, depending on accuracy required and resources available.

Aseptically weigh 25 g sample into 225 ml BHI broth (see comment on sample size on first page of this chapter). If specimen is a large mass, aseptically cut slices 0.5 cm thick. Incubate 10 min at room temperature with periodic shaking. Decant medium into 500 ml Erlenmeyer flask and incubate 3 h at 35ºC to resuscitate damaged cells. Transfer contents to 225 ml double strength TP broth in 1 liter flask. Incubate 20 h at 44.0 ± 0.2ºC.

5. Isolation of EEC
1. Direct streak. After resuscitation, streak BHI eluate to L-EMB and MacConkey agars. This approach is effective if E. coli is present at a level of 25,000 cells/g and if it constitutes at least 10% of microfloral growth on these agars. Incubate plates 20 h at 35ºC.
2. Enrichment. After incubation at 44ºC, streak to L-EMB and MacConkey agars. Incubate 20 h at 35ºC.
3. Selection. Typical lactose-fermenting biotypes on L-EMB agar correspond to description given above in F-1, Method for Fecal Coliforms and Confirmed Test for E. coli. Typical colonies on MacConkey agar appear brick red. Lactose nonfermenting biotypes on both agars produce colorless or slightly pink colonies. Because of variety of biotypes based on pathogenic potential, 20 isolates (10 typical and 10 atypical), if possible, should be recovered for further characterization.
6. Retrieval and identification (Ewing, 1986; Kreig and Holt, 1984)

Because may species can grow in the enrichment, and non- (or slow) lactose fermenters must be considered, the standard procedure for biochemical and morphological recognition of coliforms described above is inadequate for identifying E. coli. Anaerogenic, nonmotile, slow lactose fermenters may be found in several genera of Enterobacteriaceae. Most tests are performed as described above. Thus, only new or modified reactions are discussed here.

1. Primary screening. Transfer suspicious colonies to TSI agar, BAB slant, tryptone broth, arabinose broth, and urease medium. Incubate 20 h at 35ºC. Reject H₂S-positive, urease-positive, arabinose nonfermenters, and indole-negative cultures. Test ONPG reaction. Suspend growth from TSI in 0.85% saline to give detectable turbidity. Add ONPG-impregnated disk. Incubate 6 h at 35ºC. Yellow color indicates positive reaction. Reject ONPG-negative, aerogenic cultures. Some Alkalescens-Dispar (i.e., anaerogenic Escherichia) strains are negative.
2. Secondary screening (48 h incubation at 35ºC unless otherwise specified). To identify cultures, test additional reactions shown below. Use reactions in Table 14-9 to subdivide E. coli species. Since it is not known whether these additional species are of enteropathogenic significance to humans, organisms giving a typical reactions for E. coli should be further investigated. To differentiate E. coli from Shigella, examine anaerogenic, nonmotile, slow lactose fermenters for lysine decarboxylase, mucate, and acetate reactions. E. coli strains tend to give a positive response in one or more tests. Shigella sonnei, which may grow because of favorable enrichment conditions, is anaerogenic and nonmotile; it produces a negative indole reaction and slow or nonfermentation of lactose. The biochemical-physiological characteristics of E. coli are summarized in Table 14-10.

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Serological Characterization (Ewing, 1986)

Because of complex interrelationships among somatic (O), capsular (K, mainly B type), and flagellar (H) antigens and the unknown specificity of sera available commercially, the serological analysis of E. coli is somewhat more difficult than that of other Enterobacteriaceae and should not be attempted on a routine basis. Likewise, virulence cannot be completely correlated with the presence of somatic, capsular, and colonization antigens. If serological identification of an isolate seems advisable, contact specialized laboratories, such as the Centers for Disease Control and Prevention, Atlanta, GA.

Table 14-9. Differentiation of Escherichia species (Ewing, 1986; Kreig and Holt, 1984)

Reaction	E. coli typical	E. coli inactivea	E. hermanii	E. blattae	E. fergusoni	E. vulneris
IMViC	++--	++--	++--	-+-±	-+--	-+--
KCN	-	-	+	-	-	+
Glucose, gas	+	-	+	+	+
Lactose	+	-	+/-	-	-	+
Cellobiose	-	-	+	-	+
Adonitol	-	-	-	-	+
Mannitol	+	+	+	-	+/-	+
Malonate	-	-	-	+	+	+/-

^aAnaerogenic and lactose-negative.

Table 14-10. Biochemical-physiological behavior of E. coli (Ewing, 1986).

Test	Reaction
Nitrate reduction	+
Cytochrome oxidase	-
Gram-negative, short rod	+
Fermentative (TSI)	+
Mannitol	+
Lactose	+
Malonate	-
H2S	-
Urease	-
Citrate	-
Voges-Proskauer	-
Arabinose, acid	+
KCN	-
Indole	+
Acetate	+
Adonitol	-
Cellobiose	-
Glucose, gas	+
ONPG test	+
Mucate	90% +
Lysine decarboxylase	80% +
Methyl red	+

Table 14-11 lists serogroups and serotypes associated with major pathogenic groups of E. coli. Commercial sera are not available for all these pathogen-associated serogroups (Table 14-12). Along with the symptoms in disease cases, serotyping aids in suggesting which pathogenic attributes to test for, using tests described here or in other chapters.

Table 14-11. Serogroups and serotypes of enterovirulent E. coli isolated from humans with intestinal infections (APHA, 1992)

Pathogenic type	Serogroups and serotypes
Enteropathogenic (EPEC)	O18a,18c:H7 O20a,20b:H26 O26:NMa O26:H11 O28a,28c:NM O44:H34 O55:NM O55:H6 O55:H7 O86a:NM O86a:H34 O111a,111b:NM O111a,111b:H2 O111a,111b:H12 O114:H10 O1114:H32 O119:NM O119:H6 O125a,125c:H21 O126:NM O126:H27 O127:NM O127:H9 O127:H21 O128a,128b:H2 O128a,128c:H12 O142:H6 O158:H23 O159
Enterotoxigenic (ETEC)	O6:H16 O8:H9 O11:H27 O15:H11 O20:NM O25:H42 O25:NM O27:H7 O63 O78:H11 O78:H12 O128:H7 O148:H28 O149:H10 O159:H20 O167
Enteroinvasive (EIEC)	O28a,28c:NM O112a,112c:NM O124:NM O124:H30 O124:H32 O136:NM O143:NM O144:NM O152:NM O167:H4 O167:5
Enterohemorrhagic (EHEC)	O157:H7 (and O26, O111, O113, O145, and O157:H-serogroups of-EPEC)

^aNM, nonmotile.

Table 14-12. E. coli O-serogroups recognizable with commercially available sera (APHA, 1992)

EPEC	ETEC	EIEC	A-D EHEC groupa
O26:K60 O86:K61 O18:K77 O55:K59 O119:K69 O20:K61 O111:K58 O124:K72 O20:K84 O127:K63 O125:K70 O28:K73 O126:K71 O44:K74 O128:K67 O112:K66	O6 O8 O11 O78	O28:K73 O112:K66 O124:K72 O143:Kb O114:Kc	O157:H7 O1 O2 O3 O4

^aAlkalescens-Dispar group; nonmotile anaerogenic E. coli.
^bDetected with Shigella boydii 8 antiserum.
^cDetected with Shigella dysenteriae 10 antiserum.

Contents

Tests for Enteroinvasive E. coli (EIEC)

A tissue culture (HeLa cell) test (Mehlman et al., 1977) is available to screen isolates for invasive potential before confirming invasiveness by the Sereny test. An in vitro staining technique using acridine orange to stain for intracellular (invasive) bacteria in HeLa cell monolayers is also an effective assay to determine invasiveness of pathogenic E. coli (Miliotis, 1991; Miliotis and Feng, 1993).

Sereny test for Confirmation of Invasive Potential (Sereny, 1957)

1. Equipment and materials
1. Instruments for dissecting animals
2. Animal cages
2. Media: Veal infusion broth and agar (M173)
3. Diagnostic reagents
1. Guinea pigs (less than 6 months old)
2. May-Grunwald stain (R41)
3. Giemsa stain (R30)
4. Dulbecco's phosphate-buffered saline (DPBS) (R19)
4. Procedure

1. Preparation of bacteria. With needle, inoculate 30 ml veal infusion broth, using growth from veal infusion agar slant. Incubate 18-24 h at 35ºC. Centrifuge culture (20 min at 1200 x g at 18ºC). Resuspend cells in DPBS and recentrifuge. After last centrifugation, suspend total growth from 30 ml medium in 0.3 ml DPBS.
2. Performance of test. For each culture, use 3 guinea pigs, 1-6 months old. Examine the eyes for irritation or infection before use. With Pasteur pipet, transfer drop of bacterial suspension to left eye of each animal. Apply drop of uninoculated DPBS to right eye of each animal. Gently open and close eyes to spread fluids evenly over conjunctiva. Return animals to individual cages.
5. Interpretation of data

Examine animals daily for 5 d. A positive reaction is development of conjunctivitis ulceration (keratoconjunctivitis) and opacity in eye treated with bacteria, but not in control eye. Observation by veterinarian is advisable for differentiation of keratoconjunctivitis and conjunctivitis. Confirm by demonstrating intracellular location of bacteria in corneal epithelial cells, using May-Grunwald and Giemsa stains. Recovery of same culture from viruses, fungi, chlamydia, mycoplasma, and other bacteria. Consider bacteria invasive if test is positive in at least 2 of 3 trials.

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Tests for Enterotoxigenic E. coli (ETEC)

ETEC strains produce two types of toxins: heat-labile toxin (LT) and heat-stable toxin (ST). LT can be detected by the Y-l tissue culture test (see below), and ST can be detected by the infant mouse test (see below). These toxins also can be detected by ELISA, and genes coding for them can be detected by gene probes (FDA Bacteriological Analytical Manual, Chapter 24).

Y-1 Mouse Adrenal Cell Test for E. coli LT (Sack and Sack, 1975)

The validity of this method was established in a collaborative study (Lovett and Peeler, 1984). LT, which stimulates the enzyme adenylate cyclase with the production of cyclic adenosine monophosphate, is closely related to V. cholerae enterotoxin (CT) in molecular structure and mode of action. In this assay system, LT promotes conversion of elongated fibroblast-like cells into round, refractile cells.

1. Equipment and materials
1. Microtiter tissue culture plates, 96 flat-bottom wells, sterile, plastic with lid
2. Shaker incubator at 37ºC
3. SwinnexTM filter holder, 25 mm, with 0.45 µm membrane filter
4. Microtiter pipet, 0.025 ml, sterile
5. Syringe, disposable, 1 ml; 5 ml, to accommodate Swinnex filter
6. Vertical laminar flow hood (biological contaminant hood equipped with HEPATM filters) (Bellco Glass, Vineland, NJ 08360)
7. Freezer, -70 or -20ºC.
2. Media
1. Ham's F-10 medium (with glutamine and NaHCO₃ ) (M58)
2. Y-1 adrenal cell growth medium (M180)
3. Y-1 adrenal cell maintenance medium. Same as above, except reduce FBS to 1 ml.
4. Trypticase soy-yeast extract (TSYE) broth (M157)
5. Trypticase (tryptic) soy agar (TSA) (M152)
6. Casamino acids-yeast extract-salts (CAYE) broth (M34)
3. Diagnostic reagents
1. Y-1 mouse adrenal tumor cell line, American Type Culture Collection-CCL79
2. Cholera enterotoxin. Available commercially from Schwarz-Mann, Inc., Division of Mediscience, 2 Ram Ridge Road, Spring Valley, NY 10977. Before use, dilute 1:1000 in 0.01 M phosphate-buffered saline.
3. Strains of E. coli producing LT and ST are available from laboratories actively engaged in research on enteric illness.
4. Phosphate saline solution (for Y-1 assay) (R62)
4. Procedure
1. Preparation of Y-1 cell culture. Using standard cell culture techniques, grow Y-1 cells to confluence in 75 cm² plastic culture flasks at 35ºC in CO₂ incubator. To prepare microtiter plates, wash cell monolayer with 20 ml phosphate saline solution. Remove wash water with pipet and add 5 ml 0.25% trypsin. After 1 min exposure at room temperature, remove 4.5 ml trypsin and place flask in 35ºC incubator. Observe at 5 min intervals for cell detachment. When cell sheet has detached, add 5 ml growth medium and pipet repeatedly to break clumps. Pipet this cell suspension to small beaker containing 35 ml growth medium (total volume now 40 ml). Agitate cell suspension while pipetting 0.2 ml portions of cell suspension to each well of 96-well microtiter plate. Cover plates and incubate 48 h at 35ºC in CO₂ incubator.
2. Preparation of test filtrates. Inoculate TSYE broth (5 ml in 16 x 125 mm screw-cap tube) and TSA slant from each suspected E. coli colony on L-EMB agar. Examine at least 5 colonies from each subsample. Alternatively, inoculate TSYE broth from agar slant. Incubate both TSYE and TSA for 24 h at 37ºC. Transfer 0.1 ml of each TSYE culture 10 ml CAYE broth in 50 ml Erlenmeyer flask. Incubate CAYE and TSYE cultures 24 h at 34ºC in shaker incubator at 250 rpm. If growth occurs in CAYE, centrifuge culture 30 min at 1200 x g. If growth in CAYE is poor, substitute TSYE culture and treat as described for CAYE culture. Filter supernatant through 0.45 µm membrane. Heat 1 ml of each supernatant 30 min at 80ºC. Store filtrates at 4ºC.
3. Assay. Take microtiter plates prepare 48 h earlier and replace growth medium with maintenance medium. Add 0.025 ml each of heated and unheated filtrate to 4 wells of microtiter plate. Add 0.025 ml of cholera enterotoxin (CT) preparation (1 ng CT per ml) to 4 wells as positive control. Simultaneously, inoculate 4 wells with culture filtrates from known LT+ and LT- control cultures. Leave some wells uninoculated as medium controls. Incubate finished plates 30 min at 35ºC in CO₂ incubator. After 39 min remove medium and replace with fresh maintenance medium. Re-incubate 18 h at 35ºC in CO₂ incubator.
4. Interpretation. Examine all control inoculations first for proper response. A positive response is 50% or more rounded cells by visual estimate. Negative and medium controls should show 10% or less rounding. Results of this test can be confirmed only in research centers with available resources. Confirmation models are rabbit ligated ileal loop and anti-CT or anti-LT serum neutralization in Y-1 cells. To confirm identity of LT-producing cultures as E. coli, use the approach recommended under Retrieval and Identification, F, above.
Infant Mouse Test for E. coli ST (Dean et al., 1972)

The validity of this method was demonstrated in a collaborative study (Lovett and Peeler, 1984).

1. Equipment and materials
1. Balance, accurate to 0.01 g
2. Forceps, dissecting, sharp point, 4-1/2 inch (11.4 cm)
3. Needle, 27 gauge
4. Needle, animal feeding, 24 gauge, 1 inch (2.5 cm), straight
5. Tuberculin syringe, 1 ml, disposable
6. Scissors, dissecting, sharp point, 4-1/2 inch (11.4 cm)
7. Weighing boats
2. Media and reagents
1. Evans blue, 2% solution (R24)
2. Swiss albino mice, 3-5 d old
3. Procedure
1. Preparation of host. Suckling mice are commercially available from suppliers such as Charles River Breeding Laboratories, Wilmington, MA 01887.
2. Preparation of bacterial filtrates. See Y-1, Mouse Adrenal Cell Test, D-2, above. Add 2 drops 2% Evans blue to 1 ml sterile CAYE or TSYE culture broth filtrates. Use heated portion for ST assay.
3. Assay. Inject 0.1 ml culture filtrate intragastrically. Use 4 mice per filtrate. Include known ST+ and ST- controls. Keep mice 3 h at room temperature. Reject all mice not showing blue dye concentrated in the stomach or showing dye in peritoneal cavity. Sacrifice mice by CO₂ inhalation. Open abdomen and remove intestinal tract, excluding stomach and liver. Pool remaining carcasses in another tared weighing boat. Weigh both pools and compute ratio of intestinal weight to body weight.
4. Interpretation. A ratio of 0.083 or greater is considered positive; a ratio of 0.074 or less is considered negative. Filtrates giving ratios of 0.075-0.082 should be retested. Confirm identity of ST-producing cultures as E. coli by using the approach recommended under Retrieval and Identification, F, above.
Colonization Test

Virulence prerequisites for enterotoxigenic strains of E. coli include the ability to attach to the jejunal lining, to proliferate in situ, and to elaborate one or more toxins. Host specificity is manifested by possession of unique colonization factors, including antigens and lectins. At least 3 factors have been elucidated in strains of human significance: CFA I, CFA II, and 8755. Several types of mammalian cells have been proposed to show colonization: buccal, FLOW 11000, and HeLa.

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Isolation Methods for Enterohemorrhagic E. coli O157:H7

Enterohemorrhagic E. coli was first recognized as an important food-borne pathogen in 1982 (Riley et al., 1983). The organism causes hemorrhagic colitis (Riley et al., 1983), which has characteristic symptoms of bloody diarrhea and abdominal cramps; however, it may progress into hemolytic uremic syndrome (Karmali et al., 1983), a more severe complication that can result in kidney failure and death. Although there are many serotypes of EHEC, serotype O157:H7 has been most frequently implicated in food-borne diseases. In 1993, isolates of O157:H7 serotype caused numerous food-borne outbreaks, including a major outbreak in Washington state that infected about 500 persons. Most outbreaks of O157:H7 infections are caused by the consumption of contaminated ground beef; however, raw milk and other foods have also been implicated.

Several microbiological methods can be used to isolate E. coli O157:H7 from foods. Unlike typical E. coli, isolates of O157:H7 do not ferment sorbitol and are negative with the MUG assay; therefore, these criteria are commonly used for selective isolation. Sorbitol-MacConkey agar has been used extensively to isolate this organism from clinical specimens. Hemorrhagic colitis agar, a selective and differential medium, is used in a direct plating method to isolate O157:H7 from foods. A third procedure uses Sorbitol-MacConkey medium containing potassium tellurite and Cefixime. It includes an enrichment step and is a new method developed as result of the recent food-borne outbreaks. This procedure has been highly effective in isolating O157:H7 from a variety of commonly contaminated foods (Weagant et al., 1995).

1. Isolation with sorbitol-MacConkey (SMAC) agar (March and Ratnam, 1986)

Homogenize 10 g of sample in 90 ml peptone water (R56) diluent. Prepare serial tenfold dilutions in peptone water diluent in accordance with anticipated level of contamination. Pipet 0.1 ml of each dilution in duplicate onto dried surface of SMAC (M139) agar and spread evenly across each plate. Incubate plates at 35ºC and read after 18 h. Sorbitol-negative colonies are pale compared to bright pink sorbitol-positive colonies produced by E. coli and other enterics. As further confirmation, sorbitol-negative colonies can be tested for GUD activity by spotting culture on HC agar (with MUG; M62) and used to select for MUG-negative colonies (Doyle and Schoeni, 1987; Scotland et al., 1991). For definitive identification of O157:H7 serotype, test sorbitol-negative, MUG-negative colonies for agglutination with O157 and H7 antisera. CAUTION: high levels of contaminating coliforms in the sample may mask the presence of O157:H7 strains in this medium. Furthermore, isolates of Escherichia hermanii and other enterics may show similar biochemical phenotypes on SMAC and along with Citrobacter freundii may also agglutinate O157 antiserum; therefore, they may cause false-positive identifications (Bettelheim et al., 1993; Lior and Borczyk, 1987).

2. Isolation with HC agar (hemorrhagic colitis [strains of E. coli])(Szabo et al., 1986)

Strains of O157:H7 may also be isolated from foods using the HC agar (M62). Because this medium contains sorbitol and the MUG reagent, distinguishing phenotypes based on reactions to these reagents can be determined simultaneously in the same medium. The fluorescence from the MUG reaction, however, is diffusible and may spread throughout the entire plate during extended incubation. A colorimetric substrate BCIG may also be used in HC agar instead of MUG (Okrend et al., 1990). If low levels of O157:H7 are suspected, the food may be enriched first in modified trypticase soy broth (mTSB) (M156) containing novobiocin before it is plated on selective medium. Sorbitol-negative, MUG-negative colonies isolated on HC medium must be confirmed serologically with O157 and H7 antisera. The colonies may also be transferred to membranes for colony hybridization analysis for the presence of Shiga-like toxin genes. The procedures for isolation using HC agar and for enrichment in mTSB are described in Chapter 24, Identification of Food-borne Bacterial Pathogens by Gene Probes (FDA Bacteriological Analytical Manual, 8th Edition, 1998). CAUTION: Normal flora in foods may also proliferate in the mTSB enrichment medium and cause overgrowth or masking of O157:H7 colonies on the HC agar medium.

3. Isolation with Tellurite-Cefixime-Sorbitol MacConkey (TC SMAC) agar

An enrichment/isolation procedure using the TC SMAC medium was recently introduced for detecting O157:H7 in foods. Both the enrichment and the selective media contain several antibiotics which effectively suppress the growth of normal flora. Comparative analysis of the TC SMAC procedure with the HC agar method using a variety of naturally contaminated and seeded foods showed that the TC SMAC procedure was superior to the HC agar method in the recovery of O157:H7 bacteria (Weagant et al., 1995). CAUTION: Although most E. coli are sorbitol fermenters, about 6% of the isolates will not ferment sorbitol. These atypical strains may be found in foods and will appear identical to O157:H7 colonies on the TC SMAC agar. The inclusion of MUG assay in the analysis procedure should distinguish these atypical E. coli strains from the O157:H7 isolates. For additional information on the TC SMAC procedure, contact Steve Weagant, FDA, Bothell, WA (206) 483-4874.

1. Media Preparation

EHEC Enrichment Broth (EEB) - same as mTSB (M156) but with the following filter-sterilized antibiotics added after autoclaving:

Cefixime 0.05 mg/liter*
Cefsulodin 10.00 mg/liter
Vancomycin 8.00 mg/liter

TC SMAC - Sorbitol-MacConkey agar (M139) with the following filter- sterilized additives after autoclaving and tempering:

Potassium tellurite 2.50 mg/liter
Cefixime 0.05 mg/liter*
*Available from Dynal Inc., Lake Success, NY (800) 638-9416.

2. Enrichment
1. Weigh 25 g of food into 225 ml of EEB, blend or stomach briefly as necessary.
2. Incubate at 37ºC with shaking for 6 h; then after performing step "a" below, under "3. Isolation," reincubate the enrichment tube overnight.
3. Isolation
1. Spread plate 0.1 ml of 6-h EEB homogenate to a TC SMAC agar plate and streak one loopful to a second TC SMAC plate.
2. Incubate agar plates at 37ºC overnight.
3. Sorbitol-fermenting normal flora bacteria appear as pink to red colonies. Typical O157:H7 colonies are neutral/gray with a smoky center and 1-2 mm in diameter. Pick several typical O157:H7 colonies from TC SMAC onto TSAYE (M153) slants and incubate at 37ºC overnight.
4. If plates do not show typical colonies, repeat steps 1-3 again, but from the 24 h enrichment tube.
4. Confirmation
1. Screen isolates by spotting growth from TSAYE slants to a filter wetted with Kovac's reagent (spot indole test). EHEC isolates are indole-positive.
2. If indole-positive, test for O157 antigen with commercial O157 antiserum. Both Prolex E. coli O157 Latex Test Reagent kit (Pro-Lab Diagnostics, Round Rock, TX, 800-522-7744) and RIM E. coli O157:H7 Latex Test (Remel, Lenexa, KS, 800-255-6730) give satisfactory results. From the TSAYE slant, also run an API or VITEK assay to identify the isolates as E. coli.
If indole-negative, DO NOT perform latex test or further tests for Shiga-like toxin (SLT) production.
5. Results
1. If both the spot indole test and latex test kit results are positive, then confirm for the presence of SLT-I and II genes by colony hybridization (see FDA Bacteriological Analytical Manual, Chapter 24) or by polymerase chain reaction (see FDA Laboratory Information Bulletin 3811, Sept., 1993).
2. If the spot indole test is positive, but the latex test kit result is negative, confirmation for SLT production is not required.
Optional. Additional selective enrichment by use of immunomagnetic separation has been found useful for some samples (Weagant et al., 1995). Anti-O157 immunomagnetic beads are available (Dynabeads; Dynal). Immunomagnetic separation is performed on 1 ml of EEB after 6 h of incubation, following manufacturer's instructions. Beads are spread plated on TC SMAC and treated as outlined above. A partial list of commercially available rapid methods for detecting O157:H7 is given at the end of this chapter.
4. Identification of serotype O157:H7 isolates using an oligonucleotide probe

As an alternative to serological typing, isolates of serotype O157:H7 may also be identified by using the serotype-specific DNA probe, PF-27. This 18-base oligonucleotide probe, developed at CFSAN, is directed at a unique region of the uidA gene in the O157:H7 isolate. Colony hybridization analysis of 280 bacterial isolates, including E. coli, several pathogenic enteric species, other Shiga-like toxin-producing EHEC and 42 isolates of O157:H7 implicated in a recent food-borne outbreak, showed that PF-27 is highly specific only for the isolates of O157:H7 serotype (Feng, 1993). For more information on PF-27, contact Peter Feng, CFSAN, FDA, Washington, DC. Phone (202) 205-4518. For protocols on using the probe, see FDA Bacteriological Analytical Manual, Chapter 24.

Contents

Tests for Toxins of Enterohemorrhagic E. coli (EHEC)

EHEC isolates produce several toxins, but only a few have been well characterized. The major toxin is virtually identical to the Shiga toxin of Shigella dysenteriae type 1 and hence named Shiga-like toxin I (SLT-I). Another toxin, although only 60% homologous to SLT-I has been designated SLT-II. Since both toxins are cytotoxic to HeLa and Vero tissue culture cells, they are also known as verotoxin (VT) I and II. The toxins produced by EHEC are detected by tissue culture assays. However, DNA probe and polymerase chain reaction assays have also been developed to detect the presence of SLT gene in EHEC isolates.

1. Tissue culture assay for Shiga-like toxins

Equipment and materials

1. CO₂ incubator maintained at 36ºC
2. Sterile plastic plates containing 16 mm diameter wells
3. Cell counting chamber
4. Shaker incubator maintained at 37ºC
5. Centrifuge
6. Sterile 0.45 µm membrane filters
7. Inverted stage microscope

Media and reagents

1. Cell growth medium (M36)
2. Dulbecco's phosphate-buffered saline (DPBS), pH 7.2 (R19)
3. Fetal bovine serum
4. Milk serum, 2%
5. Gentamicin sulfate solution (M57)
6. Eagle's minimal essential medium MEME-L15 (M46 and M73), mixed in equal proportions

Preparation of cytotoxin-VT

Inoculate culture into 20 ml trypticase soy broth (TSB) in 250 ml Erlenmeyer flask and incubate with agitation at 37ºC for 20-24 h. Centrifuge culture at 7000 x g for 30 min to sediment bacteria. Filter supernatant through 0.45 µm membrane to remove residual bacteria. Store at 4ºC. Dilute filtrate 1:5 in DPBS, pH 7.0, before use.

Preparation of Vero monolayers

Maintain Vero culture in MEME-L15 medium containing 2% milk serum, prepared as follows: Add dry milk to double distilled water to final concentration of 10%. Add concentrate of gentamicin sulfate to level of 50 µg/ml. Stir 1 h on magnetic stirrer at room temperature. With stirring, adjust pH to 4.5 with 1 N HCl. Filter through cheesecloth. Clarify by centrifugation at 2500 rpm for 15 min. Filter again through cheesecloth. Sterilize by filtration through 0.22 µm membrane. Add milk serum to give 2% concentration by volume in MEME-L15 medium. Incubate culture in 5% CO₂ incubator held at 36ºC for 72 h. Examine culture for purity and appearance of cells. If cells are normal and not contaminated, treat with trypsin to remove monolayer. Suspend cells to density of 10 per ml in growth medium. With gentle agitation transfer 0.5 ml portions to 16 mm wells in sterile plastic dishes. Incubate 3-4 d at 36ºC in CO₂ incubator. Examine for purity and appearance of cells. Remove spent medium and replace with 0.5 ml fresh medium.

1. Toxicity test. Add 0.05 ml diluted culture filtrate to well. For control, dilute TSB 1:5 in DPBS. Add 0.05 ml to well. Incubate 4 d at 36ºC in CO₂ incubator.
2. Examine daily for cytopathic effect, i.e., rounding and shriveling of cells, including detachment. Potent preparations affect 50% of the cells in monolayer. There should be progressive increase in toxicity with increased incubation. Cytotoxicity in contrast to the cytotoxic effect of LT is not reversible if medium is changed and monolayers are re-incubated.
1. Detection of Shiga-like toxin genes in EHEC using DNA probes

The toxigenic potential of EHEC isolates may be determined by colony hybridization using oligonucleotide DNA probes that are specific for the genes that encode for SLT-I and SLT-II toxins. However, numerous other serotypes of EHEC also produce SLT. Therefore, serological or other assays are still required to identify isolates of O157:H7 serotype. For more information on these SLT probes, contact William L. Payne, CFSAN, FDA, Washington, DC, phone (202) 205-4361. For protocols on using the probes, see FDA Bacteriological Analytical Manual, Chapter 24.

2. Detection of Shiga-like toxin genes in EHEC using polymerase chain reaction (PCR) assays

In addition to probes, two different sets of PCR primers specific for SLT genes have been developed at FDA, Bothell, WA, and at CFSAN. These primers can be used to determine potential toxigenicity of EHEC isolates. Both sets of primers have been tested extensively on reference and outbreak strains of EHEC and confirmed to be very effective. For more information on these SLT primers and for PCR methods and protocols see Weagant, S.D., J.L. Bryant, and K.G. Jinneman. 1995. An improved rapid technique for isolation of Escherichia coli O157:H7 from foods. J. Food Prot. 58:7-12.; refer to FDA publication LIB 3811, Sept., 1993; or contact Walter E. Hill, SPRC, FDA, Bothell, WA, phone (206) 402-3176; or William L. Payne, CFSAN, FDA, Washington, DC, phone (202) 205-4361. Recently, a multiplex PCR procedure which can simultaneously identify the O157:H7 serotype as well as the toxin type has been developed at CFSAN and is currently under evaluation. For more information, contact Peter Feng, CFSAN, FDA, Washington, DC, phone (202) 205-4518.

CAUTION: Unlike the tissue culture assays which detect the toxins, reactivity of an EHEC isolate with SLT probes or with PCR primers merely indicates that genetic sequences for the toxins are present in that particular isolate. It does not, however, indicate that the toxins are actually produced.

Contents

Tests for Enteropathogenic E. coli (EPEC)

Enterovirulent E. coli strains that do not type as EHEC, EIEC, or ETEC strains are probably enteropathogenic E. coli (EPEC) strains, e.g., classical infantile diarrhea strains. Although there are no specific tests for EPEC strains, some methods, which are still experimental, are available (see the review by Doyle and Padhye (1989). Confirmation of a putative EPEC strain involves serogrouping and serotyping (Table 14-12) and consideration of case symptomology. Further complexity concerns the fact that some EPEC strains behave like EHEC strains (see Tables 14-1 and 14-11).

Contents

Other analytical procedures

• Bacteria and coliform counts in dairy products: Dry rehydratable film methods (AOAC, 1995a)
• Bacteria and coliform counts in milk: Dry rehydratable film methods (AOAC, 1995b)
• Coliform and Escherichia coli counts in foods: Dry rehydratable film methods (AOAC, 1995c)
• Coliforms in dairy products: Pectin gel method (AOAC, 1995d)
• Confirmed total coliform and E. coli in all foods: Substrate supporting disc method (AOAC, 1995e)
• Detection of Escherichia coli producing heat-labile enterotoxin: DNA colony hybridization method (AOAC, 1995f)
• Enterotoxigenic Escherichia coli: DNA colony hybridization method using synthetic oligodeoxyribonucleotides and paper filters (AOAC, 1995g)
• Escherichia coli enterotoxins: Mouse adrenal cell and suckling mouse assays (AOAC, 1995h)
• Escherichia coli in chilled or frozen foods: Fluorogenic assay for glucuronidase (AOAC, 1995i)
• Fecal coliforms in shellfish growing waters: Medium A-1 method (AOAC, 1995j)
• Invasiveness of mammalian cells by Escherichia coli: Microbiological method (AOAC, 1995k)
• Total coliform and Escherichia coli counts in foods: Hydrophobic grid membrane filter/MUG method (AOAC, 1995l)
• Total coliforms and Escherichia coli in water: Defined substrate technology method (AOAC, 1995m)
• Total coliforms, fecal coliforms, and Escherichia coli in foods: Hydrophobic grid membrane filter method (AOAC, 1995n)

Contents

Commercial Test Products

Table 14-13. Commercial test products for coliforms.

Test Kit	Analytical Technique	Approx. Total Test Time1	Supplier
3MTM PetrifilmTME. coli Count Plate2  [Used to enumerate E. coli and coliform bacteria]	An indicator of glucuronidase activity Dry rehydratable film method	24-48 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
ColiComplete2 [Confirms total coliforms and E. coli]	Substrate supporting disc	48 h for coliforms 30 h for E. coli	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 800/245-0113; 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com
ColiConfirm [Confirms presence of coliforms]	Substrate supporting disc	48 h	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 800/245-0113; 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com
Coliforms2	Culture	24 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
ColiGel  [For coliforms and E. coli]	Selective media, color indicator for galactosidase production (coliforms), UV fluorescence (E. coli)	28 h (coliforms)  48 h (E. coli)	Charm Sciences, Inc.  36 Franklin St.  Malden, MA  02148-4120  Phone: 781/322-1523  E-mail: charm1@charm.com  Web: www.charm.com
Colilert®2   [For coliforms and E. coil in water samples; presence/absence)	Selective media with color indicator (UV light for E. coli)	24 h	IDEXX Laboratories, Inc.  Contact: Greg Getchell  One Idexx Dr.  Westbrook, ME  04092  Phone: 207/856-0580  E-mail: greg-getchell@idexx.com  Web: www.idexx.com/fed/home/start.asp
E*Colite  [For presence/absence of coliforms and E. coli in water]	Selective media, color indicator for galactosidase production (coliforms) UV fluorescence (E. coli)	28 h (coliforms)  48 h (E. coli)	Charm Sciences, Inc.  36 Franklin St.  Malden, MA  02148-4120  Phone: 781/322-1523  E-mail: charm1@charm.com  Web: www.charm.com
ISO-GRID Method for Confirmed Coliform Count using LMG Agar2	Membrane filtration with selective and differential culture medium based on lactose fermentation	24 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
PathoGelTM  [A single assay that selectively grows, detects and quantitates coliform, E. coli and hydrogen sulfide producing Enterobacteriaceae]	Selective media, color indicator for galactosidase production (coliforms) UV fluorescence (E. coli), black precipitate (H2S production)	28 h (coliforms)  28 h (fecal coliforms)  48 h (E. coli)  48 h (H2S production)	Charm Sciences, Inc.  36 Franklin St.  Malden, MA  02148-4120  Phone: 781/322-1523  E-mail: charm1@charm.com  Web: www.charm.com
RCTTM Rapid Coliform Test	Redox reaction and color change	14 h	Applied Research Institute  Contact: Trevor R. Hopkins 3N Simm Ln.  Newton, CT 06470  Phone: 888/324-7900 E-mail: sales@arillc.com Web: www.arillc.com
SimPlateTM for Total Coliform and E. coli	MPN plate with selective media (UV light for E. coli)	24 h	IDEXX Laboratories, Inc.  Contact: Greg Getchell  One Idexx Dr.  Westbrook, ME  04092  Phone: 800/321-0207; 207/856-0496 E-mail: greg-getchell@idexx.com  Web: www.idexx.com/fed/home/start.asp
Total coliform/E. coli (estimated)	Selective media with color indicator that changes based on approximate coliform count (UV light for E. coli)	30 min for 108  10 h for 101	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

¹Includes enrichment
²AOAC Approved

Table 14-14. Commercial test products for E. coli

Test Kit	Analytical Technique	Approx. Total Test Time1	Supplier
3MTM PetrifilmTME. coli Count Plate2  [Used to enumerate E. coli and coliform bacteria]	An indicator of glucuronidase activity Dry rehydratable film method	24-48 h	3M Microbiology Products  3M Center, Building 275-5W-05  St. Paul, MN  55144-1000  Phone: 651/737-6501; 800/228-3957  E-mail: innovation@mmm.com
CHECK 3 E. coli	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
Coli ST EIA:ETC STA (Denka Seiken Co. Ltd).	EIA	18½ h	Oxoid, Inc.  Contact: Jim Bell  217 Colonnade Rd.  Nepean, Ontario K2E 7K3  Canada  Phone: 613/226-1318  E-mail: jbell@oxoid.ca
ColiComplete2 [Confirms total coliforms and E. coli]	Substrate supporting disc	30 h for E. coli 48 h for coliforms	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 800/245-0113; 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com
ColiGel  [For coliforms and E. coli)	Selective media, color indicator for galactosidase production (coliforms), UV fluorescence (E. coli)	28 h (coliforms)  48 h (E. coli)	Charm Sciences, Inc.  36 Franklin St.  Malden, MA  02148-4120  Phone: 781/322-1523  E-mail: charm1@charm.com  Web: www.charm.com
Colilert®2   [For coliforms and E. coil in water samples; presence/absence]	Selective media with color indicator (UV light for E. coli)	24 h	IDEXX Laboratories, Inc.  Contact: Greg Getchell  One Idexx Dr.  Westbrook, ME  04092  Phone: 800/321-0207; 207/856-0496  E-mail: greg-getchell@idexx.com  Web: www.idexx.com/fed/home/start.asp
E. coli2	Culture	24 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
E. coli Detection Test	Antibody-dye conjugate complex	6 h	Morningstar Diagnostics, Inc.  1832 Centre Point Cir., Ste. 103  Naperville, IL  60563  Phone: 630/577-0700  E-mail: information@mstarusa.com  web: www.mstarusa.com
E. coli Shiga-like toxins I & II	Antibody-dye conjugate complex	18-24 h	Morningstar Diagnostics, Inc.  1832 Centre Point Cir., Ste. 103  Naperville, IL  60563  Phone: 630/577-0700  E-mail: information@mstarusa.com  web: www.mstarusa.com
E*Colite  [For presence/absence of coliforms and E. coli in water]	Selective media, color indicator for galactosidase production (coliforms) UV fluorescence (E. coli)	28 h (coliforms)  48 h (E. coli)	Charm Sciences, Inc.  36 Franklin St.  Malden, MA  02148-4120  Phone: 781/322-1523  E-mail: charm1@charm.com  Web: www.charm.com
GENE-TRAK E. coli Assay	Nucleic acid hybridization	28 h	GENE-TRAK Systems  Contact: Linda Dragone  94 South St.  Hopkinton, MA  01748  Phone: 508/435-7400
ISO-GRID Method for Confirmed E. coli Count using LMG Agar and BMA Agar2	Membrane filtration with selective and differential culture medium based on lactose fermentation and b -glucuronidase	24 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
PathoGelTM  [A single assay that selectively grows, detects and quantitates coliform, E. coli and hydrogen sulfide producing Enterobacteriaceae]	Selective media, color indicator for galactosidase production (coliforms) UV fluorescence (E. coli), black precipitate (H2S production)	28 h (coliforms)  28 h (fecal coliforms)  48 h (E. coli)  48 h (H2S production)	Charm Sciences, Inc.  36 Franklin St.  Malden, MA  02148-4120  Phone: 781/322-1523  E-mail: charm1@charm.com  Web: www.charm.com
SimPlateTM for Total Coliform and E. coli	MPN plate with selective media (UV light for E. coli)	24 h	IDEXX Laboratories, Inc.  Contact: Greg Getchell  One Idexx Dr.  Westbrook, ME  04092  Phone: 800/321-0207; 207/856-0496  E-mail: greg-getchell@idexx.com  Web: www.idexx.com/fed/home/start.asp
Total coliform/E. coli (estimated)	Selective media with color indicator that changes based on approximate coliform count (UV light for E. coli)	30 min for 108  10 h for 101	Contamination Sciences LLC  Contact: Robert Steinhauser  4230 East Towne Blvd., Ste. 191  Madison, WI  53704  Phone: 608/825-6125  E-mail: bsteinha@contam-sci.com Web: www.contam-sci.com
VET-RPLA	Reversed passive latex agglutination		Oxoid, Inc.  Contact: Jim Bell  217 Colonnade Rd.  Nepean, Ontario K2E 7K3  Canada  Phone: 613/226-1318  E-mail: jbell@oxoid.ca

¹Includes enrichment
²AOAC Approved

Table 14-15. Commercial test products for E. coli O157:H7

Test Kit	Analytical Technique	Approx. Total Test Time1	Supplier
Assurance EHEC EIA [Used to detect E. coli O157]	Enzyme immunoassay	19½ h	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 800/245-0113; 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com
BAX® for Screening/E. coli O157:H7	Polymerase chain reaction	21-24 h	Qualicon, Inc.  P.O. Box 80357  Wilmington, DE  19880-0357  Phone: 800/863-6842; 302/695-9400  E-mail: info@qualicon.com  Web: www.qualicon.com
Dynabeads® anti-E. coli 0157	Immunomagnetic separation	24 h	Dynal Inc.  Contact: Technical Service  5 Delaware Dr.  Lake Success, NY  1042  Phone: 516/326-3270  E-mail: techserv@dynalusa.attmail.com  Web: www.dynal.no
E. coli O157:H72	Culture	24 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
E. coli O157 Antigen Detection Test	Antibody-dye conjugate complex	7 h	Morningstar Diagnostics, Inc.  1832 Centre Point Cir., Ste. 103  Naperville, IL  60563  Phone: 630/577-0700  E-mail: information@mstarusa.com  web: www.mstarusa.com
E. coli O157 Antigen Detection Test	Antibody-dye conjugate complex	18 h	Morningstar Diagnostics, Inc.  1832 Centre Point Cir., Ste. 103  Naperville, IL  60563  Phone: 630/577-0700  E-mail: information@mstarusa.com  web: www.mstarusa.com
E. coli 0157 Latex Test			Oxoid, Inc.  Contact: Jim Bell  217 Colonnade Rd.  Nepean, Ontario K2E 7K3  Canada  Phone: 613/226-1318  E-mail: jbell@oxoid.ca
eclipseTM  E. coli O157:H7 Rapid Color Change Test [For identifying E. coli 0157:H7 in food products, ingredients and water]	Immunoassay	8.33 or 20.33 h	Eichrom Technologies, Inc.  Contact: Cara Tomasek 8205 South Cass Ave., Suite 111  Darien, IL 60561  Phone: 630/963-0320 E-mail: info@eichrom.com Web: www.eichrom.com
EHEC-TekTM  for E. coli O157:H7 in Foods	Magnetic capture and concentration/ELISA	24 h	Organon Teknika Corp.  100 Akzo Ave.  Durham, NC 27712  Phone: 800/654-0331; 919/620-2000 E-mail: casey@orgtek.com
EIAFoss E. coli 0157	Combination ELISA and Immuno Magnetic Separation	22-24 h	Foss North America, Inc.  7682 Executive Dr.  Eden Prairie, MN  55344  Phone: 612/974-9892  E-mail: sales@fossnorthamerica.com  Web: www.fossnorthamerica.com
ImmunoCard Stat! E. coli O157:H7	Immunoassay using colloidal gold	8-24 h	Meridian Diagnostics Technical Support 3471 River Hills Dr. Cincinnati, OH  45244 Phone: 513/271-3700 E-mail: techsupport@meridiandiagnostics.com
ISO-GRID Method for E. coli O157:H7 Enumeration using SD-39 Agar	Membrane filtration with selective and differential culture medium based on lysine decarboxylase, sorbitol fermentation and b-glucuronidase	24-48 h (24 h for presumptive enumeration and 24 h additional to confirm presumptive positive results)	QA Life Sciences, Inc.  6645 Nancy Ridge Dr.  San Diego, CA  92121  Phone: 800/788-4446; 858/622-0560  E-mail: bugsy@qalife.com
NOW [for rapid detection of E. coli O157:H7]	Antibody	9 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
PATH-STICK One Step Rapid E. coli O157 Test	Immunochromatography	16-24 h	Celsis, Inc.  Contact: Susan Moffa  165 Fieldcrest Ave.  Edison, NJ 08837  Phone: 800/222-8260; 732/346-5100  E-mail: smoffa@celsis.com  Web: www.celsis.com
Premier E. coli O157	ELISA	19 h	Meridian Diagnostics Technical Support 3471 River Hills Dr. Cincinnati, OH  45244 Phone: 513/271-3700 E-mail: techsupport@meridiandiagnostics.com
Probelia PCR System [Used to detect E. coli O157:H7]	Polymerase chain reaction	24 h	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 800/245-0113; 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com
Reveal® Microbial Screening Test for E. coli O157:H7	Sandwich ELISA	8 h	Neogen Corporation  620 Lesher Pl.  Lansing, MI 48912  Phone: 517/372-9200  E-mail: NeogenCorp@aol.com  Web: www.neogen.com
RIDASCREEN Verotoxin [Used to detect E. coli VT1 and VT2]	ELISA	10-18 h	Lionheart Diagnostics  Contact: Thomas Grace  Box 998, Highland Park  Winooski, VT  05404-0998  Phone: 802/655-4740  E-mail: RbioST@voyager.net
TECRA E. coli O157 VIA [Used to detect E. coli 0157 including E. coli O157:H7]	ELISA	20 h	International BioProducts  Contact: Mike Yeager 14780 NE 95th St.  Redmond, WA  98052  Phone: 800/729-7611 425/883-1349  E-mail: myeager@intlbioproducts.com Web: www.intlbioproducts.com
Transia Card E. coli O157	Immunoassay reaction	24½ h	GENE-TRAK Systems  Contact: Linda Dragone  94 South St.  Hopkinton, MA  01748  Phone: 508/435-7400
VIDAS ECO [Used to detect E. coli O157]	Enzyme linked fluorescent assay	24 h	bioMérieux Inc. Contact: bioMérieux Industry 595 Anglum Rd.  Hazelwood, MO  63042  Phone: 314/731-8500  E-mail: usa@na.biomerieux.com Web: www.biomerieux.com
VIDAS ICE [Used to detect E. coli O157]	Immunoconcentration	24 h	bioMérieux Inc. Contact: bioMérieux Industry 595 Anglum Rd.  Hazelwood, MO  63042  Phone: 314/731-8500  E-mail: usa@na.biomerieux.com Web: www.biomerieux.com
VIP for EHEC2 [Used to detect E. coli O157:H7]	Lateral Flow Immunoassay	18 h	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 800/245-0113; 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com
VTEC-RPLA TD960   [Used to detect E. coli verotoxins VT1 and VT2]	Reversed passive latex agglutination	48 h (bacterial culture)	Oxoid, Inc.  Contact: Jim Bell  217 Colonnade Rd.  Nepean, Ontario K2E 7K3  Canada  Phone: 613/226-1318  E-mail: jbell@oxoid.ca

¹Includes enrichment
²AOAC Approved

Table 14-16. Commercial test products for E. coli enterotoxin

Test Kit	Analytical Technique	Approx. Total Test Time1	Supplier
VET-RPLA TD920  [Used to identify E. coli heat-labile enterotoxin]	Reversed passive latex agglutination	24 h (bacterial culture)	Oxoid, Inc.  Contact: Jim Bell  217 Colonnade Rd.  Nepean, Ontario K2E 7K3  Canada  Phone: 613/226-1318  E-mail: jbell@oxoid.ca

¹Includes enrichment
²AOAC Approved

Contents

References

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