Chapter Contents

The methods in this chapter can be used to test for sanitary index bacteria (the coliforms, fecal coliforms, and Escherichia coli as a coliform) and for enterovirulent E. coli (EEC) strains, of which there are several major subgroups:

Enterotoxigenic E. coli (ETEC)--gastroenteritis, traveler's diarrhea

Enteropathogenic E. coli (EPEC)--infant diarrhea

Enterohemorrhagic E. coli (EHEC)--hemorrhagic colitis

Enteroinvasive E. coli--(EIEC)--bacillary dysentery

Enteroadherent E. coli (EAEC)--newly added category

E. coli and the 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 35C (coliforms) and 45.5C (fecal coliforms and E. coli as a coliform). With all shellfish isolates, an incubation temperature of 44.5C (rather than 45.5C) is used (1,2). Some E. coli strains may be only weakly lactose-positive (delayed lactose fermentation) or even lactose-negative (5,17). Some properties of enterovirulent E. coli (EEC) subgroups are presented in Table 1, but for a full discussion, consult one of the recent reviews (13,25). Enteroadherent E. coli (EAEC), another proposed subgroup of EEC, is not fully characterized and so is not considered here. Some methods used herein correspond to AOAC's Official Methods of Analysis (6).

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 1 and current FDA field instructions on sampling and composing before proceeding with E. coli and coliform analyses.

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Conventional Method for Determining Coliforms and E. coli

1. Equipment and materials

   1. Covered water bath, with circulating system to maintain temperature of 45.5 +/- 0.2C. Water level should be above the medium in immersed tubes.

   2. Immersion-type thermometer, 1-55°C, about 55 cm long, with 0.1 subdivisions, certified by National Institute of Standards and Technology (NIST), or equivalent

   3. Incubator, 35 +/- 1°C

   4. Balance with capacity of 2 kg and sensitivity of 0.1 g

   5. Blender and blender jar (see Chapter 1)

   6. Sterile graduated pipets, 1.0 and 10.0 ml

   7. Sterile utensils for sample handling (see Chapter 1)

   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 2-5°C. 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 35°C. 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.

4. 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 35°C. Examine for gas production and record. Calculate most probable number (MPN) (see Appendix 2) of coliforms based on proportion of confirmed gassing LST tubes for 3 consecutive dilutions.

5. 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 (22). If autoclave is used to sterilize VRBA, heat small aliquots of about 100 ml no longer than 5 min at 121°C. 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 48°C 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 (1). For conventional method, pour 10 ml VRBA tempered to 48°C 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 48°C. 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. coliamong coliforms, use 100 g 4 methyl-umbelliferyl--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 35C. Incubate dairy products at 32°C (3). 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 35°C. 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 per gram 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 (29); see later section on rehydratable dry-film method.

6. 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 (1).

   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 +/- 0.2°C. 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 35°C. 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 35°C. 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 35°C. 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 (17) 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 ref. 17.
      2. Voges-Proskauer (VP)-reactive compounds. Inoculate tube of MR-VP broth and incubate 48 +/- 2 h at 35C. 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 35°C 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 35°C. Development of distinct turbidity is positive reaction.

      5. Gas from lactose. Inoculate tube of LST broth and incubate 48 +/- 2 h at 35°C. 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 35°C, (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 Appendix 2) of E. coli based on proportion of EC tubes in 3 successive dilutions that contain E. coli.

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

1. Equipment, materials, and media

   1. Covered water bath, 44.5 +/- 0.2°C

   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 +/- 0.5°C

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 +/- 0.5°C in air incubator. Transfer to water bath and incubate 21 +/- 2 h at 44.5 +/- 0.2C. 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.

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

The LST-MUG assay can presumptively identify E. coli within 24 h (19,33). 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 (33).

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 (21).

NOTE: Some foods, such as shellfish, contain natural GUD activity (36). 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 (15).

1. Equipment and materials

   1. Incubator, 35 +/- 1°C

   2. Balance with capacity of 2 kg and sensitivity of 0.1 g

   3. Blender and blender jar (see Chapter 1)

   4. Sterile graduated pipets, 1.0 and 10.0 ml

   5. Sterile utensils for sample handling (see Chapter 1)

   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 35°C 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. (33) 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 35°C. 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 35°C, (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.

EC-MUG Method for Determining E. coli MPNs in 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/liter). 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 121°C 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 35°C) 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 +/- 0.2°C 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) 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.

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

Dry Rehydratable Film Method for the enumeration of total coliforms and E.coli

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 +/- 2C and 35 +/- 2°C

   4. Water bath, 44.0 +/- 0.2°C

   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. Urea broth (M171)

   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--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 6°C. 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 35°C to resuscitate damaged cells. Transfer contents to 225 ml double strength TP broth in 1 liter flask. Incubate 20 h at 44.0 +/- 0.2°C.

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 35°C.

   2. Enrichment. After incubation at 44°C, streak to L-EMB and MacConkey agars. Incubate 20 h at 35°C.

   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 (17,24)

   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 35°C. 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 35°C. 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 35°C unless otherwise specified). To identify cultures, test additional reactions shown below. Use reactions in Table 2 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. colishould be further investigated. To differentiate E. colifrom Shigella, examine anaerogenic, nonmotile, slow lactose fermenters for lysine decarboxylase, mucate, and acetate reactions.E. colistrains tend to give a positive response in one or more tests. Shigellasonnei, 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 3.

Serological Characterization (17)

Table 4 lists serogroups and serotypes associated with major pathogenic groups of E. coli. Commercial sera are not available for all these pathogen-associated serogroups (Table 5). 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.

Tests for Enteroinvasive E. coli (EIEC)

Sereny test for Confirmation of Invasive Potential (39)

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 35°C. Centrifuge culture (20 min at 1200 x g at 18°C). 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 days. 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.

Tests for Enterotoxigenic E.coli (ETEC)

Y-1 Mouse Adrenal Cell Test for E. coli LT (37)

1. Equipment and materials

   1. Microtiter tissue culture plates, 96 flat-bottom wells, sterile, plastic with lid

   2. Shaker incubator at 37°C

   3. Swinnex^TM 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 HEPA^TM filters) (Bellco Glass, Vineland, NJ 08360)

   7. Freezer, -70 or -20°C.

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 sq cm plastic culture flasks at 35°C 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 35°C 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 35°C 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. colicolony 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 37°C. 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 34°C 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 80°C. Store filtrates at 4°C.

   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 35°C in CO₂ incubator. After 39 min remove medium and replace with fresh maintenance medium. Re-incubate 18 h at 35°C 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 (10)

1. Equipment and materials

   1. Balance, accurate to 0.01 g

   2. Forceps, dissecting, sharp point, 4-1/2 inch

   3. Needle, 27 gauge

   4. Needle, animal feeding, 24 gauge, 1 inch, straight

   5. Tuberculin syringe, 1 ml, disposable

   6. Scissors, dissecting, sharp point, 4-1/2 inch

   7. Weighing boats

2. Media and reagents

   1. Evans blue, 2% solution (R24)

   2. Swiss albino mice, 3-5 days 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

Isolation Methods for Enterohemorrhagic E. coli O157:H7

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 foodborne outbreaks. This procedure has been highly effective in isolating O157:H7 from a variety of commonly contaminated foods (41).

1. Isolation with sorbitol-MacConkey (SMAC) agar (28) 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 35°C 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 (12,38). 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 (8,26).

2. Isolation with HC agar (hemorrhagic colitis ,strains of E. coli|)(40) 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 (34). 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 Foodborne Bacterial Pathogens by Gene Probes. 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 (41). 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
      *Available from Dynal Inc., Lake Success, NY (800) 638-9416

      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 37°C 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 37C 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 37C 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.

      3. 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, Chapter 24) or by polymerase chain reaction (see LIB 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 (41). 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 in Appendix 1.

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 foodborne outbreak, showed that PF-27 is highly specific only for the isolates of O157:H7 serotype (18). For more information on PF-27, contact Peter Feng, CFSAN, FDA, Washington, DC. Phone (202) 205-4518. For protocols on using the probe, see Chapter 24.

Tests for Toxins of Enterohemorrhagic E. coli (EHEC)

Tissue culture assay for Shiga-like toxins

1. Equipment and materials

   1. CO₂ incubator maintained at 36°C

   2. Sterile plastic plates containing 16 mm diameter wells

   3. Cell counting chamber

   4. Shaker incubator maintained at 37°C

   5. Centrifuge

   6. Sterile 0.45 µm membrane filters

   7. Inverted stage microscope

2. 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 37°C 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 4°C. 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 36°C 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 10⁵ per ml in growth medium. With gentle agitation transfer 0.5 ml portions to 16 mm wells in sterile plastic dishes. Incubate 3-4 days at 36°C 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 days at 36°C 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.

Detection of Shiga-like toxin genes in EHEC using DNA probes

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

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.

Tests for Enteropathogenic E. coli (EPEC)

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3. American Public Health Association. 1985. Standard Methods for the Examination of Dairy Products, 15th ed. APHA, Washington, DC.

 
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