Chapter 20: Vibrio spp.

Updated: 8/17/00

Contents

Potential Food Safety Hazard

Contents

Vibrio spp.

The genus Vibrio includes Gram-negative, oxidase-positive (except two species), rod or curved rod-shaped facultative anaerobes. Many Vibrio spp. are pathogenic to humans and have been implicated in food-borne disease (Table 20-1). Vibrio spp. other than V. cholerae and V. mimicus do not grow in media that lack added sodium chloride, and are referred to as "halophilic" (Elliot et al., 1998)

Table 20-1. Association of Vibrio spp. with different clinical syndromesa,b.
 

Clinical Syndrome

Species

Gastroenteritis

Wound

Infection

Ear

Infection

Primary

Septicemia

Secondary

Septicemia

V. cholerae O1

+++

+

      

V. cholerae non-O1

+++

++

+

+

+

V. mimicus

++

  

+

    

V. fluvialis

++

        

V. parahaemolyticus

+++

+

+

  

+

V. alginolyticus

(+)

++

++

+

  

V. cincinnatiensis

      

+

  

V. hollisae

++

    

+

  

V. vulnificus

+

++

  

++

++

V. furnissii

(+)

        

V. damsela

  

++

      

V. metschnikovii

(+)

    

(+)

  

V. carchariae

  

+

      
a+++ = frequently reported, ++ = less common (6-100 reports); + = rare (1-5 reports), and (+) = association is unclear.
bTable taken from Pavia et al. (1989).

Contents

Vibrio cholerae

V. cholerae was first described as the cause of cholera by Pacini in 1854. Pathogenic V. cholerae produces a heat-sensitive enterotoxin that causes the characteristic cholera symptoms, including "rice water stool." The species comprises several somatic (O) antigen groups, including O-group-1, which is associated with classical and El Tor biotypes. V. cholerae Ol may have several serotypes, including Inaba, Ogawa, and Hikojima. V. cholerae non-O1 (referred to in older literature as nonagglutinable or NAG vibrios) also can cause gastrointestinal disease, though typically less severe than that caused by V. cholerae O1 (Yamamoto et al., 1983). Serotype O139 is an exception, and produces classic cholera symptoms. This serotype was first identified in 1992 (CWG, 1933) as the cause of a new epidemic of cholera in India and Bangladesh. Non-O1 V. cholerae is found more readily in estuarine waters and seafood in the United States than is the Ol serogroup; however, the 0139 serogroup has not yet been found here. Because this species can grow in media lacking sodium chloride, it is not considered a halophilic Vibrio, although traces of sodium ion are required for growth. The standard FDA method for recovery of V. cholerae is qualitative (presence/absence). Testing V. cholerae O1 and non-O1 isolates for production of cholera toxin is recommended.

Some diarrheal and otitis isolates, once thought to be atypical V. cholerae non-O1 (sucrose-negative), are now recognized as a separate species, V. mimicus (Davis et al., 1981; Shantera et al., 1983). Members of the species may produce cholera-like enterotoxins. V. mimicus can be identified by biochemical procedures used for the identification of V. cholerae (Elliot et al., 1998).

Contents

Vibrio parahaemolyticus

V. parahaemolyticus is a halophilic bacterium found naturally in estuarine waters and animals. It was first described as the cause of gastroenteritis in Japan (Fujino et al., 1951) and was first found in the United States by Baross and Liston (1968) in the estuarine waters of Puget Sound. It has a worldwide distribution in estuarine and coastal environments and has been isolated from many species of fish, shellfish, and crustaceans. V. parahaemolyticus has been implicated in numerous outbreaks of seafood-borne gastroenteritis in the United States. Between 1971 and 1978, crab, oyster, shrimp, and lobster were implicated in 14 outbreaks, which may have resulted from the consumption of raw or insufficiently heated seafood or properly cooked seafood contaminated after cooking. The FDA method of enumeration uses an MPN format (Elliott et al., 1998).

Contents

Vibrio vulnificus

V. vulnificus is a halophilic bacterium found in the estuarine environment and is similar phenotypically to V. parahaemolyticus (Oliver, 1989). The species was first described as "lactose-positive" because most strains ferment lactose and are o-nitrophenyl-b -D-galactosidase (ONPG) positive. It causes food-borne and wound disease, either of which may progress to rapidly fatal septicemia in individuals with liver disease (cirrhosis) or other underlying illnesses such as diabetes. Raw oysters are the major source of food-borne disease caused by V. vulnificus. The FDA method of enumeration uses an MPN series confirmed by biochemical testing or an immunological test, such as the ELISA, with monoclonal antibody to a species-specific intracellular antigen (Elliott et al., 1998).

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Other Vibrio species

Other halophilic Vibrio spp., including V. fluvialis, V. hollisae, V. alginolyticus, V. furnissii, and V. metschnikovii, have been associated with gastroenteritis and are present in estuarine environments along with other pathogenic and nonpathogenic species of Vibrio. V. cincinnatiensis, V. damsela, and V. carchariae have not been associated with gastroenteritis, but on rare occasions are pathogenic to humans (Table 20-1). V. anguillarum, V. damsela, and V. carchariae are pathogenic to fish. Biochemical testing is required for taxonomic speciation (Elliott et al., 1998).

Contents

Control Measures

Hazards from Vibrio can be prevented by cooking seafood thoroughly and by preventing cross-contamination once the seafood is cooked. Freezing is ineffective in killing the bacteria (Ward et al., 1997).

If V. parahaemolyticus has produced the heat-stable Kanagawa hemolysin, some cooking procedures may not destroy the hemolysin (Bradshaw et al., 1984).

The risk of V. vulnificus infection can also be reduced by rapidly refrigerating oysters from the Gulf Coast during warm-weather months. Individuals in the "high risk" groups should not consume raw molluscan shellfish (Ward et al., 1997).

Contents

FDA Guidelines

Table 20-2. FDA guidelines for Vibrio in fish.
Product 
Guideline
Reference 
Ready-to-eat fishery products (minimal cooking by consumer)  V. cholerae - presence of toxigenic 01 or non-01. FDA, 1998a 
Ready-to-eat fishery products (minimal cooking by consumer)  V. parahaemolyticus - levels equal to or greater than 1x 104/g and Kanagawa positive or negative. FDA, 1998a 
Ready-to-eat fishery products (minimal cooking by consumer)  V. vulnificus - presence of pathogenic organism showing mouse lethality. FDA, 1998a 

Contents

Recommended Microbiological Limits

Contents

ICMSF Recommended Microbial Limits

Table 20-3. Recommended microbiological limits for V. parahaemolyticus in fish (ICMSF, 1986).
Product 
n1
c2
Bacteria/gram or/cm2
m3
M4
Fresh and frozen fish and cold-smoked fish
5
2
102
103
Frozen raw crustaceans
5
1
102
103
Frozen cooked crustaceans
5
1
102
103
Cooked, chilled, and frozen crabmeat
10
1
102
103
Fresh and frozen bivalve molluscs
10
1
102
103
1Number of representative sample units.
2Maximum number of acceptable sample units with bacterial counts between m and M.
3Maximum recommended bacterial counts for good quality products.
4Maximum recommended bacterial counts for marginally acceptable quality products.

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

Contents

Canadian Food Inspection Agency Bacteriological Guidelines for Fish and Fish Products

Contents

Growth

Table 20-4. Limiting conditions for V. cholerae growth.
Parameter
Values Reported
Reference
Min. aw
.97
 FDA, 1998b
Min. pH
5
 FDA, 1998b
Max. pH
10
 Bauman et al., 1984 
Max. %NaCl
6 
 West and Colwell, 1984 
Min. temp.
10ºC (50ºF)
 FDA, 1998b
Max temp.
43ºC (109.4ºF) 
 FDA, 1998b

Table 20-5. Limiting conditions for V. parahaemolyticus growth.
Parameter
Values Reported 
Reference 
Min. aw
.94
 FDA, 1998b
Min. pH
4.8
 FDA, 1998b
Max. pH
11
 Twedt et al., 1969
Max. %NaCl
10
 Beuchat, 1974
Min. temp.
5ºC (41ºF)
 Beuchat, 1973
Max. temp.
44ºC (111ºF)
 Jackson, 1974

Table 20-6. Limiting conditions for V. vulnificus growth.
Parameter
Values Reported
Reference
Min. aw
.96
 FDA, 1998b
Min. pH
5
 FDA, 1998b
Max. pH 
10
 FDA, 1998b
Max. %NaCl
5
 FDA, 1998b
Min. temp.
8ºC (46.4ºF)
 FDA, 1998b
Max. temp.
42ºC (107.6ºF)
 FDA, 1998b

Contents

Heat Resistance

Table 20-7. Heat resistance of V. cholerae.
Temp. 
D-Value
Medium 
Reference
(ºC)
(ºF)
(min.)
    
48.9
120
9.17
Shrimp homogenate
Hinton and Grodner, 1985
49
120.2
8.15
Crabmeat
Shultz et al., 1984
54
129.2
5.02
Crabmeat
Shultz et al., 1984
54.4
129.9
0.43
Shrimp homogenate
Hinton and Grodner, 1985
60
140
2.65
Crabmeat
Shultz et al., 1984
60
140
0.39
Shrimp homogenate
Hinton and Grodner, 1985
65.5
149.9
0.32
Shrimp homogenate
Hinton and Grodner, 1985
66
150.8
1.60
Crabmeat
Shultz et al., 1984
66
150.8
1.22
Crayfish homogenate
Grodner and Hinton, 1985
71
159.8
0.30
Crabmeat
Shultz et al., 1984
71
159.8
0.30
Crayfish homogenate
Grodner and Hinton, 1985
71.1
160
0.31
Shrimp homogenate
Hinton and Grodner, 1985
76.7
170.1
0.30
Shrimp homogenate
Hinton and Grodner, 1985
77
170.6
0.27
Crayfish homogenate
Grodner and Hinton, 1985
82
179.6
0.27
Crayfish homogenate
Grodner and Hinton, 1985
82.2
180
0.28
Shrimp homogenate
Hinton and Grodner, 1985

Table 20-8. Heat resistance of V. parahaemolyticus.
Temp.
D-Value
Medium
Reference
(ºC)
(ºF)
(min.)
    
47
116.6
65.1
7.5% NaCl
Beuchat and Worthington, 1976
49
120.0
0.82
Clam homogenate
Delmore and Chrisley, 1979
51
123.8
0.66
Clam homogenate
Delmore and Chrisley, 1979
53
127.4
0.40
Clam homogenate
Delmore and Chrisley, 1979
55
131
0.29
Clam homogenate
Delmore and Chrisley, 1979

Table 20-9. Heat resistance of V. vulnificus.
Temp. 
D-Value
Medium
Reference
(ºC)
(ºF)
(min.)
    
47
116.6
2.40
Buffered saline
Cook and Ruple, 1992
50
122
1.15
Buffered saline
Cook and Ruple, 1992

Contents

Analytical Procedures

Contents

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

    5. 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

    6. 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

    7. 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.

    Contents

    V. cholerae, V. parahaemolyticus, V. vulnificus, and other Vibrio species

    NOTE: Although many of the same media are used for the enrichment and identification of various Vibrio spp., procedures have been optimized for detection of V. cholerae and for enumeration of V. parahaemolyticus and V. vulnificus. Vibrio species other than these may be encountered on thiosulfate-citrate-bile salts-sucrose (TCBS) agar and can be identified by the characteristics given in Tables 20-10, 20-11, and 20-12 and in supplementary literature such as Bergey's Manual of Systematic Bacteriology (Bauman and Schubert, 1984). All of the cultural and serological tests should be performed in conjunction with known strains for comparative purposes.

    V. hollisae does not grow readily on TCBS agar and a selective agar has not been developed. If V. hollisae is to be detected, a differential medium such as blood agar flooded with oxidase reagent after incubation (Hickman et al., 1982) or mannitol-maltose agar (Nishibuchi et al., 1988) may be used.

    1. Equipment and materials

      1. Same as for Salmonella, Chapter 17
      2. Water baths, 35-37ºC
      3. Incubator, 39-40ºC and 42ºC
      4. Commercial bacterial identification strips
      5. VET-RPLA TD20 kit (Oxoid) for cholera toxin
      6. V-bottom (conical) microwell plates for VET-RPLA kit assay
      7. Low protein-binding 0.2 µm filters (Millipore SLGV)
      8. Micropipettors or micro-repeating pipettors
      9. Tissue culture cluster plates, 96-well (CoStar, Cambridge, MA)
      10. Flat-bottomed ELISA (EIA) plates, 96-well (CoStar or Immulon 2, Dynatech Laboratories, Alexandria, VA)
      11. ELISA plate washer (optional)
      12. ELISA (EIA) plate reader
      13. Shaker incubator, 35-37ºC
      14. Sterile syringes, 1 ml
      15. Centrifuge
      16. For gene probes, see Merker (1988), Chapter 24
      17. For tissue culture: cell culture flasks; cell culture 96-well plates; CO incubator, 37ºC; inverted phase-2 contrast microscope.
      18. 10,000 M.W. cutoff ultrafiltration membranes (PM10 or YM10; Amicon, Inc., Danvers, MA) and apparatus
      19. Glass petri dishes for membrane ELISA
      20. Nitrocellulose membranes, 82 mm (Bio-Rad Labs or Schleicher and Schuell) or nylon membranes (Magnagraph), MSI (Micron Separations, Inc.)

    2. Media and reagents

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

      NOTE: Halophilic Vibrio spp. require added NaCl (2-3% final concentration). V. cholerae grow well in media with 0-3% NaCl. Add NaCl to media listed in Merker (1988), Appendix 3 to achieve a final 2-3% concentration of NaCl, with the following exception. Do not add NaCl to gelatin agar (M54) or alter the NaCl concentrations of salt tolerance testing broths (M161 and M164).

      1. AKI broth (M7)
      2. Alkaline peptone salt (APS) broth (M9)
      3. Alkaline peptone water (APW) (M10)
      4. Arginine glucose slant (AGS) (M16)
      5. Bicarbonate buffer, 0.1 M, pH 9.6 (R4)
      6. Blood agar (M20)
      7. 1% Bovine serum albumin (BSA) in ELISA buffer (R6) and in PBS (R7)
      8. Brain heart infusion (BHI) agar (M24)
      9. Bromcresol purple broth (M26) supplemented individually with sucrose, lactose, cellobiose, arabinose, D-mannitol, or D-mannose
      10. Cary-Blair transport medium (M31)
      11. Casamino acids-yeast extract (CYE) broth (M32)
      12. Chicken red blood cells, 2.5%, in physiological saline
      13. Cholera enterotoxin (Becton-Dickinson Immunodiagnostics, Orangeburg, NY 10962; Sigma Chemical Co., St. Louis, MO 63178; or List Biological Laboratories, Inc., Campbell, CA 95008)
      14. Christensen's urea agar (M40)
      15. Citric acid, 0.05 M, pH 4.0 (R13)
      16. Coating solution for V. vulnificus EIA (R15)
      17. Decarboxylase basal medium (M44), unsupplemented, and supplemented individually with arginine, lysine, or ornithine
      18. ELISA buffer for cholera toxin assay (R22)
      19. El Tor phage V (ATCC, Rockville, MD)
      20. Enzyme immunoassay (EIA) wash solution (R21) for V. vulnificus
      21. Fetal calf serum
      22. Gelatin agar (GA) (M54) and gelatin salt (GS) agar (M55)
      23. Goat antiserum to cholera toxin (CT) (List Biological Laboratories)
      24. Gram stain dyes (R32)
      25. Ham's F-10 medium with glutamine (M58), commercial preparation preferred
      26. Heart infusion broth and agar (M60)
      27. Hugh-Leifson glucose broth (M63)
      28. 1 N hydrochloric acid (HCl) (R36)
      29. Kligler iron agar (KIA) (M71)
      30. Kovacs' reagent (R38)
      31. Long-term preservation medium (M85)
      32. Mannitol-maltose agar (M96)
      33. Methyl red indicator (R44)
      34. Mineral (paraffin) oil, sterile (R46)
      35. Modified cellobiose-polymyxin B-colistin (mCPC) agar (M98)
      36. Monoclonal antibody to V. vulnificus [Available from FDA Gulf Coast Seafood Laboratory, P.O. Box 158, Dauphin Island, AL 36528-0158]
      37. Motility test medium, semisolid (M103
      38. MR-VP broth (M104)
      39. Mueller-Hinton agar (M107)
      40. Mukerjee phage IV
      41. O/129 (2,4-diamino-6,7-diisopropyl pteridine) disks, 10 and 150 µg (R51)
      42. OF medium, semisolid (M116), supplemented individually with glucose, sucrose, lactose, cellobiose, arabinose, D-mannitol, or D-mannose
      43. ONPG test reagents (R53) or commercially available disks
      44. Oxidase test reagent (R54)
      45. Penicillin-streptomycin (Pen-strep) solution (Mll9)
      46. Peroxidase-conjugated goat immunoglobulin G (anti-mouse; anti-rabbit)
      47. Peroxidase substrate solution (4-chloro-1-naphthol) for membrane ELISA (R57)
      48. Peroxidase substrate solution (ABTS) (R58)
      49. Phosphate-buffered saline (PBS), pH 7.4 (R59)
      50. Physiological saline solution, 0.85% (R63)
      51. Polymyxin B antibiotic disks, 50 units (R64) or commercially prepared
      52. Poly, Inaba, and Ogawa antisera for V. cholerae (Difco; Burroughs Wellcome). Monoclonal antibody reagents are available from Denka Seiken Co., Tokyo, Japan, through Nichimen America, Inc., 1185 Avenue of the Americas, New York, NY 10036. Monoclonal antibody reagents to V. cholerae Ol antigen are available from Global Diagnostics, One Progress Blvd., Box 33, Room N-112, Alachua, FL (904)-462-7997) and from OXOID.
      53. Rabbit antiserum to cholera toxin (contact M. Kothary)
      54. Reagents for gene-probe testing, see Merker (1988), Chapter 24
      55. Reference strains for V. cholerae classical and El Tor biotypes, serogroup O1; cytotoxin-producing strains of V. cholerae serogroup non-O1; reference strains of Kanagawa phenomenon-positive V. parahaemolyticus; cytotoxin-hemolysin-positive strains of V. vulnificus, and V. mimicus (contact M.Kothary)
      56. Sera for O and K antigen testing of O (somatic) and K (capsular) antigens of V. parahaemolyticus (Denka Seiken Co., through Nichimen America, Inc. or through OXOID or from Charles Kaysner (phone: 206-483-8788)
      57. Sodium chloride dilution water, 2% and 3% (R71)
      58. 1 N sodium hydroxide (NaOH) solution (R73)
      59. Sheep red blood cells, 5%, in physiological saline
      60. Thiosulfate-citrate-bile salts-sucrose (TCBS) agar (M147)
      61. Triple sugar iron (TSI) agar (M149)
      62. Tris-buffered saline, pH 7.5 (TBS) (R81); with 1% or 3% gelatin (R82); with 0.05% Tween 20 (TBS-Tween (R83)
      63. Triton X-100 (R86)
      64. Trypsin-EDTA solution, 1X (R87)
      65. Trypticase (tryptic) soy agar (TSA) (M152) and broth (TSB) (M154)
      66. Tryptic soy broth (TSB) without dextrose, supplemented with 1% NaCl and 24% glycerol (M155) (total 1.5% NaCl), and with 2.5% NaCl (total 3% NaCl)
      67. 1% Tryptone, 1% NaCl (T1N1) agar (M163) and T1N2 agar
      68. Tryptone broth (1%), unsupplemented (0% NaCl) (M164), or supplemented with 1, 3, 6, 8, or 10% NaCl (M161)
      69. Voges-Proskauer test reagents (R89)
      70. Wagatsuma agar (M178)
      71. Y-1 adrenal cell growth medium (M180)
      72. Y-l mouse adrenal cells or Chinese hamster ovary (CHO) cells (ATCC)

    3. Procedure for enrichment and isolation of V. cholerae and V. mimicus from foods

      4. (Figure 20-1). For stool, rectal swab, or vomitus specimens, see 4-a, below, for transport and initial inoculation procedures.

      1. Sample preparation. Aseptically weigh 25 g sample into 500 ml tared sterile blender jar or Stomacher. Cut large samples into smaller pieces before blending. Add 225 ml alkaline peptone water (APW) to jar and blend for 2 min at top speed. For oysters only (especially oysters freshly harvested from warm waters), prepare composite of 10-12 animals including shell liquor; blend to mix. Blend 50 g of this composite with 450 ml of APW. Pour 250 ml (g) of this mixture into another sterile container. Replicates for samples of oysters are incubated at different temperatures (see b, below).

        2. NOTE: Isolating specific Vibrio spp. from samples containing high concentrations of enteric bacteria may be difficult because of overgrowth. For vegetables, estuarine waters, and other environmental samples expected to have high numbers of bacteria, dilute the blended samples to a final 1:100 dilution and proceed as usual. For example, take 25 ml of blended sample and add to 225 ml APW.

        For seafood samples, especially oysters, also prepare tenfold dilutions of the blended seafood sample in 9 (or 90) ml APW blanks (1:100 and 1:1000 dilutions) and proceed as usual. Prepare 2 sets of dilution tubes for oysters. Dilutions are made to decrease competition from other vibrios.

        Dilutions may also be used to analyze for V. parahaemolyticus and V. vulnificus. If sample is to be tested for all three Vibrio species (and others), use a sample large enough to inoculate all required media, and prepare the homogenate in APW or PBS, pH 7.2-7.5. For example, if sample is to be analyzed for V. cholerae, V. parahaemolyticus, and V. vulnificus, homogenize a 50 g sample with 450 ml APW. Place 250 ml (g) of APW homogenate in sterile container and follow the method for

        V. cholerae. (If PBS is used during homogenation, transfer 250 ml (g) of PBS homogenate to 2250 ml APW.) If an MPN is to be determined with the remainder, prepare dilutions in PBS, pH 7.2-7.5, inoculate MPN tubes of APW, and incubate tubes at 35-37ºC. These tubes will serve as MPN enrichment tubes for V. parahaemolyticus and V. vulnificus, as well as V. cholerae in materials that may have high background microflora. From APW, inoculate selective plating media at 6-8 h for V. cholerae and at 18-24 h for V. cholerae, V. parahaemolyticus, and V. vulnificus. See part 4 for identification of halophilic Vibrio spp. For oyster samples to be tested for the three Vibrio species, use a sample of at least 75 g since two 250 ml (g) test portions of APW homogenate are incubated for V. cholerae analyses (one at 35-37ºC and one at 42ºC).

      2. Leave blended solutions of samples, including frozen or otherwise processed food homogenate and dilutions in jars or pour into loosely stoppered sterile 500 ml Erlenmeyer flasks and incubate jars, flasks, and dilutions 6-8 h at 35-37ºC. Plate inocula to isolation agar (see c, below), and reincubate enrichment broths for total incubation time of 18-24 h. Plate the 18-24 h enrichment broths to isolation agar.

        3. Exception. Incubate second jar or flask of oyster homogenate and one set of dilutions at 42ºC for 6-8 h (Depaola et al., 1987).

      3. After incubation, and without shaking flask, transfer 3-5 mm loopful of inoculum from pellicle (surface growth) onto at least one plate of selective plating medium: TCBS agar. (mCPC agar is optional; it may be used in addition to TCBS. Classical biotype V. cholerae is inhibited on mCPC agar by polymyxin B.) Incubate TCBS agar for 18-24 h at 35-37ºC and mCPC agar for 18-24 h at 39-40ºC.

      4. Examine plates for colony characteristics described below. Carefully pick 3 or more suspect colonies from each plate, streak each for isolation on T1N1, T1N2, or tryptic soy agar (2% total NaCl concentration), and incubate for 18-24 h at 35-37ºC. Streaking for isolation on nonselective medium may be necessary to ensure colonial purity before biochemical testing. Gelatin agar (GA) and gelatin salt (GS) agar (see e-ii, below) may also be inoculated with the same inoculum.

        1. Thiosulfate-citrate-bile salts-sucrose (TCBS) agar. On TCBS agar, V. cholerae (El Tor and classical) are large, smooth, yellow (sucrose-positive), and slightly flattened colonies with opaque centers and translucent peripheries. NOTE: Vibrio spp. do not produce tiny, creamy yellow colonies on TCBS agar. Colonies of closely related V. mimicus are green (sucrose-negative). Most other Vibrio spp. grow on TCBS agar and produce yellow or green colonies.

        2. Modified cellobiose-polymyxin B-colistin (mCPC) agar (Tamplin et al., 1991). Colonies of V. cholerae El Tor are green-to-purple (cellobiose fermentation-negative). V. vulnificus produces flattened yellow colonies with opaque centers and translucent peripheries. Most other Vibrio spp. do not grow readily on CPC agar (Massad and Oliver, 1987) or mCPC agar.

      5. Distinguish suspect vibrios from non-vibrios

        1. TSI, KIA, and arginine glucose slant (AGS). Inoculate individual colonies into TSI or KIA, and AGS media by stabbing butt and streaking slant. Incubate loosely stoppered or capped inoculated slants 18-24 h at 35-37ºC. These media are recommended because the reactions permit early presumptive differentiation between most Vibrio spp., Aeromonas spp., Plesiomonas shigelloides, and other bacteria (Tables 20-10 and 20-11).

        2. 1% Tryptone (tryptophane) broth (T1N0) and broth containing 3% NaCl (T1N3). Inoculate individual colonies into T1N0 and T1N3 broths and incubate 18-24 h at 35-37ºC. Reincubate growth-negative tubes an additional 18-24 h. V. cholerae and V. mimicus will grow in T1N0 and T1N3. Some non-vibrio bacterial species producing reactions similar to those of V. cholerae in TSI and KIA media will not grow in T1N3. Most Vibrio spp., including some V. cholerae non-O1, will grow in T1N3 only.

          3. Alternatively, gelatin agar (GA) and gelatin agar containing 3% NaCl (GS) can be used to screen isolates for salt tolerance (Smith and Goodner, 1958). Divide plates into 8 sectors. Inoculate a straight line in the center of one sector of both GA and GS plates with each isolate. Incubate 18-24 h at 35ºC. V. cholerae and V. mimicus will grow on both plates because they do not require salt. Halophilic Vibrio spp. will grow only on the GS plate. To read the gelatinase reaction, hold plate above a black surface. An opaque halo will be present around growth of gelatinase-positive organisms.

        3. Oxidation-fermentation test. Inoculate 2 tubes of Hugh-Leifson glucose broth or OF glucose medium (semisolid) with growth from an isolated colony. Overlay medium in one tube with sterile mineral oil or liquid Vaspar (50% petrolatum, 50% paraffin) to depth of 1-2 cm and incubate 1-2 d or more at 35-37ºC. Acid causes dye to change from purple to yellow in Hugh-Leifson broth, and from green to yellow in OF medium, semisolid. Vibrio spp. ferment glucose and produce acid from glucose oxidatively. Pseudomonas spp., commonly isolated from seafood by enrichment methods used for Vibrio spp., utilize glucose oxidatively only.

        4. Oxidase test. Perform oxidase test on 18-24 h growth from TSA or other medium containing no fermentable carbohydrate such as GA or GS. An easy rapid method for testing large numbers of isolates is to place a filter paper circle in a petri plate and moisten the entire filter paper with a few drops of oxidase reagent. With a sterile wooden applicator stick, toothpick, or platinum loop, pick growth from the plate and touch the moistened paper. Oxidase positive organisms will turn the paper dark purple or blue within 10 s. Pathogenic Vibrio spp. are oxidase-positive (except for V. metschnikovii).

      6. Identification and confirmation of V. cholerae O1, V. cholerae non-O1, and V. mimicus.

        1. Read results of TSI, KIA, AGS, T N and T N or GA and GS, and oxidation-1013 fermentation tests.

        2. Perform Gram stain on 18-24 h broth or agar culture.

          3. NOTE: Isolates to be carried through the remaining V. cholerae serological and biochemical tests are sucrose-positive (yellow) on TCBS agar [sucrose-negative (green) for V. mimicus)] or cellobiose-negative (green-purple) on mCPC agar. They grow in T1N0 and T1N3 broths or on GA and GS plates; show characteristic reactions (see Table 20-10) in TSI, KIA, and AGS; are gelatinase and oxidase-positive; are Gram negative rods or curved rods; and produce acid from glucose both oxidatively and fermentatively in Hugh-Leifson glucose broth or OF glucose medium, semisolid.

        3. Serological agglutination test. Use diagnostic antisera of Group O1 and subgroup Inaba (factors AC) and Ogawa (factors AB) to serotype Ol antigen and antisera or monoclonal antibodies to serotype O139 antigen to identify serogroup O139. Use 16-24 h cultures from TSA. Include positive and negative cultures and saline controls for each antiserum used. Follow directions included with antisera. Drops of 10 µl are sufficient for the test. Because antigens in different species may be related, biochemical tests must be completed before isolate is confirmed as V. cholerae O1 or non-O1.

          4. NOTE: Monoclonal antibodies are available, but anti-B and anti-C cross-react with bacteria of other species (Shimada et al., 1987). Use polyclonal sera and/or monoclonal antibodies to the A antigen of the O1 complex.

          Cultures that agglutinate in group Ol antiserum and not in plain physiological saline are V. cholerae group O1 if biochemical reactions confirm the isolate as V. cholerae. Cultures that agglutinate in this group-specific antiserum may be subtyped with Inaba and Ogawa antibodies.

          Notify Dr. Mahendra Kothary (202) 205-4454, of V. cholerae O1 isolates. Continue with biochemical characterization and toxigenicity determination of the isolates. Direct questions about methodology to Dr. LeeAnne Jackson (202) 205-4231.

          Cultures that agglutinate in poly (group O1) antiserum and in both Inaba and Ogawa antisera have all 3 factors (A, B, and C) and are serotype Hikojima.

          Cultures that agglutinate in poly antiserum but not in Inaba or Ogawa antisera cannot be typed using these antisera.

          Table 20-10. Reactionsa of certain Vibrio spp. and related microorganisms in differential tube agar media.

          Microorganism

          KIA

          TSI

          AGS

          Slant

          Butt

          Slant

          Butt

          Slant

          Butt

          V. cholerae

          K

          A

          A (K rare)

          A

          K

          a

          V. mimicus

          K

          A

          K (A rare)

          A

          K

          A

          V. parahaemolyticus

          K

          A

          K

          A

          K

          A

          V. alginolyticus

          K

          A

          A

          A

          K

          A

          V. vulnificus

          K or A

          A

          K (A rare)

          A

          K

          A

          A. hydrophila

          K or A

          A

          K or A

          A

          K

          K

          P. shigelloides

          K or A

          A

          K or A

          A

          ND

          ND
          aK, alkaline; A, acid; a, slightly acid; ND, not determined. None of the listed Vibrio spp. produce hydrogen sulfide gas in KIA, TSI, or AGS media, or gas from glucose in detectable quantities in KIA, TSI, or AGS media. Some Aeromonas spp. may produce gas from glucose in these media.

          Table 20-11. Biochemical characteristics of several of the Vibrionaceae.
           

          V. alginolyticus

          V. anguillarum

          V. carchariae

          V. cholerae

          V. cincinnatiensis

          V. damsela

          V. fluvialis

          V. furnissii

          V. harveyi

          V. hollisae

          V. metschnikovii

          V. mimicus

          V. parahaemolyticus

          V. vulnificus

          A. hydrophila

          P. shigelloides

          Photobacterium spp.

          TBS agar

          Y

          Y

          Y

          Y

          Y

          G

          Y

          Y

          Y/G

          NG

          Y

          G

          G

          G

          Y

          G

          NG/G

          MCPC agar

          NG

          NG

          nd

          P

          nd

          NG

          NG

          NG

          nd

          NG

          NG

          NG

          NG

          Y

          NG

          NG

          NG

          AGS medium

          KA

          nd

          nd

          Ka

          nd

          nd

          KK

          KK

          nd

          Ka

          KK

          KA

          KA

          KA

          KK

          nd

          nd

          Growth in:

          		                                  

          0% NaCl

          -

          -

          -

          +

          -

          -

          -

          -

          -

          -

          -

          +

          -

          -

          +

          +

          -

          3% NaCl

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          6% NaCl

          +

          +

          +

          -

          +

          V

          +

          +

          +

          +

          +

          -

          +

          +

          +

          -

          V

          8% NaCl

          +

          -

          +

          -

          -

          -

          V

          +

          V

          -

          V

          -

          +

          -

          -

          -

          -

          10% NaCl

          +

          -

          nd

          +

          -

          -

          V

          -

          V

          nd

          V

          +

          +

          +

          V

          +

          -

          Growth at 42ºC (107.6ºF)

          +

          -

          nd

          +

          -

          -

          V

          -

          V

          nd

          V

          +

          +

          +

          V

          +

          -

          Acid from:

          		                                  

          Sucrose

          +

          +

          +

          +

          +

          -

          +

          +

          V

          -

          +

          -

          -

          -

          V

          -

          V

          D-Cellobiose

          -

          +

          +

          -

          +

          +

          +

          -

          nd

          -

          -

          -

          V

          +

          +

          -

          -

          Lactose

          -

          -

          -

          -

          -

          -

          -

          -

          V

          -

          -

          -

          -

          +

          V

          -

          -

          Arabinose

          -

          V

          -

          -

          +

          -

          +

          +

          -

          +

          -

          -

          +

          -

          V

          -

          -

          D-Mannose

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          V

          -

          +

          D-Mannitol

          +

          +

          +

          +

          -

          -

          +

          +

          +

          -

          +

          +

          +

          V

          +

          -

          -

          Oxidase

          +

          +

          +

          +

          +

          +

          +

          +

          +

          +

          -

          +

          +

          +

          +

          +

          V

          ONPG

          -

          +

          -

          +

          +

          -

          +

          +

          V

          -

          +

          +

          -

          +

          +

          -

          +

          Voges-Proskauer

          +

          +

          -

          V

          +

          +

          -

          -

          -

          -

          +

          -

          -

          -

          +

          -

          V

          Arginine dihydrolase

          -

          +

          -

          -

          -

          +

          +

          +

          -

          -

          +

          -

          -

          -

          +

          +

          +

          Lysine decarboxylase

          +

          -

          +

          +

          +

          V

          -

          -

          +

          -

          +

          +

          +

          +

          V

          +

          V

          Ornithine decarboxylase

          +

          -

          +

          +

          -

          -

          -

          -

          +

          -

          -

          +

          +

          +

          -

          +

          -

          Sensitivity to:

          		                                  

          10 µg 0/129

          R

          S

          R

          S

          R

          S

          R

          R

          R

          nd

          S

          S

          R

          S

          R

          S

          V

          150 µg 0/129

          S

          S

          S

          S

          S

          S

          S

          S

          S

          nd

          S

          S

          S

          S

          R

          S

          S

          Gelantinase

          +

          +

          +

          +

          -

          -

          +

          +

          +

          -

          +

          +

          +

          +

          +

          -

          -

          Urease

          -

          -

          +

          -

          -

          +

          -

          -

          V

          -

          -

          -

          V

          -

          -

          -

          -

          Abbreviations: TCBS, thiosulfate-citrate-bile salts-sucrose; mCPC, modified cellobiose-polymyxin B-colistin; AGS, arginine-glucose slant; Y, yellow; G, green; P, purple; NG, no growth; nd, not determined; K, alkaline; A, acid; a, slightly acid; +, 80% or more of strains positive; 80% or more of strains negative (fewer than 20% of strains positive); V, variable reaction depending on species or strain; S, sensitive; R, resistant. Arginine glucose slant (AGS) reactions: slant, butt; all strains tested were hydrogen sulfide and gas negative. ONPG: o-nitro-beta-D-galactopyranoside hydrolysis by beta-galactosidase. Biochemical reactions from Brayton et al., (1986); Ewing et al., (1979); Furniss et al., (1978); Grimes et al., (1984); Madden et al., (1989); Oliver, (1989); Sakazaki, (1979); Sakazaki and Shimada, (1986); West and Colwell, (1984); West et al., (1986).

          Table 20-12. Differentiation of biotypes of V. cholerae O1a,b

          Test

          Classical

          El Tor

          Sensitivity to El Tor phage V

          -

          +

          Sensitivity to classical phage IV

          +

          -

          Sensitivity to polymyxin B, 50 units

          +

          -

          Hemolysis (sheep erythrocytes)

          -

          v

          Hemagglutination (chicken erythrocytes)

          -

          +

          Voges-Proskauer

          -

          +
          aFrom Baumann and Schubert (1984) and Madden et al. (1989). b+, Positive; -, negative; v, strains vary.

          Cultures confirmed biochemically as V. cholerae that do not agglutinate in Group Ol antiserum are V. cholerae non-O1. Test such cultures with O139 antiserum.

          Cultures that agglutinate in Group O1 antiserum and in saline cannot be typed. However, using a richer growth medium, such as heart infusion (HI) agar or BHI agar, may eliminate this autoagglutination.

        4. Biochemical reactions (Table 20-11). For specific directions for biochemical tests, including O/129 sensitivity, growth at 42ºC, and ONPG tests, see 4-c, below. Formulations for all biochemical media should include at least 2% NaCl. API strips may be used in lieu of conventional media, with 2% NaCl as diluent. For V. cholerae, use physiological saline (0.85% NaCl) as diluent.

        5. Determination of classical and El Tor biotypes. Two biotypes of V. cholerae serogroup O1 (classical and El Tor) may be distinguished by the following methods (see Table 20-12). Use more than one test to differentiate biotypes. The easiest methods are polymyxin B sensitivity, hemolysin test, and Voges-Proskauer test.

          6. Bacteriophage susceptibility. This method is a modification of that described by Finkelstein and Mukerjee (Finkelstein and Mukerjee, 1963). Inoculate HI broth with strain to be tested and incubate at 35-37ºC for 4 h. Swab surface of Mueller-Hinton agar plate with 4 h broth culture to obtain confluent bacterial growth. Let plates absorb inoculum, and place 1 loopful of appropriate test dilution of phage IV onto agar surface with 3 mm platinum loop. Observe plate after overnight incubation at 35-37ºC. Classical biotype strains are usually sensitive to this bacteriophage and will lyse on plate where phage was placed (indicated by clear plaque). El Tor biotype strains are resistant to this bacteriophage and will not be lysed (indicated by confluent growth).

          Use this same method to test for sensitivity to El Tor phage V.

          Polymyxin B sensitivity. This procedure is a modification of technique described by Han and Khie (1963). Swab surface of Mueller-Hinton agar plate with 4 h HI broth culture (35-37ºC) to obtain confluent growth. Let plates absorb inoculum and place 50 unit polymyxin B antibiotic disk on medium surface. Invert plates and incubate for 18-24 h at 35-37ºC. Classical biotype strains will demonstrate zone of inhibition around disk (10-15 mm diameter). E1 Tor biotype strains will grow to edge of disk or will be inhibited slightly (7-8 mm diameter). Alternatively, use TSA, GA, or GS agar in place of Mueller-Hinton agar.

          NOTE: If isolate was picked from mCPC, it is polymyxin B-resistant.

          Hemolysin test. Mix equal volumes (0.5 or 1 ml) of 24 h HI broth culture and 5% saline suspension of sheep red blood cells. Set up similar mixtures with portion of culture that has been heated for 30 min at 56ºC. Use known hemolytic and nonhemolytic strains of V. cholerae as controls. Incubate mixtures for 2 h in 35-37ºC water bath, then refrigerate overnight at 4-5ºC. Examine tubes for hemolysis. Low speed centrifugation may aid in detection of cell lysis. Most El Tor strains will lyse red blood cells. Heated portion of culture should produce no hemolysis because hemolysin is thermolabile.

          Classical biotypes of V. cholerae and some strains of biotype El Tor will not lyse red blood cells. Alternatively, spot inoculum onto blood agar plates containing 5% sheep red blood cells, as for Kanagawa phenomenon (see below). Incubate at 35ºC for 24 h and check for $hemolysis surrounding colonies.

          Chicken red blood cell agglutination. Prepare thick, milky bacterial suspension in physiological saline from 18 to 24 h TSA culture. On clean glass slide, mix 1 loopful of washed chicken red blood cells (2.5% in physiological saline) with suspension of bacterial culture to be tested. Visible clumping of red blood cells indicates El Tor biotype. Classical strains usually do not agglutinate red blood cells. Perform positive and negative controls.

          Voges-Proskauer (VP) test. Perform test in MR-VP broth after 18-24 h incubation at 22ºC. El Tor biotype strains are usually positive; classical strains are negative.

        6. Minimal characters for biochemical identification of V. cholerae. The following characteristics are presumptive of V. cholerae:

          • Morphology: Gram-negative asporogenous rod or curved rod
          • TSI or KIA appearance: Acid slant/acid butt, gas production negative, H2S-negative
          • Hugh-Leifson test: Glucose fermentation and oxidation-positive
          • Cytochrome oxidase: Positive
          • Arginine dihydrolase test: Negative
          • Lysine decarboxylase test: Positive
          • Voges-Proskauer test: El Tor biotype-positive, classical biotype-negative; V. mimicus negative
          • Growth at 42ºC: Positive
          • Halophilism test: 0% NaCl-positive; 3% NaCl-positive; 6% NaCl- usually negative. Some strains of V. cholerae non-O1 may not grow in 0% NaCl.
          • Sucrose fermentation: Positive (negative for V. mimicus)
          • ONPG test: Positive
          • Arabinose fermentation: Negative
          • O/129 sensitivity: Sensitive to 10 and 150 µg O/129

      7. Detection of cholera enterotoxin (CT) and cytotoxin

        8. Test isolate determined to be V. cholerae (including O1, O139, and other non-O1 serogroups) or V. mimicus biochemically and/or serologically for CT by direct or immunological test. Direct tests include the effect of toxin in vitro on Y-l mouse adrenal cells or Chinese Hamster Ovary (CHO) cells, and the in vivo suckling mouse assay (Baselski et al., 1977). Immunological methods include ELISAs and latex agglutination tests. DNA probes are available for CT-like enterotoxin gene sequences (see Merker (1988), Chapter 24).

        1. Y-l Adrenal cell or Chinese Hamster Ovary (CHO) cell assay for enterotoxin (CT) and cytotoxin

          2. Cell culture flasks. Using standard cell culture techniques, grow Y-l or CHO cells on surface of 25 cm2 plastic cell culture flasks, using 5 ml Y-l cell growth medium at 37ºC in 5% CO2 incubator. Replace medium after 48 and 96 h and observe appearance of cells, using inverted phase-contrast microscope. Test cells before they become totally confluent in flask. Before preparing fresh flasks or monolayers in wells of microwell plates, wash monolayer with 5 ml sterile 0.85% saline or PBS. Add 0.5 ml lX trypsin-EDTA solution and incubate at 37ºC for 15 min. Cells can be dislodged from surface by tapping flask against hand. To stop trypsin activity, add 4.5 ml Y-l growth medium (total volume in flask, 5 ml), wash cells from surface of flask, and transfer cell suspension to sterile tubes. Let large clumps of cells settle for 2 min. To prepare new flask of cells, add 1 ml cell suspension and 4 ml Y-l growth medium to new 25 cm2 tissue culture flask.

          Preparation of microwell plates. Add 15 ml Y-l growth medium to 5 ml cell suspension prepared above. Transfer 0.2 ml of this diluted cell suspension to each of 96 wells. Incubate plates in CO2 incubator at 37ºC. When cell monolayers are confluent (usually within 3 d), plate is ready for use.

          Preparation and concentration of test filtrates. Use AKI medium to enhance CT production by V. cholerae O1 El Tor (Iwanaga and Yamamoto, 1985). Inoculate 15 ml AKI tubes and incubate 4 h without shaking at 35-37ºC. Then transfer entire volume to 250 ml flask and incubate 16 h with shaking (200 rpm) at 35-37ºC. Centrifuge culture at 900 x g for 30 min in refrigerated centrifuge. Discard cells. Filter-sterilize supernatant, using 0.22 µm low protein-binding membrane, before testing. If large volumes of supernatant are collected, concentrate by ultrafiltration through 10,000 M.W. exclusion membrane, such as Amicon PM10 or YM10.

          Optional broth medium: Inoculate CYE broth with isolate and incubate for 18-24 h at 35-37ºC. Transfer 0.1-0.2 ml of this culture into 125 ml flask containing 25 ml CYE broth. Incubate with shaking (100-200 rpm) for 24 h at 35ºC.

          Prepare supernatant as described above.

          Assay. Remove old Y-l medium and drain microwell plate by inverting on sterile towel. Add 0.1 ml fresh medium per well. Add 0.05 ml of each filter-sterilized bacterial culture supernatant to microtiter plate wells. For CT assay, use 0.05 ml of cholera enterotoxin (5 ng/ml) for positive control. For cytotoxin assay, use 0.05 ml of V. cholerae 2194C culture supernatant for positive control. Use sterile CYE medium for negative control. For second negative control well, boil culture supernatant from V. cholerae 2194C for 5 min. Incubate overnight at 35-37ºC in CO2 incubator.

          Interpretation. Examine wells at 100 or 200X magnification, using inverted stage phase microscope. Compare test wells to positive control wells. For CT assay, a positive well contains more than 10% rounded Y-l cells. For cytotoxin assay, a positive well contains 50% or more dead, lysed, and/or detached cells. El Tor strains are typically hemolytic (cytotoxic). Consequently, CT cannot be detected unless its concentration exceeds that of cytotoxin, and the sample is titered (various dilutions are used in assay).

          Chinese hamster ovary (CHO) cell alternative. CHO cells may be used rather than Y-l cells. CHO cells elongate when exposed to CT.

        2. Immunological detection of cholera enterotoxin (CT): microwell plate and membrane ELISAs.

          3. Screen suspect colonies for production of these toxins with appropriate dilutions of specific antibodies against CT, using membrane ELISA, a 96-well microELISA, or reversed-passive-latex agglutination (RPLA) assay kit in 96-well format. As with all immunological tests, when suppliers or stocks of antibody or conjugate change, the new reagents must be titered to determine optimum test dilutions.

          Grow isolates in AKI medium at 35-37ºC, as described for the Y-l assay (CYE medium optional). Centrifuge at 900 x g for 20 min in refrigerated centrifuge. Decant supernatant into sterile flask and discard cells. To obtain clear supernatants, filter through low protein-binding membrane. Supernatants need not be sterilized and may be frozen at -20ºC before use.

          To detect low amounts of toxin in culture supernatant, concentrate toxin using method of Yamamoto et al. (Yamamoto et al., 1983).

          1. Micro ELISA

            2. Coat each well of a flat-bottomed, 96-well ELISA plate with 50 µ1 of appropriate dilution (e.g., 1:1000) of goat anti-cholera toxin diluted in 0.1 M bicarbonate buffer, pH 9.6. Cover and place at 4ºC overnight. Wash plates 3 times with ELISA buffer. To reduce nonspecific binding, block wells with 200 µ1 1% bovine serum albumin (BSA) in ELISA buffer for 30 min at 25ºC. Wash 3 times with ELISA buffer.

            Add 100 µ1 test substance to each well. Run positive controls (enterotoxin-producing bacterial strains) and negative controls (uninoculated growth medium). Cover and incubate plates for 1 h at 37ºC. Wash plates 3 times with ELISA buffer.

            Add 100 µ1 of appropriately diluted (in 1% BSA in ELISA buffer) rabbit anti-CT to each well. Cover and incubate plates at 37ºC for 1 h. Wash plates 3 times with ELISA buffer. NOTE: Titrate rabbit anti-CT before use. A 1:500 dilution has been used successfully with some preparations.

            Add 100 µ1 of appropriately diluted goat anti-rabbit peroxidase conjugate (e.g., 1:1000 in 1% BSA in ELISA buffer) solution to each well. Cover plates and incubate at 37ºC for 1 h. Wash plates 3 times with ELISA buffer.

            Add 100 µ1 of ABTS solution to each well. Cover and incubate plates at 37ºC for 10-30 min. If necessary, reincubate plates at 37ºC to obtain darker reactions.

            Results. Read optical density of each well at 410 nm on spectrophotometer [ELISA (EIA) reader]. Culture supernatants yielding optical density 0.1 unit greater than background are positive for production of either CT or cytotoxin.

          2. Reversed-passive-latex agglutination (RPLA) assay (Oxoid VET-RPLA TD20). This assay tests supernatants after simple centrifugation or filtration of 24 h, 35-37ºC AKI or CYE medium culture. See kit for instruction on assay set-up following supernatant preparation.

          3. Membrane ELISA

            4. Fill large petri dish with 50 ml TBS. Handle nitrocellulose membrane with forceps and gloves. Mark membrane with pencil for future orientation. Place membrane at angle into TBS buffer to wet thoroughly. Remove after 10 min and place on filter paper for 5 min. Displace 2 µ1 of each culture supernatant, including positive and negative control cultures and uninoculated medium, to tip of micropipet and touch to membrane surface. Place each sample 6-8 mm apart. To avoid increasing spot size, let membrane dry completely before applying additional 2 µ1 aliquots to spots. Let membrane dry completely for 5 min after application of last sample. About 75 cultures can be analyzed on a single membrane 9 cm in diameter. Immerse membrane in 50 ml TBS-3% gelatin solution for 1 h. Agitate solution intermittently or place on rotary shaker.

            Remove membrane from TBS-3% gelatin and transfer to 50 ml solution of rabbit anti-CT antiserum diluted 1:100 (or other appropriate dilution) in TBS-1% gelatin. Incubate 2-3 h at 25ºC with gentle agitation. Briefly rinse membrane in 50 ml double distilled water. Wash membrane 5 min with 50 ml TBS-Tween-20. Repeat washing procedure 3 more times.

            Transfer membrane from wash solution to 50 ml goat anti-rabbit IgG-peroxidase conjugate diluted 1:3000 (or other appropriate dilution) with TBS-1% gelatin. Incubate at 25ºC for 2 h with gentle agitation. Remove and wash membrane as above.

            Prepare R57 peroxidase color development solution immediately before use. Transfer membrane from wash solution into color development solution. CT-positive spots will appear as purple dots within 5 min. Avoid prolonged color development beyond 15-30 min. If precipitate forms in color development solution, prepare fresh solution and use immediately. Immerse membrane in distilled water to stop color development.

            Interpretation of data. Spots containing 1 ng or greater concentration of CT become visible as purple dots within 2-5 min. Negative control cultures may give a very faint spot if color development proceeds beyond 5 min. High titer sera should be diluted (1:200 or 1:400) to minimize background color changes that may be observed with CT-negative control cultures.

      8. Report. The following should appear in the final report: identification of isolate based on biochemical test results; serological results (Ol, Inaba, Ogawa, Hikojima, Non-O1, O139); biotype of V. cholerae Ol (El Tor or classical); and toxigenicity results (toxigenic or nontoxigenic, and cytotoxic or noncytotoxic if tissue culture was used). Notify Dr. Mahendra Kothary, FDA, telephone (202) 205-4454, of V. cholerae O1 and O139 isolates.
    4. Procedure for enrichment, isolation, and enumeration of V. parahaemolyticus, V. vulnificus, and other halophilic Vibrio spp. (see Figure 20-2)

      1. Stool sample. The importance of obtaining blood, tools, rectal swabs, or vomitus specimens in outbreaks cannot be overemphasized. Obtain specimens at the earliest opportunity because the carrier state is short-lived. Transport specimens in Cary-Blair medium, or inoculate immediately into APW enrichment broth, or streak onto TCBS and mCPC agar plates, depending on transit time. Incubate TCBS agar at 35ºC and mCPC agar at 39-40ºC for 18-24 h.

        If transit time will be longer than 8 h, place stool specimen in Cary-Blair transport medium. In the laboratory, streak loopful onto TCBS and mCPC agars.

        If transit time is 8 h or less, place stool specimen in APW. After 12-16 h incubation at 35-37ºC, streak enrichment broth on both TCBS and mCPC agars. Incubation times longer than 16 h result in progressively lowered bacterial viability.

        Sample rectal swab specimen (preferably with polyester fiber-tipped swab), contained in 7 ml Cary-Blair transport medium or APW, by streaking onto both TCBS and mCPC agars.

        Examine plates and proceed to biochemical identification.

      2. Food sample

        1. Sample composition

          2. Fish: surface tissues, gut, or gills

          Shellfish: entire interior contents of animal; pool 10-12 animals, homogenize, and remove 50 g from composite for test sample

          Crustaceans: entire animal, if possible, or central portion of animal, including gills and gut

        2. Test sample preparation

          3. V. parahaemolyticus. Aseptically prepare 1:10 dilution of 50 g seafood in 2 or 3% NaCl dilution water or PBS, pH 7.2-7.5, in sterile, tared blender jar. Blend 2 min at high speed. For example, blend 50 g seafood with 450 ml of 2 or 3% NaCl dilution water or PBS, OR blend 50 g seafood with 50 ml of dilution liquid (1:2); then make 1:5 dilution (20 g of 1:2 dilution in 80 ml dilution liquid) of the homogenate for a 1:10 total dilution. Prepare tenfold dilutions in 2 or 3% NaCl dilution water or PBS, pH 7.2-7.5. Inoculate 3-tube, multiple dilution, alkaline peptone water (APW) or alkaline peptone salt (APS) broth MPN series (i.e., add 1 ml portions of each 1:10 and higher dilution to sets of 3 tubes containing 10 ml APW or APS). Incubate tubes 16-18 h at 35-37ºC. NOTE: Inoculations of MPN tubes must be completed within 15-20 min of dilution preparation.

          V. vulnificus. Prepare APW original and 10-fold dilutions in PBS, pH 7.2-7.5. Inoculate MPN series as described for V. parahaemolyticus and incubate at 35-37ºC for 12-16 h.

          A rapid, specific enumeration technique for V. vulnificus, using a nonradioactive probe (Wright et al., 1993, Appl. Environ. Microbiol. 59:541-546) is currently being evaluated.

          NOTE: If enrichment is for both V. parahaemolyticus and V. vulnificus, use PBS, pH 7.2-7.5 and APW enrichment broth.

        3. After incubation do not shake tubes. For isolation of V. parahaemolyticus, for all dilutions containing a turbid tube, and at least one dilution higher, streak TCBS agar with 1 loopful from top 1 cm of each enrichment broth. For isolation of V. vulnificus streak on mCPC (Tamplin et al., 1991) agar. Also streak APW broth cultures of verified V. parahaemolyticus and V. vulnificus strains on TCBS and mCPC agar plates as controls for subsequent tests. Incubate TCBS at 35-37ºC and mCPC at 39-40ºC for 18-24 h. Optional: Use blood agar or mannitol-maltose agar, incubated at 35-37ºC for 18-24 h, to detect V. hollisae.

          4. TCBS agar. On TCBS agar, V. parahaemolyticus, V. vulnificus, V. mimicus, and V. harveyi are round, 2-3 mm diameter, green or blue-green colonies. V. alginolyticus, V. fluvialis, V. cholerae, V. metschnikovii, and some V. vulnificus colonies are larger and yellow (acid from sucrose fermentation).

          mCPC agar. On mCPC agar, V. vulnificus colonies are flat and yellow (acid from cellobiose fermentation) with opaque centers and translucent peripheries, about 2 mm in diameter. This is a presumptive identification of V. vulnificus. Non-cellobiose fermenters, such as V. cholerae El Tor, appear as purple or green, raised colonies. V. parahaemolyticus rarely grows on mCPC. Other species of Vibrio do not grow readily on mCPC agar. Pseudomonads produce purple or green colonies and are frequently observed at low dilutions of sample.

          For rapid confirmed identification of V. vulnificus, transfer colonies from mCPC agar to APW for the monoclonal antibody-EIA (Tamplin et al., 1991) (see 6-b, Serology, below). Use isolates confirmed by EIA to compute MPN of V. vulnificus in sample.

          A specific gene probe method, available for detection of cytotoxin-hemolysin gene of V. vulnificus, may be used as an additional presumptive identification procedure (see Chapter 24).

          Blood agar. Flood 18-24 h plate with oxidase reagent and pick oxidase-positive colonies (Hickman et al., 1982). Because V. hollisae does not grow on TCBS or mCPC agars, this nonselective method may isolate the organism. However, overgrowth by other bacteria may be a problem.

          Mannitol-maltose agar. On this nonselective medium, V. hollisae colonies are round, shiny, and purple (non-mannitol, non-maltose fermenting), whereas other Vibrio spp. are yellow (acid from mannitol and/or maltose fermentation) (Nishibuchi et al., 1988). Overgrowth by other bacteria may be a problem.

          Enumeration. After suspect colonies are identified biochemically or serologically with EIA, apply MPN tables (Chapter 10) for final enumeration of species.

      3. Procedure for biochemical identification

        1. Read GS and GA plates. Halophilic Vibrio spp. will grow only on GS plates. Most Vibrio spp. are gelatinase-positive and will form an opaque halo around growth.

        2. Oxidase test. Use growth from GS plate (or other medium with no fermentable carbohydrate) for oxidase test. Place 2 or 3 drops of oxidase test reagent on bacterial growth, or transfer small amount of growth with sterile toothpick or platinum loop to filter paper moistened with oxidase reagent. (Do not use nickel chromium loops.) Dark blue color should develop rapidly (within 2 min) for positive reaction. V. metschnikovii is the only oxidase-negative, pathogenic, halophilic Vibrio spp.

        3. From isolated colonies, inoculate motility test medium, AGS, TSI, TSB, TSA slant, and TSA plate for O/129 sensitivity test (all with final 2% NaCl concentration) and incubate 18-24 h at 35-37ºC. Use various tests in Tables 20-10 and 20-11 for identification.

          4. NOTE: Before proceeding, make sure culture does not grow on GA, is gelatinase-positive, and is pure. Vibrio spp. cultures often have 2 colony morphologies, which may or may not be stable.

          Motility test medium. Stab inoculum in center and to 2/3 the depth of motility test medium. Diffuse circular bacterial growth from line of stab is a positive test. V. vulnificus, V. parahaemolyticus, and related Vibrio spp. are motile. After 24 h, tightly cap tube and store at 20-25ºC to preserve culture.

          Arginine-glucose slant. Streak slant and stab butt of AGS, modified from Kaper et al. (Kaper et al., 1980). Vibrio spp. do not produce H2 S or gas. Typical reactions of V. parahaemolyticus and V. vulnificus are alkaline (purple) slant and acidic butt (yellow) (Tables 20-10 and 20-11).

          Triple sugar iron. Streak slant and stab butt of TSI agar. Vibrio spp. produce acidic butt (yellow) and do not produce gas or H2S. V. parahaemolyticus produces alkaline slant (red). V. vulnificus usually produces an alkaline slant (red) (Table 2). Use this or other medium containing lactose as source of inoculum for ONPG test.

          Inoculate tubes of TSA and TSB or motility test medium as source of inoculum for further testing.

          O/129 Vibriostat sensitivity. Use the disk diffusion method described above for polymyxin B sensitivity of V. cholerae O1 or place disks on densely streaked area of an isolation plate (TSA with 2% NaCl final concentration). Use disks containing 10 and 150 µg of vibriostat O/129. Vibrio spp. are sensitive to 150 µg of O/129, but some are resistant to 10 µg of O/129. See Table 3 for differentiation based on sensitivity to 10 µg of O/129. Disks are commercially available or can be prepared in the lab. Alternatively, use TSA agar containing 10 or 150 µg of O/129 per ml.

        4. Continue identification tests after finding typical reactions in media inoculated previously (Tables 20-10 and 20-11). Compute MPN of V. parahaemolyticus (see Chapter 10), based on number of tubes containing V. parahaemolyticus.

          5. ONPG test. Perform ONPG test using portion of culture from TSI or other medium containing lactose. Use conventional tube test (preferred) in fume hood, or commercially available disks. Strip tests for ONPG are sometimes unreliable for Vibrio spp. V. vulnificus is ONPG-positive; V. parahaemolyticus is ONPG-negative.

          Rapid test strips. Use multiwell (e.g., API 20E) strips as alternative to conventional tube format for biochemical tests. However, some Vibrio spp. will not grow in commercial test strip media when physiological saline (0.85% NaCl) is used as diluent. Use 2% NaCl as diluent, since most halophilic Vibrio spp. require higher concentration of NaCl (MacDonell et al., 1982). If commercial test strips do not allow identification, continue with conventional tests.

          Hugh-Leifson glucose broth or OF glucose medium, semisolid. Inoculate 2 tubes of medium with growth from TSA. Overlay medium in 1 tube with sterile mineral oil to a depth of 1-2 cm, and incubate 2 d at 35 ± 2ºC. Vibrio spp. ferment glucose and produce acid oxidatively. Acid causes dye to change from purple to yellow in Hugh-Leifson glucose broth and from green to yellow in OF glucose medium.

          Arginine dihydrolase, 1ysine decarboxylase, and ornithine decarboxylase. Inoculate 1 tube of each of the 3 media containing amino acid and 1 tube lacking amino acid. (The arginine reaction can also be read from the AGS tube: acid butt (yellow) from glucose fermentation means isolate is negative for arginine dihydrolase.) Overlay each tube with sterile mineral oil 1-2 cm thick, and incubate 4 d at 35-37ºC. Examine tubes every day. Alkaline pH resulting from decarboxylation of amino acids turns medium purple (positive). Yellow color results from acid production from glucose fermentation (negative). Control tubes containing no amino acid should be yellow. Purple color medium in control tubes indicates no growth. Most V. parahaemolyticus and V. vulnificus strains are arginine dihydrolase-negative, lysine decarboxylase-positive, and ornithine decarboxylase-positive. Some V. vulnificus and V. parahaemolyticus are ornithine decarboxylase-negative. Rare strains of V. vulnificus are lysine decarboxylase-negative.

          Salt tolerance. From TSB culture, inoculate 1 tube each of 1% tryptone broth containing 0, 1, 3, 6, 8, or 10% NaCl (T1N0, T1N1, T1N3, T1N6, T1N8, or T1N10), and incubate 18-24 h at 35-37ºC. Consider only profuse growth as positive. Halophilic Vibrio spp. do not grow in broth containing 0% NaCl, but all Vibrio spp. grow in broth containing 3% NaCl. Various species have different salt tolerances that can be used for identification (Table 20-12).

          Growth at 42ºC. Inoculate prewarmed tube of TSB containing 2% NaCl with small loopful of 24 h TSB-2% NaCl culture. Incubate in 42ºC water bath for 24 h. Consider only profuse growth as positive. V. cholerae, V. parahaemolyticus, V. alginolyticus, and V. vulnificus grow at 42ºC.

          Voges-Proskauer (VP) test. Inoculate MR-VP broth containing NaCl with growth from TSA slant and incubate 2 d at 35-37ºC. Perform VP test. V. parahaemolyticus, V. vulnificus, and V. fluvialis are VP-negative.

          Carbohydrate fermentation. From growth on TSA slant, inoculate 1 tube each of bromcresol purple broth or OF medium containing NaCl, semisolid, containing sucrose, lactose, D-mannitol, mannose, arabinose, or cellobiose. Overlay medium with sterile mineral oil to depth of 1-2 cm and incubate at 35-37ºC for 4-5 d. Acidic fermentation turns medium yellow. Check tubes every 24 h and compare reactions to those in Table 3. Occasional strains of V. vulnificus are mannitol-negative.

          Urea hydrolysis. Test presumptive V. parahaemolyticus for urea hydrolysis by inoculating Christensen's urea agar tubes or plates and incubating at 35-37ºC for 18 h. V. parahaemolyticus strains vary in ability to hydrolyze urea. Urea hydrolysis may be correlated with certain somatic (O-antigen) groups.

          NOTE: Urease-positive strains give API codes not found in the ID book. Call API for confirmation of strain.

          Culture preservation. Inoculate semisolid, long-term preservation medium or motility test medium by stabbing deeply into agar. Incubate 24 h at 35-37ºC. Tighten caps after 24 h to prevent dehydration. Alternatively, add layer of sterile mineral oil to 24 h cultures in motility test medium. Store cultures at room temperature after initial growth. DO NOT REFRIGERATE. For long-term preservation, place 1 ml of 6-12 h TSB-2% NaCl culture and 0.1 ml sterile glycerol into sterile cryotubes. Freeze immediately at -70ºC or in liquid nitrogen.

        5. Kanagawa phenomenon. The Kanagawa reaction demonstrates the presence of a specific thermostable direct hemolysin (TDH) on Wagatsuma agar. A positive reaction correlates closely with pathogenicity of V. parahaemolyticus isolates. Strains recovered from seafood are usually Kanagawa-negative.

          6. Some clinical isolates of V. parahaemolyticus produce related hemolysins but not TDH. Two other hemolysins, having sequence homology with TDH but exhibiting no hemolysis on Wagatsuma agar, were recently identified and purified (Honda et al., 1988).

          Fresh human or rabbit red blood cells (within 24 h of draw) are necessary for preparation of Wagatsuma agar.

          Spot droplet from 18 h TSB-3% NaCl culture on duplicate plates of well-dried Wagatsuma agar. Spot several cultures including verified positive and negative controls in circular pattern on plate. Incubate at 35-37ºC and observe results in 24 h.

          A positive test is zone of ß-hemolysis, i.e., sharply defined zone of transparent clearing of red blood cells around colony, without multiple concentric rings or greening.

          Measure zone of hemolysis from edge of colony to outer edge of zone. Isolates that produce clear zone of hemolysis 3 mm or larger are considered Kanagawa phenomenon-positive and are presumed to be pathogenic. Isolates that produce clear zones of hemolysis of less than 3 mm may be weakly pathogenic and should be tested in rabbit ileal loop assay (Twedt et al., 1980). No observation of plate beyond 24 h is valid.

          A gene probe method for detecting TDH of V. parahaemolyticus is available (see Merker (1988), Chapter 24). V. hollisae is positive for TDH by gene probe, but its hemolysin cannot be detected on Wagatsuma agar. Request gene probe sequence tdh-3 from servicing laboratory.

    5. Characteristics for biochemical identification of V. parahaemolyticus and V. vulnificus

      6. The following characteristics are presumptive of V. parahaemolyticus or V. vulnificus:

      • Morphology: Gram-negative asporogenous rod
      • TSI appearance: V. parahaemolyticus, alkaline slant/acid butt, gas production-negative, H2S-negative; V. vulnificus, alkaline slant (rarely acidic)/acid butt, gas production-negative, H2S-negative
      • Hugh-Leifson test: Glucose oxidation and fermentation-positive
      • Cytochrome oxidase: Positive
      • Arginine dihydrolase test: Negative
      • Lysine decarboxylase test: Positive (rare V. vulnificus are lysine decarboxylase-negative)
      • Voges-Proskauer test: Negative
      • Growth at 42ºC: Positive
      • Halophilism test: V. parahaemolyticus: 0% NaCl-negative; 3, 6, and 8% NaCl-positive; 10% NaCl-negative or poor. V. vulnificus: 0% NaCl-negative; 3, 6% NaCl-positive; 8% NaCl-negative
      • Sucrose fermentation: Negative (rare V. vulnificus are positive)
      • ONPG test: V. parahaemolyticus, negative; V. vulnificus, positive
      • Arabinose fermentation: V. parahaemolyticus, usually positive (variable); V. vulnificus, negative
      • Sensitivity to O/129: V. parahaemolyticus: sensitive to 150 µg, resistant to 10 µg; V. vulnificus: sensitive to 10 and 150 µg.

      Table 20-13. Antigenic scheme of V. parahaemolyticusa

      O Group

      K Antigen

      1

      1, 25, 26, 32, 38, 41, 56, 58, 64, 69

      2

      3, 28

      3

      4, 5, 6, 7, 29, 30, 31, 33, 37, 43, 45, 48, 54, 57, 58, 59, 65

      4

      4, 8, 9, 10, 11, 12, 13, 34, 42, 49, 53, 55, 63, 67

      5

      15, 17, 30, 47, 60, 61, 68

      6

      18, 46

      7

      19

      8

      20, 21, 22, 39, 70

      9

      23, 44

      10

      19, 24, 52, 66, 71

      11

      36, 40, 50, 51, 61
      aFrom Twedt (1989), R. Sakazaki (1986).

    1. Serology

      1. Serological identification of V. parahaemolyticus. Determination of somatic (O) and capsular (K) serotypes of V. parahaemolyticus is not required for identification. Serotyping antisera are expensive and few, if any, FDA labs have the complete set. Contact Charles Kaysner (206) 483-8788 for possible referral of isolates. For those who wish to determine the O and K antigens (Table 20-13), the following protocol is offered.

        1. Inoculate 2 slants of TSA-2% NaCl; incubate at 35-37ºC for 18-24 h.

        2. Somatic (O) antigen

          3. Preparation. Wash growth from one TSA-2% NaCl slant with solution containing 2% NaCl and 5% glycerol; transfer to autoclavable centrifuge tube. Autoclave suspension at 121ºC for 1 h. Centrifuge suspension at 4000 rpm for 15 min. Resuspend the packed cells in 2% NaCl. A heavy suspension is best for this slide agglutination test.

          Determination. With wax pencil, divide microscope slide into 12 equal compartments. Place small drop of heavy suspension into each compartment. Add 1 drop of the 11 O-group antisera to separate compartments. Add 1 drop of 2% NaCl to 12th compartment (autoagglutination control). Tilt slide gently to mix all components, and rock slide back and forth for 1 min. Positive agglutination may be read immediately.

          If no agglutination occurs with any of the 11 O antisera, autoclave the suspension at 121ºC again for 1 h and retest. If agglutination is still negative, the O antigens of the culture are unknown.

        3. Capsular (K) antigen

          4. Preparation. Capsular (K) antigen. Wash growth from one TSA-2% NaCl slant with 2% NaCl solution to make a smooth heavy suspension of cells.

          Determination. Test first with pooled K antisera (I-IX), and then with each of the monovalent K antisera within the pool showing agglutination. (Each pool consists of 8-10 flagellar agglutinins.)

          On slide, mark off appropriate number of compartments plus control compartment. Place small drop of heavy cell suspension and add 1 drop of appropriate K antiserum to individual compartments. Add 1 drop of 2% NaCl to autoagglutination control. Tilt slide gently to mix components, and rock slide back and forth for 1 min. Positive agglutination may be read immediately.

      2. V. vulnificus EIA (Tamplin et al., 1991). Use EIA specific for intracellular antigen to confirm identity of V. vulnificus isolates directly from mCPC agar (yellow translucent colonies with opaque centers).

        3. Prepare log phase cultures. Transfer 2 typical V. vulnificus colonies from each inoculated plate and confirmed culture of V. vulnificus, using sterile wooden sticks, toothpicks, or inoculating loop, to individual wells of 96-well plate (tissue culture cluster plate) containing 100 µ1 APW per well. Incubate 3-4 h, or until turbid, at 35-37ºC.

        Coat enzyme immunoassay (EIA) plates. After incubating microtiter plates, transfer 25 µ1 from each cluster plate well to one well of a 96-well EIA plate. Add 25 µ1 EIA coating solution (0.02% Triton X-100) to each well. Place EIA plates in dry 35ºC incubator overnight to evaporate samples in wells.

        Optional: To store isolates after transfer to EIA plates, add equal volume sterile TSB supplemented with 1% NaCl and 24% glycerol to each well of tissue culture plate. Isolates can be stored indefinitely at -70ºC.

        Block binding sites. Remove dried EIA plates from incubator. To reduce nonspecific binding of reagents, add 200 µ1 of 1% BSA in PBS to each well. Incubate at room temperature for 1 h.

        Discard BSA. Remove BSA solution by firmly slapping plates onto countertop covered with absorbent towels.

        Add monoclonal antibody. Prepare diluted (e.g., 1:4) monoclonal antibody specific for V. vulnificus in PBS. Add 50 µ1 to test wells. Control wells receive antibody with specificity other than V. vulnificus, tissue culture media, or PBS. Incubate at room temperature for 1 h. Wash plate 3 times with wash solution.

        Add conjugate. Dilute peroxidase-conjugated goat anti-mouse IgG with PBS. Add 50 µ1 to each well and incubate in dark at room temperature for 1 h. Wash 5 times.

        Add substrate. Add 100 µ1 freshly prepared ABTS substrate solution to each well. Incubate about 10 min at room temperature, or until maximum color develops (usually less than 30 min). Compare negative controls to respective test wells for positive reactions. A well is usually considered positive if its optical density is 0.200 above that of negative control. An EIA plate reader is normally not required to differentiate reactions, but if used, read optical density at 410 nm.

    2. Gene probes

      3. Gene probes (oligonucleotides) for V. cholerae enterotoxin (CTX All), V. parahaemolyticus thermostable direct hemolysin (TDH-3), and V. vulnificus cytotoxin-hemolysin are available from Dr. Joseph Madden, FDA, 200 C St., SW, Washington, DC 20204, or from Fannie Harrell, HFR-MW460, MCI, MIN-DO, 240 Hennepin Ave., Minneapolis, MN 55401.

      These probes are for genes associated with pathogenicity or species specificity. See Merker (1988), Chapter 24 for gene probe methods.

    3. Fatty acid analysis

      4. Vibrio spp. may be identified by gas chromatographic analysis of cellular fatty acids. Warren Landry, FDA, Dallas District Office (214) 655-5308, has developed a computer library for identification of many bacterial species, including Vibrio spp., using the Hewlett-Packard Microbial Identification System. The equipment is not available in all FDA laboratories, but unusual Vibrio spp. isolates may be sent to the Dallas laboratory for study and confirmation.

Contents

Other analytical procedures

Contents

Commercial Test Products

Table 20-14. Commercial test products for V. cholerae.

Test Kit

Analytical Technique

Approx. Total Test Time1

Supplier

CHECK 3 Vibrio sp.

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

ISO-GRID Method for Vibrio parahaemolyticus Count using VSP agar

Membrane filtration with selective and differential culture medium using sucrose 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

VET-RPLA TD920
[Used to identify V. cholerae 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

Contents

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