Part 1: Detection of Halophilic Vibrio Species in Seafood

Laboratory Procedure MFLP-37
October 2006

Health Products and Food Branch
Ottawa

Microbiological Methods Committee
Microbiology Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Health Products and Food Branch, Health Canada
Postal Locator: 2204A1
Ottawa, Ontario K1A 0L2

E-mail: Don_Warburton@sc-hc.gc.ca

1. APPLICATION

The method may be used for the isolation and enumeration of Vibrio parahaemolyticus, Vibrio vulnificus and other halophilic Vibrio spp. in seafood to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. This revised method replaces MFHPB-15 dated April 1997 and MFLP-57 dated September 1996.

2. DESCRIPTION

The genus Vibrio consists of Gram negative, rod- or curved-rod shaped facultative anaerobes. Most species are oxidase positive and halophilic; that is, they require additional sodium chloride in growth media. The exceptions are V. cholera and V. mimicus (non-halophilic). Many Vibrio spp., including Vibrio cholera, Vibrio parahaemolyticus and Vibrio vulnificus are human pathogens.

3. PRINCIPLE

Detection of Vibrio parahaemolyticus and Vibrio vulnificus, as well as other halophilic vibrios, requires three successive phases: (i) enrichment in selective medium, (ii) plating out onto isolation agars and presumptive identification, and (iii) confirmation with biochemical, serological and pathogenicity tests. This method is based on a procedure published in the Bacteriological Analytical Manual (BAM) (8.9).

4. DEFINITION OF TERMS

See Appendix A of Volume 3

5. COLLECTION OF SAMPLES

See Appendix B of Volume 3

6.0 PROCEDURE

Each sample unit may be analyzed individually or the analytical units may be combined where requirements of the applicable sampling plan can be met.

Salt content of media follows in square brackets [NaCl%].

1. Alkaline peptone salt broth (APS) [3%]
2. Phosphate buffered saline (PBS) [0.85%]
3. Alkaline peptone water (APW) [1%]
4. Thiosulphate citrate bile salts agar (TCBS) [1%]
5. Modified cellobiose-polymyxin-B-colistin (mCPC) [2%]
6. Blood agar (optional) [0.5%] - optional to add NaCl for halophiles
7. Mannitol-maltose agar (optional) [2%]
8. T₁N₂ agar (1% tryptone and 2% NaCl) (optional)
9. 1% Tryptone broths containing 0, 1, 3, 6, 8 and 10% NaCl (T₁N₀, T₁N₁, T₁N₃, T₁N₆, T₁N₈, T₁N₁₀)
10. Trypticase soy agar (TSA) [0.5%] - add 1.5% NaCl for halophiles
11. Arginine glucose slant (AGS) [2%]
12. Gelatin agar (GA) [0%]
13. Gelatin agar with 3% NaCl (GS)
14. Decarboxylase basal medium containing arginine, lysine, ornithine - optional to add NaCl for halophiles
15. MR-VP broth and V-P reagents - optional to add NaCl to broth for halophiles
16. Carbohydrates in bromcresol purple broth or OF broth, semi-solid: sucrose, lactose, D-mannitol, mannose, arabinose, cellobiose [0.5%] - optional to add NaCl for halophiles
17. Wagatsuma agar [7%]
18. Hugh-Leifson glucose broth [3%] OR OF glucose medium, semi-solid [0.5%]
19. Christensen's urea agar slants [0.5%] - optional to add NaCl for halophiles
20. Gram stain solutions
21. Motility test medium [0.5%] - optional to add NaCl for halophiles
22. Triple sugar iron agar (TSI) [0.5%] - optional to add 2.5% NaCl for halophiles
23. API 20E identification strips (bioMérieux Inc.) or equivalent
24. Oxidase reagent or commercially available test strips
25. O-nitrophenyl-ß-D galactoside (ONPG) test solution or commercial product
26. Sterile mineral oil
27. Positive and negative control cultures (ATCC or equivalent)
28. 2% NaCl in distilled water (for dilution and inoculation of API)
29. Incubators, 35°C, 40°C and 42°C
30. Blender, stomacher or equivalent

7. PROCEDURE

7.1 Sample composition:

1. Fish: surface tissues, gut or gills
2. Shellfish: entire interior contents of animal; pool 10-12 animals plus more if needed, blend, not stomach, and remove 50 g from composite for test sample
3. Crustaceans: entire animal, if possible, or central portion of animal, including gills and gut

7.2 Handling of Sample Units

7.2.1

In the laboratory, prior to analysis, keep sample units refrigerated (4-8°C), at room temperature or frozen, depending on the nature of the product. Thaw the frozen samples in a refrigerator or under time and temperature conditions which prevent microbial growth or death.

7.2.2

Analyze thawed sample units as soon as possible after their receipt in the laboratory to minimize overgrowth and to reduce the potential for viable but non-culturable vibrios (VBNC).

7.3 Preparation of Dilutions

7.3.1 V. parahaemolyticus:

7.3.1.1 Aseptically prepare a 1:10 dilution by combining 50 g of seafood with 450 mL of phosphate buffered saline (PBS), pH 7.2-7.5 or 2% NaCl in a sterile blender jar or stomacher bag. Blend shellfish for 2 min at high speed. Blend or stomach other samples for 2 min.

Alternately: Blend 50 g of seafood with 50 mL of dilution liquid (1:2); then make a 1:5 dilution (20 g of a 1:2 dilution in 80 mL dilution liquid) of the homogenate for a 1:10 total dilution.

7.3.1.2 Prepare tenfold dilutions in 2% NaCl or PBS, pH 7.2-7.5. Inoculate a 3-tube, multiple dilution, MPN series using alkaline peptone water (APW) or APS broth (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°C. Inoculation of MPN tubes must be completed within 15-20 min of dilution preparation.

7.3.2 V. vulnificus.

7.3.2.1 Prepare initial 1:10 dilution in APW and subsequent 10-fold dilutions in PBS, pH 7.2-7.5. Inoculate MPN series as described for V. parahaemolyticus and incubate at 35°C for 12-16 h.

7.4 Plating

7.4.1

After incubation do not shake culture tubes.

7.4.2

For isolation of V. parahaemolyticus, examine the tubes for turbidity. Streak all dilutions showing visible turbidity plus the next highest (non-turbid) dilution, by taking a loopful of culture from the top 1 cm of each broth. Streak onto thiosulphate citrate bile salts agar (TCBS)

7.4.3

For isolation of V. vulnificus, streak broths onto modified cellobiose-polymyxin-B-colistin (mCPC) using the technique described in 7.4.2.

7.4.4

Streak APS/APW control cultures of V. parahaemolyticus and V. vulnificus onto TCBS and mCPC.

7.4.5

Incubate TCBS at 35°C and mCPC at 40°C for 18-24 h.

7.4.6 Optional:

V. hollisae

Use blood agar or mannitol-maltose agar, incubated at 35°C for 18-24 h, to detect V. hollisae.

7.5 Identification

7.5.1

Examine TCBS and mCPC agars for typical V. parahaemolyticus and V. vulnificus colonies, as well as for characteristics of other Vibrio spp..

7.5.1.1 Thiosulfate citrate bile salts sucrose (TCBS) agar:

On TCBS agar, V. parahaemolyticus, V. vulnificus, V. mimicus, and V. mimicus are round, 2-3 mm in 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).

7.5.1.2 Modified cellobiose-polymyxin B-colistin (mCPC) agar

On mCPC agar, V. vulnificus colonies are flat and yellow (acid from cellobiose fermentation) with opaque centres 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.

7.5.1.3 Blood agar

Flood 18-24 h plate with oxidase reagent and pick oxidase positive (dark blue) colonies (8.15). Because V. hollisae does not grow on TCBS or mCPC agars, this non-selective method may isolate the organism. However, overgrowth by other bacteria may be a problem.

7.5.1.4 Mannitol-maltose agar

On this non-selective 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). Overgrowth by other bacteria may be a problem.

7.5.2

Pick 3 or more typical or suspicious colonies from each medium and streak T₁N₂ agar (1% tryptone and 2% NaCl) or tryticase soy agar (TSA) + 1.5% NaCl (final 2% NaCl concentration) for isolation. Incubate 18-24 h at 35°C.

7.5.3 Salt tolerance: gelatin agar (GA) and gelatin agar with 3% NaCl (GS)

Using the same colonies, test for salt tolerance by dividing GA and GS plates into 8 sectors. Inoculate a straight line in the centre of one sector of both GA and GS plates with each isolate. Incubate 18-24 h at 35°C. 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 the plate above a black surface. An opaque halo will be present around the growth of gelatinase positive organisms. Most Vibrio spp. are gelatinase positive.

7.5.4 Preliminary Biochemical identification:

7.5.4.1 Rapid identification tests

API 20E strips or equivalent rapid identification kits may be used as an alternative to conventional tube media for biochemical tests. However, some Vibrio spp. will not grow in commercial test strip media when physiological saline (0.85% NaCl) is used as the diluent. Use 2% NaCl as the diluent. If commercial test strips do not allow identification, continue with conventional tests.

7.5.4.2 Oxidase test

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

7.5.4.3

From isolated colonies, inoculate motility test medium, AGS, triple sugar iron agar slant (TSI). Also inoculate trypticase soy broth (TSB), TSA slant and TSA plate all with a final 2% NaCl concentration required for additional tests. Incubate for 18-24 h at 35°C. Use various tests in Table 2 and 3 for identification.

a) Motility test medium
Stab inoculum in centre and to 2/3 the depth of motility test medium. Incubate 18-24 h at 35°C. 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 20-25°C to preserve culture.

b) Arginine-glucose slant
Streak slant and stab butt of AGS. Incubate tubes loosely stoppered or capped for 18-24 h at 35°C. Vibrio spp. do not produce H₂S or gas. Typical reactions of V. parahaemolyticus and V. vulnificus are alkaline (purple) slant and acidic butt (yellow). Refer to Table 1 and 2 for interpretation of results.

c) Triple sugar iron agar slant
Streak slant and stab butt of TSI agar. Incubate tubes loosely stoppered or capped for 18-24 h at 35°C. 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). Refer to Table 1 and 2 for interpretation of results.

Use this or other medium containing lactose as the inoculum source for the ONPG test.

7.5.4.4 O/129 Vibriostat sensitivity

Place disks containing 10 and 150 µg of vibriostat O/129 on a densely streaked area of a TSA with 2% NaCl final concentration. Invert plates and incubate for 18-24 h at 35°C. Vibrio spp. are sensitive to 150 µg of O/129, but some are resistant to 10 µg of O/129. Refer to Table 2 for interpretation of results.

7.5.5

If typical reactions are observed, continue with identification tests. Compute MPN of V. parahaemolyticus (see Table 4), based on the number of tubes containing V. parahaemolyticus.

7.5.6 Other Biochemical Tests

7.5.6.1 ONPG test

Perform ONPG test using inoculum from the TSI culture. or other medium containing lactose. Use conventional tube test (preferred) in the fume hood or commercially available disks. V. vulnificus is ONPG positive; V. parahaemolyticus is ONPG negative.

7.5.6.2 Oxidation-fermentation test

Inoculate 2 tubes of Hugh-Leifson glucose broth or OF glucose medium, semi-solid with growth from an isolated colony. Overlay medium in one tube with sterile mineral oil or liquid Vaspar (50% petrolatum, 50% parafin) to a depth of 1-2 cm and incubate 1-2 days or more at 35°C. Acid causes the dye to change from purple to yellow in Hugh-Leifson broth and from green to yellow in OF medium, semi-solid. 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.

7.5.6.3 Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase

Inoculate 1 tube of each of the 3 amino acid media and 1 control tube without amino acid. (The arginine reaction can also be read from the AGS tube: acid butt (yellow) from glucose fermentation means the isolate is negative for arginine dihydrolase.) Overlay each tube with 1-2 cm of sterile mineral oil and incubate 4 days at 35°C. Examine tubes each day. Decarboxylation of amino acids results in an alkaline pH which turns the medium purple (positive). A yellow colour is caused by acid production from glucose fermentation (negative). Control tubes containing no amino acid should be yellow. Purple colour 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.

7.5.6.4 Salt tolerance

From the TSB culture, inoculate 1 tube each of 1% tryptone broth containing 0, 1, 3, 6, 8 and 10% NaCl (T₁N₀, T₁N₁, T₁N₃, T₁N₆, T₁N₈, T₁N₁₀), and incubate 18-24 h at 35°C. 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 (see Table 2).

7.5.6.5 Growth at 42°C

Inoculate a pre-warmed tube of TSB containing 2% NaCl with a small loopful of 24 h TSB-2% NaCl culture. Incubate in a 42°C waterbath or incubator for 24 h. Consider only profuse growth as positive. V. cholerae, V. parahaemolyticus, V. alginolyticus, and V. vulnificus grow at 42°C.

7.5.6.6 Voges-Proskauer (V-P) test

Inoculate MR-VP broth containing NaCl with growth from a TSA slant and incubate 2 days at 35°C. Perform the V-P test. V. parahaemolyticus, V. vulnificus, and V. fluvialis are V-P negative.

7.5.6.7 Carbohydrate fermentation

From growth on the TSA slant, inoculate 1 tube each of the following carbohydrates: sucrose, lactose, D-mannitol, mannose, arabinose and cellobiose. Prepare the media in bromcresol purple broth or OF medium, semisolid, with NaCl. Overlay medium with sterile mineral oil to a depth of 1-2 cm and incubate at 35°C for 4-5 days. Acidic fermentation turns medium yellow. Check tubes every 24 h. Occasional strains of V. vulnificus are mannitol negative. See Table 2 for interpretation of results.

7.5.6.8 Urea hydrolysis

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

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

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

1. Morphology: Gram negative asporogenous rod
2. 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
3. Oxidation-fermentation: Glucose oxidation and fermentation positive
4. Cytochrome oxidase: Positive
5. Arginine dihydrolase test: Negative
6. Lysine decarboxylase test: Positive (rare V. vulnificus are lysine decarboxylase negative)
7. Voges-Proskauer test: Negative
8. Growth at 42°C: Positive
9. 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
10. Sucrose fermentation: Negative (rare V. vulnificus are positive)
11. ONPG test: V. parahaemolyticus negative; V. vulnificus positive
12. Arabinose fermentation: V. parahaemolyticus, usually positive (variable); V. vulnificus negative
13. Sensitivity to O/129: V. parahaemolyticus: sensitive to 150 µg, resistant to 10 µg; V. vulnificus: sensitive to 10 and 150 µg.

7.7 Enumeration

After suspect colonies are identified, apply MPN tables (Table 4) for final enumeration of species.

7.8 Pathogenicity test

7.8.1 Kanagawa phenomenon (optional)

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.

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

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

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

Measure the zone of hemolysis from the edge of the colony to the outer edge of the zone. Isolates that produce a 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 (8.32).

7.9 Serology

Serological identification of V. parahaemolyticus

Determination of somatic (O) and capsular (K) serotypes (Table 3) of V. parahaemolyticus is not required for identification. Serotyping antisera are difficult to obtain.

Method:

7.9.1

Inoculate 2 slants of TSA-2% NaCl; incubate at 35°C for 18-24 h.

7.9.2 Somatic (O) antigen

1. Preparation: Wash growth from one TSA-2% NaCl slant with a solution containing 2% NaCl and 5% glycerol; transfer to an autoclavable centrifuge tube. Autoclave suspension at 121°C 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.
2. With a wax pencil, divide microscope slide into 12 equal compartments. Place a 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 12^th compartment (autoagglutination control). Tilt the slide gently to mix all components and rock slide back and forth for 1 min. Positive agglutination may be read immediately.
3. If no agglutination occurs with any of the 11 O antisera, autoclave the suspension at 121°C again for 1 h and retest. If agglutination is still negative, the O antigens of the culture are unknown.

7.9.3 Capsular (K) antigen

1. Wash growth from one TSA-2% NaCl slant with 2% NaCl solution to make a smooth heavy suspension of cells.
2. 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.)
3. On slide, mark off appropriate number of compartments plus control compartment. Place a 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.

7.10 Culture preservation

Inoculate semi-solid, long-term preservation medium or motility test medium by stabbing deeply into the agar. Incubate 24 h at 35°C. Tighten caps after 24 h to prevent dehydration. Alternatively, add a 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 -70°C or in liquid nitrogen.

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8.30 Tisan, D.L. 1999. Vibrio. In: Manual of Clinical Microbiology, 7^th ed. P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, R.H. Yolken (eds.), ASM Press, Washington, D.C.

8.31 Twedt, R.M. 1989. Vibrio parahaemolyticus, pp. 543-568. In: Foodborne Bacterial Pathogens. M.P. Doyle (ed). Marcel Dekker, New York.

8.32 Twedt, R.M., J.T. Peeler and P.L. Spaulding. 1980. Effective ileal loop dose of Kanagawa-positive Vibrio parahaemolyticus. Appl. Environ. Microbiol. 40:1012-1016.

8.33 West, P.A. and R.R. Colwell. 1984. Identification and classification of Vibrionaceae--an overview, pp. 285-363. In: Vibrios in the environment. R.R. Colwell (ed). John Wiley & Sons, New York.

8.34 West, P.A., P.R. Brayton, T.N. Bryant and R.R. Colwell. 1986. Numerical taxonomy of vibrios isolated from aquatic environment. Int. J. Syst. Bacteriol. 36:531-543.

Table 1. Reactions ^a of certain Vibrio spp. and related microorganisms in differential tube agar media

Microorganism	KIA b		TSI b		AGS b
	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
V. vulnificus	K or A	A	K (A rare)		K	A
A. hydrophila c	K or A	A	K or A	A	K	K
P. shigelloides	K or A	A	K or A	A	ND	ND

Table 2. Biochemical characteristics of selected Vibrionaceae

	V. cholerae	V. hollisae	V. mimicus	V. parahaemolyticus	V. vulnificus
*TCBS agar	Y	NG	G	G	G
mCPC agar	P	NG	NG	NG	Y
AGS medium	Ka	Ka	KA	KA	KA
Growth in:
0% NaCl	+	-	+	-	-
3% NaCl	+	+	+	+	+
6% NaCl	-	+	-	+	+
8% NaCl	-	-	-	+	-
10% NaCl	-	-	-	-	-
Growth at 42°C	+	nd	+	+	+
Acid from:
Sucrose	+	-	-	-	-
D-Cellobiose	-	-	-	V	+
Lactose	-	-	-	-	+
Arabinose	-	+	-	+	-
D-Mannose	+	+	+	+	+
D-Mannitol	+	-	+	+	V
Oxidase	+	+	+	+	+
ONPG	+	-	+	-	+
Voges-Proskauer	V	-	-	-	-
Arginine dihydrolase	-	-	-	-	-
Lysine decarboxylase	+	-	+	+	+
Ornithine	+	-	+	+	+
Sensitivity to:
10 µg 0/129	S	nd	S	R	S
150 µg 0/129	S	nd	S	S	S
Gelatinase	+	-	+	+	+
Urease	+	V	-	-	V

Table 3. Antigenic scheme of V. parahaemolyticus ^a

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

Table 4. For 3 tubes each at 0.1, 0.01, and 0.001 g inocula, the MPNs per gram and 95 percent confidence intervals

Pos. tubes			MPN/g	Conf. lim.		Pos. tubes			MPN/g	Conf. lim.
0.10	0.01	0.001		Low	High	0.10	0.01	0.001		Low	High
0	0	0	<3.0	--	9.5	2	2	0	21	4.5	42
0	0	1	3.0	0.15	9.6	2	2	1	28	8.7	94
0	1	0	3.0	0.15	11	2	2	2	35	8.7	94
0	1	1	6.1	1.2	18	2	3	0	29	8.7	94
0	2	0	6.2	1.2	18	2	3	1	36	8.7	94
0	3	0	9.4	3.6	38	3	0	0	23	4.6	94
1	0	0	3.6	0.17	18	3	0	1	38	8.7	110
1	0	1	7.2	1.3	18	3	0	2	64	17	180
1	0	2	11	3.6	38	3	1	0	43	9	180
1	1	0	7.4	1.3	20	3	1	1	75	17	200
1	1	1	11	3.6	38	3	1	2	120	37	420
1	2	0	11	3.6	42	3	1	3	160	40	420
1	2	1	15	4.5	42	3	2	0	93	18	420
1	3	0	16	4.5	42	3	2	1	150	37	420
2	0	0	9.2	1.4	38	3	2	2	210	40	430
2	0	1	14	3.6	42	3	2	3	290	90	1,000
2	0	2	20	4.5	42	3	3	0	240	42	1,000
2	1	0	15	3.7	42	3	3	1	460	90	2,000
2	1	1	20	4.5	42	3	3	2	1100	180	4,100
2	1	2	27	8.7	94	3	3	3	>1100	420	--

Source: BAM (8.8)

---

PART 2: DETECTION OF VIBRIO CHOLERAE

1. APPLICATION

The method may be used for the detection of Vibrio spp. in foods to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act.

2. DESCRIPTION

Vibrio cholerae is a Gram negative, oxidase positive, rod- or curved-rod shaped facultative anaerobe. Unlike most Vibrio spp., Vibrio cholerae is non-halophilic; i.e, it does not require additional sodium chloride in growth media. The species is comprised of several somatic (O) antigenic groups and subgroups.

3. PRINCIPLE

Detection of Vibrio cholerae requires three successive phases: (i) enrichment in selective medium, (ii) plating onto isolation agars and presumptive identification, and (iii) confirmation with biochemical, serological, and toxigenicity tests. This method is based on a procedure published in the Bacteriological Analytical Manual (BAM) (8.7).

4. DEFINITION OF TERMS

See Appendix A of Volume 3.

5. COLLECTION OF SAMPLES

See Appendix B of Volume 3.

6. MATERIALS AND SPECIAL EQUIPMENT

The following media and reagents (1-24) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 and reference 8.1 for the formula of individual media.

Salt content of media follows in square brackets [NaCl %].

1. Alkaline peptone water (APW) [1%]
2. Phosphate buffered saline (PBS) (optional) [0.85%]
3. Thiosulphate citrate bile salts agar (TCBS) [1%]
4. Modified cellobiose-polymyxin-B-colistin (mCPC) (optional) [2%]
5. Tryptone salt (T₁N₁) agar (1% tryptone and 1% NaCl) OR T₁N₂ agar (1% tryptone and 2% NaCl) OR Tryptic soy agar with 2% total sodium chloride concentration
6. 1% Tryptone broth (no NaCl) (T₁N₀) and 1% Tryptone broth with 3% NaCl (T1N3) OR Gelatin agar (GA) [0%] and Gelatin agar with 3% NaCl (GS)
7. Triple sugar iron agar (TSI) OR Kligler iron agar (KIA) slants [0.5%]
8. Hugh-Leifson glucose broth [3%] OR OF glucose medium (semi-solid) [0.5%]
9. Heart infusion (HI) broth [0.5%]
10. Heart Infusion (HI) agar [0.5%] or Brain Heart Infusion (BHI) agar [0.5%] or equivalent
11. Mueller-Hinton agar plates [0%]
12. Tryticase soy agar (TSA) [0.5%]
13. Sheep red blood cells
14. Chicken red blood cells
15. Gram stain solutions
16. Physiological saline
17. Oxidase reagent or commercially available test strips
18. MR-VP broth [0%] and V-P reagents
19. API 20E identification strips or equivalent
20. Positive and negative control cultures (ATCC or equivalent)
21. Antisera
22. Phage IV and El Tor phage V (optional)
23. Polymyxin B antibiotic discs, 50 units
24. Vibriostat O/129 discs, 10 and 150 µg
25. Stomacher, blender or equivalent
26. Waterbath, 35°C
27. Incubators, 35°C, 42°C; 39-40°C (optional)

7. PROCEDURE

7.1 Handling of Sample Units

7.1.1

In the laboratory, prior to analysis, keep sample units refrigerated (4 to 8°C) or frozen, depending on the nature of the product. Thaw the frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.

7.1.2

Analyze sample units as soon as possible after their receipt in the laboratory to minimize overgrowth and to reduce the potential for viable but non-culturable vibrios (VBNC).

7.2 Preparation for Analysis

7.2.1

Have ready sterile alkaline peptone water (APW) at correct concentrations.

7.2.2

For sample preparation, aseptically weigh out 25 g of sample into a 500 mL tared sterile blender jar or stomacher bag. Cut large samples into smaller pieces before blending. Add 225 mL of APW to the jar or stomacher bag and process for 2 min at top speed.

For oysters only, especially those freshly harvested from warm waters, prepare a composite of 10-12 animals including shell liquor; blend, not stomach, 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 7.4 below)

7.3 Preparation of Dilutions

7.3.1

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.

7.3.2

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.

7.3.3 Optional:

Dilutions may also be used to analyze for V. parahaemolyticus and V. vulnificus. If the 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 phosphate buffered saline (PBS), pH 7.2-7.5. For example, if the 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 a 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°C. 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 1 for identification of halophilic Vibrio spp. For oyster samples to be tested for the three Vibrio species, use a samples of at least 75 g since two 250 mL (g) test portions of APW homogenate are incubated for V. cholera analyses (one at 35°C and one at 42°C)

7.4 Plating and Incubation

7.4.1

Leave sample solutions, including frozen or otherwise processed food homogenates and dilutions, in jars or stomacher bags or pour into loosely stoppered sterile 500 mL Erlenmeyer flasks and incubate jars, bags, flasks, and dilutions for 6-8 h at 35°C. Plate inocula onto thiosulphate citrate bile salts agar (TCBS) and modified cellobiose-polymyxin-B-colistin (mCPC) (optional) agar (see 7.4.2 below), and reincubate enrichment broths for a total incubation time of 18-24 h. Plate the 18-24 h enrichment broths to isolation agar.

Exception: Incubate second sample of oyster homogenate and one set of dilutions at 42°C for 6-8 h.

7.4.2

After incubation, and without shaking sample container, transfer a 3-5 mm loopful of inoculum from the pellicle (surface growth) onto TCBS agar and optionally onto mCPC. Incubate TCBS agar for 18-24 h at 35°C and mCPC agar for 18-24 h at 40°C.

7.5 Identification

7.5.1

Examine plates for colony characteristics described below. Carefully pick 3 or more suspect colonies from each plate, streak each for isolation onto T₁N₁, T₁N₂, or tryptic soy agar (2% total NaCl concentration), and incubate for 18-24 h at 35°C. Streaking for isolation on non-selective medium, may be necessary to ensure colonial purity before biochemical testing. Gelatin agar (GA) and gelatin salt (GS) agar may also be inoculated with the same inoculum (see 7.5.2.2 b).

7.5.1.1 Thiosulfate citrate bile salts sucrose (TCBS) agar:

After 18-24 h incubation on TCBS agar, V. cholerae (E1 Tor and classical) (and other sucrose-fermenting vibrios) appear as medium-sized, smooth, yellow colonies with opaque centres and translucent peripheries. Vibrios which do not ferment sucrose are green in color.

7.5.1.2 Modified cellobiose-polymyxin B-colistin (mCPC) agar:

Colonies of V. cholerae E1 Tor are green-to-purple (cellobiose fermentation-negative). V. vulnificus produces flattened yellow colonies with opaque centres and translucent peripheries. Most other Vibrio spp. do not grow readily on CPC agar or mCPC agar.

7.5.2 Distinguishing suspect vibrios from non-vibrios:

7.5.2.1 Triple sugar iron agar slant (TSI) or Kligler iron agar slant (KIA) and arginine glucose slant (AGS):

Inoculate individual colonies into AGS media and either TSI or KIA by stabbing the butt and streaking the slant. Incubate loosely stoppered or capped slants for 18-24 h at 35°C. These media are recommended because the reactions permit early presumptive differentiation between most Vibrio spp., Aeromonas spp., Plesiomonas shigelloides and other bacteria (see Table 5).

7.5.2.2 Screening for salt tolerence:

1. 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°C. 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.
   
   Alternatively:

2. Gelatin agar (GA) and gelatin agar with 3% NaCl (GS):

   GA and GS can be used to screen isolates for salt tolerance. 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 35°C. 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.

7.5.2.3 Oxidation-fermentation test:

Inoculate 2 tubes of Hugh-Leifson glucose broth or OF glucose medium (semi-solid) with growth from an isolated colony. Overlay medium in one tube with sterile mineral oil or liquid Vaspar (50% petrolatum, 50% paraffin) to a depth of 1-2 cm and incubate 1-2 days or more at 35°C. Acid causes the dye to change from purple to yellow in Hugh-Leifson broth and from green to yellow in OF medium, semi-solid. 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.

7.5.2.4 Oxidase test:

Perform the oxidase test on 18-24 h growth from tryticase soy agar (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 seconds. Pathogenic Vibrio spp. are oxidase-positive (except for V. metschnikovii).

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

7.6.1

Read results of TSI or KIA, AGS and oxidation-fermentation; as well as T₁N₀ and T₁N₃ or GA and GS.

7.6.2

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

7.6.3

Test results for isolates which are to be carried through the remaining V. cholerae serological and biochemical tests are as follows:

1. Gram-negative rods or curved rods
2. Sucrose-positive (yellow) on TCBS agar [sucrose-negative (green) for V. mimicus]
3. Cellobiose-negative (green-purple) on mCPC agar
4. Growth in T₁N₀ and T₁N₃ broths or on GA and GS plates
5. Show characteristic reactions (see Table 5) in TSI, KIA, and AGS
6. Gelatinase and oxidase-positive
7. Produce acid from glucose both oxidatively and fermentatively in Hugh-Leifson glucose broth or OF glucose medium, semisolid

7.7 Serological agglutination

7.7.1

Use diagnostic antisera of Group O1 and subgroup Inaba (factors AC) and Ogawa (factors AB) to serotype O1 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 an isolate is confirmed as V. cholerae O1 or non-O1.

7.7.2

Cultures that agglutinate in group O1 antiserum and not in plain physiological saline are V. cholerae group O1 if biochemical reactions confirm the isolate as V. cholerae.

7.7.3

Cultures that agglutinate in this group-specific antiserum may be subtyped with Inaba and Ogawa antibodies.

7.7.4

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.

7.7.5

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

7.7.6

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

7.7.7

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.

7.8 Additional Biochemical reactions

For additional biochemical tests, O/129 sensitivity, growth at 42°C and ONPG tests, as well as specific directions for performing these tests, see Part 1, section 7.5 and Table 3. Formulations for biochemical media should include at least 2% NaCl. API strips may be used in lieu of conventional media. For V. cholerae, use physiological saline (0.85% NaCl) as diluent.

7.9 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 6). Use more than one test to differentiate biotypes. The easiest methods are polymyxin B sensitivity, hemolysin test and Voges-Proskauer test. See descriptions below.

7.9.1 Bacteriophage susceptibility.

This method is a modification of that described by Finkelstein and Mukerjee (8.9). Inoculate HI broth with the strain to be tested and incubate at 35°C for 4 h. Swab surface of Mueller-Hinton agar plate with a 4 h broth culture to obtain confluent bacterial growth. Let plates absorb inoculum and place 1 loopful of the appropriate test dilution of phage IV onto the agar surface with a 3 mm loop. Observe the plate after overnight incubation at 35°C. Classical biotype strains are usually sensitive to this bacteriophage and will lyse on the plate where the 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.

7.9.2 Polymyxin B sensitivity:

This procedure is a modification of a technique described by Han and Khie (8.11). Swab the surface of a Mueller-Hinton agar plate with a 4 h HI broth culture (35°C) to obtain confluent growth. Let plates absorb inoculum and place a 50 unit polymyxin B antibiotic disk on medium surface. Invert plates and incubate for 18-24 h at 35°C. Classical biotype strains will demonstrate a zone of inhibition around the disk (10-15 mm diameter). EI Tor biotype strains will grow to the edge of the disk or will be inhibited slightly (7-8 mm diameter).

Alternatively, use TSA, GA, or GS agar in place of Mueller-Hinton agar.

7.9.3 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 a portion of culture that has been heated for 30 min at 56°C. Use known hemolytic and non-hemolytic strains of V. cholerae as controls. Incubate mixtures for 2 h in a 35°C waterbath, then refrigerate overnight at 4-5°C. 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. Incubate at 35°C for 24 h and check for beta-hemolysis surrounding colonies.

7.9.4 Chicken red blood cell agglutination:

Prepare a thick, milky bacterial suspension in physiological saline from an 18 to 24 h TSA culture. On a clean glass slide, mix 1 loopful of washed chicken red blood cells (2.5% in physiological saline) with a suspension of the 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.

7.9.5 Voges-Proskauer (VP) test:

Perform test in MR-VP broth after 18-24 h incubation at 22°C. El Tor biotype strains are usually positive; classical strains are negative.

7.10 Minimal characteristics for biochemical identification of V.cholerae

The following characteristics are presumptive for V. cholerae:

1. Morphology: Gram-negative asporogenous rod or curved rod.
2. TSI or KIA appearance: Acid slant/acid butt, gas production negative, H₂S negative
3. Fermentation test: Glucose fermentation and oxidation positive
4. Cytochrome oxidase: Positive
5. Arginine dihydrolase test: Negative
6. Lysine decarboxylase test: Positive
7. Voges-Proskauer test: El Tor biotype positive, classical biotype negative; V. mimicus negative
8. Growth at 42°C: Positive
9. 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.
10. Sucrose fermentation: Positive (negative for V.mimicus)
11. ONPG test: Positive
12. Arabinose fermentation: Negative
13. O/129 sensitivity: Sensitive to 10 and 150 µg O/129

8. REFERENCES

8.1 Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.

8.2 Baselski, V., R. Briggs and C. Parker. 1977. Intestinal fluid accumulation induced by oral challenge with Vibrio cholerae or cholera toxin in infant mice. Infect. Immun. 15:704-712.

8.3 Baumann, P. and R.H.W. Schubert. 1984. Section 5. Facultatively anaerobic Gram-negative rods, Family II. Vibrionaceae, pp. 516-550. In: Bergey's Manual of Systematic Bacteriology, Vol. 1. J.G. Holt and N.R. Krieg (eds). Williams & Wilkins, Baltimore.

8.4 Cholera Working Group. 1933. Large epidemic of cholera-like disease in Bangladesh caused by Vibrio cholerae O139 synonym Bengal. Lancet 342:387-390.

8.5 Davis, B.R., G.R. Fanning, J.M. Madden, A.G. Steigerwalt, H.B. Bradford, Jr., H.L. Smith, Jr. and D.J. Brenner. 1981. Characterization of biochemically atypical Vibrio cholerae strains and designation of a new pathogenic species, Vibrio mimicus. J. Clin. Microbiol. 14:631-639.

8.6 DePaola, A., C.A. Kaysner and R.M. McPhearson. 1987. Elevated temperature method for recovery of Vibrio cholerae from oysters (Crassostrea gigas). Appl. Environ. Microbiol. 53:1181-1182.

8.7 Elliot, E.L., C. A. Kaysner, L. Jackson and M. L. Tamplin. 1998. Vibrio cholerae, V. parahaemolyticus, V. vulnificus, and other Vibrio spp. In: Bacteriological Analytical Manual, 8^th Edition, Revision A, 1998. Chapter 9. Published by AOAC International, Gaithersburg. MD.

8.8 Ewing, W.H., K.M. Tomfohrde and P.J. Naudo. 1979. Isolation and identification of Vibrio cholerae and certain related vibrios: an outline of methods. Species 2:10-22.

8.9 Finkelstein, R.A. and S. Mukerjee. 1963. Hemagglutination: a rapid method for differentiating Vibrio cholerae and El Tor vibrios. Proc. Soc. Exp. Biol. Med. 112:355-359.

8.10 Fujino, T., Y. Okuno, D. Nakada, A. Aoyama, K. Fukai, T. Mukai and T.Ueho. 1951. On the bacteriological examination of Shirasu food poisoning. J. Jpn. Assoc. Infect. Dis. 35:11-12.

8.11 Han, G.K. and T.D. Khie. 1963. A new method for the differentiation of Vibrio comma and Vibrio El Tor. Am. J. Hyg. 77:184-186.

8.12 Iwanaga, M. and K. Yamamoto. 1985. New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor. J. Clin.Microbiol. 22:405-408.

8.13 MacDonell, M.T., F.L. Singleton and M.A. Hood. 1982. Diluent decomposition of use of API 20E in characterizing marine and estuarine bacteria. Appl. Environ. Microbiol. 44:423-427.

8.14 Madden, J.M., B.A. McCardell and J.G. Morris, Jr. 1989. Vibrio cholerae, pp. 525-542. In: Foodborne Bacterial Pathogens. M.P. Doyle(ed). Marcel Dekker, New York.

8.15 Massad, G., and J.D. Oliver. 1987. New selective and differential medium for Vibrio cholerae and Vibrio vulnificus. Appl. Environ. Microbiol. 53:2262-2264.

8.16 Sakazaki, R. 1979. Vibrio infections, pp. 173-209. In: Foodborne Infections and Intoxications, 2nd ed. H. Riemann and R. Bryan (eds). Academic Press, New York.

8.17 Sakazaki, R. and T. Shimada. 1986. Vibrio species as causative agents of foodborne infections, pp. 123-151. In: Developments in Food Microbiology - 2. R.K. Robinson (ed). Elsevier Applied Science, New York.

8.18 Shimada, T., R. Sakazaki and M. Oue. 1987. A bioserogroup of marine vibrios possessing somatic antigen factors in common with Vibrio cholerae O1. J. Appl. Bacteriol. 62:452-456.

8.19 Smith, Jr., H.L. and K. Goodner. 1958. Detection of bacterial gelatinases by gelatin-agar plate methods. J. Bacteriol. 76:662-665.

8.20 West, P.A. and R.R. Colwell. 1984. Identification and classification of Vibrionaceae--an overview, pp. 285-363. In: Vibrios in the Environment. R.R. Colwell (ed). John Wiley & Sons, New York.

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Table 5. Reactions ^a of certain Vibrio spp. and related microorganisms in differential tube agar media

Microorganism	KIA b		TSI b		AGS b
	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 c	K or A	A	K or A	A	K	K
P. shigelloides	K or A	A	K or A	A	ND	ND

Table 6. Differentiation of biotypes of V. cholerae O1 ^{a b}

Test	El Tor	Classical
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	+	-