GB 4789.7-2013National food safety standard - Food Microbiological Examination - Vibrio parahaemolyticus (English PDF)

3.2 thiosulfate-citrate-bile salt-sucrose (TCBS) agar. see A.2 of Annex A

3.3 3% sodium chloride trypsin soybean agar. see A.3 of Annex A

3.6 3% sodium chloride mannitol test medium. see A.6 of Annex A

3.7 3% sodium chloride lysine decarboxylase test medium. see A.7 of

3.8 3% sodium chloride MR-VP medium. see A.8 of Annex A

3.9 3% sodium chloride solution. see A.9 of Annex A

5.1 Sample preparation

5.1.1 After the collection of non-frozen sample, immediately store it in a 7°C

~ 10°C refrigerator, and carry out the early inspection as soon as possible.

The frozen sample should be thawed at 45°C for not more than 15 min or at

2°C to 5°C for not more than 18 h.

5.1.2 Take surface tissue, gut or gill for fish and cephalopods. For shellfish,

take all contents, including shellfish and body fluids.

5.1.3 Take 25 g (mL) of the sample aseptically. Add 225 mL of 3% sodium

chloride alkaline peptone. Homogenize at 8000 r/min for 1 min with a rotary

blade homogenizer. Or slap with slam homogenizer for 2 min.

5.2 Enrichment

5.2.1 Qualitative detection

Incubate the 1.10 liquid sample prepared in 5.1.3 at 36°C ± 1°C for 8 h to 18

h.

5.2.2 Quantitative detection

5.3 Separation

5.3.1 For all enrichment solutions with growth, use the inoculation ring in the

distance of 1 cm below the surface to take a ring of enrichment solution.

Scribe and separate on TCBS plate or Vibrio color medium plate, a test tube

for a flat plate. Cultivate it at 36°C ± 1°C for 18 h ~ 24 h.

5.4 Pure culture

Pick up three or more suspicious colonies. Scribe 3% sodium chloride trypsin

soybean agar plate. Cultivate at 36°C ± 1°C for 18h ~ 24h.

5.5 Preliminary identification

5.5.1 Oxidase test. select a single culture of pure culture for oxidase test;

Vibrio parahaemolyticus shall be oxidase positive.

5.5.4 Halophilic test. pick purely cultured single suspicious colony;

respectively inoculate 0%, 6%, 8% and 10% peptone water of different

concentrations of sodium chloride; cultivate at 36°C ± 1°C for 24h observation;

observe the liquid turbidity. Vibrio parahaemolyticus shall not grow or grow

weakly in cisplatin without sodium chloride and of 10% sodium chloride. It

shall grow vigorously in peptone water of 6% sodium chloride and 8% sodium

chloride.

5.6 Determination of identification

Take pure culture and respectively inoculate mannitol test medium containing

3% sodium chloride, lysine decarboxylase test medium, MR-VP medium.

6.1 Preparation

Inoculate two tubes of 3% sodium chloride trypsin soy agar test tube slope.

Cultivate at 36°C ± 1°C for 18h ~ 24h. Use 5% glycerol solution containing 3%

sodium chloride to wash 3% sodium chloride trypsin soy agar slant culture so

as to obtain a strong bacterial suspension.

6.2 Identification of K Antigen

Take a tube of 6.1 well-prepared suspension. First, use polyvalent K

antiserum for detection.

6.3 Identification of O antigen

Transfer another tube of bacteria suspension into the centrifuge tube for 1 h

sterilization at 121°C. After sterilization, carry out the centrifugation at 4000

r/min for 15 min. Discard the upper liquid. Wash the precipitate three times

with physiological saline, centrifugation at 4000 r/min for 15 min each time.

After the last centrifugation, it shall stay a little upper liquid. Well mix the

bacteria into a suspension.