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GB 4789.7-2013: National Food Safety Standard -- Food Microbiological Examination -- Vibrio parahaemolyticus
GB 4789.7-2013
GB
NATIONAL STANDARD OF
THE PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Food Microbiological
Examination - Vibrio parahaemolyticus
ISSUED ON. NOVEMBER 29, 2013
IMPLEMENTED ON. JUNE 1, 2014
Issued by. National Health and Family Planning Commission of China
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative references ... 4 
3 Mediums and reagents ... 4 
4 Inspection procedures ... 5 
5 Requirements ... 6 
6 Serological typing (optional) ... 8 
7 Kanagawa test (optional) ... 10 
8 Results and reports ... 10 
Annex A Medias and reagents ... 12 
Annex B Vibrio parahaemolyticus most probable number (MPN) search table19 
National Food Safety Standard - Food Microbiological
Examination - Vibrio parahaemolyticus
1 Scope
This Standard specifies the inspection method for Vibrio parahaemolyticus in
food.
This Standard is applicable to the inspection of Vibrio parahaemolyticus in
food.
2 Normative references
In addition to microbial laboratory routine sterilization and culture equipment,
other equipment and materials are as follows.
a) constant temperature incubator. 36°C ± 1°C;
b) refrigerator. 2°C ~ 5°C, 7°C ~ 10°C;
c) constant temperature water bath. 36°C ± 1°C;
d) homogenizer or sterile mortar;
e) balance. resolution of 0.1 g;
f) sterile test tubes. 18mm × 180mm, 15mm × 100mm;
g) sterile straws. 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or
micro-pipettes and tips;
h) sterile conical flasks. volumes of 250 mL, 500 mL, 1000 mL;
j) sterile Petri dish. diameter of 90 mm;
j) automatic microbial biochemical identification system;
k) sterile surgical scissors, tweezers.
3 Mediums and reagents
3.1 3% sodium chloride alkaline peptone water. see A.1 of Annex A
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
3.4 3% sodium chloride trisaccharide iron agar. see A.4 of Annex A
3.5 salt test medium. see A.5 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
Annex A
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
3.10 Wagstsuma blood agar. see A.10 of Annex A
3.11 oxidase reagents. see A.11 of Annex A
3.12 gram stain solution. see A.12 of Annex A
3.13 ONPG. see A.13 of Annex A
3.14 Voges-Proskauer (V-P) reagent. see A.14 of Annex A
3.15 vibrio color development medium
3.16 biochemical identification kit
4 Inspection procedures
See Figure 1 for Vibrio parahaemolyticus test procedures.
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.
5.5.2 Smear microscopy. smear the suspected colony; carry out Gram
stain; perform the microscopic examination on observation morphology.
Vibrio parahaemolyticus shall be Gram-negative, rod-shaped, curved, oval,
without spores, with flagella.
5.5.3 Pick purely cultured single suspicious colony. Transfect 3% sodium
chloride trisaccharide agar slope and puncture the bottom layer. Cultivate at
36°C ± 1°C for 24h to observe the results. The reaction of Vibrio
parahaemolyticus in 3% sodium chloride trisaccharide iron agar shall be that
the bottom turns yellow not black, without bubbles; slope color shall be
unchanged or red becomes deeper, with power.
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.
Observe the results after cultivating at 36°C ± 1°C for 24h ~ 48h. Use 3%
sodium chloride trisaccharide agar overnight culture for ONPG test. It can
select biochemical identification kit or automatic microbial biochemical
identification system.
6 Serological typing (optional)
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
Distilled water 1000.0 mL
A.3.2 Method
Dissolve the ingredients in A.3.1 in distilled water. Correct pH to 7.3 ± 0.2.
Sterilize at high temperature of 121°C for 15 min.
A.4 3% sodium chloride trisaccharide iron agar
A.4.1 Ingredient
Peptone 15.0 g
Proteose peptone 5.0 g
Beef jelly 3.0 g
Yeast extract 3.0 g
Sodium chloride 30.0 g
Lactose 10.0 g
Sucrose 10.0 g
Glucose 1.0 g
Ferrous sulfate (FeSO4) 0.2 g
Phenol red 0.024 g
Sodium thiosulfate (Na2S2O3) 0.3 g
Agar 12.0 g
Distilled water 1000.0 mL
A.4.2 Method
Dissolve the ingredients in A.4.1 in distilled water. Correct pH to 7.4 ± 0.2.
Sub-fill into the test tubes of appropriate capacities. Sterilize at high
temperature of 121°C for 15 min. Made high-level slope of which the slope
length is 4 cm ~ 5 cm; high depth is 2 cm ~ 3 cm.
A.5 Salt test medium
A.5.1 Ingredient
Tryptone 10.0 g
Sodium chloride Add in different amounts
Distilled water 1000.0 mL
A.5.2 Method
Dissolve the ingredients in A.5.1 in distilled water. Correct pH to 7.2 ± 0.2.
Prepare 5 bottles in total, 100 mL of each bottle. Each bottle shall be added
with varying amounts of sodium chloride. (1) none; (2) 3 g; (3) 6 g; (4) 8 g; (5)
10 g. Sub-fill into the test tubes. Sterilize at high temperature of 121°C for 15
min.
A.6 3% sodium chloride mannitol test medium
Dissolve N, N, N ', N'-tetramethyl-p-phenylenediamine hydrochloride in
distilled water. Store in 2°C ~ 5°C refrigerator from light. Use within 7d.
A.11.3 Test method
Use fine glass rods or one-time inoculation needle to select fresh (24 h)
colony. Coat on a filter paper moistened by oxidase reagents. If the filter
paper within 10 s shows pink or purple, it shall be positive for oxidase test. No
discoloration shall be negative for oxidase test.
A.12 Gram stain solution
A.12.1 Crystal violet dyeing solution
A.12.1.1 Ingredient
Crystal violet 1.0 g
95% ethanol 20.0 mL
1% aqueous ammonium oxalate solution 80.0 mL
A.12.1.2 Method
Completely dissolve the crystal violet in ethanol and then mix with ammonium
oxalate solution.
A.12.2 Gram iodine solution
A.12.2.1 Ingredient
Iodine 1.0 g
Potassium iodide 2.0 g
Distilled water 1000.0 mL
A.12.2.2 Method
Mix iodine with potassium iodide. Add distilled water to shake a little. After
being completely dissolved, add distilled water to 300 mL.
A.12.3 Safranin complex liquid
A.12.3.1 Ingredient
Safranin 0.25 g
95% ethanol 10.0 mL
Distilled water 1000.0 mL
A.12.3.2 Method
Dissolve safranin n ethanol and then dilute wi...