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GB 4789.44-2020: National food safety standard - Food microbiological examination - Vibrio vulnificus
GB 4789.44-2020
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard -
Food microbiological examination - Vibrio vulnificus
ISSUED ON: SEPTEMBER 11, 2020
IMPLEMENTED ON: MARCH 11, 2021
Issued by: National Health Commission of the PRC;
State Administration for Market Regulation.
Table of Contents
1 Scope ... 3 
2 Apparatus and materials ... 3 
3 Media and reagents ... 4 
4 Examination procedure ... 5 
5 Operation procedure ... 5 
6 Results and reports ... 14 
Appendix A Media and reagents ... 15 
National food safety standard -
Food microbiological examination - Vibrio vulnificus
1 Scope
This Standard specifies the examination method for Vibrio vulnificus in aquatic
products.
This Standard applies to the examination of Vibrio vulnificus in aquatic products
such as fish, shrimp, crab, shellfish.
2 Apparatus and materials
In addition to the conventional sterilization and culture apparatus in the
microbiology laboratory, other apparatus and materials are as follows:
2.1 Constant-temperature incubator: 36 °C±1 °C.
2.2 Refrigerator: 2 °C~5 °C, 7 °C~10 °C.
2.3 Constant-temperature water bath.
2.4 Homogenizer or sterile mortar.
2.5 Balance: Sensitivity is 0.1 g.
2.6 PCR instrument.
2.7 Electrophoresis apparatus or capillary electrophoresis apparatus.
2.8 Gel electrophoresis imaging system or UV detector.
2.9 Biosafety cabinet.
2.10 High-speed centrifuge (maximum speed is at least 15000 r/min).
2.11 Vortex oscillator.
2.12 Micro-adjustable pipette (range 2.5 μL, 10 μL, 100 μL, 1000 μL) and
matching tips.
2.13 Precision pH test paper or pH meter.
be stored at 7 °C~10 °C (because Vibrio vulnificus is very easy to form a living
and uncultivable state at 4 °C, samples must not be placed at 4 °C). And, as far
as possible, complete the examination within 24 h. Frozen samples shall be
thawed under warm conditions not exceeding 45 °C. The thawing time shall not
exceed 15 min.
5.1.2 Sampling: For fish and cephalopods, take surface tissues, intestines and
gills. For shellfish, take the entire contents (including meat and body fluids). For
crustaceans, take the whole animal or its central part (including intestines and
gills). For shellfish or hard shell crustaceans, it shall first use running tap water
to rinse the shell; use filter paper to absorb the surface moisture. Then the shell
shall be opened aseptically; the corresponding part shall be taken according to
the above requirements.
5.1.3 USE aseptic operation to take 25 g of the above-treated sample; ADD 225
mL of PNCC enrichment broth; USE a rotating blade homogenizer to
homogenize at 8000 r/min for 1 min. Or use a flapping homogenizer to
homogenize for 2 min; mix thoroughly to prepare a 1:10 sample homogenate.
If there is no homogenizer, put the sample in a sterile mortar and grind it
thoroughly. TAKE 25 g of the ground sample; TRANSFER it into a 500 mL
sterile conical flask; ADD 225 mL of PNCC enrichment broth and shake and
mix well, to prepare a sample homogenate of 1:10.
5.2 Enrichment
Incubate the 1:10 sample homogenate prepared in 5.1.3 at 36 °C±1 °C for 18
h±1 h.
5.3 PCR testing
The environmental conditions and process control of PCR test shall be
implemented in accordance with the provisions of GB/T 27403 "Criterion on
quality control of laboratories - Molecular biological testing of food"; the same
below.
5.3.1 DNA template preparation
At a distance of 1 cm below the surface of the PNCC enrichment broth, pipette
1 mL into a 1.5 mL EP tube; CENTRIFUGE at 9000 r/min for 3 min and discard
the supernatant. ADD 1 mL of PBS to the precipitate to suspend it and wash it
thoroughly; CENTRIFUGE at 9000 r/min for 3 min and discard the supernatant.
FOLLOW this step to repeatedly wash the precipitate 2 to 3 times; DISCARD
the last supernatant. ADD 1 mL of sterile ultrapure water; BOIL at 100 °C for 10
min; then centrifuge at 12000 r/min for 5 min; the supernatant is used for PCR
analysis. If it cannot be analyzed in time, store it at -20 °C for later use.
5.3.3 Electrophoresis
Gel electrophoresis is used to detect PCR products. USE 0.5×TBE buffer to
prepare 1.5% agarose gel (containing DNA dyes such as EB 0.5 μg/mL or
Goldview 5 μL/100 mL or Gelred 5 μL/50 mL). TAKE 5 μL of PCR amplification
product; MIX it with 1 μL of 6× nucleic acid electrophoresis loading buffer; apply
the sample; at the same time, add a DNA molecular weight standard (range
100 bp~1000 bp) to a hole. According to the following formula: the distance
between the positive and negative electrodes of the electrophoresis tank
(cm)×5 V/cm, calculate and set the voltage. USE 0.5×TBE buffer constant-
pressure electrophoresis. According to the moving position of bromophenol
blue, determine the electrophoresis time. USE the gel imaging system or
ultraviolet detector to observe and record the results.
5.3.4 Result determination
Quality control system: Negative control and blank control have no amplified
bands; and, the positive control has amplified bands of expected size (519 bp);
then the detection system is normal. Otherwise, if any kind of control has
abnormal results, the test shall be repeated. The pollution factor shall be
eliminated at the same time.
Positive result: When the quality control system is normal, an amplified band of
the expected size (519 bp) appears in the sample to be tested, then the PCR
result is determined to be positive.
Negative result: When the quality control system is normal, the sample to be
tested does not show an amplified band of the expected size (519 bp), then the
PCR result is determined to be negative.
5.4 Separation
USE a 10 μL inoculation loop to dip a loop of enrichment broth at 1 cm below
the surface of the PNCC enrichment broth. Respectively streak-inoculate on
CC and mCPC plates; incubate at 36 °C±1 °C for 18 h±1 h. The typical
morphology of Vibrio vulnificus on CC and mCPC plates is: round and flat;
yellow to orange colonies which are transparent, or are opaque in center but
transparent at edges under light. The diameter of the colony is 1 mm~2 mm. A
yellow halo may appear (or not appear) around the colony.
5.5 Isolated pure culture
PICK at least 5 suspicious Vibrio vulnificus colonies from each of the CC plate
and mCPC plate (select all if less than 5); respectively inoculate them on 3%
sodium chloride tryptone soy agar plates; incubate at 36 °C±1 °C for 18 h±1 h
for subsequent identification. The colony morphology of Vibrio vulnificus on the
Appendix A
Media and reagents
A.1 Peptone-sodium chloride-cellobiose-polymyxin E (PNCC) enrichment
broth
A.1.1 Solution 1
A.1.1.1 Composition
Peptone: 50.0 g
Sodium chloride: 10.0 g
Distilled water: 900.0 mL
A.1.1.2 Preparation method
DISSOLVE the components in A.1.1.1 in distilled water; USE 1 mol/L
hydrochloric acid solution and 1 mol/L sodium hydroxide solution to adjust the
pH to 8.5±0.2; autoclave at 121 °C for 10 min.
A.1.2 Solution 2
A.1.2.1 Composition
Cellobiose: 0.8 g
Polymyxin E: 1000 U
Distilled water: 100.0 mL
A.1.2.2 Preparation method
DISSOLVE cellobiose in distilled water; HEAT it slightly to completely dissolve;
after cooling, add antibiotics; USE a 0.22 μm microporous membrane to filter
and sterilize for later use.
PNCC enrichment broth is obtained by mixing solution 1 with solution 2.
A.2 Cellobiose-polymyxin E (CC) agar medium
A.2.1 Solution 1
A.2.1.1 Composition
Sodium chloride: 20.0 g
Bromothymol blue: 0.04 g
Cresol red: 0.04 g
Agar: 15.0 g
Distilled water: 900.0 mL
A.3.1.2 Preparation method
DISSOLVE the components in A.3.1.1 in distilled water; HEAT and boil until
completely dissolved; USE 1 mol/L hydrochloric acid solution and 1 mol/L
sodium hydroxide solution to adjust the pH to 7.6±0.2; COOL to 48 °C~55 °C
for later use.
A.3.2 Solution 2
A.3.2.1 Composition
Cellobiose: 10.0 g
Polymyxin B: 100000 U
Polymyxin E: 400000 U
Distilled water: 100.0 mL
A.3.2.2 Preparation method
DISSOLVE cellobiose in 100.0 mL of distilled water; HEAT it slightly until it is
completely dissolved; after cooling, add antibiotics; USE a 0.22 μm microporous
membrane to filter and sterilize for use.
MIX solution 2 and solution 1 and pour on the plate.
A.4 3% sodium chloride tryptone soy agar
A.4.1 Composition
Tryptone: 15.0 g
Soy peptone: 5.0 g
Sodium chloride: 30.0 g
Agar: 15.0 g
Sodium thiosulfate (Na2S2O3): 0.3 g
Agar: 12.0 g
Distilled water: 1000.0 mL
A.6.2 Preparation method
DISSOLVE the various components in A.4.1 in distilled water; USE 1 mol/L
hydrochloric acid solution and 1 mol/L sodium hydroxide solution to adjust the
pH to 7.4±0.2. After autoclaving at 121 °C for 15 min, separate them into test
tubes, to make a high-level slant with a length of 4 cm~5 cm and a depth of 2
cm~3 cm for use.
A.7 Halophilic test medium
A.7.1 Composition
Tryptone: 10.0 g
Sodium chloride: ADD the corresponding amount in turn according to the
different concentration
Distilled water: 1000.0 mL
A.7.2 Preparation method
DISSOLVE the various components in A.5.1 in distilled water. After using 1
mol/L hydrochloric acid solution and 1 mol/L sodium hydroxide solution to adjust
the pH to 7.2±0.2, dispense into 300 mL conical flasks. Each flask contains 100
mL. A total of 5 flasks are prepared. ADD different amounts of sodium chloride:
(1) 0 g; (2) 3 g; (3) 6 g; (4) 8 g; (5) 10 g. Then, the sodium chloride solution of
each concentration is divided into test tubes of appropriate capacity. Each tube
contains 10 mL. Autoclave at 121 °C for 15 min for later use.
A.8 3% sodium chloride lysine decarboxylase test medium
A.8.1 Composition
Peptone: 5.0 g
Yeast powder: 3.0 g
Glucose: 1.0 g
Bromocresol purple: 0.02 g
L-lysine: 5.0 g
A.11.1 Composition
N,N,N',N'-tetramethylp-phenylenediamine hydrochloride: 1.0 g
Distilled water: 100.0 mL
A.11.2 Preparation method
Oxidase reagent shall be prepared in small amounts. After preparation, it shall
be stored in a refrigerator at 2 °C~5 °C in the dark; and be used within 7 d.
A.12 Gram staining solution
A.12.1 Crystal violet staining solution
A.12.1.1 Composition
Crystal violet: 1.0 g
95% ethanol: 20.0 mL
1% ammonium oxalate aqueous solution: 80.0 mL
A.12.1.2 Preparation method
Completely dissolve the crystal violet in ethanol; then mix it with the ammonium
oxalate solution.
A.12.2 Gram iodine solution
A.12.2.1 Composition
Iodine: 1.0 g
Potassium iodide: 2.0 g
Distilled water: 300.0 mL
A.12.2.2 Preparation method
MIX iodine and potassium iodide first; ADD a little distilled water and shake well.
When it is completely dissolved, add distilled water to 300.0 mL.
A.12.3 Safranin counterstaining solution
A.12.3.1 Composition
Safranin: 0.25 g