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GB 4789.10-2016 English PDF (GB4789.10-2016)

GB 4789.10-2016 English PDF (GB4789.10-2016)

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GB 4789.10-2016: National Food Safety Standard -- Food Microbiological Examination -- Examination of Staphylococcus Aureus
This Standard specifies the examination method for Staphylococcus aureus in foods. Method I of this Standard applies to the qualitative examination of Staphylococcus aureus in foods; Method II applies to the counting of Staphylococcus aureus in foods with high Staphylococcus aureus content; Method III applies to the counting of Staphylococcus aureus in foods with low Staphylococcus aureus content.
GB 4789.10-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard -
Food microbiological examination -
Staphylococcus aureus
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the
People’s Republic of China;
China Food and Drug Administration.
3. No action is required - Full-copy of this standard will be automatically and immediately delivered to your EMAIL address in 0~60 minutes.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Equipment and materials ... 4
3 Medium and reagents ... 5
4 Examination procedure ... 5
5 Operating procedure ... 6
6 Result and report ... 8
7 Examination procedure ... 8
8 Operating procedure ... 8
9 Result calculation ... 10
10 Report ... 11
11 Examination procedure ... 12
12 Operating procedure ... 12
13 Result and report ... 13
Annex A Medium and reagents ... 14
Annex B Staphylococcal enterotoxin detection ... 19
Annex C Retrieval table for most probable number (MPN) of staphylococcus aureus ... 24
Foreword
This Standard replaces GB 4789.10-2010 “National food safety standard - Food microbiological examination. Staphylococcus aureus”, SN/T 0172-2010
“Determination of Staphylococcus aureus in foods for import and export”, SN/T 2154- 2008 “Determination of coagulase-positive staphylococci in import and export food - Technique using rabbit plasma fibrinogen agar medium”.
Compared with GB 4789.10-2010, the main changes of this standard are as follows. - The enrichment fluid for test is modified to 7.5 % sodium chloride broth. National food safety standard -
Food microbiological examination –
Staphylococcus aureus
1 Scope
This Standard specifies the examination method for Staphylococcus aureus in foods. Method I of this Standard applies to the qualitative examination of Staphylococcus aureus in foods; Method II applies to the counting of Staphylococcus aureus in foods with high Staphylococcus aureus content; Method III applies to the counting of Staphylococcus aureus in foods with low Staphylococcus aureus content.
2 Equipment and materials
In addition to routine sterilization and culture equipment for microbiological laboratories, other equipment and materials are as follows.
2.1 Constant temperature incubator. 36 °C ± 1 °C.
2.2 Refrigerator. 2 °C ~ 5 °C.
2.3 Constant temperature water bath. 36 °C ~ 56 °C.
2.4 Balance. with division of 0.1 g.
2.5 Homogenizer.
2.6 Oscillator.
2.7 Sterile pipette. 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or micro pipette and tip.
2.8 Sterile Erlenmeyer flask. with capacity of 100 mL and 500 mL.
2.9 Sterile Petri dish. with diameter of 90 mm.
2.10 Spreader.
2.11 pH meter or pH colorimetric tube or precision pH test paper.
5.4 Preliminary identification
Staphylococcus aureus on the Baird-Parker plate is round, smooth-surface, raised, moist, with colony diameter of 2 mm ~ 3 mm, color of gray black to black color, shiny, and usually light (non-white) edge, around the opaque circle (precipitation) and a clear band outside. When the inoculating needle touches the colony, there is a butter-like sticky feeling. Sometimes strains of fats can be decomposed can be seen, except opaque circle and clear band, the appearance is basically the same. Colonies isolated from long-term stored frozen or dehydrated foods are often with lighter black than typical colonies, and more rough appearance and more dry texture. On the blood plate, the colony is larger, round, smooth raised, moist, golden (sometimes white), with transparent hemolytic circle seen around the colony. Pick the above-mentioned suspicious colonies for Gram’s stain microscopic examination and plasma coagulase test.
5.5 Confirmation identification
5.5.1 Stain microscopic examination. Staphylococcus aureus is Gram’s positive cocci, arranged in grape-like, no spores, no capsule, with the diameter of about 0.5 μm ~ 1 μm.
5.5.2 Plasma coagulase test. PICK at least 5 suspicious colonies (select all when there are less than 5) on Baird-Parker plate or blood plate; INOCULATE them to 5 mL of BHI and nutrient agar slants respectively; CULTURE at 36 °C ± 1 °C for 18 h ~ 24 h. TAKE 0.5 mL of newly prepared rabbit plasma into a small test tube, ADD 0.2 mL ~ 0.3 mL of BHI culture, SHAKE well; PLACE it in temperature box or water bath box at 36 °C ± 1 °C; OBSERVE every half hour for a total of 6 h; if there is solidification (that is, when the test tube is tilted or inverted, there are clots) or if the solidification volume is greater than half the original volume, it is determined to be positive results. While the broth culture of the positive and negative Staphylococcus aureus strains of plasma coagulase test is used as a control. It can also use commercial reagents, operate according to the instructions, carry out the plasma coagulase test.
If the result is suspicious, pick the colonies on the nutrient agar slant into 5 mL of BHI and culture at 36 °C ± 1 °C for 18 h ~ 48 h.
5.6 Staphylococcal enterotoxin examination (optional)
For identification of suspicious food poisoning samples or Staphylococcus aureus strains producing staphylococcal enterotoxin, Staphylococcal enterotoxin shall be tested according to Annex B.
T - the number of Staphylococcus aureus colonies in the sample;
A - the total number of typical colonies of a certain dilution;
B - the number of colonies identified as positive of a certain dilution; C - the number of colonies used for identification test of a certain dilution; d - the dilution factor.
Equation (2).
where.
T - the number of Staphylococcus aureus colonies in the sample;
A1 - the total number of typical colonies of the first dilution (low dilution factor); B1 - the number of colonies identified as positive of the first dilution (low dilution factor);
C1 - the number of colonies used for identification test of the first dilution (low dilution factor);
A2 - the total number of typical colonies of the second dilution (high dilution factor); B2 - the number of colonies identified as positive of the second dilution (high dilution factor);
C2 - the number of colonies used for identification test of the second dilution (high dilution factor);
1.1 - the calculation coefficient;
d - the dilution factor (first dilution).
10 Report
According to the calculation results of the equations in Clause 9, report the number of Staphylococcus aureus in per g (mL) of sample, expressed as CFU/g (mL); if the T value is 0, report the lowest dilution factor multiplies by less than 1. 95 % ethanol 20.0 mL
1 % aqueous ammonium oxalate solution 80.0 mL
A.8.1.2 Preparation method
Completely DISSOLVE the crystal violet in ethanol, and then MIX with ammonium oxalate solution.
A.8.2 Gram’s liquid iodine
A.8.2.1 Ingredients
Iodine 1.0 g
Potassium iodide 2.0 g
Distilled water 300 mL
A.8.2.2 Preparation method
MIX iodine and potassium iodide first; ADD a small amount of distilled water and SHAKE well; after completely dissolved, ADD distilled water to 300 mL.
A.8.3 Safranin counter stain
A.8.3.1 Ingredients
Safranin 0.25 g
95 % ethanol 10.0 mL
Distilled water 90.0 mL
A.8.3.2 Preparation method
DISSOLVE safranin in ethanol, and then DILUTE with distilled water.
A.8.4 Staining method
a) FIX the smear on the flame, DROP crystal violet stain, STAIN for 1 min, RINSE. b) DROP Gram’s liquid iodine, REACT for 1 min, RINSE.
c) DROP 95 % ethanol to decolorize for about 15 s ~ 30 s, until the stain is rinsed away, do not over-decolorize, RINSE.
d) DROP counter stain to stain for 1 min, RINSE and DRY for microscopic Annex B
Staphylococcal enterotoxin detection
B.1 Reagents and materials
Unless otherwise specified, the reagents used are analytical regents, and the test water shall comply with the provisions of Grade I water in GB/T 6682.
B.1.1 A, B, C, D, E-type Staphylococcal enterotoxin ELISA kit.
B.1.2 pH test paper, with the range of 3.5 ~ 8.0, and the accuracy of 0.1. B.1.3 0.25 mol/L Tris buffer with pH 8.0. DISSOLVE 121.1 g of Tris in 800 mL of deionized water; after cooling to room temperature, ADD 42 mL of concentrated HCL to pH 8.0.
B.1.4 Phosphate buffer with pH 7.4. WEIGH 0.55 g of NaH2PO4...
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