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GB 4789.4-2016 PDF in English

GB 4789.4-2016 (GB4789.4-2016) PDF English
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GB 4789.4-2016: PDF in English

GB 4789.4-2016
National Food Safety Standard – Food
Microbiological Examination – Salmonella Test
Issued by. National Health and Family Planning Commission of PRC;
China Food and Drug Administration
3. No action is required - Full-copy of this standard will be automatically &
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Table of Contents
Foreword ... 3
1 Scope ... 4
2 Devices and Materials ... 4
3 Medium and Reagent ... 5
4 Test Procedures ... 5
5 Operation Procedures ... 7
6 Results and Reports ... 13
Appendix A Medium and Reagent ... 14
Appendix B Common Salmonella Antigens ... 26
This Standard replaced GB 4789.4-2010 National Food Safety Standard – Food
Microbiological Examination – Salmonella Test, SN 0170-1992 Method for Detection
of Salmonella (Including Arizona) in Food for Export, and SN/T 2552.5-2010
Microbiological Examination Method for Milk and Milk Products Hygiene – Part 5.
Detection of Salmonella.
Compared with GB 4789.4-2010, the combined standard has the major changes as
--- Modify the detection process and serological detection operation procedures;
--- Modify the Appendix A and Appendix B.
National Food Safety Standard – Food
Microbiological Examination – Salmonella Test
1 Scope
This Standard specifies the detection method of Salmonella in food.
This Standard is applicable to the test of salmonella in food.
2 Devices and Materials
In addition to the microbial laboratory routine sterilization and cultivation device, other
devices and materials are as follows.
2.1 Refrigerator. 2°C~5°C.
2.2 Constant temperature incubator. 36°C±1°C, 42°C±1°C.
2.3 Homogenizer.
2.4 Oscillator.
2.5 Electronic balance. sensitivity 0.1g.
2.6 Sterile conical flask. capacity of 500mL and 250mL.
2.7 Sterile pipettes. 1mL (with 0.01mL scale), 10ml (with 0.1ml scale) or micro pipette
and sucker.
2.8 Sterile culture dish. diameter of 60mm and 90mm.
2.9 Sterile test tube. 3mm × 50mm, 10mm × 75mm.
2.10 pH meter or pH colorimetric tube or precision pH test paper.
2.11 Automatic microbe biochemical identification system.
2.12 Sterile capillary tube.
3 Medium and Reagent
3.1 Buffer Petone Water (BPW). see A.1.
3.2 Tetrathionate Broth (TTB). see A.2.
3.3 Selenite Cystine (SC) Broth. see A.3.
3.4 Bismuth Sulfite (BS) Agar. see A.4.
3.5 HE agar. see A.5.
3.6 Xylose lysine Desoxycholate (XLD) Agar. see A.6.
3.7 Salmonella chromogenic medium.
3.8 Triple Sugar Iron (TSI) Agar. see A.7.
3.9 Petone water, indole reagent. see A.8.
3.10 Urea agar (pH7.2). see A.9.
3.11 Potassium cyanide (KCN) medium. see A.10.
3.12 Lysine decarboxylase test medium. see A.11.
3.13 Sugar fermentation tube. see A.12.
3.14 O-Nitrophenyl β-D galactopyranoside (ONPG) medium. see A.13.
3.15 Semi-solid agar. see A.14.
3.16 Sodium malonate medium. see A.15.
3.17 Salmonella O, H and Vi diagnosed serum.
3.18 Biochemical identification reagent kit.
4 Test Procedures
Salmonella test procedure is shown in Figure 1.
Generally, use 1.2%~1.5% agar culture as the antigens for the slide agglutination test.
Firstly, remove the self-agglutination reaction; drop one drop of saline on the clean
slide; mix the to-be-tested culture into the saline drops; so that it becomes uniform
turbid suspension; shake the slide gently for 30s~60s; observe the reaction under the
black background (if necessary, use magnifier to observe); if there is visible O
agglutination, then it is considered to have self-agglutination; otherwise it is considered
to have no self-agglutination. The culture without self-agglutination shall take the
serological identification as per the following method.
5.5.2 Identification of polyvalent bacterial antigen (O)
Draw 2 zones with size of 1cm×2cm; pick up 1-ring of to-be-tested bacteria; separately
place 1/2-ring on the upper part of each zone on the slide; thereof, the lower part of
one zone is added 1 drop of polyvalent bacterial (O) antiserum; the lower part of the
other zone is added 1 drop of saline to control. Then use sterile inoculation ring or
needle to separately grind the bacteria moss on two zones into emulsion. Tilt the slide
to shake and mix for 1min; observe against the black background; any degree of
agglutination was positive reaction. When O serum is not agglutinated, inoculate the
strains onto the medium with higher agar content (e.g. 2%~3%) to re-check; if the O
agglutination reaction is prevented due to the presence of Vi antigen, pick up bacteria
moss to make concentrated bacteria liquid in 1mL of saline; boiling on the alcohol lamp
flame then check.
5.5.3 Identification of polyvalent flagellum antigen (H)
The operation is the same as 5.5.2. When H antigen was poorly developed, inoculate
the strain into the center of 0.55%~0.65% semi-solid agar plate; when colonies were
growing, take bacteria from the edge to check; or inoculate the strain with the small
glass tube containing 0.3%~0.4% semi-solid agar for once or twice, take bacteria from
the far end, culture and then check.
5.6 Serological classification (optional)
5.6.1 Identification of O antigen
Use A~F polyvalent O serum to do the slide agglutination test; meanwhile use saline
to control. The self-agglutination substances in the saline is rough strain, which can’t
be classified.
The substance that is agglutinated by A~F polyvalent O serum shall successively use
O4; O3, O10; O7; O8; O9; O2 and O11 factor serum to do the agglutination test. Judge
the O groups according to the test results. The strains that are agglutinated by O3,
O10 serum shall use O10, O15, O34, O19 single factor serum to do the agglutination
test; judge the subgroups of E1, E4; the final determination of each O antigen
composition shall be based on the test results of O single factor serum; If there is no
O single factor serum, use two O complex factor serum to check.
H polyvalent 3 k, r, y, z, z10, lv, lw, lz13, lz28, lz40
H polyvalent 4, 1, 2; 1, 5; 1, 6; 1, 7; z6
H polyvalent 5 z4z23, z4z24, z4z32, z29, z35, z36, z38
H polyvalent 6 z39, z41, z42, z44
H polyvalent 7 z52, z53, z54, z55
H polyvalent 8 z56, z57, z60, z61, z62
The final determination of each H antigen composition shall be based on the test
results of H single factor serum; if there is no H single factor serum, then use two H
complex factor serum to verify.
When detecting H antigen in Phase-1 and failing to detect H antigen in Phase-2, or
when detecting H antigen in Phase-2 and failing to detect H antigen in Phase-1, 1
generation ~ 2 generations can be inoculated on the agar slope then check. If there is
still one-phase H antigen is found out, then use phase variation method to check
another phase. Single-phase bacteria don’t have to do phase variation test.
The phase variation test method is as follows.
Simple plate method. dry the surface moisture on the 0.35%~0.4% semi-solid agar
plate; pick up 1-ring of factor serum to drop onto the semi-solid plate surface; stand for
a moment; when serum is absorbed into agar, dibble the to-be-tested strains in the
center of serum; after culturing, take bacteria from the edge of growing bacteria moss
to test.
Small glass tube method. melt the semi-solid tube (each tube about 1mL~2mL) onto
the alcohol lamp; and cool off to 50°C; take 0.05mL ~ 0.1mL of known phase H factor
serum, add it into the molten semi-solid substance; after mixing evenly; use capillary
pipette to absorb and place into small glass tube for phase variation test; after
coagulating, use inoculation needle to pick up to-be-tested bacteria; inoculate onto one
end. Place the small glass tube horizontally onto the plate; put wet cotton beside it, so
that prevent the moisture is vaporized and dry shrink; check the result every day; after
the other phase bacteria dissociation, the bacteria can be picked up from the other end
to check. The concentration of serum in the medium shall have appropriate proportion,
when it is too high, the bacteria can’t grow; when it is too low, the same phase bacteria
power can’t be suppressed. Generally, it is added with serum amount of 1.200~1.800.
Small inverted tube method. place the small glass tube (the lower-end opening shall
remain a gap rather than flush) with two ends open into the semi-solid tube; the upper
end of small glass tube shall be higher than the medium surface; backup after
sterilization. Heating and melting on the temporarily-used alcohol lamp; cool off to 50°C;
pick up 1-ring of factor serum; add into the semi-solid substance of the small casing;
Appendix A
Medium and Reagent
A.1 Buffer Peptone Water (BPW)
A.1.1 Compositions
Peptone 10.0g
Sodium chloride 5.0g
Disodium hydrogen phosphate (containing 12 crystal water) 9.0g
Potassium dihydrogen phosphate ...
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.