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食品安全国家标准 食品微生物学检验 沙门氏菌检验
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GB 4789.4-2016
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标准编号: GB 4789.4-2016 (GB4789.4-2016)
中文名称: 食品安全国家标准 食品微生物学检验 沙门氏菌检验
英文名称: National food safety standard -- Food microbiological examination -- Salmonella Test
行业: 国家标准
中标分类: X09
字数估计: 22,275
发布日期: 2016-12-23
实施日期: 2017-06-23
旧标准 (被替代): GB 4789.4-2010; SN 0170-1992; SN/T 2552.5-2010
标准依据: 国家卫生和计划生育委员会公告2016年第17号
GB 4789.4-2016: 食品安全国家标准 食品微生物学检验 沙门氏菌检验
GB 4789.4-2016 英文名称: National food safety standard -- Food microbiological examination -- Salmonella Test
1 范围
本标准规定了食品中沙门氏菌(Salmonella)的检验方法。
本标准适用于食品中沙门氏菌的检验。
2 设备和材料
除微生物实验室常规灭菌及培养设备外,其他设备和材料如下:
2.1 冰箱:2℃~5℃。
2.2 恒温培养箱:36℃±1℃,42℃±1℃。
2.3 均质器。
2.4 振荡器。
2.5 电子天平:感量0.1g。
2.6 无菌锥形瓶:容量500mL,250mL。
2.7 无菌吸管:1mL(具0.01mL刻度)、10mL(具0.1mL刻度)或微量移液器及吸头。
2.8 无菌培养皿:直径60mm,90mm。
2.9 无菌试管:3mm×50mm、10mm×75mm。
2.10 pH计或pH比色管或精密pH试纸。
2.11 全自动微生物生化鉴定系统。
2.12 无菌毛细管。
3 培养基和试剂
3.1 缓冲蛋白胨水(BPW):见A.1。
3.2 四硫磺酸钠煌绿(TTB)增菌液:见A.2。
3.3 亚硒酸盐胱氨酸(SC)增菌液:见A.3。
3.4 亚硫酸铋(BS)琼脂:见A.4。
3.5 HE琼脂:见A.5。
3.6 木糖赖氨酸脱氧胆盐(XLD)琼脂:见A.6。
3.7 沙门氏菌属显色培养基。
3.8 三糖铁(TSI)琼脂:见A.7。
3.9 蛋白胨水、靛基质试剂:见A.8。
3.10 尿素琼脂(pH7.2):见A.9。
3.11 氰化钾 (KCN)培养基:见A.10。
3.12 赖氨酸脱羧酶试验培养基:见A.11。
3.13 糖发酵管:见A.12。
3.14 邻硝基酚β-D半乳糖苷(ONPG)培养基:见A.13。
3.15 半固体琼脂:见A.14。
3.16 丙二酸钠培养基:见A.15。
3.17 沙门氏菌O、H和Vi诊断血清。
3.18 生化鉴定试剂盒。
4 检验程序
沙门氏菌检验程序见图1。
5 操作步骤
5.1 预增菌
无菌操作称取25g(mL)样品,置于盛有225mLBPW的无菌均质杯或合适容器内,以8000r/min~
10000r/min均质1min~2min,或置于盛有225mLBPW 的无菌均质袋中,用拍击式均质器拍打
1min~2min。若样品为液态,不需要均质,振荡混匀。如需调整pH,用1mol/mL无菌 NaOH 或
HCl调pH至6.8±0.2。无菌操作将样品转至500mL锥形瓶或其他合适容器内(如均质杯本身具有无
孔盖,可不转移样品),如使用均质袋,可直接进行培养,于36℃±1℃培养8h~18h。
如为冷冻产品,应在45℃以下不超过15min,或2℃~5℃不超过18h解冻。
5.2 增菌
轻轻摇动培养过的样品混合物,移取1mL,转种于10mLTTB内,于42℃±1℃培养18h~24h。
同时,另取1mL,转种于10mLSC内,于36℃±1℃培养18h~24h。
5.3 分离
分别用直径3mm的接种环取增菌液1环,划线接种于一个BS琼脂平板和一个XLD琼脂平板(或HE
琼脂平板或沙门氏菌属显色培养基平板),于36℃±1℃分别培养40h~48h(BS琼脂平板)或18h~24h
(XLD琼脂平板、HE琼脂平板、沙门氏菌属显色培养基平板),观察各个平板上生长的菌落,各个平板
上的菌落特征见表1。
5.4 生化试验
5.4.1 自选择性琼脂平板上分别挑取2个以上典型或可疑菌落,接种三糖铁琼脂,先在斜面划线,再于
底层穿刺;接种针不要灭菌,直接接种赖氨酸脱羧酶试验培养基和营养琼脂平板,于36℃±1℃培养
18h~24h,必要时可延长至48h。在三糖铁琼脂和赖氨酸脱羧酶试验培养基内,沙门氏菌属的反应结
果见表2。
5.4.2 接种三糖铁琼脂和赖氨酸脱羧酶试验培养基的同时,可直接接种蛋白胨水(供做靛基质试验)、
尿素琼脂(pH7.2)、氰化钾(KCN)培养基,也可在初步判断结果后从营养琼脂平板上挑取可疑菌落接
种。于36℃±1℃培养18h~24h,必要时可延长至48h,按表3判定结果。将已挑菌落的平板储存
于2℃~5℃或室温至少保留24h,以备必要时复查。
5.4.2.1 反应序号A1:典型反应判定为沙门氏菌属。如尿素、KCN和赖氨酸脱羧酶3项中有1项异
常,按表4可判定为沙门氏菌。如有2项异常为非沙门氏菌。
GB 4789.4-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard – Food
Microbiological Examination – Salmonella Test
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
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 &
immediately delivered to your EMAIL address in 0~60 minutes.
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
Foreword
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
follows.
--- 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.
3.18 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
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
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.
5.6.1 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
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
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
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
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.1.1 Compositions
Peptone 10.0g
Sodium chloride 5.0g
Disodium hydrogen phosphate (containing 12 crystal water) 9.0g
Potassium dihydrogen phosphate ...
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