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食品安全国家标准 食品微生物学检验 金黄色葡萄球菌检验
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GB 4789.10-2016
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标准编号: GB 4789.10-2016 (GB4789.10-2016)
中文名称: 食品安全国家标准 食品微生物学检验 金黄色葡萄球菌检验
英文名称: National Food Safety Standard -- Food Microbiological Examination -- Examination of Staphylococcus Aureus
行业: 国家标准
中标分类: C53
国际标准分类: 07.100.30
字数估计: 17,177
发布日期: 2016-12-23
实施日期: 2017-06-23
旧标准 (被替代): GB 4789.10-2010; SN/T 0172-2010; SN/T 2154-2008
标准依据: National Health and Family Planning Commission Notice No.17 of 2016
发布机构: 中华人民共和国国家卫生和计划生育委员会、国家食品药品监督管理总局
范围: 本标准规定了食品中金黄色葡萄球菌(Staphylococcus aureus)的检验方法。本标准第一法适用于食品中金黄色葡萄球菌的定性检验;第二法适用于金黄色葡萄球菌含量较高的食品中金黄色葡萄球菌的计数;第三法适用于金黄色葡萄球菌含量较低的食品中金黄色葡萄球菌的计数。
GB 4789.10-2016: 食品安全国家标准 食品微生物学检验 金黄色葡萄球菌检验
GB 4789.10-2016 英文名称: National Food Safety Standard -- Food Microbiological Examination -- Examination of Staphylococcus Aureus
1 范围
本标准第一法适用于食品中金黄色葡萄球菌的定性检验;第二法适用于金黄色葡萄球菌含量较高的
食品中金黄色葡萄球菌的计数;第三法适用于金黄色葡萄球菌含量较低的食品中金黄色葡萄球菌的计数。
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 恒温培养箱:36℃±1℃。
2.2 冰箱:2℃~5℃。
2.3 恒温水浴箱:36℃~56℃。
2.4 天平:感量0.1g。
2.5 均质器。
2.6 振荡器。
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 无菌锥形瓶:容量100mL、500mL。
2.9 无菌培养皿:直径90mm。
2.10 涂布棒。
2.11 pH计或pH比色管或精密pH试纸。
3 培养基和试剂
3.1 7.5%氯化钠肉汤:见A.1。
3.2 血琼脂平板:见A.2。
3.3 Baird-Parker琼脂平板:见A.3。
3.4 脑心浸出液肉汤(BHI):见A.4。
3.5 兔血浆:见A.5。
3.6 稀释液:磷酸盐缓冲液:见A.6。
3.7 营养琼脂小斜面:见A.7。
3.8 革兰氏染色液:见A.8。
3.9 无菌生理盐水:见A.9。
第一法 金黄色葡萄球菌定性检验
4 检验程序
金黄色葡萄球菌定性检验程序见图1。
图1 金黄色葡萄球菌检验程序
5 操作步骤
5.1 样品的处理
称取25g样品至盛有225mL7.5%氯化钠肉汤的无菌均质杯内,8000r/min~10000r/min均质
1min~2min,或放入盛有225mL7.5%氯化钠肉汤无菌均质袋中,用拍击式均质器拍打1min~
2min。若样品为液态,吸取25mL样品至盛有225mL7.5%氯化钠肉汤的无菌锥形瓶(瓶内可预置适
当数量的无菌玻璃珠)中,振荡混匀。
5.2 增菌
将上述样品匀液于36℃±1℃培养18h~24h。金黄色葡萄球菌在7.5%氯化钠肉汤中呈混浊
生长。
5.3 分离
将增菌后的培养物,分别划线接种到Baird-Parker平板和血平板,血平板36℃±1℃培养18h~
24h。
5.4 初步鉴定
金黄色葡萄球菌在Baird-Parker平板上呈圆形,表面光滑、凸起、湿润、菌落直径为2mm~3mm,
颜色呈灰黑色至黑色,有光泽,常有浅色(非白色)的边缘,周围绕以不透明圈(沉淀),其外常有一清晰
带。当用接种针触及菌落时具有黄油样黏稠感。有时可见到不分解脂肪的菌株,除没有不透明圈和清
晰带外,其他外观基本相同。从长期贮存的冷冻或脱水食品中分离的菌落,其黑色常较典型菌落浅些,
且外观可能较粗糙,质地较干燥。在血平板上,形成菌落较大,圆形、光滑凸起、湿润、金黄色(有时为白色),菌落周围可见完全透明溶血圈。挑取上述可疑菌落进行革兰氏染色镜检及血浆凝固酶试验。
5.5 确证鉴定
5.5.1 染色镜检:金黄色葡萄球菌为革兰氏阳性球菌,排列呈葡萄球状,无芽胞,无荚膜,直径约为
0.5μm~1μm。
5.5.2 血浆凝固酶试验:挑取Baird-Parker平板或血平板上至少5个可疑菌落(小于5个全选),分别接
种到5mLBHI和营养琼脂小斜面,36℃±1℃培养18h~24h。
取新鲜配制兔血浆0.5mL,放入小试管中,再加入BHI培养物0.2mL~0.3mL,振荡摇匀,置
36℃±1℃温箱或水浴箱内,每半小时观察一次,观察6h,如呈现凝固(即将试管倾斜或倒置时,呈现
凝块)或凝固体积大于原体积的一半,被判定为阳性结果。同时以血浆凝固酶试验阳性和阴性葡萄球菌
菌株的肉汤培养物作为对照。也可用商品化的试剂,按说明书操作,进行血浆凝固酶试验。
结果如可疑,挑取营养琼脂小斜面的菌落到5mLBHI,36℃±1℃培养18h~48h,重复试验。
5.6 葡萄球菌肠毒素的检验(选做)
可疑食物中毒样品或产生葡萄球菌肠毒素的金黄色葡萄球菌菌株的鉴定,应按附录B检测葡萄球
菌肠毒素。
6 结果与报告
6.1 结果判定:符合5.4、5.5,可判定为金黄色葡萄球菌。
6.2 结果报告:在25g(mL)样品中检出或未检出金黄色葡萄球菌。
第二法 金黄色葡萄球菌平板计数法
7 检验程序
金黄色葡萄球菌平板计数法检验程序见图2。
图2 金黄色葡萄球菌平板计数法检验程序
8 操作步骤
8.1 样品的稀释
8.1.1 固体和半固体样品:称取25g样品置于盛有225mL磷酸盐缓冲液或生理盐水的无菌均质杯
内,8000r/min~10000r/min均质1min~2min,或置于盛有225mL稀释液的无菌均质袋中,用拍
击式均质器拍打1min~2min,制成1∶10的样品匀液。
8.1.2 液体样品:以无菌吸管吸取25mL样品置于盛有225mL磷酸盐缓冲液或生理盐水的无菌锥形
瓶(瓶内预置适当数量的无菌玻璃珠)中,充分混匀,制成1∶10的样品匀液。
8.1.3 用1mL无菌吸管或微量移液器吸取1∶10样品匀液1mL,沿管壁缓慢注于盛有9mL磷酸盐
缓冲液或生理盐水的无菌试管中(注意吸管或吸头尖端不要触及稀释液面),振摇试管或换用1支1mL
无菌吸管反复吹打使其混合均匀,制成1∶100的样品匀液。
吸头。
8.2 样品的接种
根据对样品污染状况的估计,选择2个~3个适宜稀释度的样品匀液(液体样品可包括原液),在进
行10倍递增稀释的同时,每个稀释度分别吸取1mL样品匀液以0.3mL、0.3mL、0.4mL接种量分别
加入三块Baird-Parker平板,然后用无菌涂布棒涂布整个平板,注意不要触及平板边缘。使用前,如
Baird-Parker平板表面有水珠,可放在25℃~50℃的培养箱里干燥,直到平板表面的水珠消失。
8.3 培养
在通常情况下,涂布后,将平板静置10min,如样液不易吸收,可将平板放在培养箱36℃±1℃培
养1h;等样品匀液吸收后翻转平板,倒置后于36℃±1℃培养24h~48h。
8.4.1 金黄色葡萄球菌在Baird-Parker平板上呈圆形,表面光滑、凸起、湿润、菌落直径为2mm~
3mm,颜色呈灰黑色至黑色,有光泽,常有浅色(非白色)的边缘,周围绕以不透明圈(沉淀),其外常有一
清晰带。当用接种针触及菌落时具有黄油样黏稠感。有时可见到不分解脂肪的菌株,除没有不透明圈
和清晰带外,其他外观基本相同。从长期贮存的冷冻或脱水食品中分离的菌落,其黑色常较典型菌落浅
些,且外观可能较粗糙,质地较干燥。
8.4.2 选择有典型的金黄色葡萄球菌菌落的平板,且同一稀释度3个平板所有菌落数合计在20CFU~
200CFU之间的平板,计数典型菌落数。
8.4.3 从典型菌落中至少选5个可疑菌落(小于5个全选)进行鉴定试验。分别做染色镜检,血浆凝固
酶试验(见5.5);同时划线接种到血平板36℃±1℃培养18h~24h后观察菌落形态,金黄色葡萄球菌
9 结果计算
9.1 若只有一个稀释度平板的典型菌落数在20CFU~200CFU之间,计数该稀释度平板上的典型菌
落,按式(1)计算。
9.2 若最低稀释度平板的典型菌落数小于20CFU,计数该稀释度平板上的典型菌落,按式(1)计算。
9.3 若某一稀释度平板的典型菌落数大于200CFU,但下一稀释度平板上没有典型菌落,计数该稀释
度平板上的典型菌落,按式(1)计算。
9.4 若某一稀释度平板的典型菌落数大于200CFU,而下一稀释度平板上虽有典型菌落但不在
20CFU~200CFU范围内,应计数该稀释度平板上的典型菌落,按式(1)计算。
9.5 若2个连续稀释度的平板典型菌落数均在20CFU~200CFU 之间,按式(2)计算。
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 &
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
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,
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
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;
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);
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
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
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
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
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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