化妆品微生物标准检验方法细菌总数测定 PDF.doc

化妆品微生物标准检验方法 细菌总数测定Standard methods of microbiological examination for determination of aerobic bacterial count中华人民共和国国家标准化妆品微生物标准检验方法细菌总数测定UDC 668：576 .85.07GB7918.287Standard methods ofmicrobiological examination for cosmetics Standard plate countThe peoples Republic of China National Standard for cosmeticsstandard methods of microbiological examination for UDC 668:576methods of microbiological examination for.85.07GB7918.287Standard cosmetics Standard plate count 细菌总数系指1g或1ml化妆品中所含的活菌，数量。测定细菌总数可用来判明化妆品被细菌污染的程度，以及生产单位所用的原料、工具设备、工艺流程、操作者的卫生状况，是对化妆品进行卫生学评价的综合依据。本标准采用标准平板计数法。1 方法提要 化妆品中污染的细菌种类不同，每种细菌都有它一定的生理物质性，培养时对营养要求，培养温度、培养时间、pH值、需氧性质等均有所不同。在实际工作中，不可能做到满足所有菌的要求，因此所测定的结果，只包括在本方法所使用的条件下（在卵磷脂、吐温80营养琼脂上，于37培养48h)生长的一群嗜中温的需氯及兼性厌氧的细菌总数。2 培养基和试剂2.1 生理盐水：见GB 7918-87化妆品微生物标准检验方法 总则。2.2 卵磷脂、吐温80-营养琼脂培养基成分： 蛋白胨 20g牛肉膏3g氯化钠 5g琼脂 15g卵磷脂 1g吐温80 7g蒸馏水 1000ml制法：先将卵磷脂加到少量蒸馏水中，加热溶解，加入吐温80将其他成分(除琼脂外)加到其余蒸馏水中，溶解。加入已溶解的卵磷脂、吐温80，混匀，调pH值为7.17.2，加入琼脂，121(15 1b)20min高压灭菌，储存于冷暗处备用。3 仪器3.1 锥形烧瓶。3.2 量筒。3.3 pH计或精密pH试纸。3.4 高压消毒锅。3.5 试管。3.6 灭菌平皿：直径9cm。3.7灭菌刻度吸管：10ml、2ml、1ml。3.8 酒精灯。3.9 恒温培养箱。3.10 放大镜。4 操作步骤4.1 用灭菌吸管吸取1:10稀释的检样2ml，分别注入到两个灭菌平甲内，每皿1ml。另取1ml注入到9ml灭菌生理盐水试管中(注意勿使吸管接触液面)，更换一支吸管，并充分混匀，使成1:100稀释液。吸取2ml,分别注入到两个灭菌平皿内，每皿1ml。如样品含菌量高，还可再稀释成1:1000，1:10000，等，每种稀释度应换1支吸管。4.2 将熔化并冷至4550的卵磷脂、吐温80、营养琼脂培养基倾注平皿内，每皿约15ml，另倾注一个不加样品的灭菌空平皿，作空白对照。随即转动平皿，使样品与培养基充分混合均匀，待不琼脂凝固后，翻转平皿，置37培养箱内培养48h。5 菌落计数方法先用肉眼观察，点数菌落数，然后再用放大510倍的放大镜检查，以防遗漏。记下各平皿的菌落数后。求出同一稀释度各平皿生长的平均菌落数。若平皿中有连成片状的菌落或花点样菌落蔓延生长时，该平皿不宜计数。若片状菌落不到平皿中的一半，面其余一半中菌落数分布又很均匀，则可将此半个平皿菌落计数后乘2，以代表全皿菌落数。6 菌落计数及报告方法6.1 首先选取平均菌落数在30300之间的平皿，作为菌落总数测定的范围。当只有一个稀释度的平均菌落数符合此范围时，即以该平皿菌落数乘其稀释倍数（见表中例）。6.2 若有两个稀释度，其平均菌落数均在30300个之间，则应求出两者菌落总数之比值来决定。若其比值小于或等于2应报告其平均数，若大于2则报告其中较小的菌落数(见表中例2及例3)。6.3 若所有稀释度的平均菌落数均大于300个，则应按稀释度最高的平均菌落数乘以稀释倍数报告之(见表中例4)。6.4 若所有稀释度的平均菌落数均少于30个，则应按稀释度最低的平均菌落数乘以稀释倍数报告之(见表中例5)。6.5 若所有稀释度的平均菌落数均不在30300个之间，其中一个稀释度大于300个，而相邻的另一稀释度小于30个时，则以接近30或300的平均菌落数乘以稀释倍数报告之(见表中例6)。6.6 若所有的稀释度均无菌生长，报告数为每克或每毫升小于10个。6.7 菌落计数的报告，菌落数在10以内时，按实有数值报告之，大于100时，采用二位有效数字，在二位有效数字后面的数值，应以四舍五入法计算。为了缩短数字后面零的个数，可用10的指数来表示(见下表报告方式栏)。在报告菌落数为“不可计”时，应注明样品的稀释度。细菌计数结果及报告方法The total number of bacteria refers to that of 1G or 1ml cosmeticscontaining live bacteria, number. Determination of the total number of bacteria can be used to ascertain cosmetic is the degree of bacterial contamination, as well as production units used by the raw materials,tools, equipment, process flow, the health status of cosmetic,hygienic evaluation on the basis of the comprehensive. This standarduses the standard plate count method. The 1 method cosmeticcontaminated with bacteria of different types, each species of bacteria has its certain physiological material, when cultured onnutritional requirements, culture temperature, culture time, pH value,oxygen properties are different. In real work, is impossible to satisfy all demands of the bacteria, the results of determination, including only the conditions ( in lecithin, Twain 80 Nutrient Agar, in 3748h )growth of a mesophilic the chlorine and facultative anaerobicbacteria. 2 culture media and reagents 2.1 saline: see GB 7918-87 standard methods of microbiological examination for . 2.2 lecithin,Twain 80- nutrient agar culture medium composition: beef extractpeptone 20g 3G sodium chloride 5g agar 15g lecithin 1g Twain 80 7g1000ml distilled water preparation method : first the lecithin to small amounts of distilled water, heating to dissolve, joined Twain 80 otheringredients ( except agar outside) to the rest of the distilled water,dissolved. Add dissolved lecithin, Twain 80, blending, regulating pH value was 7.1 7.2, add the agar, 121 ( 15 1b ) 20min high pressure sterilization, storage in the cold dark alternate. The 3instruments of the 3.1 conical flask. 3.2 cylinder. 3.3 pH or pH testprecision. 3.4 high pressure sterilizing pot. 3.5 tube. 3.6 sterile Petri dish: diameter of 9cm. 3.7: 10ml, 2ml sterilization scale straw, 1ml. 3.8alcohol lamp. The 3.9 thermostatic culture box. 3.10 magnifier. 4steps of 4.1 sterilization straw draw a 1:10 dilution of the sample 2ml,injected into the two sterilization flat A, each dish 1ml. Another 1ml into the 9ml of sterile saline in vitro ( pay attention not to make strawcontact surface), replace a straw, and mixed thoroughly, make 1:100 dilution. Draw 2ml, injected into the two sterile Petri dish, each dish 1ml. Such as samples of bacteria content high, can be diluted to1:1000, 1:10000, each dilution should change 1 straw. 4.2 will bemelted and cooled to 45 50 lecithin, Twain 80, nutrient agar pourplate, each dish is about 15ml, the other into a sample of sterilizedempty Petri dish, as blank control. Then turning Petri dish, make the sample and the medium are fully mixed evenly, when agar solidified,turning Petri dish, is 37 incubator culture 48h. In 5 the colony counting method to observed with the naked eye, number ofcolonies, then a magnification of 5 10 times magnificationendoscopy, to prevent the omission. Write down the plate after thecolony number. The same dilution of the Petri dish growth averagebacterial count. If the dish is connected into a lamellar colony or spending like colony spreading growth, not counting the Petri dish. If the lamellar colony to Petri dish in half, face the remaining half colony number in distribution is very uniform, may bring the half a Petri dishbacterium count by 2, to represent the whole dish colony number. 6colony counting and reporting method first selects 6.1 averagecolony number in 30 300 between the plate, as the colony determination range. When only a dilution of the average colonynumber is consistent with this range, namely in the Petri dish colony number by the dilution factor (see Table 1 ). 6.2 if two dilution, the average colony number all is in 30 300 between, should the colony total ratio to decide. If the ratio is less than or equal to 2 shall reportthe average, if greater than 2 report which smaller colonies (seeTable 2 and 3 ). 6.3 if all the dilution degree average colony numberis greater than 300, it should be according to the dilution of the highest average colony number multiplied by the dilution multiplereport (see Table 4 ). 6.4 if all the dilution degree average colony number less than 30, they should be the dilution of the lowest average colony number multiplied by the dilution multiple report (seeTable 5 ). 6.5 if all the dilution degree average colony number is in 30 300 between, wherein a dilution is greater than 300, and the other adjacent dilutions less than 30, to close to 30 or 300 in theaverage colony number multiplied by the dilution multiple report (seeTable 6 ). 6.6 if all dilution are sterile growth, report number per gram or per ml of less than 10. 6.7 count report, colony number in 10 when, according to the actual numerical report, more than 100, with two significant digits, in two digits following numerical, should be fourto five homes in calculation. In order to reduce the numbers behindthe number of zero, can be used 10 index to express (see table below report column ). In the report as not counting colonies ,should be marked sample dilution degree. Bacterial counting result and report method 执行标准：GB/T 7918-1987Executive standard: GB/T 7918-1987细菌总数系指1g或1ml化妆品中所含的活菌数量。测定细菌总数可用来判明化妆品被细菌污染的程度，以及生产单位所用的原料、工具设备、工艺流程、操作者的卫生状况，是对化妆品进行卫生学评价的综合依据。细菌总数系指1g或1ml化妆品中所含的活菌数量。测定细菌总数可用来判明化妆品被细菌污染的程度，以及生产单位所用的原料、工具设备、工艺流程、操作者的卫生状况，是对化妆品进行卫生学评价的综合依据。The total number of bacteria refers to 1g or 1ml cosmetics contained in the living bacterium number. Determination of the total number of bacteria can be used to ascertain cosmetic is the degree of bacterial contamination, as well as production units used by the raw materials,tools, equipment, process flow, the health status of cosmetic,hygienic evaluation on the basis of the comprehensive. The total number of bacteria refers to 1g or 1ml cosmetics contained in the living bacterium number. Determination of the total number of bacteria can be used to ascertain cosmetic is the degree of bacterial contamination, as well as production units used by the raw materials,tools, equipment, process flow, the health status of cosmetic,hygienic evaluation on the basis of the comprehensive.本标准采用标准平板计数法。1方法提要 化妆品中污染的细菌种类不同，每种细菌都有它一定的生理特性，培养时对营养要求，培养温度、培养时间、pH值、需氧性质等均有所不同。在实际工作中，不可能做到满足所有菌的要求，因此所测定的结果，只包括在本方法所使用的条件下（在卵磷脂、吐温80营养琼脂上，于37培养48h）生长的一群嗜中温的需氧及兼性厌氧的细菌总数。2培养基和试剂This standard uses the standard plate count method. The 1 methodcosmetic contaminated with bacteria of different types, each species of bacteria has its certain physiological characteristics, cultivation ofnutritional requirements, culture temperature, culture time, pH value,oxygen properties are different. In real work, is impossible to satisfy all demands of the bacteria, the results of determination, including only the conditions ( in lecithin, Twain 80 Nutrient Agar, in 3748h )growth of a group of mesophilic aerobic and facultative anaerobicbacteria. 2 culture media and reagents21生理盐水：见GB 7918187化妆品微生物标准检验方法总则。2.1 saline: see GB 7918.1 - 87 standard methods of microbiological examination for .22卵磷脂、吐温80-营养琼脂培养基成分：蛋白胨20g2.2 lecithin, Twain 80- nutrient agar culture medium composition:peptone 20g牛肉膏3g氯化钠5gBeef extract 3G sodium chloride 5g琼脂15g15g agar卵磷脂1gLecithin 1g吐温807gTwain 80 7g蒸馏水1000ml1000ml distilled water制法：先将卵磷脂加到少量蒸馏水中，加热溶解，加入吐温80将其它成分（除琼脂外）加到其余的蒸馏水中，溶解加入已溶解的卵磷脂、吐温80，混匀，调pH值为7174，加入琼脂，121（15 lb）20min高压灭菌，储存于冷暗处备用。Method: the lecithin added small amounts of distilled water, heating to dissolve, joined Twain 80 other ingredients ( except agar outside) tothe rest of the distilled water, dissolved by adding dissolved lecithin,Twain 80, blending, regulating pH value was 7.1 7.4, add the agar,121 ( 15 lb ) 20min high pressure sterilization stored in the dark,cold standby.3仪器3 instruments31锥形烧瓶。3.1 conical flask.32量筒。3.2 cylinder.33pH计或精密pH试纸。3.3 pH or pH test precision.34高压消毒锅。3.4 high pressure sterilizing pot.35试管。3.5 tube.36灭菌平皿：直径9cm。3.6 sterile Petri dish: diameter of 9cm.37灭菌刻度吸管：10ml、2ml、1ml。3.7: 10ml, 2ml sterilization scale straw, 1ml.38 酒精灯。3.8 alcohol lamp.39恒温培养箱。The 3.9 thermostatic culture box.310放大镜。3.10 magnifier.4操作步骤4 steps41用灭菌吸管吸取110稀释的检样2ml，分别注入到两个灭菌平皿内，每皿1ml。另取1ml注入到9ml灭菌生理盐水试管中（注意勿使吸管接触液面），更换一支吸管，并充分混匀，使成1100稀释液。吸取2ml，分别注入到两个灭菌平皿内，每皿1ml。如样品含菌量高，还可再稀释成11000，110000，等，每种稀释度应换1支吸管。The 4.1 sterilization straw draw 1 10 dilution of the sample 2ml,injected into the two sterile Petri dish, each dish 1ml. Another 1ml into the 9ml of sterile saline in vitro ( pay attention not to make strawcontact surface), replace a straw, and fully mixing, to 1: 100 diluted solution. Draw 2ml, injected into the two sterile Petri dish, each dish 1ml. Such as samples of bacteria content high, can be diluted to 1 1000, 1 10000, . . And so, each dilution should change 1 straw.42将熔化并冷至4550的卵磷脂、吐温80、营养琼脂培养基倾注平皿内，每皿约15ml，另倾注一个不加样品的灭菌空平皿，作空白对照。随即转动平皿，使样品与培养基充分混合均匀，待琼脂凝固后，翻转平皿，置37培养箱内培养48h。4.2 will be melted and cooled to 45 50 lecithin, Twain 80, nutrient agar pour plate, each dish is about 15ml, the other into a sample ofsterilized empty Petri dish, as blank control. Then turning Petri dish,make the sample and the medium are fully mixed evenly, the agarsolidified, turning Petri dish, is 37 incubator culture 48h.5菌落计数方法In 5 the colony counting method先用肉眼观察，点数菌落数，然后再用放大510倍的放大镜检查，以防遗漏。记下各平皿的菌落数后。求出同一稀释度各平皿生长的平均菌落数。若平皿中有连成片状的菌落或花点样菌落蔓延生长时，该平皿不宜计数。若片状菌落不到平皿中的一半，而其余一半中菌落数分布又很均匀，则可将此半个平皿菌落计数后乘2，以代表全皿菌落数。First observed with the naked eye, number of colonies, then a magnification of 5 10 times magnification endoscopy, to prevent the omission. Write down the plate after the colony number. The samedilution of the Petri dish growth average bacterial count. If the dish is connected into a lamellar colony or spending like colony spreadinggrowth, not counting the Petri dish. If the lamellar colony to Petri dishin half, while the remaining half colony number distribution is uniform,may bring the half a Petri dish bacterium count by 2, to represent thewhole dish colony number.6菌落计数及报告方法6 colony counting and reporting method61首先选取平均菌落数在30300之间的平皿，作为菌落总数测定的范围。当只有一个稀释度的平均菌落数符合此范围时，即以该平皿菌落数乘其稀释倍数（见表中例1）。6.1 first select the average colony number in 30 300 between theplate, as the colony determination range. When only a dilution of theaverage colony number is consistent with this range, namely in thePetri dish colony number by the dilution factor (see Table 1 ).62若有两个稀释度，其平均菌落数均在30300个之间，则应求出两者菌落总数之比值来决定。若其比值小于或等于2，应报告其平均数，若大于2则报告其中较小的菌落数（见表中例2及例3）。6.2 if two dilution, the average colony number all is in 30 300between, should the colony total ratio to decide. If the ratio is less than or equal to 2, it shall report its average, if greater than 2 reportwhich smaller colonies (see Table 2 and 3 ).63若所有稀释度的平均菌落数均大于300个，则应按稀释度最高的平均菌落数乘以稀释倍数报告之（见表中例4）。6.3 if all the dilution degree average colony number is greater than300, it should be according to the dilution of the highest averagecolony number multiplied by the dilution multiple report (see Table 4).64若所有稀释度的平均菌落数均少于30个，则应按稀释度最低的平均菌落数乘以稀释倍数报告之（见表中例5）。6.4 if all the dilution degree average colony number less than 30,they should be the dilution of the lowest average colony numbermultiplied by the dilution multiple report (see Table 5 ).65若所有稀释度的平均菌落数均不在30300个之间，其中一个稀释度大于300个，而相邻的另一稀释度小于30个时，则以接近30或300的平均菌落数乘以稀释倍数报告之（见表中例6）。6.5 if all the dilution degree average colony number is in 30 300between, wherein a dilution is greater than 300, and the other adjacent dilutions less than 30, to close to 30 or 300 in the averagecolony number multiplied by the dilution multiple report (see Table 6).66若所有的稀释度均无菌生长，报告数为每克或每毫升小于10个。6.6 if all dilution are sterile growth, report number per gram or per mlof less than 10.67菌落计数的报告，菌落数在10以内时，按实有数值报告之，大于100时，采用二位有效数字，在二位有效数字后面的数值，应以四舍五入法计算。为了缩短数字后面零的个数，