美国微生物测试USP51和USP61+62
微生物计数检查法USP61中英对照版
非无菌产品微生物学检查：微生物计数检查法61中英对照版<61> :非无菌产品微生物学检查：微生物计数检查法导言.以下所描述的这些检测将使得对在有氧的条件下生长的嗜温性细菌和真菌进行定量计数成为可能。
a . , , , .这些检测主要设计用于测定一种物质或制备品是否符合已确立的微生物质量标准。
当用于此类目的时，需遵照以下所给的说明，包括待取样品的数量，并且按照下面所述解释结果。
.这些方法不适用于以活菌作为活性成分的产品。
, , , .可以使用替代的微生物规程，包括自动化方法，只要已经证明它们与药典方法具同等作用。
通用规程12 . .在经过设计可避免外来微生物污染供试产品的条件下，进行此项测定。
用于避免污染的这些预防措施是必须做到，它们不会影响任何试图在此项检验中揭示的微生物。
, , , . , .如果供试产品具有抗菌活性，则此活性需在尽可能的范围内去除或中和。
如果将灭活剂用于这个目的，则必须证实它们的功效和对微生物不具毒性。
, .如果表面活性物质用于样品制备，则必须证实它们对微生物不具毒性以及与所使用的任何灭活剂的兼容性。
计数法, . () ; , , .按规定，使用膜过滤法或多个平板计数法中的一种。
液体稀释法（）通常对微生物计数而言是最不准确的方法；然而，对具备非常低生物载荷的特定产品类型，它可能是最合适的方法。
a . a . .方法的选择基于某些因素，例如产品的性质和微生物的要求限度。
所选的方法必须能够对充足的样本量进行检测，以判断与质量标准的符合性。
所选方法的适用性必须被建立。
1.表1. 供试微生物的制备和使用生长促进在产品存在的情况下计数方法的适用性微生物供试菌株的制备总好氧微生物计数总酵母菌和霉菌计数总好氧微生物计数总酵母菌和霉菌计数6538, 9518, 4.83, 13276金黄色葡萄球菌如： 6538, 9518, 4.83, 或 13276300-350 18-24大豆酪蛋白消化物琼脂培养基或大豆酪蛋白消化物肉汤培养基300-350 18-24 小时≤100 300-350≤3大豆酪蛋白消化物培养基和大豆酪蛋白消化物肉汤培养基≤100 300-350 ≤3 天≤100 300-350≤3大豆酪蛋白消化物培养基（最大几率数）大豆酪蛋白消化物肉汤培养基≤100 300-350 ≤3 天9027, 8626 82.118, 13275绿脓杆菌如 9027, 8626 82.118, 或 13275300-350 18-24大豆酪蛋白消化物琼脂培养基或大豆酪蛋白消化物肉汤培养基300-350 18-24 小时≤100 300-350≤3大豆酪蛋白消化物琼脂培养基和大豆酪蛋白消化物肉汤培养基≤100 300-350≤3 天≤100 300-350≤3大豆酪蛋白消化物琼脂培养基（最大几率数）大豆酪蛋白消化物肉汤培养基≤100 300-350≤3 天6633, 8054, 52.62, 3134枯草芽孢杆菌如 6633, 8054, 52.62, 或 3134300-350 18-24大豆酪蛋白消化物琼脂培养基或大豆酪蛋白消化物肉汤培养基300-350 18-24 小时≤100 300-350≤3大豆酪蛋白消化物琼脂培养基和大豆酪蛋白消化物肉汤培养基≤100 300-350≤3 天≤100 300-350≤3大豆酪蛋白消化物琼脂培养基（最大几率数）大豆酪蛋白消化物肉汤培养基≤100 300-350≤3 天10231, 3179, 48.72, 1594白色念珠菌如 10231, 3179, 48.72, 或 1594200-250 2-3（沙氏）葡萄糖琼脂培养基或（沙氏）葡萄糖肉汤培养基≤100 300-350≤5 大豆酪蛋白消化物琼脂培养基≤100 300-350≤5 天≤100 200-250≤5（沙氏）葡萄糖琼脂培养基≤100 200-250≤5 天≤100 300-350≤5 :大豆酪蛋白消化物琼脂培养基≤100 300-350≤5 天 (最大几率数)：不适用≤100 200-250≤5（沙氏）葡萄糖琼脂培养基≤100 200-250≤5 天16404, 149007, 1431.83, 9455黑曲霉如 16404, 149007, 1431.83, 或 9455200-250 5-7 ,（沙氏）葡萄糖琼脂培养基或马铃薯葡萄糖琼脂培养基200-250 5-7天,或直到实现良好的产孢≤100 300-350≤5大豆酪蛋白消化物琼脂培养基≤100 300-350≤5 天≤100 200-250≤5（沙氏）葡萄糖琼脂培养基≤100 200-250≤5 天≤100 300-350≤5 :大豆酪蛋白消化物琼脂培养基≤100 300-350≤5 天(最大几率数)：不适用≤100 200-250≤5（沙氏）葡萄糖琼脂培养基≤100 200-250≤5 天生长促进试验和计数方法的适用性通用考虑因素.在供试产品存在的情况下，必须确立检测微生物的试验能力。
美国微生物测试USP51和USP61+62
美国微生物测试USP51 和USP61＋62 介绍(2009/12/22 18:31)美国微生物测试USP51 和USP61＋62 介绍玩具需作微生物测试的材料：用于玩具中的化妆品、液体、糊状物、油灰（putties)、凝胶和粉末（艺术品材料，如粉笔、蜡笔、墨水等除外）1. USP<61>微生物限量测试*1- 测试目的: 检验材料本身受微生物污染的情况（也即微生物洁净度情况）- USP<61>包括以下六个微生物测试：(1) Total aerobic microbial Count 菌落总数(定量）(2) Mould and Yeast Count 霉菌和酵母菌数(定量）(3) Staphylococcus aureus 金黄色葡萄球菌(定性）(4) Pseudomonas aeruginosa 绿脓杆菌（定性）(5) Salmonella 沙门氏菌（定性）(6) Escherichia Coli 大肠杆菌（定性）USP61 Limit Requirement:(1)+(2) < 5000 CFU/g ( if the material was used in baby product or eye area product, the limit should be < 500 CFU/g)(3)/(4)/(5)/(6) should be “ABSENT per 10g “2. USP<51>防腐剂抗菌效力测试- 测试目的为防止的材料在保存过程中或使用过程中发生微生物*现象，须在材料中加入适量的防腐剂，而防腐剂效果如何则需通过USP《51》测试进行评价。
-USP<51>测试简述：将标准指定编号的以下菌种接入样品，然后在第7 天，第14 天，第28 天分别检验每种菌的存活数量，存活数量越少，其防腐剂抗菌效果越好。
Staphylococcus aureus 金黄色葡萄球菌Escherichia coli 大肠杆菌Pseudomonas aeruginosa 绿脓杆菌Candida albicans 白色念株菌Aspergillus niger 黑曲霉USP51 Limit requirement (for toy):细菌(Staphylococcus aureus/ Escherichia coli / Pseudomonas aeruginosa)14 天细菌减少≥2.0 LOG (99%)28 天不再繁殖真菌(Candida albicans /Aspergillus niger)14 天和28 天不再繁殖Remark: LOG REDUCTION = LOG10 (INITIAL COUNT / NO. OFMICRO-ORGANISM RECOVERED)*1 Remark:最近，美国药典（USP）发布了重大调整，原第61章“微生物限量测试”被拆分成两部分，即第61章“非灭菌产品中微生物测试：微生物计数检测”和第62章“非灭菌产品中微生物测试：特定微生物检测”。
USP微生物检测精要
100
100
药物制剂和药用物质的接受标准
非无菌原料的微生物质量标准
剂型 TAMC TCYMC 无控制微生物 （cfu/g or mL）（cfu/g or mL） （1g or mL）
部分药物成分， 1000 辅料
100
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药物制剂和药用物质的接受标准
除了表中的微生物，其他需检查的微生 物的重要性应根据下述评估：
USP概述 USP与法律
如果一个产品或器械声称或将其描述为官方药 典中已认可的药物，但是其效力与药典标准不 符，或其质量或纯度低于药典标准，将被认为 是次级品。（联邦食品、药品、化妆品法案 第 501（b）章节）
USP概述 USP微生物学内容
与其总的使命一致，USP在微生物学方 面的角色是，在微生物方面连同其他要 求来发展公共标准，保证产品质量的一 致性——药品、辅料和原料药。
药物制剂和药用物质的接受标准
有害微生物：
洋葱伯克氏菌
需氧的非芽孢杆菌。存在于水中，土壤及植物 中，如水果和蔬菜。伯克氏菌属中有两种常见 人体致病菌，洋葱伯克氏菌及类鼻疽伯克氏菌。 洋葱伯克氏菌是患有囊肿性纤维化和慢性肉芽 肿疾病的患者体内重要的呼吸道致病菌。以前 属于假单胞菌菌属。
重要区域环控用培养基必须
——双层包装 ——最终灭菌，否则进行全数预培养及用前检查
微生物实验室规范
培养基质控测试
所有待用的培养基应检查：
——生长能力
每批制备的培养基均需进行 测试菌种根据药典，供应商说明及环境中分离得到的菌株——无菌 ——pH，±0.2 ——容器/平皿的完整性 ——抑制或指示能力
生产商必须按GMP的指导进行制剂产品的生产 储存和分发，以保证最终制剂仅带有低水平的 微生物。
USP通则目录整理
USP〈21〉温度计USP〈31〉容量仪器USP〈41〉称量与天平USP〈51〉抗菌有效性测试USP〈55〉生物指示剂-耐受力测试USP〈61〉非无菌产品微生物检测：微生物计数测试USP〈62〉非无菌产品微生物检测：特定微生物测试USP〈71〉无菌测试USP〈85〉细菌内毒素测试USP〈87〉体外生物反应测试USP〈151〉热源测试USP〈191〉鉴别USP〈231〉重金属-背景和主题USP〈232〉重金属杂质-限度USP〈233〉重金属杂质-检测规程USP〈467〉残留溶剂USP〈695〉多晶型USP〈741〉熔点USP〈1035〉生物指示剂USP〈1072〉消毒剂与杀菌剂USP〈1111〉非无菌药品的微生物评估USP〈1112〉水分活性应用USP〈1113〉微生物特性确定、鉴定及菌株分型USP〈1116〉无菌工艺环境的微生物控制与监测USP〈1117〉微生物实验室规范USP〈1151〉药物剂型（pharmaceutical dosage forms）USP〈1196〉药典协调USP〈1207〉无菌产品包装-完整性评估USP〈1208〉无菌测试-隔离系统验证USP〈1209〉灭菌化学和物理化学指示剂及综合指示剂USP〈1211〉灭菌与无菌保证USP〈1222〉最终灭菌药品-参数放行USP〈1223〉微生物替代方法验证USP〈1224〉转移USP〈1225〉验证USP〈1226〉确认USP〈1227〉药物的微生物回收率验证USP〈1231〉制药用水USP〈2021〉微生物计数测试-营养和食品补充剂USP〈2022〉控制菌的微生物检查-营养和食品补充剂USP〈2023〉非无菌营养和食品补充剂的微生物评估USP〈2232〉试行版膳食补充剂中的重金属元素污染整理人*****************。
USP微生物试验说明
美國藥典微生物試驗說明Essentials of USP Microbiological Testing研討會大綱I.基礎微生物學II.良好微生物實驗室規範III.藥典協同之改變IV.IV.滅菌滅菌滅菌與無菌保證與無菌保證V.微生物方法之確效微生物學研究微小生命形式的生物學分支研究微小生命形式的生物學分支。
典型的微小生命形式是單細胞的生命形式的微小生命形式是單細胞的生命形式，，也包括非常少也包括非常少量量的多細胞生物體 新陳代謝基本需求能量(如光、硫、一氧化碳或氨一氧化碳或氨))碳、氮、鈣、磷、硫、鎂、鉀和鈉 水(即使真菌也需要0.6 Aw 以上的游以上的游離離水)分類學生物環境多樣性嗜低溫菌嗜中溫菌嗜高溫菌生物環境多樣性嗜酸性菌嗜中性菌嗜鹼性菌依DNA在細胞中的情況可將細胞分為兩大類：真核細胞Eukaryotic cells原核細胞Prokaryotic cells原核生物Prokaryotic cells大部分細菌對人體無害如: 乳酸菌但, 也有微生物會致病細菌(分2界) 古細菌 真細菌原核生物革原核生物革蘭蘭氏分類氏分類法法Hans C.J. Gram –丹麥細菌學家(1853-1938).發現發現了了細胞學上的染色和染色技術細胞學上的染色和染色技術（（革蘭氏染色染色），），），用於區分細菌的分用於區分細菌的分用於區分細菌的分類類學組群學組群。
脂多糖(相對耐熱)如果進入人體，能導致發燒、白血球減少、腹瀉、休克（熱原Pyrogen）(內毒素Endotoxin)革蘭氏陽性結構革蘭氏陰性結構什麼是芽孢?在惡劣惡劣的環境條件下產生的有再生能的環境條件下產生的有再生能的環境條件下產生的有再生能力力的細胞或細胞群的細胞或細胞群。
特徵特徵：：–厚壁–耐惡劣惡劣環境環境–非常緩慢的代謝速非常緩慢的代謝速度度分4界原生動物 動物植物真菌真菌-特徵皆為化學異異營菌皆為化學具強大的酵素 (腐生或寄生腐生或寄生) ) ) 具強大的酵素pH5））酸性的生活環境（（pH5 比細菌比細菌更更酸性的生活環境高鹽、、高糖環境中生長高鹽黴菌和蕈是多細胞酵母是單細胞新陳代謝Metabolism 依據分解代謝是否能夠使用O2為什麼了解微生物對新陳代謝的需求很重要？為的是想培養微生物生長更為的是防止微生物生長USP微生物相關務必遵守章節<51> 防腐效力/Antimicrobial Preservatives –Effectiveness耐受力力測試<55> 生物指示劑–耐受Biological Indicators –Resistance Performance Tests微生物計數數試驗非無菌產品的微生物檢驗：：微生物計<61> 非無菌產品的微生物檢驗Microbiological Examination of Nonsterile Products:Microbial Enumeration Tests<62> 特定菌的微生物檢查/ Microbiological Proceduresfor Absence of Specified Microorganisms<71> 無菌試驗/ Sterility Tests<85> 細菌內毒素試驗Bacterial Endotoxins TestUSP微生物相關章節建議遵循<1035> 滅菌生物指示劑<1072> 消毒劑與殺菌劑<1111> 非無菌產品的微生物評估<1112> 水活性應用<1116> 清淨室和其他管制環境的微生物評估<1117> 微生物實驗室規範 <1207> 無菌產品包裝–完整性評估 <1209> 滅菌–化學和物理化學指示劑，及綜合指示劑<1211> 滅菌與無菌保證<1222> 最終滅菌藥品–參數放行 <1223> 微生物方法確效<1227> 藥物的微生物回收率確效 <1231> 製藥用水<2021> 微生物計數試驗–營養和膳食補充劑<2022> 特定菌的微生物檢查–營養和膳食補充劑<2023> 非無菌營養和膳食補充劑的微生物評估EP & USP & JP 藥典方法的國際協合International Harmonization of Pharmacopoeia時限–現行的EP 只維持到2008年12月31日–USP 將維持至2009年4月影響行適用性試驗並需重新進行–各藥廠將必需提早因應,並需重新進批產品來來測試其一致性.( Suitability Test),以至少3 批產品此協合後的方法不不僅在產品( non-sterile)的稀釋液此協合後的方法及預試驗,生長試驗上有改變,在部份培養基的組成有所改變,甚至新增一些檢驗項目與培養基,在有些品管菌株及接種量量都有改變品管菌株及接種微生物限量(USP <61> & EP 2.6.12) Microbial Enumeration TestTAMC= Total aerobic microbial count (on TSA, incl. fungi)TYMC= Total combined yeast/mould count (on Sab.4%-Dextrose Agar, incl. bacteria)特定微生物USP <62> & EP 2.6.13 Tests for Specific Microorganisms目前–Escherichia coli–Salmonella–Pseudomonasaeruginosa–Staphylococcusaureus 協調後–Escherichia coli–Salmonella–Pseudomonas aeruginosa–Staphylococcus aureus–Clostridia 產芽孢梭菌–Bile-tolerant Gram-negative bacteria 耐膽鹽革耐膽鹽革蘭蘭氏陰性菌–Candida albicans白色白色念念珠菌Test for Escherichia coli:Presence/Absence (Current USP Method)Test for Escherichia coli:Presence/Absence (Harmonized Method)<1112> 水活性應用水活性是水活性是什什麼?–產品中水的蒸氣壓P 與相同溫與相同溫度度下純水中的蒸氣壓Po 的比的比率率。
微生物检测方法-USP
<61>非无菌产品微生物检查：微生物计数检查简介：下述方法能够对在有氧条件下生长的嗜温性细菌和真菌进行定量计数。
该检查主要是为了确定某种物质或剂型是否符合既定的微生物质量标准。
当以此为目的时，按照下面给出的规定进行，包括所需的样品数量以及按照下文规定的要求对结果进行描述。
此方法不适用于用活微生物作为活性成分的产品。
若使用了其他的微生物程序，包括自动方法，则应提供其等同于药典方法的证明。
一般程序为了避免外来微生物的影响，必须在设定的条件下进行微生物检查。
为避免此污染所采取的预防措施必须不影响所要进行检查的微生物。
如果待测样品具有抗微生物活性，则在可能的情况下，移除或中和这种抗微生物活性。
如果因此而是用了钝化剂，则必须证明其有效性及对微生物无毒性。
如果在处理样品时使用了表面活性剂，必须证明其对微生物无毒性及与证明其所使用的钝化剂的兼容性。
计数方法用薄膜过滤法和平皿计数法。
最大机率法（MPN）一般来说最不准确的微生物计数方法。
但是对于生物负荷非常小的确定的产品来说可能是最适合的方法。
对检查方法的选择是基于像产品的本质和微生物的限度这样一些因素。
所选择的方法必须能够用足够的样品来判断出其是否符合标准规定。
必须证明所选择的方法的适用性。
生长促进试验、计数方法的适用性和阴性对照一般原则必须证明检测方法能够从携带微生物的样品中检测出此微生物。
如果在检测过程中有了改变或会影响检测结果的产品的改变，则必须证明其适用性。
测试菌株的准备用标准化的测试菌株的稳定混悬液或用下述规定的方法准备测试菌株。
使用菌种培养维护技术（菌种系统），以使从原始主菌株中移除的用于接种的微生物不大于5个片段。
按照表1中所描述的方法分别对细菌和真菌的测试菌株进行培养。
用PH 7.0 的氯化钠-蛋白胨缓冲液或PH 7.2 磷酸盐缓冲液来配制测试混悬液；制备A. brasiliensis 孢子混悬液，可加入0.05%的聚山梨酯80。
在2小时内使用该混悬液或如果在2°－8°储存则在24小时内使用。
USP-51抗菌效力测试(完整资料).doc
【最新整理，下载后即可编辑】51ANTIMICROBIAL EFFECTIVENESS TESTING抗菌的效力测试Antimicrobial preservatives are substances added to nonsterile dosage forms toprotect them from microbiological growth or from microorganisms that areintroduced inadvertently during or subsequent to the manufacturing process. Inthe case of sterile articles packaged in multiple-dose containers, antimicrobialpreservatives are added to inhibit the growth of microorganisms that may beintroduced from repeatedly withdrawing individual doses.抗菌防腐剂是实际被添加到有菌的剂量配方里的物质，使他们不受微生物滋长或是被无意中引进到生产过程中的微生物侵害。
在无菌包装多剂量配方的容器里，抗菌防腐剂被添加用来抑制由重复添加单剂量所引进的微生物的滋长。
Antimicrobial preservatives should not be used as a substitute for goodmanufacturing practices or solely to reduce the viable microbial population of anonsterile product or control the presterilization bioburden of multidoseformulations during manufacturing. Antimicrobial preservatives in compendialdosage forms meet the requirements for Added Substancesunder Ingredients and Processesin the General Notices.抗菌防腐剂不应被作为在良好生产操作中的替代方式使用，或仅是用来减少一个有菌产品的微生物群数量，或是在生产时控制多剂量的微生物量。
哪里可以做微生物测试microbiological USP6162USP51
USP51 Limit requirement (for toy): 细菌 (Staphylococcus aureus/ Escherichia coli / Pseudomonas aeruginosa) 14 天细菌减少≥2.0 LOG (99%) 28 天不再繁殖 真菌 (Candida albicans /Aspergillus niger)? 14 天 和28= LOG10 (INITIAL COUNT / NO. OF MICRO-ORGANISM RECOVERED) *1 Remark: 最 近，美国药典（USP）发布了重大调整，原第61章“微生物限量测试” 被拆分成两部分，即第61章“非灭菌产品中微生物测试：微生物计数检 测”和第62章“非灭菌产品中微生物测试：特定微生物检测”。调整后 的两章（请参见下表），同时出口欧美的客户其测试成本将大大节约。 新USP61+62章节将于2009年5月1日生效。
USP61+62不断更新中。。
微生物测试microbiological limitsUSP616251
微生物测试USP51 和USP61＋62 玩具需作微生物测试的材料： 用于玩具中的化妆品、液体、糊状物、油 灰（putties)、凝胶 和 粉末 （艺术品材料，如粉笔、蜡笔、墨水等除外） 1. USP微生物限量测试*1 - 测试目的: 检验材料本身受微生物污染的情况 （也即微生物洁净度情况） - USP包括以下六个微生物测试： (1) Total aerobic microbial Count 菌落总数 (定量） (2) Mould and Yeast Count 霉 菌和酵母菌数 (定量） (3) Staphylococcus aureus 金黄色葡萄球菌 (定性） (4) Pseudomonas aeruginosa 绿脓杆菌（定性） (5) Salmonella 沙门氏 菌 （定性） (6) Escherichia Coli 大肠杆菌 （定性）
微生物计数USP61中文版
微生物计数检测需氧菌总计数USP<61>USP<61>中文版美国药典微生物限量61）微生物限度检测（MICROBIAL LIMIT TESTS）此章提供方法来检测可能存在的好氧微生物其他制药过程中可能出现的微生物的数量，包括原材料和成品中的。
如果经过验证确认可以得到相同或更好的检测结论，也允许采用自动化的检测方法。
在样品检测过程中须进行无菌操作。
若无特别说明，则“培养（incubate）”一词指在30—35℃的培养箱培养24至48小时；“生长（growth）”一词用于专门的判定，说明“存在和可能存在活的微生物”。
准备实验 (Preparatory Testing)本章涉及实验结果的有效性取决于：提供的被检测样品本身在实验条件下，被充分证明不会抑制可能存在的微生物的生长。
因此，在准备样品时，需要正规的实验操作和符合要求的实验条件，接种稀释样品到含有以下（微生物）培养物的培养基：金黄色（奥里斯）葡萄球菌（Staphylococcus aureus）,大肠埃希氏菌（Escherichia coli）, 铜绿假单胞菌(Pseudomonas aeruginosa), 和沙门氏菌（Salmonella）。
方法如下：将用肉汤培养基培养24小时后的（微生物）不小于 10-3 稀释的微生物培养物，加 1 ml（微生物）培养液到磷酸(盐)缓冲液(pH 7.2)，液体大豆酪蛋白消化物培养基（Fluid Soybean-Casein Digest Medium），或者液体乳糖培养基（Fluid Lactose Medium）。
相应培养基培养失败则需要采取以下方法更改检测程序：（1）增加稀释液体积，检测样品加入量仍维持不变；或者（2）中和一定数量的干扰因子；或者（3）结合（1）、（2）得出适当条件，使接种物得以生长。
以下是一些物质的成分和浓度，该物质及浓度可用于加入培养基、阻止物质发挥抑菌作用：大豆卵磷脂（soy lecithin, 0.5%）或者聚山梨醇酯 20（polysorbate 20, 4.0%）。
各国药典微生物方法对比
各国药典微生物方法对比本篇文章旨在比较各国药典中微生物检测方法的不同之处。
微生物检测对于保证药品的质量和安全至关重要，各国药典都提供了相应的指导和标准。
然而，由于各国的文化、法律和技术差异，各国的药典在微生物检测方法上存在一定的差异。
以下将以中括号内的内容为主题，详细介绍各国药典中微生物方法对比。
[美国药典（USP）微生物方法]美国药典（USP）是全球最主要的药典之一，其微生物检测方法有着广泛的应用。
USP推荐的微生物方法主要基于美国食品药品监督管理局（FDA）的要求，主要包括细菌计数、限度测试和特定微生物检测。
其方法的特点在于简单易行、结果可靠且易于验证。
其中，细菌计数方法主要采用菲斯特计数法或冷凝液计数法。
而在限度测试方面，则主要采用的方法是逐级稀释、涂布法和培养法。
USP还明确规定了一些特定微生物的检测方法，如大肠菌群、铜绿假单胞菌和金黄色葡萄球菌等，其检测方法主要依赖于PCR技术和传统的培养法。
[欧洲药典（Ph. Eur.）微生物方法]欧洲药典（Ph. Eur.）是欧洲地区药典的统一标准，其微生物方法与美国药典存在一定的差异。
Ph. Eur.对微生物检测也有着详细的规定，包括细菌计数、限度测试和特定微生物检测。
在细菌计数方面，Ph. Eur.主要采用薄膜过滤法或蔗糖凝胶法。
而在限度测试方面，Ph. Eur.则主要采用稀释平板法、滚珠法和过滤膜方法。
与USP 不同的是，Ph. Eur.也明确规定了一些特定微生物的检测方法，如霉菌和酵母菌等。
这些检测方法涵盖了PCR技术、酶联免疫吸附试验（ELISA）和传统的培养法。
[中国药典（ChP）微生物方法]中国药典（ChP）是中国主要的药典标准，其微生物方法也存在一定的特点。
ChP的微生物检测主要分为总菌落计数、限度测试和特定微生物检测。
与美欧药典不同的是，ChP对微生物检测方法的规定相对较为简洁。
在细菌计数方面，ChP主要采用的方法有薄膜过滤法、落下法和滚珠法等。
美国药典微生物检测精要
美国药典微生物检测精要引言微生物检测在药品生产中起着至关重要的作用。
药品的微生物污染可能对患者的健康造成严重威胁。
为了确保药品的安全性、可靠性和有效性，美国药典（United States Pharmacopeia, USP）制定了一系列用于微生物检测的指南和标准。
本文将概述美国药典微生物检测的精要内容。
检测方法目视法目视法是最简单、最常用的微生物检测方法之一。
该方法通过对样品进行肉眼观察，检验人员可以检测到样品中是否存在明显的微生物污染。
目视法的优点是操作简单，不需要特殊的设备和试剂，适用于多种药品和原料的微生物检测。
然而，目视法存在主观性和局限性，无法检测到微生物的低水平污染。
营养物质法营养物质法是一种常用的培养基法。
该方法以适宜的培养基为基础，通过对样品进行培养，利用微生物的生长特性来检测样品中是否存在微生物污染。
营养物质法可以检测到微生物的低水平污染，并且可以进一步鉴定和计数微生物。
然而，营养物质法需要一定的培养时间，且存在一定的培养基选择性和适应性的局限性。
生物化学法生物化学法是一种通过检测微生物代谢产物来判断样品中是否存在微生物污染的方法。
该方法利用微生物的代谢反应，检测样品中的特定代谢产物的存在与否。
生物化学法可以快速、准确地检测微生物污染，并且不受培养基选择性和适应性的限制。
然而，生物化学法需要特定的试剂和设备，并且对检测人员的技术要求较高。
检测标准美国药典对药品的微生物检测制定了一系列的标准和要求。
这些标准主要包括微生物限度试验、总微生物数试验、鉴别试验和特定微生物试验等。
微生物限度试验微生物限度试验旨在确定样品中存在的微生物数量的限度。
该试验要求将样品接种在适当的培养基中，经过一定的培养时间后，观察并计数生长的微生物数量。
美国药典针对不同类型的药品和原料制定了不同的微生物限度指标，以确保药品的微生物安全性。
总微生物数试验总微生物数试验用于确定样品中所有可培养的微生物的数量。
该试验要求将样品接种在适当的培养基中，经过一定的培养时间后，观察并计数生长的微生物数量。
美国药典微生物试验说明
更為的是防止微生物生長
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USP微生物相關
務必遵守章節
<51> 防腐效力/Antimicrobial Preservatives – Effectiveness <55> 生物指示劑–耐受力測試
Biological Indicators – Resistance Performance Tests <61> 非無菌產品的微生物檢驗：微生物計數試驗
而是通過適當滅菌工程或無菌充填過程確效， 並嚴格執行製程規定（cGMP）才可得到的。
–環境的微生物控制 –原料和組成分的微生物控制 –所有微生物控制的整合 –人員的微生物控制 –製程的微生物控制 –最終成品的微生物控制
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無菌的概率possibility
每批產品的無菌性是根據概率推定的，即有產 品被污染的可能性也是微乎其微的
通常被接受的無菌保證度（SAL）是 10-6 的 微生物存活率或更低，即保證經滅菌的物品或 劑型中有活的微生物的可能性不超過百萬分之 一。
我們能做到比 10-6 更好嗎?
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滅菌前的生物負荷
對熱不安定的物品，透過了解滅菌前物品的生物負 荷，經過 適當的時間週期，及足夠的滅菌前產品批 數，才足以建立滅 菌製程。
<2023> 非無菌營養和膳食補充劑 的微生物評估
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EP & USP & JP 藥典方法的國際協合
International Harmonization of Pharmacopoeia
United States Pharmacopeia
European Pharmacopoeia
美国药典微生物限度检测
61微生物限度检测（MICROBIAL LIMIT TESTS）此章提供方法来检测可能存在的好氧微生物其他制药过程中可能出现的微生物的数量，包括原材料和成品中的。
如果经过验证确认可以得到相同或更好的检测结论，也允许采用自动化的检测方法。
在样品检测过程中须进行无菌操作。
若无特别说明，则“培养（incubate）”一词指在30—35℃的培养箱内培养24至48小时；“生长（growth）”一词用于专门的判定，说明“存在和可能存在活的微生物”。
准备实验 (Preparatory Testing)本章涉及实验结果的有效性取决于：提供的被检测样品本身在实验条件下，被充分证明不会抑制可能存在的微生物的生长。
因此，在准备样品时，需要正规的实验操作和符合要求的实验条件，接种稀释样品到含有以下（微生物）培养物的培养基：金黄色（奥里斯）葡萄球菌（Staphylococcus aureus）,大肠埃希氏菌（Escherichia coli）, 铜绿假单胞菌(Pseudomonas aeruginosa), 和沙门氏菌（Salmonella）。
方法如下：将用肉汤培养基培养24小时后的（微生物）不小于10-3稀释的微生物培养物，加1 ml（微生物）培养液到磷酸(盐)缓冲液(pH 7.2)，液体大豆酪蛋白消化物培养基（Fluid Soybean-Casein Digest Medium），或者液体乳糖培养基（Fluid Lactose Medium）。
相应培养基培养失败则需要采取以下方法更改检测程序：（1）增加稀释液体积，检测样品加入量仍维持不变；或者（2）中和一定数量的干扰因子；或者（3）结合（1）、（2）得出适当条件，使接种物得以生长。
以下是一些物质的成分和浓度，该物质及浓度可用于加入培养基、阻止物质发挥抑菌作用：大豆卵磷脂（soy lecithin, 0.5%）或者聚山梨醇酯20（polysorbate 20, 4.0%）。
USP61-微生物限度微生物计数
58〈55〉 Biological Indicators—Resistance Performance Tests / Microbiological TestsUSP 36It is preferable to use the same method as that defined by ENUMERATION METHODSthe biological indicator manufacturer to determine D values.The use of a different method can result in differences that Use the Membrane Filtration method or one of the Plate-are more an artifact of the method than a variation in the Count Methods , as directed. The Most-Probable-Number performance of the biological indicator.(MPN) Method is generally the least accurate method for microbial counts; however, for certain product groups with very low bioburden, it may be the most appropriate Survival Time and Kill Timemethod.The choice of a method is based on factors such as the Take two groups, each consisting of 10 specimens of the nature of the product and the required limit of microorgan-relevant biological indicator, in their original, individual con-isms. The method chosen must allow testing of a sufficient tainers. Place the specimens of a group in suitable specimen sample size to judge compliance with the specification. The holders that permit each specimen to be exposed to the suitability of the chosen method must be established.sterilizing conditions at a specific location in the BIER cham-ber.Expose the specimens for the required survival time, enter GROWTH PROMOTION TEST, SUITABILITY the chamber, and remove the holder(s) containing the 10OF THE COUNTING METHOD AND NEGATIVEspecimens. Repeat the above procedure immediately, or CONTROLSpreheat if a substantial interval has elapsed, so as to subject the second holder(s) containing 10 specimens similarly to the first conditions, but for the required kill time.The Survival time and kill time for all monographed biolog-General Considerationsical indicators is described in the official monograph under the heading for each.The ability of the test to detect microorganisms in the presence of product to be tested must be established.Suitability must be confirmed if a change in testing per-formance or a change in the product that may affect the outcome of the test, is introduced.Preparation of Test Strains〈61〉 MICROBIOLOGICAL Use standardized stable suspensions of test strains or pre-EXAMINATION OF NONSTERILE pare as stated below. Seed-lot culture maintenance tech-niques (seed-lot systems) are used so that the viable micro-PRODUCTS:MICROBIAL organisms used for inoculation are not more than fivepassages removed from the original master seed-lot. Grow ENUMERATION TESTSeach of the bacterial and fungal test strains separately as described in Table 1.Use Buffered Sodium Chloride–Peptone Solution pH 7.0 or Phosphate Buffer Solution pH 7.2 to make test suspensions; to suspend A. brasiliensis spores, 0.05% of polysorbate 80 may INTRODUCTIONbe added to the buffer. Use the suspensions within 2hours,or within 24hours if stored between 2° and 8°. As an alter-The tests described hereafter will allow quantitative enu-native to preparing and then diluting a fresh suspension of meration of mesophilic bacteria and fungi that may grow vegetative cells of A. brasiliensis or B. subtilis, a stable spore under aerobic conditions.suspension is prepared and then an appropriate volume of The tests are designed primarily to determine whether a the spore suspension is used for test inoculation. The stable substance or preparation complies with an established speci-spore suspension may be maintained at 2° to 8° for a vali-fication for microbiological quality. When used for such pur-dated period of time.poses, follow the instructions given below, including the number of samples to be taken, and interpret the results as stated below.Negative ControlThe methods are not applicable to products containing viable microorganisms as active ingredients.To verify testing conditions, a negative control is per-Alternative microbiological procedures, including auto-formed using the chosen diluent in place of the test prepa-mated methods, may be used, provided that their equiva-ration. There must be no growth of microorganisms. A neg-lence to the Pharmacopeial method has been demonstrated.ative control is also performed when testing the products as described under Testing of Products . A failed negative control requires an investigation.GENERAL PROCEDURESGrowth Promotion of the MediaCarry out the determination under conditions designed to avoid extrinsic microbial contamination of the product to be Test each batch of ready-prepared medium and eachexamined. The precautions taken to avoid contamination batch of medium prepared either from dehydrated medium must be such that they do not affect any microorganisms or from the ingredients described.that are to be revealed in the test.Inoculate portions/plates of Soybean–Casein Digest Broth If the product to be examined has antimicrobial activity,and Soybean–Casein Digest Agar with a small number (not this is, insofar as possible, removed or neutralized. If inac-more than 100 cfu) of the microorganisms indicated in Ta-tivators are used for this purpose, their efficacy and their ble 1, using a separate portion/plate of medium for each.absence of toxicity for microorganisms must be Inoculate plates of Sabouraud Dextrose Agar with a small demonstrated.number (not more than 100 cfu) of the microorganisms in-If surface-active substances are used for sample prepara-dicated in Table 1, using a separate plate of medium for tion, their absence of toxicity for microorganisms and their each. Incubate according to the conditions described in Ta-compatibility with any inactivators used must be ble 1.demonstrated.USP 36Microbiological Tests / 〈61〉 Microbiological Examination59Table 1. Preparation and Use of Test MicroorganismsSuitability of Counting Method inGrowth Promotion the Presence of ProductPreparation of Test Total Aerobic Total Yeasts and Total Aerobic Total Yeasts and Microorganism Strain Microbial Count Molds Count Microbial Count Molds Count Staphylococcus aureus Soybean–Casein Digest Soybean–Casein Soybean–Caseinsuch as ATCC 6538,Agar or Soybean–Casein Digest Agar and Digest Agar/MPNNCIMB 9518, CIP 4.83,Digest Broth Soybean–Casein Soybean–Caseinor NBRC 1327630°–35°Digest Broth Digest Broth18–24 hours≤100 cfu≤100 cfu30°–35°30°–35°≤3 days≤3 daysPseudomonas aeruginosa Soybean–Casein Digest Soybean–Casein Soybean–Caseinsuch as ATCC 9027,Agar or Soybean–Casein Digest Agar and Digest Agar/MPNNCIMB 8626, CIP 82.Digest Broth Soybean–Casein Soybean–Casein118, or NBRC 1327530°–35°Digest Broth Digest Broth18–24 hours≤100 cfu≤100 cfu30°–35°30°–35°≤3 days≤3 daysBacillus subtilis such as Soybean–Casein Digest Soybean–Casein Soybean–CaseinATCC 6633, NCIMB Agar or Soybean–Casein Digest Agar and Digest Agar/MPN8054, CIP 52.62, or Digest Broth Soybean–Casein Soybean–CaseinNBRC 313430°–35°Digest Broth Digest Broth18–24 hours≤100 cfu≤100 cfu30°–35°30°–35°≤3 days≤3 daysCandida albicans such as Sabouraud Dextrose Agar Soybean–Casein Sabouraud Soybean–Casein Sabouraud ATCC 10231, NCPF or Sabouraud Dextrose Digest Agar Dextrose Agar Digest Agar Dextrose Agar 3179, IP 48.72, or NBRC Broth≤100 cfu≤100 cfu≤100 cfu≤100 cfu 159420°–25°30°–35°20°–25°30°–35°20°–25°2–3 days≤5 days≤5 days≤5 days≤5 daysMPN: not applica-bleAspergillus brasiliensis such Sabouraud Dextrose Agar Soybean–Casein Sabouraud Soybean–Casein Sabouraud as ATCC 16404, IMI or Potato–Dextrose Agar Digest Agar Dextrose Agar Digest Agar Dextrose Agar 149007, IP 1431.83, or20°–25° 5–7 days, or≤100 cfu≤100 cfu≤100 cfu≤100 cfu NBRC 9455until good sporulation is30°–35°20°–25°30°–35°20°–25°achieved≤5 days≤5 days≤5 days≤5 daysMPN: not applica-bleFor solid media, growth obtained must not differ by a pared) in Buffered Sodium Chloride–Peptone Solution pH 7.0, factor greater than 2 from the calculated value for a stan-Phosphate Buffer Solution pH 7.2, or Soybean–Casein Digest dardized inoculum. For a freshly prepared inoculum, growth Broth. A surface-active agent such as 1g per L of polysor-of the microorganisms comparable to that previously ob-bate 80 may be added to assist the suspension of poorly tained with a previously tested and approved batch of me-wettable substances. If necessary, adjust to a pH of 6 to 8. dium occurs. Liquid media are suitable if clearly visible Further dilutions, where necessary, are prepared with the growth of the microorganisms comparable to that previ-same diluent.ously obtained with a previously tested and approved batch Fatty Products—Dissolve in isopropyl myristate sterilized of medium occurs.by filtration, or mix the product to be examined with theminimum necessary quantity of sterile polysorbate 80 or an-other noninhibitory sterile surface-active reagent heated, if Suitability of the Counting Method in thenecessary, to not more than 40° or, in exceptional cases, to Presence of Product not more than 45°. Mix carefully and if necessary maintainthe temperature in a water bath. Add a sufficient quantity ofthe prewarmed chosen diluent to make a 1 in 10 dilution ofthe original product. Mix carefully, while maintaining the PREPARATION OF THE SAMPLEtemperature for the shortest time necessary for the forma-tion of an emulsion. Further serial 10-fold dilutions may be The method for sample preparation depends on the phys-prepared using the chosen diluent containing a suitableical characteristics of the product to be tested. If none ofconcentration of sterile polysorbate 80 or another noninhib-the procedures described below can be demonstrated to beitory sterile surface-active reagent.satisfactory, a suitable alternative procedure must beFluids or Solids in Aerosol Form—Aseptically transfer developed.the product into a membrane filter apparatus or a sterile Water-Soluble Products—Dissolve or dilute (usually a 1container for further sampling. Use either the total contents in 10 dilution is prepared) the product to be examined inor a defined number of metered doses from each of the Buffered Sodium Chloride–Peptone Solution pH 7.0, Phosphatecontainers tested.Buffer Solution pH 7.2, or Soybean–Casein Digest Broth. If nec-Transdermal Patches—Remove the protective cover essary, adjust to a pH of 6 to 8. Further dilutions, wheresheets (“release liners”) of the transdermal patches and necessary, are prepared with the same diluent.place them, adhesive side upwards, on sterile glass or plastic Nonfatty Products Insoluble in Water—Suspend thetrays. Cover the adhesive surface with a suitable sterile product to be examined (usually a 1 in 10 dilution is pre-60〈61〉 Microbiological Examination / Microbiological Tests USP 36porous material (e.g., sterile gauze) to prevent the patches This information serves to indicate that the article is not from sticking together, and transfer the patches to a suita-likely to be contaminated with the given species of the mi-ble volume of the chosen diluent containing inactivators croorganism. However, it is possible that the product inhib-such as polysorbate 80 and/or lecithin. Shake the prepara-its only some of the microorganisms specified herein, but tion vigorously for at least 30minutes.does not inhibit others not included among the test strainsor those for which the latter are not representative. Then,perform the test with the highest dilution factor compatible INOCULATION AND DILUTION with microbial growth and the specific acceptance criterion.Add to the sample prepared as directed above and to acontrol (with no test material included) a sufficient volume RECOVERY OF MICROORGANISMS IN THE PRESENCE OF of the microbial suspension to obtain an inoculum of not PRODUCTmore than than 100 cfu. The volume of the suspension ofthe inoculum should not exceed 1% of the volume of di-For each of the microorganisms listed, separate tests are luted product.performed. Only microorganisms of the added test strain To demonstrate acceptable microbial recovery from the are counted.product, the lowest possible dilution factor of the prepared Membrane Filtration—Use membrane filters having a sample must be used for the test. Where this is not possible nominal pore size not greater than 0.45 µm. The type of due to antimicrobial activity or poor solubility, further ap-filter material is chosen in such a way that the bacteria-propriate protocols must be developed. If inhibition of retaining efficiency is not affected by the components of the growth by the sample cannot otherwise be avoided, the sample to be investigated. For each of the microorganisms aliquot of the microbial suspension may be added after neu-listed, one membrane filter is used.tralization, dilution, or filtration.Transfer a suitable quantity of the sample prepared as de-scribed under Preparation of the Sample, Inoculation and Dilu-tion, and Neutralization/Removal of Antimicrobial Activity NEUTRALIZATION/REMOVAL OF ANTIMICROBIAL ACTIVITY(preferably representing 1g of the product, or less if largenumbers of cfu are expected) to the membrane filter, filter The number of microorganisms recovered from the pre-immediately, and rinse the membrane filter with an appro-pared sample diluted as described in Inoculation and Dilutionpriate volume of diluent.and incubated following the procedure described in Recov-For the determination of total aerobic microbial countery of Microorganisms in the Presence of Product, is compared(TAMC), transfer the membrane filter to the surface of the to the number of microorganisms recovered from the con-Soybean–Casein Digest Agar. For the determination of total trol preparation.combined yeasts and molds count (TYMC), transfer theIf growth is inhibited (reduction by a factor greater thanmembrane to the surface of the Sabouraud Dextrose Agar. 2), then modify the procedure for the particular enumera-Incubate the plates as indicated in Table 1. Perform thetion test to ensure the validity of the results. Modification ofcounting.the procedure may include, for example,Plate-Count Methods—Perform plate-count methods at (1)An increase in the volume of the diluent or cultureleast in duplicate for each medium, and use the mean count medium;of the result.(2)Incorporation of a specific or general neutralizingagents into the diluent;Pour-Plate Method—For Petri dishes 9cm in diameter,(3)Membrane filtration; or add to the dish 1mL of the sample prepared as described(4)A combination of the above measures.under Preparation of the Sample, Inoculation and Dilution,and Neutralization/Removal of Antimicrobial Activity and 15 to Neutralizing Agents—Neutralizing agents may be used20mL of Soybean–Casein Digest Agar or Sabouraud Dextrose to neutralize the activity of antimicrobial agents (see TableAgar, both media maintained at not more than 45°. If larger 2). They may be added to the chosen diluent or the me-Petri dishes are used, the amount of agar medium is in-dium preferably before sterilization. If used, their efficacycreased accordingly. For each of the microorganisms listed and their absence of toxicity for microorganisms must bein Table 1, at least two Petri dishes are used. demonstrated by carrying out a blank with neutralizer andIncubate the plates as indicated in Table 1. Take the arith-without product.metic mean of the counts per medium, and calculate thenumber of cfu in the original inoculum.Table 2. Common Neutralizing Agents/Methods forSurface-Spread Method—For Petri dishes 9cm in diame- Interfering Substancester, add 15 to 20mL of Soybean–Casein Digest Agar orPotential Neutralizing Sabouraud Dextrose Agar at about 45° to each Petri dish, Interfering Substance Agents/Method and allow to solidify. If larger Petri dishes are used, the vol-Glutaraldehyde, mercurials Sodium hydrogen sulfite ume of the agar is increased accordingly. Dry the plates, for(Sodium bisulfite)example, in a laminar-airflow cabinet or in an incubator. For Phenolics, alcohol, aldehydes,Dilution each of the microorganisms listed in Table 1, at least two sorbate Petri dishes are used. Spread a measured volume of not lessthan 0.1mL of the sample, prepared as directed under Prep-Aldehydes Glycinearation of the Sample, Inoculation and Dilution, and Neutrali-Quaternary ammonium com-Lecithinzation/Removal of Antimicrobial Activity over the surface of pounds (QACs), parahydroxy-the medium. Incubate and count as directed for Pour-Plate benzoates (parabens), bis-Method.biguanidesMost-Probable-Number (MPN) Method—The precision QACs, iodine, parabens Polysorbateand accuracy of the MPN Method is less than that of the Mercurials Thioglycollate Membrane Filtration method or the Plate-Count Method. Un-Mercurials, halogens, aldehydes Thiosulfate reliable results are obtained particularly for the enumeration EDTA (edetate)Mg or Ca ions of molds. For these reasons, the MPN Method is reserved forthe enumeration of TAMC in situations where no othermethod is available. If the use of the method is justified,If no suitable neutralizing method can be found, it can beproceed as follows.assumed that the failure to isolate the inoculated organismPrepare a series of at least three serial 10-fold dilutions of is attributable to the microbicidal activity of the product.the product as described for Preparation of the Sample, Inoc-USP 36Microbiological Tests / 〈61〉 Microbiological Examination61ulation and Dilution, and Neutralization/Removal of Antimicro-RESULTS AND INTERPRETATIONbial Activity. From each level of dilution, three aliquots of1g or 1mL are used to inoculate three tubes with 9 to When verifying the suitability of the Membrane Filtration 10mL of Soybean–Casein Digest Broth. If necessary a surface-method or the Plate-Count Method, a mean count of any of active agent such as polysorbate 80, or an inactivator of the test organisms not differing by a factor greater than 2 antimicrobial agents may be added to the medium. Thus, if from the value of the control defined in Inoculation and Dilu-three levels of dilution are prepared, nine tubes are inocu-tion in the absence of product must be obtained. When lated.verifying the suitability of the MPN Method, the calculated Incubate all tubes at 30° to 35° for not more than 3 days.value from the inoculum must be within 95% confidenceIf reading of the results is difficult or uncertain owing to the limits of the results obtained with the control.nature of the product to be examined, subculture in the If the above criteria cannot be met for one of more of the same broth or in Soybean–Casein Digest Agar for 1 to 2 days organisms tested with any of the described methods, theat the same temperature, and use these results. From Table method and test conditions that come closest to the criteria 3, determine the most probable number of microorganisms are used to test the product.per g or mL of the product to be examined.TESTING OF PRODUCTS Table 3. Most-Probable-Number Values of MicroorganismsObservedCombinations Amount Used for the Test of Numbers of Tubes MPN per g or95%Showing Growth in per mL of ConfidenceUnless otherwise directed, use 10g or 10mL of the prod-Each Set Product Limitsuct to be examined taken with the precautions referred to Number of g or mL of above. For fluids or solids in aerosol form, sample 10 con-Product per Tube tainers. For transdermal patches, sample 10patches.0.10.010.001The amount to be tested may be reduced for active sub-000<30–9.4stances that will be formulated in the following conditions: 00130.1–9.5the amount per dosage unit (e.g., tablet, capsule, injection)is less than or equal to 1mg, or the amount per g or mL 01030.1–10(for preparations not presented in dose units) is less than 011 6.1 1.2–171mg. In these cases, the amount of sample to be tested is 020 6.2 1.2–17not less than the amount present in 10 dosage units or 10g0309.4 3.5–35or 10mL of the product.100 3.60.2–17For materials used as active substances where the sample 1017.2 1.2–17quantity is limited or batch size is extremely small (i.e., lessthan 1000mL or 1000g), the amount tested shall be 1% of 102114–35the batch unless a lesser amount is prescribed or justified 1107.4 1.3–20and authorized.111114–35For products where the total number of entities in a batch 120114–35is less than 200 (e.g., samples used in clinical trials), the 121155–38sample size may be reduced to two units, or one unit if the 130165–38size is less than 100.Select the sample(s) at random from the bulk material or 2009.2 1.5–35from the available containers of the preparation. To obtain 201144–35the required quantity, mix the contents of a sufficient num-202205–38ber of containers to provide the sample.210154–38211205–38Examination of the Product212279–94220215–40221289–94MEMBRANE FILTRATION 222359–94230299–94Use a filtration apparatus designed to allow the transfer of 231369–94the filter to the medium. Prepare the sample using a300235–94method that has been shown to be suitable as described in 301389–104Growth Promotion Test and Suitability of the Counting Method, 3026416–181 transfer the appropriate amount to each of two membranefilters, and filter immediately. Wash each filter following the 310439–181procedure shown to be suitable.3117517–199For the determination of TAMC, transfer one of the mem-31212030–360brane filters to the surface of Soybean–Casein Digest Agar.31316030–380For the determination of TYMC, transfer the other mem-3209318–360brane to the surface of Sabouraud Dextrose Agar. Incubate 32115030–380the plate of Soybean–Casein Digest Agar at 30° to 35° for 3to 5 days and the plate of Sabouraud Dextrose Agar at 20°32221030–400to 25° for 5 to 7 days. Calculate the number of cfu per g or 32329090–990per mL of product.33024040–990When examining transdermal patches, separately filter 33146090–198010% of the volume of the preparation described for Prepara-3321100200–4000tion of the Sample through each of two sterile filter mem-333>1100branes. Transfer one membrane to Soybean–Casein DigestAgar for TAMC and the other membrane to Sabouraud Dex-trose Agar for TYMC.62〈61〉 Microbiological Examination / Microbiological Tests USP 36 PLATE-COUNT METHODS〈62〉 MICROBIOLOGICAL Pour-Plate Method—Prepare the sample using a method EXAMINATION OF NONSTERILE that has been shown to be suitable as described in GrowthPromotion Test and Suitability of the Counting Method. Pre-PRODUCTS:TESTS FORpare for each medium at least two Petri dishes for each levelof dilution. Incubate the plates of Soybean–Casein Digest SPECIFIED MICROORGANISMS Agar at 30° to 35° for 3 to 5 days and the plates ofSabouraud Dextrose Agar at 20° to 25° for 5 to 7 days. Se-lect the plates corresponding to a given dilution and show-ing the highest number of colonies less than 250 for TAMCand 50 for TYMC. Take the arithmetic mean per culture me-INTRODUCTIONdium of the counts, and calculate the number of cfu per gor per mL of product.The tests described hereafter will allow determination of Surface-Spread Method—Prepare the sample using a the absence of, or limited occurrence of, specified microor-method that has been shown to be suitable as described in ganisms that may be detected under the conditions Growth Promotion Test and Suitability of the Counting described.Method. Prepare at least two Petri dishes for each medium The tests are designed primarily to determine whether a and each level of dilution. For incubation and calculation of substance or preparation complies with an established speci-the number of cfu, proceed as directed for the Pour-Plate fication for microbiological quality. When used for such pur-Method.poses, follow the instructions given below, including thenumber of samples to be taken, and interpret the results asstated below.MOST-PROBABLE-NUMBER METHODAlternative microbiological procedures, including auto-mated methods, may be used, provided that their equiva-Prepare and dilute the sample using a method that haslence to the Pharmacopeial method has been demonstrated. been shown to be suitable as decribed in Growth PromotionTest and Suitability of the Counting Method. Incubate alltubes for 3 to 5 days at 30° to 35°. Subculture if necessary,GENERAL PROCEDURESusing the procedure shown to be suitable. Record for eachlevel of dilution the number of tubes showing microbial The preparation of samples is carried out as described in growth. Determine the most probable number of microor-Microbiological Examination of Nonsterile Products: Microbial ganisms per g or mL of the product to be examined from Enumeration Tests 〈61〉.Table 3.If the product to be examined has antimicrobial activity,this is insofar as possible removed or neutralized as de-Interpretation of the Results scribed in Microbiological Examination of Nonsterile Products:Microbial Enumeration Tests 〈61〉.The total aerobic microbial count (TAMC) is considered to If surface-active substances are used for sample prepara-be equal to the number of cfu found using Soybean–Casein tion, their absence of toxicity for microorganisms and their Digest Agar; if colonies of fungi are detected on this me-compatibility with any inactivators used must be demon-dium, they are counted as part of TAMC. The total com-strated as described in Microbiological Examination of Non-bined yeasts and molds count (TYMC) is considered to be sterile Products: Microbial Enumeration Tests 〈61〉.equal to the number of cfu found using Sabouraud DextroseAgar; if colonies of bacteria are detected on this medium,GROWTH-PROMOTING AND INHIBITORY they are counted as part of TYMC. When the TYMC is ex-pected to exceed the acceptance criterion due to the bacte-PROPERTIES OF THE MEDIA, SUITABILITY OF rial growth, Sabouraud Dextrose Agar containing antibiotics THE TEST AND NEGATIVE CONTROLS may be used. If the count is carried out by the MPNMethod, the calculated value is TAMC.The ability of the test to detect microorganisms in the When an acceptance criterion for microbiological quality presence of the product to be tested must be established.is prescribed, it is interpreted as follows:Suitability must be confirmed if a change in testing perfor-—101 cfu: maximum acceptable count = 20;mance or a change in the product that may affect the out-—102 cfu: maximum acceptable count = 200;come of the test is introduced.—103 cfu: maximum acceptable count = 2000;and so forth.Preparation of Test StrainsThe recommended solutions and media are described inTests for Specified Microorganisms 〈62〉.Use standardized stable suspensions of test strains asstated below. Seed-lot culture maintenance techniques(seed-lot systems) are used so that the viable microorgan-isms used for inoculation are not more than five passagesremoved from the original master seed-lot.AEROBIC MICROORGANISMSGrow each of the bacterial test strains separately in con-tainers containing Soybean–Casein Digest Broth or onSoybean–Casein Digest Agar at 30° to 35° for 18 to 24hours.Grow the test strain for Candida albicans separately onSabouraud Dextrose Agar or in Sabouraud Dextrose Broth at20° to 25° for 2 to 3 days.。
USP 61 微生物限度检测——计数法
Use the Membrane Filtration method or one of the Plate-Count Methods, as directed. The Most-
Probable-Number (MPN) Method is generally the least accurate method for microbial counts; however, for certain product groups with very low bioburden, it may be the most appropriate method.
2. GENERAL PROCEDURES通用规程
Carry out the determination under conditions designed to avoid extrinsic microbial contamination of the product to be examined. The precautions taken to avoid contamination must be such that they do not affect any microorganisms that are to be revealed in the test. 微生物计数环境应能防止外来微生物对供试品的污染。防止污染的措施不能影响供试品中微生物 的检出。
<61>MICONSTERILE PRODUCTS: MICROBIAL ENUMERATION TESTS
非无菌产品微生物限度检查：微生物计数法
1. INTRODUCTION导言
The tests described hereafter will allow quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions. 微生物计数法系用于能在有氧条件下生长的嗜温细菌和霉菌的定量计数。
美国药典微生物试验说明
實驗數據判讀
不合格原因
–實驗室錯誤 –產品失敗 無論上述何種情況，管理階層必須立即被告知
OOS調查
–數據偏離的程度(嚴重性？) –實驗室環境 –取樣方法 –物料的特性(存活的污染微生物？) –留樣的評估
32/9
<1211>滅菌和無菌保證
每批產品不是通過<71>無菌試驗就可得到無 菌保證。
<1209> 滅菌–化學和物理化學指 示劑，及綜合指示劑
<1211> 滅菌與無菌保證
<1222> 最終滅菌藥品–參數放行
<1223> 微生物方法確效
<1227> 藥物的微生物回收率確效
<1231> 製藥用水
<2021> 微生物計數試驗–營養和 膳食補充劑
<2022> 特定菌的微生物檢查–營 養和膳食補充劑
Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests <62> 特定菌的微生物檢查 / Microbiological Procedures for Absence of Specified Microorganisms <71> 無菌試驗 / Sterility Tests <85> 細菌內毒素試驗 Bacterial Endotoxins Test
劑型 (投藥途徑) 口服固體 口服液劑
外用或鼻腔用藥
陰道用藥
直腸用藥 藥品主成分&賦型劑
<61> TAMC [CFU/g or CFU/ml]
USP61非无菌产品的微生物检查：微生物的计数检查
＜61＞微生物试验（61）非无菌产品的微生物检查：微生物计数试验序言后面描述的试验对可在好氧条件下生长的嗜温细菌和真菌进行定量计数。
设计此试验主要是为了确定一种原料药和制剂的微生物质量是否符合已建立的质量标准。
作此用途时，遵循下面给出的说明，包括采样编号，并按照下面的说明判定结果。
此方法不适用于以活的微生物作为活性成份的产品。
也可以使用其它的微生物规程，包括自动化方法，只要它们和已经证明的药典方法等效。
通用规程在设计好的条件下进行检定，避免供试品受到外部微生物的污染。
避免污染所采取的预防措施必须不影响试验中出现的任何微生物。
如果供试品中含有抑菌活性，那么应当尽可能的将其去除或中和。
如果用灭活剂来达到此目的，那么必须证明其有效性以及对微生物不具有毒性。
如果表面活性剂用于样品制备，那么必须证明其对微生物的无毒性及与所用灭活剂的相容性。
计数方法如下所示，使用膜过滤法或平板计数法其中一种。
通常微生物计数用最大几率计数（MPN）法是最不精确的；但是，对生物负荷极低的特定产品组，它可能是最为适宜的方法。
选择方法取决于这些因素，如产品性质及要求的微生物限度。
选择的方法必须允许对足量的样品量进行试验，从而判断是否符合质量标准。
必须建立所选方法的适用性。
促生长试验和计数方法的适用性一般考虑必须建立实验检出供试品中所含微生物的能力。
如果试验操作或产品方面引入了可能影响试验结果的变更，则必须确认适用性。
试验菌株的制备使用标准化的稳定试验菌悬液，或者按下述说明制备。
使用种子批培养保存技术（生产菌种系统）以便从原始主种子批中取出用于接种的活菌传代不超过5次。
每种细菌和真菌试验菌株的生长情况在表1中分别详细说明。
使用pH 7.0的氯化钠-蛋白胨缓冲液或pH 7.2的磷酸盐缓冲液制备试验悬液；为了悬浮黑曲霉孢子，可加入0.05%的聚山梨醇酯80（吐温80）。
在2小时内使用悬液，如果在2°到8°保存，可在24小时内使用。
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美国微生物测试USP51 和USP61＋62 介绍(2009/12/22 18:31)
美国微生物测试USP51 和USP61＋62 介绍
玩具需作微生物测试的材料：
用于玩具中的化妆品、液体、糊状物、油灰（putties)、凝胶和粉末（艺术品材料，如粉笔、蜡笔、墨水等除外）
1. USP<61>微生物限量测试*1
- 测试目的: 检验材料本身受微生物污染的情况（也即微生物洁净度情况）
- USP<61>包括以下六个微生物测试：
(1) Total aerobic microbial Count 菌落总数(定量）
(2) Mould and Yeast Count 霉菌和酵母菌数(定量）
(3) Staphylococcus aureus 金黄色葡萄球菌(定性）
(4) Pseudomonas aeruginosa 绿脓杆菌（定性）
(5) Salmonella 沙门氏菌（定性）
(6) Escherichia Coli 大肠杆菌（定性）
USP61 Limit Requirement:
(1)+(2) < 5000 CFU/g ( if the material was used in baby product or eye area product, the limit should be < 500 CFU/g)
(3)/(4)/(5)/(6) should be “ABSENT per 10g “
2. USP<51>防腐剂抗菌效力测试
- 测试目的
为防止的材料在保存过程中或使用过程中发生微生物*现象，须在材料中加入适量的防腐剂，而防腐剂效果如何则需通过USP《51》测试进行评价。
-USP<51>测试简述：
将标准指定编号的以下菌种接入样品，然后在第7 天，第14 天，第28 天分别检验每种菌的存活数量，存活数量越少，其防腐剂抗菌效果越好。
Staphylococcus aureus 金黄色葡萄球菌
Escherichia coli 大肠杆菌
Pseudomonas aeruginosa 绿脓杆菌
Candida albicans 白色念株菌
Aspergillus niger 黑曲霉
USP51 Limit requirement (for toy):
细菌(Staphylococcus aureus/ Escherichia coli / Pseudomonas aeruginosa)
14 天细菌减少≥2.0 LOG (99%)
28 天不再繁殖
真菌(Candida albicans /Aspergillus niger)
14 天和28 天不再繁殖
Remark: LOG REDUCTION = LOG10 (INITIAL COUNT / NO. OF
MICRO-ORGANISM RECOVERED)
*1 Remark:
最近，美国药典（USP）发布了重大调整，原第61章“微生物限量测试”被拆分成两部分，即第61章“非灭菌产品中微生物测试：微生物计数检测”和第62章“非灭菌产品中微生物测试：特定微生物检测”。
调整后的两章（请参见下表），同时出口欧美的客户其测试成本将大大节约。
新USP61+62章节将于2009年5月1日生效。
USP 61章：
非灭菌产品中微生物测试：微生物计数测试微生物
(1) 好氧微生物总数Total Aerobic Microbial count
(2) 酵母菌和霉菌总数Total Yeasts and Molds count
USP 62章：
非灭菌产品中微生物测试：特定微生物检测
(3) 耐胆汁酸革兰氏阴性菌Bile-tolerant Gram-negative bacteria（新!）
(4) 埃希氏大肠杆菌Escherichia Coli
(5) 沙门氏菌Salmonella
(6) 铜绿假单胞菌Pseudomonas Aeruginosa
(7) 金黄色葡萄球菌Staphylococcus Aureus
(8) 梭菌Clostridia（新!）
(9) 白色假丝酵母菌Candida albicans（新!）。