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2．6．12非无菌产品的微生物限度检查 (有活力的需氧菌总数的计数)本附录给出了两种测试方法第一种给出与专论相符合的相关测试方法除非说明采用第2种测试。
参照本章的内容采用第１种测试方法。
考虑到申请上市许可的需要。
第2种测试方法也是欧洲药典正式方法的组成部分。
一旦相关部分修改，用第２种测试方法代替第１种。
第二种方法为了与日本药局方和美国药典的要求一致A .欧洲药典的方法下述检验方法是对有氧条件下生长的嗜温细菌和真菌数的计数检查。
这些试验设计主要用来检查样品是否符合药典对微生物限度的要求，用于这一目的时要遵守以下指导原则，包括样品的抽样数选取及结果的解释。
这些试验也可以用于药典中抗菌防腐剂效力的检验(5.1.3)。
它们可进一步用来监测原料质量，药物制剂的微生物质量监测(5.1.4)。
当用于这些目的时，例如生产者用于原料和／或成品的检测或者用于认证过程评价时，进行测试的方法包括抽取样品数及结果的解释是生产者和主管当局间协议中的事项。
检定应该在样品不会被污染的条件下进行。
用来防止污染的预防措施必须不影响在试验中微生物的检出。
如果被检测的产品含有抗菌活性，它必须被充分中和，并证明所用的灭活剂是有效的和对微生物是无毒性的本章描述了薄膜滤过法，培养板记数法测定有活力的需氧菌总数当没有其它方法进行细菌计数时可用最大可能数法。
方法的选择基于产品的性质及预期的微生物数量这些因素。
任何被选用的方法必须被适当地验证。
当5．1．3章或5．1．4章联合应用时，可以使用平皿法(pour—plate)，表面涂布平皿计数法(surface—spread)及薄膜滤过法。
供试品的准备抽样计划：抽样必须遵循明确的抽样计划。
抽样计划由一批样品的数量，与不被接受的高污染产品相关的健康危险，产品的特性，预期污染水平等因素而定。
除另有规定外，取10g或10ml样品或制剂，按上述方法预处理。
从原料中或从制剂包装中随机地选取样品。
根据样品(原料或制剂)的特性，必要时可从多个包装中取样，混合后，得到所需检品量。
非无菌样品的微生物检测——总好氧活菌计数（EP6）2.6.12 非无菌样品的微生物检测（总好氧活菌计数）这章有两套测试方法。
第一套给出的鉴定方法与原来专论中的方法保持一致。
在专论研究中参考此章，一般采用第一套方法，除非准许使用第二套方法。
第二套方法也是欧洲药典的官方方法，也可以作为参考，值得注意的是在上市授权申请时的使用。
一旦药典中相关专论的修订完成，那么计划用第二套方法代替第一套方法。
第二套方法已经与JP和USP达成一致要求。
A、欧洲药典方法这里描述的方法允许对好氧嗜温细菌和好氧真菌的定量计数。
检测主要是为了确定药典专论中提及的物质是否符合药典专论规定的微生物要求。
当为了此目的而应用该方法时，应遵循下述指导，包括取样数目和按照下述规定进行的结果解释。
这些测试方法可用于《抗微生物防腐剂的有效性》（5.1.3），也可用于原料的质量监控，并与《微生物学特性》（5.1.4）指导原则联合使用。
如果是用于这类目的时，比如生产商对原材料和/或成品的监控或者是工艺验证，那么包括取样数目和结果解释在内的检测过程，都应是生产商与主管部门的协议问题。
本检测必须在特定的条件下进行，以避免供试品在检查过程中受意外污染；同时所采取的措施必须对检测所涉及的微生物无影响。
如果供试品具有抗菌活性，必须充分中和这些活性。
如果实验中用到了灭活剂，必须证明它们对微生物有效并且无毒。
按照专论所述，用薄膜过滤或者平皿计数法测定总好氧活菌数。
最大概率法（MPN）是用于细菌计数的，当没有其他方法时可以采用此法。
对于方法的选择，要根据各种因素，如供试品的特性和预计微生物数目。
所选择的任何方法都必须经过合理验证。
当与5.1.3或5.1.4联合使用时，可以使用倾注平皿法、表面铺展法，或者薄膜过滤法。
供试品的准备取样方法取样必须遵守规定的取样方法，取样方法取决于一些因素如批量、被高度污染后的供试品所引起的健康危害、供试品的性质和预计污染水平。
除另有规定外，一般供试品的检验量为10g或10ml，取样应在上述防范污染的措施下进行。
欧洲药典标准-回复欧洲药典标准——提高医药品质的指南导论：欧洲药典标准是制药和医学领域的一项重要参考，为确保制药工业生产过程的质量和安全性提供了明确指导。
本文将逐步介绍欧洲药典标准，包括其背景、作用、更新过程以及对制药行业和患者的影响。
第一部分：背景1. 欧洲药典的起源欧洲药典由欧洲药典委员会（EDQM）负责管理，成立于1964年。
其初衷是通过制定和推广共享卓越的药物质量控制方法，促进药品的质量、安全性和疗效。
2. 欧洲药典的目标欧洲药典的目标是确保欧洲范围内生产和分发的药品质量符合高标准，以保障患者使用的药品的有效性、安全性和一致性。
第二部分：作用1. 药物质量控制欧洲药典标准提供了一套严格的化学、物理和生物学测试方法，用于检验制药品质量。
药品生产商可以依据这些标准确保其产品的质量和一致性。
2. 环境和设备要求欧洲药典标准还规定了制药工业所需的环境和设备要求，以确保药品在生产、包装和储存过程中不受到污染。
3. 药品注册要求欧洲药典标准是制药行业在欧洲市场上注册药品所必需遵守的要求之一。
符合这些标准的药品可以获得欧洲药品注册的批准。
第三部分：更新过程1. 欧洲药典更新委员会欧洲药典标准每年都会进行更新和修订，以跟上科学和技术的发展。
这项工作由欧洲药典更新委员会负责，该委员会由来自各个国家的专家组成。
2. 更新内容和程序更新的内容包括新增的药品、检验方法的修订和新的法规要求。
更新程序包括对提案的评估、公开咨询和最终修订版本的制定。
第四部分：对制药行业和患者的影响1. 制药企业欧洲药典标准的遵循是制药企业获得药品注册批准的前提。
遵守这些标准有助于企业确保产品质量和合规性，提升企业的竞争力。
2. 患者利益欧洲药典标准的严格要求确保了药品的品质和一致性，提高了患者对药品疗效和安全性的信任。
患者能够获得符合标准的高质量药品，从而更有效地治疗和管理疾病。
结论：欧洲药典标准是欧洲范围内制药和医学领域的重要参考文献。
欧洲药典标准-回复药典是一种被广泛应用的药物品质标准，旨在确保药物的安全有效性和一致性。
在欧洲，欧洲药典（European Pharmacopoeia，简称EP）是药物品质标准的权威参考。
本文将详细介绍欧洲药典标准，并探讨其制定过程、应用范围和质量保证体系。
欧洲药典是一个权威的多语种国际标准，用于指导欧洲范围内的药品质量控制和动植物药材的使用。
它的主要目标是保护公众的健康和药物质量的一致性。
欧洲药典通过提供详细的技术要求和试验方法，确保药物的安全性、质量和有效性。
这些标准是法律要求的，对于药品的生产、检验、注册和销售等环节都具有强制性。
欧洲药典的制定过程是一个复杂而严格的过程，确保其科学性、可靠性和可操作性。
制定药典标准的首要任务是提供关于药物成分、成分含量、制剂质量和肆无忌惮等方面的详细要求。
制定标准的过程主要涉及以下几个步骤：1. 专家委员会的选任和组成：欧洲药典委员会由各成员国的专家组成，他们代表了各个与药品质量相关的领域。
委员会通过逐步审查和修订主题相关的信息以达成一致的看法。
2. 信息搜集和分析：药典编委会通过收集国内外研究成果、提取其他药典的信息以及接受专业机构的建议来获取丰富的药物信息。
然后对这些信息进行仔细分析，并根据实际需要确定关注的主题。
3. 提议书的准备和评议：基于对信息的分析和专家的经验意见，制定药典委员会针对具体主题的提议书。
这些提议书将被交给各个专家评审小组进行严格的评审和审查。
在评审期间，专家组将讨论并提出修改或澄清建议。
4. 决策和讨论：在评审小组的讨论和决策下，撰写新的或修改现有的章节，并形成正式的标准。
这些标准将提交给药典委员会的会议进行讨论和投票，以确保标准的内容是一致且明确的。
5. 公众咨询和意见征询：在标准定稿后，将公开征询公众、行业机构和利益相关者的意见。
这些意见将被认真分析，并对标准进行必要的修改和完善。
6. 标准的正式出版：最终的标准将被编写成正式的文档，并在欧洲药典的备案中登记。
04/2010:206132.6.13.非无菌产品的微生物检测: 特定微生物的检测（4）1.介绍以下描述的几项检测可用于检测不得存在或限量存在的特定微生物，这些微生物可在规定的条件下检出。
设计这些检测,主要用于鉴定一种物质或制剂是否符合一个已建立的微生物质量规定。
用于此目的时，可按如下规定确定取样数量，判定结果。
如果能证明它等同于药典的方法，可采用替代性的微生物的检测程序（包括自动化的方法）。
2 .一般性操作规程按通用章节2.6.12所述制备样品。
若供试品有抑菌性活性，需按通用章节2.6.12所述尽可能的减少其抑菌性或中和。
如果样品制备过程中使用了表面活性剂，则必须按通用章节2.6.12所述证明表面活性剂对微生物的无毒性和它与所使用的钝化剂的相容性。
3. 培养基的促生长性和抑制性以及检测的适用性必须验证在有供试品存在的条件下检测方法检出微生物的能力。
如果检测过程或产品发生了变化，且该变化可能影响检测的结果，则需证明其适用性。
3-1. 检测菌株的制备使用稳定的标准检测菌株混悬液或按以下规定制备。
采用种子菌培养保持技术，确保原代菌连续传代不超过5次。
3-1-1.需氧的微生物。
将每种细菌检测菌株分别在大豆酪蛋白消化物肉汤培养基或大豆酪蛋白消化琼脂培养基上于30 - 35 ℃培养18 - 24小时。
将白色念珠菌的检测菌株在分别在萨布罗右旋糖琼脂培养基上或萨布罗右旋糖肉汤培养基上于20 - 25 ℃培养2 - 3天。
－金黄色葡萄球菌如：ATCC 6538，NCIMB 9518，CIP 4.83或NBRC 13276；（4）本章已通过药典协调，见章5.8.药典协调－铜绿假单胞菌如：ATCC 9027，NCIMB 8626，CIP 82.118或NBRC 13275；－大肠杆菌如：ATCC 8739，NCIMB 8545，CIP 53.126或NBRC 3972；－肠道沙门菌：血清型为salmonella enterica subsp.enterica serptype typhimutium,如ATTC 14028，或其替代品salmonella enterica subsp.enterica serotype abony如NBRC 100797，NCTC 6017或CIP 80.39;－白色念珠菌如：ATCC 10231，NCPF 3179，IP 48.72或NBRC 1594；用pH 7.0的氯化钠-蛋白胨缓冲溶液或pH 7.2的磷酸缓冲溶液配制供试混悬液。
2.6.12非无菌产品的微生物限度检查（总的需氧菌菌落计数）本章包括两种检验方法。
第一种方法给出的是测定适用性的参考方法。
因些在一个专论中依照本法就意味着依从第一种方法，除非特别指出要使用第二种方法。
第二种方法也是欧洲药典的官方组成部分，并且值得注意的是第二种方法特别适用于销售认可。
一旦该相关专论被修订就意味着将用第二种方法替代第一种方法。
第二种方法包含日本药典和美国药典的共同部分使其能够协调。
A．欧洲药典方法该试验方法所讲的是在需氧条件下生长的嗜温性细菌和真菌类微生物的定量检测。
设计该试验的最初目的是以审疑的态度为了测定一种物质按照欧洲药典的检测方法是否符合微生物的特定限定要求。
如果是以这种目的来测定的话，根据下述方法，包括所需的样品数和按照下面方法来解释结果。
该法也用来测定药典所描述的抗菌保留的有效性（5.1.3）。
此外，该法也可以用来监测原料质量同时也可以用来作为微生物质量检测的指导方针（5.1.4）。
如果是用来指导以下目的，如生产厂家的原料检测和/或终产品的检测或终点判定，则包括所需样品数和结果解释的试验方法就必须在生产厂家和主管当局之间达成一致。
执行该方法所设计的实验条件就必须避免所测产品受到意外污染。
避免污染所采取的措施不侧够影响任何所测的微生物。
如果所测产品有抗菌活性，则该产品活性必须补充分中和掉。
如果因为该种目的使用灭活剂则必须证明该灭活剂对微生物的有效性和无毒性。
测定总的需氧微生物数通过微孔滤膜法或专论中所描述的平板菌落计数法。
当没有其它方法可以釆用来测定细菌数时保留最大可能数（Most Probable Number, MPN）.选择哪种应该根据产品的特性和可预计的微生物数等因素来选择。
任何选择的方法必须通过合理的验证。
当根据5.1.3或5.1.4使用时，可以使用平板浇注法和表面扩散法，和微孔滤膜法。
样品制备：抽样检验方法：所选样品必须遵循抽样样品规则。
抽样检验的结果依赖于下列因素：如样品批号，不可接受的高污染产品的健康危害，产品特性，可预料的污染程度等。
阅读欧洲药典时需要注意的问题阅读欧洲药典时需要注意的问题1. ContentFor a non-specific assay (for example, titrimetry) the assay limits are usually 99.0-101.0% percent (unless otherwise justified). For a specific assay using a separation technique (for example, liquid or gas chromatography), the upper assay limit is normally 102.0 percent; the lower assay limit will take any necessary account of the impurities present and may therefore be lower than 98.0 percent.2. Characteristic / AppearanceThe term “white” is not used without qualification since, if viewed against a standard white material, very few pharmaceutical materials will appear truly white. It is, of course, not intended that such a comparison be made but experience shows that certain users of the Pharmacopoeia may insist on doing so as part of a purchasing contract. The term “white or almost white” is used instead.In English, the dominant is placed second, whereas in French, it is place first. Expressions such as lemon-yellow, buff, salmon-pink are to be avoided.3. IdentificationNormally, a single set of tests for identification is given. Where justified, in order to give users of the Pharmacopoeia a choice between methods requiring complex instrumentation and other methods, two sets of identification tests may be included.This is usually the case when the substance is used in hospital and/or community pharmacies. Monographs then have subdivisions entitled “First identification” and “Second identification”. The test or tests that constitute the “Second identification” may be used instead of the test or tests of the “Fist identification” provided it can be demonstrated that the substance or medicinal product is fully traceable to a batch certified to comply with all the requirements of the monograph.3.1 PolymorphismThe general chapter allows for recrystallisation before recording of the spectrum. Where a monograph mentions polymorphism then a method for recrystallisation is described unless it is the intention to limit the scope of the monograph to the crystalline form represented by the chemical reference substance in the latter case the monograph indicates that the spectrum is recorded “without recrystallisation”.3.2 Ultraviolet and Visible Absorption SpectrophotometryThe concentration of the solution to be examined is such that the absorbance preferably lies between 0.5 and 1.5 measured in a 1 cm cell.3.3 Specific Optical RotationWhen an enantiomer is described in a monograph, a test for optical rotation is given in the IDENTIFICATION section or a cross-reference is made to the test for enantiometic purity in the TESTS section. When both the racemate (or the racemic mixture) and the enantiomer are available then, in the monograph of the racemate, an angle of rotation will be given in the TESTS sectionand will be referred to in the IDENTIFICATION section. When only the racemate is available the angle of rotation will be given in the TESTS section, provided the specific optical rotation of the chiral form is known and is of sufficient magnitude to provide a meaningful test for racemic character.4. Test4.1 Appearance of SolutionThis test makes it possible to ascertain the general purity of a substance by the detection of impurities insoluble in the solvent selected, or of colored impurities.The “Appearance of solution” test is practically always prescribed for substances intended for preparations for parenteral use. Apart from this it is to be applied only if it yields useful information concerning the general purity of the substance.It can comprise both tests or one only, namely:l Clarity and degree of opalescence of liquids (2.2.1);l Degree of coloration of liquids (2.2.2).4.1.1 Clarity and degree of opalescenceThis test is mainly performed on colorless substances or those which give only slightly colored solutions in order to permit valid comparison with reference suspensions.4.1.2 Degree of colorationThe test applies to essentially colorless substances that contain, or may degrade to form, colored impurities that can be controlled by limiting the color of solution of the substance.4.2 Related SubstancesMonographs should include acceptance criteria for:l Each specified impurity;l Unspecified impurities (previously referred to as “any other impurities”), normally set at the identification threshold;l Total impurities.Impurities to be controlled include: intermediates and by-products of synthesis; co-extracted substances in products of natural origin; degradation products. Monographs on organic chemicals usually have a test entitled “Related substances” (or a test with equivalent purpose under a different title), designed for control of organic impurities. Inorganic impurities are usually covered, where applicable, by other tests. Residual solvents are covered by specific provisions.The acceptance criterion for specified impurities may be set at the identification threshold for the substance.The acceptance criteria for specified impurities take account of both:1. qualification data, where applicable, the limit being set at a level not greater than that at which the impurity is qualified; the information on qualification is provided by the producer and the compatibility of the limit with the qualification data and approved specifications is checked by the competent authorities during elaboration of the monograph and/or during the Pharmeuropa comment phase; and2. batch analysis data, the acceptance criteria being set to take account of normal production; data is provided by the producer for typical batches and checked during elaboration of the monograph on not fewer than 3 batches.4.3 Heavy MetalsThe heavy metals detected by the general methods are those that precipitate at pH 3.5 in the form of dark-colored sulphides through the action of sulphide ions or reagents capable of producing them: lead, copper, silver, mercury, cadmium, bismuth, ruthenium, gold, platinum, palladium, vanadium, arsenic, antimony, tin and molybdenum.In routine practice, Methods A, B, C, D, F and G are unsuitable for establishing limits below 5 ppm, unless filtration is prescribed. For lower limits, Method E can be used, which makes it possible to go down to 0.5 ppm. In order to ensure limit contents of less than 0.5 ppm, it is necessary to resort to tests specific for each metal which are frequently based on atomic spectrophotometry.Tests for individual metal catalysts are included where it is known that they are used in the synthesis of an active substance or excipient. Limits take account of toxicity, the route of administration and the manufacturing process.4.4 Loss on DryingGenerally an upper limit for loss on drying is given. If the substance is a hydrate (or solvate) upper and lower limits are indicated. Drying is carried out to constant mass, unless a drying time is specified in the monograph. When a drying time is prescribed, adequate validation data must be provided. Where the drying temperature is indicated using a single value, a tolerance of ±2°C is understood. For temperatures higher than 105 °C, a larger tolerance should be indicated in the monograph, if necessary.Limits less than 10 percent should be given with 2 significantfigures and limits of 10 percent or greater should be given with 3 significant figures. The sample size is chosen to give a difference of 5-50mg before/after drying and is indicated with 4 significant figures.4.5 ThermogravimetryLoss on drying can be determined by this method when the amount of substance has to be restricted, for example to reduce exposure for the analyst or if the substance is very expensive.4.6 Sulphated AshThis test is usually intended for the global determination of foreign cations present in organic substances and in those inorganic substances which themselves are volatilized under the conditions of the test. Thus the test will be of little value as a purity requirement for the majority of inorganic salts of organic substances, due to the resulting high bias.The limit in a test for Sulphated ash is usually set at 0.1 percent, unless otherwise justified. The amount of substance prescribed for the test must be such that a residue corresponding to the limit will weigh not less than 1.0mg and the prescribed mass of substance is then given with the appropriate precision (1.0g).5. AssayWhen the identification and purity tests are sufficiently characteristic and searching, a non-specific but precise assay may be used rather than a specific and less precise assay.。
欧洲药典附录2.6.12非无菌产品的微生物限度检查之一(有活
力的需氧菌总数的计数)
张新妹;胡昌勤;王淑兰
【期刊名称】《中国药品标准》
【年(卷),期】2003(004)004
【摘要】@@ 下述检验方法是对需氧条件下生长的嗜温细菌和真菌数的计数检查.【总页数】4页(P59-61,17)
【作者】张新妹;胡昌勤;王淑兰
【作者单位】中国药品生物制品检定所抗生素室,北京,100050;中国药品生物制品检定所抗生素室,北京,100050;中国药品生物制品检定所所办,北京,100050
【正文语种】中文
【中图分类】R927.1
【相关文献】
1.《美国药典》35版附录"《62》非无菌产品微生物检查:控制菌检查法"中两个问题的探讨 [J], 刘洪祥;曹晓云
2.《美国药典》( USP31/NF26)附录非无菌产品的微生物计数检查 [J], 李秋菲;闵红;宋愿智
3.《美国药典》(USP31/NF26)附录非无菌产品的微生物检查-控制菌检查法 [J], 李秋菲;由亚宁;高飞;王小亮
4.欧洲药典附录2.6.13非无菌产品的微生物限度检查之二(对特定微生物的检查)
[J], 张新妹;胡昌勤;王淑兰
5.中职“非无菌产品微生物限度检查”微课在教学中的实践应用 [J], 何慧贞;因版权原因，仅展示原文概要，查看原文内容请购买。
07/2010:206122.6.12非无菌产品微生物检测：总需氧活菌计数(2)1.简介以下描述的检测方法可用于在有氧条件下生长的嗜温性细菌和真菌的定量计数。
本检测方法主要用来判定某物质或制剂是否符合微生物质量规定。
用于此目的时，可按如下规定确定取样数量和判定结果。
本法不适用于以活性菌作有效成分的产品。
可使用替代性的微生物检测程序（包括自动化方法），只要证明其与已经过论证的药典方法是等效的。
2.一般程序在设计用于避免供试品受外界微生物污染的规定条件下进行测定。
检测过程中防污染的措施必须不影响微生物的检出。
若供试品有抑菌性活性，必须尽可能的减少其抑菌性或中和。
如果使用了灭活剂，则必须证明灭活剂的有效性及对微生物的无毒性。
如果样品制备过程中使用了表面活性剂，则必须证明表面活性剂对微生物的无毒性和它与使用的灭活剂的相容性。
3.计数方法按规定使用薄膜过滤法或平板计数法。
MPN法通常是准确性最差的微生物的计数方法，但对微生物量很低的产品可以是最适用的方法。
方法的选择基于如药品特性、微生物限度等因素。
所选方法必需能通过检验足量样品来判定其符合规定。
方法的适用性必须验证。
4.生长促进检测与计数方法适用性和阴性对照4-1. 总体考虑必须建立在有供试品存在条件下检测微生物的能力。
若检测操作或产品改变时，且这些改变可能会影响检测的结果，则须证实适用性。
4-2. 测试菌株的制备采用稳定的标准测试菌株混悬液或按以下规定制备。
采用种子菌培养保持技术，确保原代菌连续传代不超过5次。
按表2.6.12.-1.的描述分别培养细菌株和真菌株。
采用pH 7.0 氯化钠-蛋白胨缓冲液或pH 7.2的磷酸缓冲液制备供测试菌混悬液。
悬浮黑曲霉（A.brasiliensis）孢子时可加入0.05%的吐温80。
该混悬液在2小时内(2) 本章已通过药典协调，见章5.8.药典协调使用或在2 - 8 ℃贮存条件下24小时内使用。
作为制备并稀释新鲜黑曲霉（A.brasiliensis）和枯草芽孢杆菌营养细胞悬浮液的一种替代方法，可用适量体积的稳定的孢子悬浮液作为供试菌接种。
Frequently asked questions on chapter 2.6.12 Microbiological examination of non-sterile product: total viable aerobic countB. Harmonised methodPARAGRAPHCONCERNEDQUESTIONS ANSWERSCan I use other strains those that are cited in the Ph.Eur.? You must use the micro-organisms that are cited in this chapter or equivalent strains from other culture collections.4.2 Preparation of teststrainIs there a method that allows to verify that there is 100 CFU in the inoculum? You may establish a turbidimetric calibration curve or use another suitable method and then you will be able to determine rapidly the concentration of your inoculum.You can also use already-prepared certified strains.4.3 Negative control When are you actuallysupposed to do thenegative control: whentesting the suitability of themethod, or when testingthe product, or in bothsituations? You are supposed to do the negative control at the same time as when you are testing the product.What is the purpose of the negative control? The purpose of this negative control is to show that there is no contamination during the testing of the product.If a positive result is obtained with a negative control, the test can be regarded as invalid and may be repeated.4.3 Negative controlDoes it have to be done every time the product is tested or during the method validation or is it possible to do it periodically? It is preferable to do a negative control every time that the product is tested.Is it necessary to test the growth promotion on all received batches or does it serve just for microbiological validation? Do we have to test the growth promotion of diluted broth? Growth promotion must be tested for each new batch of medium, either by the supplier or the analyst. Growth promotion must be tested on agar media and nutritive broth but not on diluted broth.4.4 Growth promotion ofthe mediaDo we have to test systematically in parallel a previous and approved batch in order to compare with the new batch? You do not have to test a previous batch in parallel. You can do the comparison ‘on paper’ if growth was clearly described.Why have growth promotion tests to be performed on sabouraud-dextrose agar (SDA) and casein soya bean digest agar (CSA) for C. albicans and A. niger? Is it, because colonies of fungi detected on CSA are counted as part of TAMC? As bacteria growing on SDA are also counted as part of TYMC, why have the growth promotion tests not to be performed on SDA with the bacterial strains? This is a matter of definition. TAMC by definition includes yeast and moulds. Therefore the media have to be checked with these micro-organisms. TYMC is by definition yeasts and moulds count so growth promotion with bacteria is not essential.SDA with antibiotics may be used as an alternative.4.4 Growth promotion ofthe mediaWhat does the factor of 2 mean?How is this factor calculated? It means that the result can be twice that of the inoculum. For example with an inoculum of 100 CFU, acceptable counts are : 100/2 = 50 CFU to 100 x 2 = 200 CFU.The factor is introduced to take account of the variability of the method.4.5.2 Inoculation anddilution What is the sufficientvolume of the microbialsuspension of not morethan 100 CFU? What does100 CFU refer to?The micro-organisms are tobe added to thediluted/suspended product atthe end of the preparation orafter the neutralisation (in thelast fraction of the rinsingfluid in the case of filtration orsimultaneously with thepreparation in/on the Petridish in the case of the platecount method).The 100 CFU refers to theinoculum (eg what will be onthe filter or on the plate).4.5.2 Inoculation anddilution Problem of comprehensionof table 2.6.12-2.If I am going to validate aTAMC: should I use bothC. albicans and A. nigerfor my qualification?And if I am validating foryeast and mould countshould both C. albicansand A. niger be used forthe qualification?Yeast and moulds must berecovered on both CSA andSDA.Yes, you must validate withC. albicans and A. niger (seealso 4.4).4.5.3 Neutralisation/removal of antimicrobial activity Why is it necessary todemonstrate the efficacyof neutralising agents?Has this to be performedby comparison of buffersolutions with and withoutneutralising agents? Fromour point of view it is notnecessary, if recovery ofmicro-organisms in thepresence of product isgiven. It is very timeconsuming to test differentcombinations ofneutralising agents prior tothe validation test.It is good practice to test theefficacy and the absence oftoxicity of the neutralisingagent. Effectiveness ischecked by performingincubation of the product withand without neutralisingagent, absence of toxicity isperformed on a blank withneutraliser and withoutproduct. This can be done inparallel.4.5.4-1 Membranefiltration Why is the use of 0.45 µmfilter deemed appropriate,in view of the fact that a0.22 µm filter is regardedrequired for sterilisationupon filtration? The use ofa 0.45 µm filter would notbe sufficient to retain themicrobiological micro-organisms that could bepresent; instead a 0.22 µmfilter would be sufficient.In the sterility testmembranes with a pore sizenot greater than 0.45 µm areused as well. The reasonswhy filters with a larger poresize have been chosen are:1. Filtration is easier (faster)particularly in case of viscousproducts.2. If the filters are placed on anutrient medium, thenutrients must diffuse throughthe filter. This diffusion isfaster as the pore size islarger. Thus, growth can bedetected earlier on a 0.45 µmfilter as compared to a0.22 µm filter.If colonies of bacteria aredetected on SDA they arecounted as part of theTYMC. Then bacteria arecounted twice (TAMC andTYMC). Could you tell methe reason?If a product hasacceptance criteria forTYMC, why the bacteriadetected on SDA must beincluded in the fungiresults (TYMC)?TAMC is Total AerobicMicrobial Count, whichinclude both bacteria andfungi (yeast and moulds). Forthe TYMC, bacteria mightoccasionally grow on SDAplates, especially if they arepresent in the product inreasonably high numbers.Antibiotics can be included toresult in a product failing tomeet TYMC acceptancecriteria.5.3 Interpretation of theresultsWhat is the signification of “102CFU: maximum acceptable count: 200”. It means that if the specification in a product monograph is 102micro-organisms, the product could be release if up to 200 micro-organisms are counted.。
