Bioburden Test 生物负荷检查.doc

TVC：total viable count 菌落总数TAMC：total aerobic microbial count 总好氧微生物计数TYMC：total combined yeasts and molds count 总霉菌酵母菌计数Bioburden Tests 生物负荷检查A bioburden test is referred to as a total viable count (TVC) test for estimation of viable aerobic mesophilic microorganisms in products or articles not purported to be sterile. In essence, a bioburden test can be carried out as the USP TAMC test, or as the total of the TAMC and TYMC tests (TVC = TAMC + TYMC), or using a medium of choice with either single- or dual-temperature incubation conditions. In general, bioburden tests are performed for the estimation of microorganisms in samples such as containers, product contact surfaces, water, in-process samples, final bulk products prior to sterilization, and any other type of material that require assessment of their bioload, with no reference to a defined compendial requirement for sample amount. Bioburden tests are typically performed during cleaning validation studies, and the samples can be processed directly using a device containing a nutrient medium (e.g., contact plate). Alternatively, samples can be collected using swabs, swatches, or rinse fluid and then processed using the chosen microbial recovery method.一个生物负荷检查被称为总活菌计数（TVC），用于评价非无菌产品的嗜温好氧微生物。本质上讲，生物负荷检查可以作为USP中TAMC检查进行，或者作为TAMC和TYMC的结合进行，或者用一种培养基可以选择单个温度或者两个温度点培养。通常，生物负荷检查用于对容器、与产品直接接触的表面、水、中控样品、终产品灭菌前和没有明确的样本量作参考的，需要评估生物负载的任何其他材料作微生物评价。生物负荷检查一般在清洁验证时进行，样品可以用含有营养培养基的设备直接处理（如接触碟）。样品可以用棉签、样品块和冲洗液收集，之后经过微生物回收处理。Two-Media Bioburden Test 基于两种培养基的生物负荷检查As explained, a bioburden test can be performed as the USP TAMC and TYMC tests by the plate-count method (pour-plate or spread-plate) or membrane filtration method. At the end of the incubation period, the counts obtained from the TAMC and TYMC tests are added and reported as the TVC. An attempt should be made to characterize colonies isolated from both media so that the same type of organism is not counted twice.正如上面解释的，生物负荷检查可以作为USP中的TAMC和TYMC项目用平板计数法（倒平板或者扩散平板）或薄膜过滤法检查。培养周期结束时，TAMC和TYMC获得的菌落总数合并在一起作为TVC报告。应该对菌落特征进行鉴别防止两种培养基上的相同微生物被重复计数。One-Medium, Dual-Temperature Incubation Bioburden Test 基于一种培养基两个温度点培养的生物负荷检查The one-medium, dual-temperature incubation method is designed for the detection of low bioburden of both bacteria and fungi surviving in an oligotrophic environment. Although the microbial limit tests call for the use of the sabouraud dextrose medium for the recovery of fungi, studies performed over the years have demonstrated that all-purpose media, such as SCD, are capable of recovering a wide range of bacteria, yeasts, and molds 1, 2. The test can be performed as the USP TAMC or as a modification to the TAMC test using alternate media and alternate incubation conditions. However, a more popular approach is to modify the TAMC method and perform the test using SCD agar or microbial content test agar (MCTA), and incubate the test samples at two temperature ranges for the optimum recovery of both bacteria and fungi. This test approach is widely used in the pharmaceutical industry for microbial monitoring of environments, surfaces, and equipment, and is suggested in the Association for the Advancement of Medical Instrumentation (AAMI) guidelines for articles expected to have a low bioburden. Samples collected are typically incubated at 3035C for 23 d followed by a 57 d incubation period at 2025oC. It is recommended that plates should be observed for microbial growth at the end of the initial incubation period for detection of possible spreaders and to prevent plate overgrowth.一种培养基两个温度点培养的方法用于检测低生物负荷的能在贫营养环境中生存的细菌和真菌。虽然微生物限度检查要求使用沙氏培养基回收真菌，但经年的研究证明诸如SCD的万能培养基可以回收细菌、酵母菌和霉菌。该项检查可以按照USP的TAMC进行，或者参照TAMC用其它替代培养基和其它培养条件进行。然而，一个更流行的方法是参照TAMC方法用SCD琼脂培养基或者微生物含量测试琼脂培养基（MCTA）并在对细菌和真菌有最佳回收率的两个温度范围培养样品。该方法在制药工业广泛应用于环境、表面和仪器的微生物监控。该方法还是AAMI（医疗器械发展协会）指导方针建议针对生物负载低的物品使用的。收集的样品一般在3035培养23天之后在2025培养57天。建议在前一个培养周期结束时检查平板，观察微生物生长情况，防止在第二个培养周期出现过度生长情况。Variations of the dual-temperature incubation bioburden test include length of incubation of test plates as well as the order of incubation temperature range: some methods specify a low-temperature incubation period initially, followed by moderate- temperature incubation. This issue has actually been a topic of debate in the industry and among regulatory inspectors for several years. The concern was that there could be a risk of possible low recovery of fungi and certain psychrophilic organisms if plates were to be incubated initially at a moderate temperature range (3035C) because faster-growing mesophilic bacteria could overcrowd the plates. Nowadays, the general consensus is that the order of temperature of incubation is not a critical factor for most environmental organisms, including the typical contaminants in pharmaceutical products and facilities, especially when low-level bioburden is expected. Many studies have been performed by companies in support of a dual-temperature incubation method, and results do not show significant difference in results. In fact, most environmental fungi grow very well at 3035C; fastidious bacteria remain viable at 2025C and are readily recovered when incubated at 3035C. However, using an initial higher incubation temperature does have a compliance advantage: this approach is referenced in the AAMI guidelines, which, to the authors knowledge, may be the only published reference on this subject.两个温度点培养的方法的变更既包括平板培养时间的长度也包括培养温度范围的制定：一些方法先指定一个低温培养周期，接着进行中温培养。这个问题其实已在同行业的监管检查人员之间争论数年。大家担心的是该方法可能存在的风险：如果平板先在中温范围（3035）培养会因为嗜温菌快速增长造成平板过度拥挤，从而使真菌和某些嗜冷微生物的回收率低。如今，普遍的共识是对于大多数环境微生物来说培养温度不是一个关键因素，包括制药企业中产品、设备的典型的污染，特别是应该为低生物负荷的情况。支持两个温度点培养的公司进行了很多研究，结果并没有显著差别。事实上，大多数环境真菌在3035生长良好；苛刻的细菌在2025仍然可以生存并且在3035培养时仍然可以回收。然而，初始培养时采用高一些的温度确实存在优势：该方法参考了AAMI指导方针，该仿真也许是唯一一个关于该问题的出版的参考文件。One point of consideration when choosing this test approach is the need to use a mixed inoculum composed of representative test organisms when performing method suitability studies. The study design must demonstrate that the various types of challenge organisms can be recovered on the same medium without the inhibitory or masking effects of one species over another.选择该检查方法时需要考虑的一点是，进行方法实用性研究时需要使用由典型的测试微生物组成的混合接种体。研究设计必须证明各种类型的挑战微生物都可以在同种培养基上回收，没有抑制现象或者一种微生物盖过另一种的遮蔽效应。Over the years, the USP Chapter has undergone several revisions, and theUSP has received many comments from pharmaceutical companies concerning thecontents of this chapter. Although the bioburden methods recommended by the USPare not ideal for the detection of stressed and starved organisms, they are still recognizedas appropriate techniques for establishing trends in bioburden in water systemsin a timely manner. The USP also states that other recovery methods, includingmedia and incubation conditions, and larger sample volumes may be used for theoptimal recovery of microorganisms found in various types of water systems. Infact, most highly purified water systems are extremely effective in the removal andprevention of biofilm formation; thus, a sample size of 1.0 mL is not appropriate fortesting and trending the microbial quality of the water produced.When using sample volumes larger than 1.0 mL, the membrane filtration methodshould be used; a membrane filter with a rating of 0.45 m is generally the preferredmethod for testing liquid samples for bioburden. This is especially true for watersamples because the filtration process allows retention and recovery of a high numberof small cells (e.g., Gram-negative