BioburdenTest生物负荷检查 - 图文

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 microorga ni sms in products or articles not purported to be sterile. In esse nee, a bioburde n test can be carried out as the USP TAMC test, or as the total of the TAMC and TYMC tests (TVC = TAMC + TYMC), or us ing a medium of choice with either single- or dual-temperature incubation conditions. In general, bioburde n tests are performed for the estimati on of microorga ni sms in samples such as contain ers, product con tact surfaces, water, i n-process samples, final bulk products prior to

sterilization, and any other type of material that require assessment of their bioload, with no refere nee to a defi ned compe ndial requireme nt for sample amount. Bioburden tests are typically performed during cleaning validation studies, and the samples can be processed directly using a device containing a nu trie nt medium (e.g., con tact plate). Alter natively, samples can be collected using swabs, swatches, or rinse fluid and the n processed using the chose n 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 expla in ed, a bioburden test ca n be performed as the USP TAMC and TYMC tests by the plate-co unt method (pour-plate or spread-plate) or membra ne filtrati on method. At the end of the in cubati on period, the counts obta ined 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 orga nism is not coun ted 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 on e-medium, dual-temperature in cubati on method is desig ned for the detect ion 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 fun gi, studies performed over the years have dem on strated that all-purpose media, such as SCD, are capable of recoveri ng 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 con diti ons. 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 in cubate the test samples at two temperature ran ges for the optimum recovery of both bacteria and fungi. This test approach is widely used in the pharmaceutical industry for microbial mon itori ng of en vir onmen ts, surfaces, and equipme nt, and is suggested in the Associati on for the Adva nceme nt of Medical In strume ntati on (AAMI) guideli nes for articles expected to have a low bioburden. Samples collected are typically in cubated at 30435 °C for 2 — d followed by a 5 — d in cubation period at 20 —

5oC. It is recomme nded 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30~35C培养2~3天之后在20~25C培养5~7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 in cubati on period in itially, followed by moderatetemperature in cubatio n. This issue has actually bee n a topic of debate in the in dustry and among regulatory in spectors 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 (30 —5 °C) becausefaster-grow ing mesophilic

bacteria could overcrowd the plates. Nowadays, the gen eral consen sus is that the order of temperature of in cubatio n 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 in cubati on method, and results do not show sig ni fica nt differe nee in results. In fact, most environmental fungi grow very well at 30 ￡5°C; fastidious bacteria rema in viable at 20 -25°C and are readily recovered whe n in cubated at 30 435°C. However, using an initial higher incubation temperature does have a complia nee adva ntage: this approach is refere need in the AAMI guideli nes, which, to the author ' s knowledge, may be the only publishedrerfee 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（30~35C）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 30~35C生长良

好；苛刻的细菌在20~25C仍然可以生存并且在30~35C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 -- 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 ino culum composed of represe ntative test orga ni sms whe n perform ing method suitability studies. The study desig n must dem on strate that the various types of challe nge orga ni sms can be recovered on the same medium without the in hibitory or mask ing effects of one species over ano ther.

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 <1231> has un derg one several revisi ons, and the USP has

received many comme nts from pharmaceutical compa nies concerning the contents of this chapter. Although the bioburde n methods recomme nded by the USP are not ideal for the detect ion of

stressed and starved orga ni sms, they are still recog ni zed as appropriate tech niq ues for establish ing trends in bioburde n in water systems in a timely manner. The USP also states that other recovery methods, in clud ing media and in cubati on con diti ons, and larger sample volumes may be used for the optimal recovery of microorga nisms found in various types of water systems. In fact, most highly purified water systems are extremely effective in the removal and preve nti on of biofilm formati on; thus, a sample size of 1.0 mL is not appropriate for test ing and trending the microbial quality of the water produced.

Whe n using sample volumes larger tha n 1.0 mL, the membra ne filtrati on method should be used; a membra ne filter with a rati ng of 0.45 卩 m is gen erally the preferred

method for testi ng liquid samples for bioburde n. This is especially true for water samples because the filtrati on process allows rete nti on and recovery of a high nu mber of small cells (e.g., Gram-negative and starved microorganisms) typically found in water systems. Based on the expected

bioburde n of the samples collected, most pharmaceutical compa nies have chose n the membra ne filtrati on method for test ing purified waters and the pour-plate method, using a 1.0 mL sample volume, for the testi ng of feedwater. The MilliflexR system (Millipore Corporati on, www.millipore. com) see n in Figure 4.2 is a membra ne filtrati on system that offers faster filtrati on by using filters that have up to twice the surface area of sta ndard 47-mm membra nes. Water samples are processed using sterile filter un its that comb ine a funnel and a

grid membra ne filter in one device and plated with agar-based media contained in ready-to-use

cassettes. Figure 4.3 shows bacterial growth on the membra ne filter cassette follow ing a specified in cubati on period.