BioburdenTest生物負(fù)荷檢查

1、TVC : total viable count 菌落總數(shù)TAMC : total aerobic microbial count 總好氧微生物計(jì)數(shù)TYMC : total combined yeasts and molds count總霉菌酵母菌計(jì)數(shù)Bioburden Tests生物負(fù)荷檢查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 n

2、ot 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 f

3、or 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

4、 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 pro

5、cessed using the chose n microbial recovery method.一個(gè)生物負(fù)荷檢查被稱(chēng)為總活菌計(jì)數(shù)(TVC ),用于評(píng)價(jià)非無(wú)菌產(chǎn)品的嗜溫好氧 微生物。本質(zhì)上講，生物負(fù)荷檢查可以作為USP中TAMC檢查進(jìn)行，或者作為T(mén)AMC和TYMC的結(jié)合進(jìn)行，或者用一種培養(yǎng)基可以選擇單個(gè)溫度或者兩個(gè)溫度 點(diǎn)培養(yǎng)。通常，生物負(fù)荷檢查用于對(duì)容器、與產(chǎn)品直接接觸的表面、水、中控樣 品、終產(chǎn)品滅菌前和沒(méi)有明確的樣本量作參考的，需要評(píng)估生物負(fù)載的任何其他材料作微生物評(píng)價(jià)。生物負(fù)荷檢查一般在清潔驗(yàn)證時(shí)進(jìn)行，樣品可以用含有營(yíng)養(yǎng) 培養(yǎng)基的設(shè)備直接處理(如接觸碟)。樣品可以用棉簽、樣品塊和沖

6、洗液收集， 之后經(jīng)過(guò)微生物回收處理。Two-Media Bioburden Test基于兩種培養(yǎng)基的生物負(fù)荷檢查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

7、 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.正如上面解釋的，生物負(fù)荷檢查可以作為 USP中的TAMC和TYMC項(xiàng)目用平板 計(jì)數(shù)法(倒平板或者擴(kuò)散平板)或薄膜過(guò)濾法檢查。培養(yǎng)周期結(jié)束時(shí)，TAMC和TYMC獲得的菌落總數(shù)合并在一起作為T(mén)VC報(bào)告。應(yīng)該對(duì)菌落特征進(jìn)行鑒別防止兩種培養(yǎng)

8、基上的相同微生物被重復(fù)計(jì)數(shù)。One-Medium, Dual-Temperature Incubation Bioburden Test 基于一種培養(yǎng)基 兩個(gè)溫度點(diǎn)培養(yǎng)的生物負(fù)荷檢查T(mén)he 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 ca

9、ll 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 modifica

10、tion 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 recove

11、ry 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 t

12、o 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

13、plate overgrowth.一種培養(yǎng)基兩個(gè)溫度點(diǎn)培養(yǎng)的方法用于檢測(cè)低生物負(fù)荷的能在貧營(yíng)養(yǎng)環(huán)境中生 存的細(xì)菌和真菌。雖然微生物限度檢查要求使用沙氏培養(yǎng)基回收真菌，但經(jīng)年的研究證明諸如SCD的萬(wàn)能培養(yǎng)基可以回收細(xì)菌、酵母菌和霉菌。該項(xiàng)檢查可以 按照USP的TAMC進(jìn)行，或者參照TAMC用其它替代培養(yǎng)基和其它培養(yǎng)條件進(jìn) 行。然而，一個(gè)更流行的方法是參照 TAMC方法用SCD瓊脂培養(yǎng)基或者微生物 含量測(cè)試瓊脂培養(yǎng)基(MCTA)并在對(duì)細(xì)菌和真菌有最佳回收率的兩個(gè)溫度范圍 培養(yǎng)樣品。該方法在制藥工業(yè)廣泛應(yīng)用于環(huán)境、表面和儀器的微生物監(jiān)控。該方 法還是AAMI (醫(yī)療器械發(fā)展協(xié)會(huì))指導(dǎo)方針建議針對(duì)生物

14、負(fù)載低的物品使用的。 收集的樣品一般在3035C培養(yǎng)23天之后在2025C培養(yǎng)57天。建議在前一 個(gè)培養(yǎng)周期結(jié)束時(shí)檢查平板，觀察微生物生長(zhǎng)情況，防止在第二個(gè)培養(yǎng)周期出現(xiàn) 過(guò)度生長(zhǎng)情況。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

15、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

16、 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, includi

17、ng 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 env

18、ironmental fungi grow very well at 30 5C; fastidious bacteria rema in viable at 20 -25C and are readily recovered whe n in cubated at 30 435C. 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 t

19、he author s knowledge, may be the only publishedrerfee on this subject.兩個(gè)溫度點(diǎn)培養(yǎng)的方法的變更既包括平板培養(yǎng)時(shí)間的長(zhǎng)度也包括培養(yǎng)溫度范圍 的制定：一些方法先指定一個(gè)低溫培養(yǎng)周期，接著進(jìn)行中溫培養(yǎng)。這個(gè)問(wèn)題其實(shí)已在同行業(yè)的監(jiān)管檢查人員之間爭(zhēng)論數(shù)年。大家擔(dān)心的是該方法可能存在的風(fēng) 險(xiǎn)：如果平板先在中溫范圍（3035C）培養(yǎng)會(huì)因?yàn)槭葴鼐焖僭鲩L(zhǎng)造成平板過(guò) 度擁擠，從而使真菌和某些嗜冷微生物的回收率低。如今，普遍的共識(shí)是對(duì)于大多數(shù)環(huán)境微生物來(lái)說(shuō)培養(yǎng)溫度不是一個(gè)關(guān)鍵因素，包括制藥企業(yè)中產(chǎn)品、設(shè)備的 典型的污染，特別是應(yīng)該為低生物負(fù)

20、荷的情況。支持兩個(gè)溫度點(diǎn)培養(yǎng)的公司進(jìn)行 了很多研究，結(jié)果并沒(méi)有顯著差別。事實(shí)上，大多數(shù)環(huán)境真菌在3035C生長(zhǎng)良好；苛刻的細(xì)菌在2025C仍然可以生存并且在3035C培養(yǎng)時(shí)仍然可以回收。 然而，初始培養(yǎng)時(shí)采用高一些的溫度確實(shí)存在優(yōu)勢(shì)：該方法參考了AAMI指導(dǎo)方針，該仿真也許是唯個(gè)關(guān)于該問(wèn)題的出版的參考文件。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

21、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.選擇該檢查方法時(shí)需要考慮的一點(diǎn)是，進(jìn)行方法實(shí)用性研究時(shí)需要使用由典型的 測(cè)試微生物組成的混合接種體。研究設(shè)計(jì)必須證明各種類(lèi)型的

22、挑戰(zhàn)微生物都可以 在同種培養(yǎng)基上回收，沒(méi)有抑制現(xiàn)象或者一種微生物蓋過(guò)另一種的遮蔽效應(yīng)。Over the years, the USP Chapter 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

23、 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 vol

24、umes 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

25、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 preferredmethod 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 st