成品預(yù)裝培養(yǎng)基平皿實(shí)用技術(shù)手冊（連載二）：潔凈區(qū)微生物檢測方法
發(fā)布時間：
2022-12-27
作者：
培養(yǎng)基訂購網(wǎng)站
制藥企業(yè)生產(chǎn)車間、實(shí)驗(yàn)室潔凈區(qū)的微生物污染與污染源密切相關(guān)，細(xì)菌、真菌、病毒性污染等較為常見，主要由潔凈環(huán)境和設(shè)施管理不當(dāng)、人員不規(guī)范的行為與交叉污染、操作菌株的泄露、原輔料混入、設(shè)備與容器污染等原因造成。因而選擇一種可靠的潔凈區(qū)微生物檢測方法，可以有效監(jiān)控潔凈區(qū)的環(huán)境，控制微生物污染的風(fēng)險(xiǎn)，確保微生物監(jiān)測結(jié)果的可靠性和準(zhǔn)確性，體現(xiàn)潔凈區(qū)環(huán)境微生物控制的真實(shí)水平。
培養(yǎng)基是微生物試驗(yàn)的基礎(chǔ)，直接影響微生物試驗(yàn)結(jié)果。適宜的培養(yǎng)基制備方法、貯藏條件和質(zhì)量控制試驗(yàn)是提供優(yōu)質(zhì)培養(yǎng)基的保證。
在微生物檢測時，我們應(yīng)該高度重視所使用的培養(yǎng)基（包括購置不同批號的成品預(yù)裝培養(yǎng)基、不同批次的脫水培養(yǎng)基干粉、按照處方使用不同批次的原材料自行配制培養(yǎng)基等）、制備程序（包括水質(zhì)控制、配制方法、滅菌程序等）、貯藏條件（溫濕度、時間及盛裝培養(yǎng)基的容器條件等）等是否滿足微生物檢測用要求。
無論是做潔凈區(qū)沉降菌、浮游菌，還是表面微生物，所使用的培養(yǎng)基平皿在使用之前都必須對使用批次的培養(yǎng)基平皿做促生長試驗(yàn)，這也是培養(yǎng)基的性能試驗(yàn)，更是培養(yǎng)基的內(nèi)在質(zhì)量標(biāo)準(zhǔn)。它是用已知的標(biāo)準(zhǔn)菌株按照規(guī)程和標(biāo)準(zhǔn)測定培養(yǎng)基性能是否符合要求。
在這個實(shí)驗(yàn)中，我們不能忽略的一個環(huán)節(jié)，就是需要使用對照培養(yǎng)基。關(guān)于對照培養(yǎng)基，在《中國藥典》中借鑒歐美藥典，引入培養(yǎng)基適用性檢查以保障微生物限度檢查結(jié)果的準(zhǔn)確性和可靠性。
但是，對于歐美藥典中的“之前驗(yàn)證過的培養(yǎng)基”的采納與否，存在很大爭議，集中在兩點(diǎn)。第一，我國藥品微生物實(shí)驗(yàn)室小而分散，數(shù)以千計(jì)的企業(yè)和基層藥檢所難以有效地進(jìn)行對照培養(yǎng)基的驗(yàn)證、評價(jià)工作；第二、在監(jiān)督檢查中，難以有效評估企業(yè)微生物實(shí)驗(yàn)室對對照培養(yǎng)基的驗(yàn)證情況，以統(tǒng)一的、經(jīng)過充分評價(jià)和驗(yàn)證過的對照培養(yǎng)基進(jìn)行培養(yǎng)基的適用性試驗(yàn)。
所以我們在使用對照培養(yǎng)基過程中，應(yīng)該注意：
在保證配制和滅菌過程無誤的條件下，對照培養(yǎng)基可以不經(jīng)適用性實(shí)驗(yàn)檢查直接用于樣品檢查。
不同處方培養(yǎng)基的替代使用不能通過培養(yǎng)基適用性試驗(yàn)簡單確定，要按標(biāo)準(zhǔn)規(guī)定使用培養(yǎng)基。
不建議使用經(jīng)過對照培養(yǎng)基進(jìn)行適用性試驗(yàn)檢查合格的其它培養(yǎng)基作為實(shí)驗(yàn)室自用的“對照培養(yǎng)基”。
如果沒有特殊規(guī)定，培養(yǎng)基配制采用蒸餾水或純化水均可，但應(yīng)進(jìn)行檢測控制。
干粉培養(yǎng)基或培養(yǎng)基原料變質(zhì)不應(yīng)再用；成品培養(yǎng)基儲存過程發(fā)現(xiàn)變色、長菌、嚴(yán)重脫水等也不應(yīng)再用。
2、潔凈區(qū)微生物檢測
制藥企業(yè)在評估生產(chǎn)潔凈環(huán)境的微生物狀況時，需對潔凈環(huán)境進(jìn)行動態(tài)或靜態(tài)監(jiān)測，使用沉降菌平皿、空氣浮游菌平皿和表面接觸皿對潔凈區(qū)微生物檢測。其中浮游菌和沉降菌的檢測根據(jù)GB16293-2010《醫(yī)藥工業(yè)潔凈室(區(qū))浮游菌的測試方法》和GB16294-2010《醫(yī)藥工業(yè)潔凈室(區(qū))沉降菌的測試方法》規(guī)定進(jìn)行檢測。表面微生物檢測方法有擦拭法和表面接觸碟法。
2.1沉降菌取樣
沉降菌檢測方法采用沉降法，即通過自然沉降原理收集在空氣中的生物粒子于培養(yǎng)基平皿，經(jīng)過若干時間，在適宜的條件下讓其繁殖到可見的菌落進(jìn)行計(jì)數(shù)，以平板培養(yǎng)基皿中的菌落數(shù)來判斷結(jié)晶環(huán)境內(nèi)活微生物數(shù)，并以此來評定潔凈區(qū)的潔凈度。
取樣方法：環(huán)境監(jiān)控人員用消毒劑消毒雙手，取出TSA平皿，按照采樣點(diǎn)布置圖逐個放置，然后從里到外逐個打開培養(yǎng)皿蓋，并將蓋內(nèi)側(cè)向下放在培養(yǎng)皿底座旁邊，使培養(yǎng)基表面暴露在空氣中。為了避免污染培養(yǎng)基表面，手和手臂不要經(jīng)過已開蓋的培養(yǎng)皿上方。
2.2浮游菌取樣
浮游菌取樣是通過空氣采樣儀收集懸浮在空氣中的生物性粒子于專門的培養(yǎng)基，經(jīng)若干時間和適宜的生長條件讓其繁殖到可見的菌落并計(jì)數(shù)，以判定該潔凈區(qū)的微生物濃度。
浮游菌采樣器一般采用撞擊法機(jī)理，可分為狹縫式采樣器、離心式或針孔式采樣器。
狹縫式采樣器由內(nèi)部風(fēng)機(jī)將氣流吸入，通過采樣器的狹縫式平板，將采集的空氣噴射并撞擊到緩慢旋轉(zhuǎn)的平板培養(yǎng)基表面上，附著的活微生物粒子經(jīng)培養(yǎng)后形成菌落。
離心式采樣器由于內(nèi)部風(fēng)機(jī)高速旋轉(zhuǎn)，氣流從采樣器前部吸入從后部流出，在離心力的作用下，空氣中的活微生物粒子有足夠的時間撞擊到專用的固形培養(yǎng)條上，附著的活微生物粒子經(jīng)培養(yǎng)后形成菌落。
針孔式采樣器是氣流通過一個金屬蓋吸入，蓋子上是密集的經(jīng)過機(jī)械加工的特制小孔，通過風(fēng)機(jī)收集到細(xì)小的空氣流直接撞擊到平板培養(yǎng)基表面上，附著的活微生物粒子經(jīng)培養(yǎng)后形成菌落。
目前大部分制藥企業(yè)采用針孔式采樣器進(jìn)行浮游菌采樣，故介紹一下其取樣方法。
取樣方法（針孔式采樣器）：
環(huán)境監(jiān)控人員應(yīng)首先確定空氣采樣器在校驗(yàn)合格的范圍內(nèi)；
采樣前，用經(jīng)消毒劑浸濕的無菌布擦拭空氣采樣器的外表面，并讓其自然揮干；
準(zhǔn)備好一個已滅菌好的采樣器蓋子，滅菌后的采樣蓋子僅能使用一次；
環(huán)境監(jiān)控人員用消毒劑消毒雙手，取出TSA平皿，將培養(yǎng)皿正置于頂部的支撐面上，將蓋子打開放置在一邊，打開消毒袋，取出多孔采集蓋，蓋到采樣器上，開始采集空氣樣品；
每個取樣點(diǎn)，用一塊TSA培養(yǎng)基平皿，取樣量1000升；
取樣結(jié)束后，在培養(yǎng)基平皿上標(biāo)示相關(guān)信息，如房間號、檢測項(xiàng)目、級別及班次、取樣日期等。
注意：在采樣過程中，不要將任何物體（包括操作人員的手和頭）置于培養(yǎng)基平皿上方，避免在取樣點(diǎn)附近活動。
2.3表面微生物取樣
表面微生物的取樣有兩種方法，即擦拭法和表面接觸碟法。
擦拭法是一種常用的表面微生物取樣方法，在沒有表面接觸平皿法之前，潔凈區(qū)表面微生物取樣幾乎都采用這種方法，常用于設(shè)備表面取樣，優(yōu)點(diǎn)是能對彎曲表面直接取樣。通過考察有代表性部位的微生物水平評價(jià)設(shè)備污染狀況，另外還常用于設(shè)備的清潔驗(yàn)證中。但擦拭取樣法有其局限性，它的微生物回收率通常受取樣工具、溶解溶劑、取樣人員、檢測方法等因素的影響。
具體操作方法：將棉簽按在取樣表面上，用力使其彎曲，平穩(wěn)而緩慢的擦拭取樣表面，在向前移動的同時將其從一邊移動到另一邊。擦拭過程應(yīng)覆蓋整個表面，翻轉(zhuǎn)棉簽，讓棉簽的另一面也進(jìn)行擦拭，但與前次擦拭移動方向垂直，至少需擦拭25cm2的區(qū)域。
表面接觸碟法是在擦拭法的基礎(chǔ)上發(fā)展起來的，采用直徑為55mm的接觸皿對被取樣表面進(jìn)行接觸取樣。平皿應(yīng)裝滿一種體積可控的瓊脂培養(yǎng)基（根據(jù)目標(biāo)微生物選擇），為表面采樣特制。瓊脂應(yīng)在培養(yǎng)皿上形成凸起彎月面，即檢測人員打開平皿后，用培養(yǎng)基表面輕輕接觸取樣點(diǎn)表面，注意不要旋轉(zhuǎn)，取樣完畢后立即蓋上平皿蓋，完全接觸過程大約5s，取樣結(jié)束后用消毒劑浸濕的無菌潔凈布擦拭取樣點(diǎn)。
3、注意事項(xiàng)
對于單向流潔凈區(qū)或送風(fēng)口，采樣器采樣口朝向應(yīng)正對氣流方向；對非單向流潔凈區(qū)，采樣口向上。
布置采樣點(diǎn)時，至少應(yīng)盡量避開塵粒較集中的回風(fēng)口。
采樣時，環(huán)境監(jiān)控人員應(yīng)站在采樣口的下風(fēng)側(cè)，并盡量少動。
應(yīng)采取一切措施防止采樣過程的污染和其他可能對樣品的污染。
培養(yǎng)皿在用于檢測時，為避免培養(yǎng)皿運(yùn)輸或搬動過程造成的影響，宜同時進(jìn)行陰性對照試驗(yàn)，每次或每個區(qū)域取一個對照皿，與采樣皿同法操作，但不需暴露采樣，然后與采樣后的培養(yǎng)皿（TSA、SDA）一起放入培養(yǎng)箱內(nèi)培養(yǎng)，結(jié)果應(yīng)無菌落生長。
日水生物 qdrishui.cn
Chapter ii methods of microbial detection in clean areas
Pharmaceutical enterprise production workshop, laboratory clean area is closely related to the microbial pollution and pollution sources, such as bacteria, fungi, viral pollution is relatively common, mainly by the clean environment and facility management, personnel is not standard behavior to the leak of cross contamination, operating strains, such as raw materials, equipment and containers with pollution causes. So choose a reliable clean areas microbial detection methods, can effectively monitor the clean area environment, control the risk of microbial contamination, to ensure the reliability of the microbiological monitoring results and accuracy, manifests the clean area environment real level of microbial control.
1. Medium
Culture medium is the basis of microorganism test, which affects the result of microorganism test directly. Suitable preparation method, storage condition and quality control test are the guarantee to provide high quality medium.
In microbial detection, we should attach great importance to the use of culture medium (including the purchase of different batch of finished product with culture medium, different batches of dehydrated medium dry powder, according to the prescription with different batches of raw materials prepared by medium, etc.), preparation program (including water quality control, preparation methods, the sterilization procedures, etc.), storage conditions (temperature and humidity conditions, time and culture medium of containers, etc.), such as whether satisfy with microbial detection requirements.
Whether do clean areas settlement bacteria, planktonic bacteria, microorganisms, or surface of the medium used AGAR before use must be on the use of batch culture medium plate do the growth test, performance test, it is also a medium is culture medium internal quality standards. It USES known standard strains to determine whether the performance of the medium meets the requirements in accordance with the regulations and standards.
One of the things that we can't ignore in this experiment is the need for a controlled culture medium. As for the control medium, the applicability test of the medium was introduced to ensure the accuracy and reliability of the microbial limit test results in the Chinese pharmacopoeia.
However, there is much controversy over the adoption of "previously validated culture media" in the European and American pharmacopoeia, focusing on two points. First, China's pharmaceutical microbiology laboratory is small and scattered, and it is difficult for thousands of enterprises and grassroots drug testing laboratories to effectively verify and evaluate the control media. Second, in the supervision and inspection, difficult to effectively evaluate enterprise microbiology laboratory for verification of the contrast medium, in a unified, after full evaluation and verification of the applicability of the contrast medium to medium test.
Therefore, we should pay attention to:
Under the condition of ensuring the correct preparation and sterilization process, the control medium can be directly used for sample inspection without the applicability test.
The alternative use of different prescription media cannot be simply determined by the applicability test of the media.
It is not recommended to use a controlled medium as a "controlled medium" for laboratory use if it has been tested for suitability.
If there is no special requirement, distilled water or purified water can be used in the preparation of media, but detection and control should be carried out.
The dry powder medium or medium raw material should not be used again after metamorphosis. Discoloration, long bacteria, severe dehydration, etc. should not be used in the storage of finished media.
2. Microbial detection in the clean area
Pharmaceutical companies in the evaluation of the production of clean environment the micro-organisms, need for dynamic or static monitoring of clean environment, using settlement bacteria AGAR, air planktonic bacteria AGAR and surface contact dish of clean areas microbial detection. Of planktonic bacteria and settlement bacteria detection according to GB16293-2010 "pharmaceutical industry clean room (area) test method of planktonic bacteria and GB16294-2010" pharmaceutical industry clean room (area) settlement bacteria test method "provision for testing. Surface microbial detection methods include wiping method and surface contact plate method.
2.1 sampling of sedimentary bacteria
Settlement bacteria detection method using sedimentation method, namely through collecting natural sedimentation principle in the biological particles in the air in the medium plate, after some time, under the condition of suitable for its breeding to visible colony count, the number of colonies in a dish with flat medium to judge the crystallization environment inside living microbial number, and to assess the clean area cleanliness.
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Sampling methods: environmental monitoring personnel with disinfectant hands, take out the TSA AGAR, according to the sample point arrangement placed individually, and then from the inside out thru a dish cover, and put a dish cover down the inside of the base, make medium surface exposed to air. To avoid contaminating the surface of the culture medium, hands and arms should not pass over the open plate.
2.2 sampling of phytoplankton
Planktonic bacteria in the sample is collected through air sampling instrument biological particles suspended in the air in the special medium, after some time and suitable growing conditions for its breeding to visible colonies and count, to determine the microorganism concentration in clean.
Phytoplankton samplers generally adopt impact mechanism, which can be divided into slit samplers, centrifugal samplers or pinhole samplers.
Slit type sampler by internal fan will air suction, through the slit type tablet sampler, will be collected by the air jet and the impact to the slow rotating plate culture medium on the surface, adhesion of live microorganisms particles form colonies after training.
High-speed rotating centrifugal sampler due to internal fan, air flow from the front of sampler inhaled from the rear, under the action of centrifugal force, the live microorganisms in air particles have enough time to hit the special article training on solid form, attached to living microbial particles form colonies after training.
Pinhole type sampler is inhaled air through a metal cover, the lid is populated after machining of special holes, through collecting fan of tiny air flow that have a direct impact on plating medium surface, adhesion of live microorganisms particles form colonies after training.
At present, most pharmaceutical companies use pinhole sampler for phytoplankton sampling, so its sampling method is introduced.
Sampling method (pinhole sampler) :
The environmental monitoring personnel should first determine that the air sampler is within the range of verification.
Before sampling, wipe the outside surface of the air sampler with a sterile cloth soaked with disinfectant and let it dry naturally.
Prepare a sterilized sampler cover and use the sterilized sampler cover only once.
Environmental monitoring personnel with disinfectant hands, take out the TSA AGAR, the dishes are placed on the top of the support surface, open the lid placed on one side, open the sterilization bags, remove the porous collecting cover, cover on the sampler, began to collect air samples;
At each sampling point, a TSA culture medium plate was used to sample 1000 liters.
After the sampling, relevant information, such as room number, test items, class and shift, sampling date, are marked on the petri dish.
Note: during the sampling process, do not place any object (including operator's hand and head) above the plate of culture medium to avoid the movement near the sampling point.
2.3 surface microbial sampling
There are two methods for sampling surface microbes, namely wiping method and surface contact plate method.
Cleaning method is a commonly used surface sampling method, before there is no surface contact method of culture dish, clean areas on the surface of nearly all the sampling in this approach, often used for sampling equipment surface, advantage is able to directly sampling curved surface. It is often used in equipment cleaning verification to evaluate the pollution status of equipment by examining the microbial level of representative parts. However, wiping sampling method has its limitations, and its microbial recovery rate is usually affected by sampling tools, dissolution solvents, sampling personnel, detection methods and other factors.
Specific operation method: press the cotton swab on the sampling surface, bend it vigorously, wipe the sampling surface smoothly and slowly, and move it from one side to the other while moving forward. The wiping process should cover the entire surface, turn over the cotton swab and allow the other side of the swab to be wiped. However, the swab should be vertical to the previous wipe movement direction, and at least wipe the area of 25cm2.
The surface contact disc method was developed on the basis of the wiping method. The contact plate with a diameter of 55mm was used to sample the sampled surface. The plate should be filled with a volume controlled agar-agar medium (selected by the target microbe) for surface sampling. AGAR should be in a petri dish, bump is formed on the meniscus, namely open testing of AGAR, gently with the surface of the medium contact surface of sampling point, be careful not to rotate, after the sampling immediately cover plate cover, completely about 5 s contact process, the end of the sampling with disinfectant soaking sterile clean cloth to wipe sampling points.
3. Notes
For the one-way flow clean area or the air supply port, the sampling port of the sampler should be in the direction of the air flow. For non-one-way flow clean area, the sampling port is up.
When arranging sampling points, at least try to avoid the dust particles concentration of the return air outlet.
During sampling, environmental monitoring personnel should stand on the leeward side of the sampling port and move as little as possible.
All measures should be taken to prevent contamination of the sampling process and other possible contamination of samples.
Petri dish when used to detect, in order to avoid a dish transport or move process, appropriate negative contrast experiment was carried out at the same time, each time or each area take a control plate, and the sampling with the method of plating operations, but not to be exposed to the sampling, and then with the sampling (TSA, SDA) in the culture dish after cultivation in the box, the results should be no growth of colonies.
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