<th id="hta7t"><sup id="hta7t"></sup></th>

<th id="hta7t"><sup id="hta7t"></sup></th>

    <th id="hta7t"><video id="hta7t"></video></th>
  1. <th id="hta7t"><option id="hta7t"></option></th><object id="hta7t"></object>
    1. <code id="hta7t"></code>
    2. <output id="hta7t"></output>

      <code id="hta7t"><em id="hta7t"></em></code>

      成品預裝培養基平皿實用技術手冊(連載二):潔凈區微生物檢測方法

      發布時間:

      2022-12-27

      作者:

      培養基訂購網站


      制藥企業生產車間、實驗室潔凈區的微生物污染與污染源密切相關,細菌、真菌、病毒性污染等較為常見,主要由潔凈環境和設施管理不當、人員不規范的行為與交叉污染、操作菌株的泄露、原輔料混入、設備與容器污染等原因造成。因而選擇一種可靠的潔凈區微生物檢測方法,可以有效監控潔凈區的環境,控制微生物污染的風險,確保微生物監測結果的可靠性和準確性,體現潔凈區環境微生物控制的真實水平。
      培養基是微生物試驗的基礎,直接影響微生物試驗結果。適宜的培養基制備方法、貯藏條件和質量控制試驗是提供優質培養基的保證。
       
      在微生物檢測時,我們應該高度重視所使用的培養基(包括購置不同批號的成品預裝培養基、不同批次的脫水培養基干粉、按照處方使用不同批次的原材料自行配制培養基等)、制備程序(包括水質控制、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。
       
      無論是做潔凈區沉降菌、浮游菌,還是表面微生物,所使用的培養基平皿在使用之前都必須對使用批次的培養基平皿做促生長試驗,這也是培養基的性能試驗,更是培養基的內在質量標準。它是用已知的標準菌株按照規程和標準測定培養基性能是否符合要求。
       
      在這個實驗中,我們不能忽略的一個環節,就是需要使用對照培養基。關于對照培養基,在《中國藥典》中借鑒歐美藥典,引入培養基適用性檢查以保障微生物限度檢查結果的準確性和可靠性。
       
      但是,對于歐美藥典中的“之前驗證過的培養基”的采納與否,存在很大爭議,集中在兩點。第一,我國藥品微生物實驗室小而分散,數以千計的企業和基層藥檢所難以有效地進行對照培養基的驗證、評價工作;第二、在監督檢查中,難以有效評估企業微生物實驗室對對照培養基的驗證情況,以統一的、經過充分評價和驗證過的對照培養基進行培養基的適用性試驗。
       
      所以我們在使用對照培養基過程中,應該注意:
       
      在保證配制和滅菌過程無誤的條件下,對照培養基可以不經適用性實驗檢查直接用于樣品檢查。
      不同處方培養基的替代使用不能通過培養基適用性試驗簡單確定,要按標準規定使用培養基。
      不建議使用經過對照培養基進行適用性試驗檢查合格的其它培養基作為實驗室自用的“對照培養基”。
      如果沒有特殊規定,培養基配制采用蒸餾水或純化水均可,但應進行檢測控制。
      干粉培養基或培養基原料變質不應再用;成品培養基儲存過程發現變色、長菌、嚴重脫水等也不應再用。
      2、潔凈區微生物檢測
      制藥企業在評估生產潔凈環境的微生物狀況時,需對潔凈環境進行動態或靜態監測,使用沉降菌平皿、空氣浮游菌平皿和表面接觸皿對潔凈區微生物檢測。其中浮游菌和沉降菌的檢測根據GB16293-2010《醫藥工業潔凈室(區)浮游菌的測試方法》和GB16294-2010《醫藥工業潔凈室(區)沉降菌的測試方法》規定進行檢測。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基平皿,經過若干時間,在適宜的條件下讓其繁殖到可見的菌落進行計數,以平板培養基皿中的菌落數來判斷結晶環境內活微生物數,并以此來評定潔凈區的潔凈度。 
      成品預裝培養基平皿實用技術手冊(連載二):潔凈區微生物檢測方法
      取樣方法:環境監控人員用消毒劑消毒雙手,取出TSA平皿,按照采樣點布置圖逐個放置,然后從里到外逐個打開培養皿蓋,并將蓋內側向下放在培養皿底座旁邊,使培養基表面暴露在空氣中。為了避免污染培養基表面,手和手臂不要經過已開蓋的培養皿上方。
      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基,經若干時間和適宜的生長條件讓其繁殖到可見的菌落并計數,以判定該潔凈區的微生物濃度。
       
      浮游菌采樣器一般采用撞擊法機理,可分為狹縫式采樣器、離心式或針孔式采樣器。
       
      狹縫式采樣器由內部風機將氣流吸入,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的平板培養基表面上,附著的活微生物粒子經培養后形成菌落。
       
      離心式采樣器由于內部風機高速旋轉,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條上,附著的活微生物粒子經培養后形成菌落。
       
      針孔式采樣器是氣流通過一個金屬蓋吸入,蓋子上是密集的經過機械加工的特制小孔,通過風機收集到細小的空氣流直接撞擊到平板培養基表面上,附著的活微生物粒子經培養后形成菌落。
       
      目前大部分制藥企業采用針孔式采樣器進行浮游菌采樣,故介紹一下其取樣方法。
       
      取樣方法(針孔式采樣器):
      環境監控人員應首先確定空氣采樣器在校驗合格的范圍內;
      采樣前,用經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;
      環境監控人員用消毒劑消毒雙手,取出TSA平皿,將培養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,用一塊TSA培養基平皿,取樣量1000升;
      取樣結束后,在培養基平皿上標示相關信息,如房間號、檢測項目、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)置于培養基平皿上方,避免在取樣點附近活動。
      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,潔凈區表面微生物取樣幾乎都采用這種方法,常用于設備表面取樣,優點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設備污染狀況,另外還常用于設備的清潔驗證中。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。擦拭過程應覆蓋整個表面,翻轉棉簽,讓棉簽的另一面也進行擦拭,但與前次擦拭移動方向垂直,至少需擦拭25cm2的區域。 
      表面接觸碟法是在擦拭法的基礎上發展起來的,采用直徑為55mm的接觸皿對被取樣表面進行接觸取樣。平皿應裝滿一種體積可控的瓊脂培養基(根據目標微生物選擇),為表面采樣特制。瓊脂應在培養皿上形成凸起彎月面,即檢測人員打開平皿后,用培養基表面輕輕接觸取樣點表面,注意不要旋轉,取樣完畢后立即蓋上平皿蓋,完全接觸過程大約5s,取樣結束后用消毒劑浸濕的無菌潔凈布擦拭取樣點。
      3、注意事項
      對于單向流潔凈區或送風口,采樣器采樣口朝向應正對氣流方向;對非單向流潔凈區,采樣口向上。
      布置采樣點時,至少應盡量避開塵粒較集中的回風口。
      采樣時,環境監控人員應站在采樣口的下風側,并盡量少動。
      應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。
      培養皿在用于檢測時,為避免培養皿運輸或搬動過程造成的影響,宜同時進行陰性對照試驗,每次或每個區域取一個對照皿,與采樣皿同法操作,但不需暴露采樣,然后與采樣后的培養皿(TSA、SDA)一起放入培養箱內培養,結果應無菌落生長。
      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.
      -- -- 7 -- --
      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.
      暫無數據

      暫無數據

      一本色道久久综合亚洲精品,综合久久给合久久狠狠狠97色,一本色道久久综合一区,亚洲精品人成无码中文毛片
      <th id="hta7t"><sup id="hta7t"></sup></th>

      <th id="hta7t"><sup id="hta7t"></sup></th>

        <th id="hta7t"><video id="hta7t"></video></th>
      1. <th id="hta7t"><option id="hta7t"></option></th><object id="hta7t"></object>
        1. <code id="hta7t"></code>
        2. <output id="hta7t"></output>

          <code id="hta7t"><em id="hta7t"></em></code>