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      培養基平皿應用中常見問題和解答

      發布時間:

      2022-12-24

      作者:


      培養基平皿應用中常見問題和解答
       
      1. 目前我國尚未有一次性平皿統一的質量標準,作為用戶在購置時該如何選擇廠家?
      答:首先,根據國食藥監械[2008]535號文件摘選中培養基產品分類界定,平板被劃分在醫療器械管理范疇。
      其次、2013年11月26日,國家食品藥品監督總局在《食品藥品監管總局關于印發體外診斷試劑分類子目錄的通知》中將培養基類產品劃分在《6840體外診斷試劑分類子目錄(2013版)》。
      第三、2014年9月5日,國家食品藥品監督總局在《關于醫療器械生產質量管理規范執行有關事宜的通告》第四點中指出“自2018年1月1日起,所有醫療器械生產企業應當符合醫療器械生產質量管理規范的要求”。
      綜合以上三點,說明我國已從法規上對這類生產作出明確規定,所以在選一次性平皿的生產廠家供應商時,首先要選擇合法合規的生產企業。
       
      2. 成品預裝培養基平皿是否需要預培養?
      答:根據2015版《中國藥典》第四部9203藥品微生物實驗室質量管理指導原則中要求:“用于環境監控的培養基須特別防護,最好要雙層包裝和終端滅菌,如果不能采用終端滅菌的培養基,那么在使用前應進行100%的預培養,以防止外來的污染物帶到環境中及避免出現假陽性結果”。目前市場上的一次性培養基平皿產品都是最終滅菌產品,所以使用前不需要預培養。
       
      3. 若三層真空包裝的一次性培養基平皿在使用過程中發現漲袋,能否繼續使用?
      答:建議不要帶進潔凈區使用,特別是A、B級區,會給潔凈區環境帶來污染風險。
       
      4. 實驗室自制培養皿滅菌后為何會出現水汽太多的現象?
      答:原因有兩個:第一、澆注平皿時培養基溫度太高,而澆注后沒有冷卻至室溫就蓋上蓋子;第二、滅菌柜的干燥性能不佳造成。
       
      5.一次性培養基平皿沉降菌采樣后為何會出現培養基縮板的現象?
      答:如果潔凈區的溫濕度(溫度:18℃~22℃;相對濕度控制在45%~65%;風速小于0.54m/s)沒有特殊要求,沉降菌采樣后縮板,可能是一次性平皿的質量問題;
      如果潔凈區的溫濕度有特殊要求,則要根據驗證來確定在沉降菌采樣時平皿的暴露時間。
       
      6. 使用前為何會出現冷凝水很多的現象?
      答:培養基平皿中水分占95%左右。正常情況下瓊脂表面是光滑、潤濕的,冷凝水太多,其原因主要是培養基平皿所處的環境溫差太大,特別是由于運輸環境或庫房保存不當造成的,所以必須確保平皿所處的環境溫差不要太大。
       
      7. 用于水系統監控的R2A平皿是否在使用前必須進行預培養?
      答:如果不能采用終端滅菌的培養基,那么在使用前應進行100%的預培養。
       
      8. 在環境監測沉降菌采樣時,為了使平皿完全暴露,平皿蓋子如何放置比較合適?如果在百級層流下取樣,蓋子的面向上或向下有差異嗎?
      答:一般情況向下放置皿蓋,但也允許向上放置,相當于多取樣監測一塊平皿,環境監測更嚴格一些。
       
      9. 環境監測沉降菌的培養結果沒有長菌是以0還是以小于1表示結果?
      答:建議用0表示結果,以便于今后的趨勢分析。
       
      10. 生化培養箱、超凈工作臺、滅菌鍋等設備每年都需要做IQ、PQ、OQ嗎?
      答:2015版《中國藥典》第四部9203藥品微生物實驗室質量管理指導原則中要求:應由有資質的人員進行生物安全柜、層流超凈工作臺及高效過濾器的安裝與更換,要按照確認的方法進行現場生物和物理的檢測,并定期進行再驗證。
      實驗室生物安全柜和層流超凈工作臺的通風應符合微生物風險級別及符合安全要求。應定期對生物安全柜、層流超凈工作臺進行監測,以確保其性能符合相關要求。實驗室應保存檢查記錄和性能測試結果。
      《GMP指南-實驗室》中對培養箱要求是:新設備應該在進行IQ、PQ、OQ合格之后方可使用。使用時應進行溫度監測,建議在OQ時進行多點溫度分布的確認,并應定期進行校正和設備的表面清潔。
      確認周期均為硬性要求,一般一年一次,如果延長周期需要進行相應的風險評估。
       
      11. 在做培養平皿促生長實驗時,經常會發現個別實驗菌回收率小于50%,是什么原因?
      答:2015版《中國藥典》1105中規定:按表1規定,接種不大于100cfu的菌液至胰酪大豆胨液體培養基管或胰酪大豆胨瓊脂培養基平板或沙氏葡萄糖瓊脂培養基平板,
      置表1規定條件下培養。每一試驗菌株平行制備2管或2個平皿。同時,用相應的對照培養基替代被檢培養基進行上述試驗。
       
      被檢固體培養基上的菌落平均數與對照培養基上的菌落平均數的比值應在0.5~2范圍內,且菌落形態大小應與對照培養基上的菌落一致;
      如果回收率小于50%,主要是實驗誤差導致,為了確保每次接種量的濃度相當,最好采用同一支試管的菌液或定量菌株。
       
      12. 微生物方法驗證必須做3個批次嗎?如果只有一個批次產品,下一個批次產品要等2個月以后,那現在這個批號的產品報告書是否等3個批次的驗證完之后才能出呢?
      答:目前藥典沒有規定必須做3個批次產品,所以用一個批次產品做三次也是允許的,而且現在的方法學適用性試驗也在考慮這個問題,目的是為了與驗證的概念有更好的區分。
       
      13. 成品預裝培養基平皿儲存在2~8℃冰箱內,對平皿質量有影響嗎?
      答:首先根據所購置的培養基平皿產品的儲存環境要求選擇合適的溫度保存,其次要保證您所用的冰箱溫度分布均勻,在運行過程中不會超出接觸碟儲存溫度范圍。如果溫度低于0℃,會使其中的瓊脂凝膠強度受到影響而導致平板質量不符合要求。
       
      14. 對于GMP附錄一《無菌藥品》中規定的潔凈區級別中,D 級潔凈區的浮游菌和沉降菌是否都需要進行監測?
      答:企業可根據各工序污染的風險高低,評估決定D級潔凈區的動態微生物監測頻次。一般情況下,由于采樣的對象不同,浮游菌和沉降菌監測都是需要的。
       
      15. GMP附錄一《無菌藥品》中“注(2)單個沉降碟的暴露時間可以少于 4 小時,同一位置可使用多個沉降碟連續進行監測并累積計數。”這里描述的是如何累積的?
      答:用這個點的所有結果相加,并除以實際小時數,再乘上4小時。監測時間應涵蓋生產時間,但如果實際生產時間短于4小時,監測時間就沒有必要達到4小時。例如,如果某工序操作總時間不足4 小時,如2小時,累計共檢出5cfu(菌落數),換算方法是:5/2*4=10cfu/4h。
       
      16. 附錄一 中 D 級微生物檢測方法,是三種方法任選一種呢?還是都需要做呢?
      答:三種方法都需要做。因為沉降菌、浮游菌和表面微生物三種監測原理是不同的,針對不同的微生物,不能完全相互替代。不同企業應根據廠房、產品特性、工藝流程及設備的特點(例如暴露區域及時間)評估結果制定監測方案與頻次。
       
      17. 間歇式非最終滅菌制劑生產前,對環境微生物情況進行監測,是浮游菌、沉降菌二選一還是都必須做?
      答:檢測微生物時,浮游菌、沉降菌都應該做。應注意的是,所有無菌藥品生產的潔凈區空氣凈化系統應當保持連續運行,維持相應的潔凈度級別。因故停機再次開啟空氣凈化系統,應當進行必要的測試以確認仍能達到規定的潔凈度級別要求。
       
      18. 潔凈區的人員密度限度為多少?
      答:《采暖通風與空氣調節設計規范》GB50019—2003中第3.1.9 條第2款:工業建筑應保證每人不小于30m3/h的新風量;《醫藥工業潔凈廠房設計規范》GB50457—2008中第9.1.3 條第2款:潔凈室內每人新鮮空氣量不應小于40m3/h。主要考慮每個人的平均新風量,所以4~6平方米/人。
       
      19. 無菌原料藥培養基模擬試驗是否必須做?
      答:無菌原料藥參照無菌制劑管理,應該進行培養基模擬灌裝驗證,并應注意以下問題:
      1.對于開放系統,企業最好選擇分段式模擬方案,以利于尋找污染來源;
      2.應密切關注模擬過程中使用的抑菌溶劑和抑菌工藝條件;
      3.應根據最終成品的最大分裝劑量制定驗證可接受標準。
       
      20. 模擬實驗中用到的培養基,要做哪些適用性檢查?
      答:應考慮以下實驗或以下實驗的一部分:無菌性試驗,促生長試驗,滅菌適應性、流動性實驗、溶解性實驗和觀察適用性等。
       
      21. 培養基平皿傳入無菌區,一般采用什么方式?
      答:大多選擇逐層脫外包的方法,在生產開始前進行。
      方法:傳到潔凈區的設備,一是需要嚴格的保護設施,如培養皿用專用密閉的不繡鋼桶或呼吸袋盛裝培養皿;二是要有一套合理的滅菌處理方式,如擦拭(殺孢子劑)、照射(臭氧或紫外線)、汽化雙氧水等;三是效果經驗證,是可以從普通區傳入潔凈區。

       
      Common problems and solutions in the application of culture medium plate
      1. at present, there is no uniform quality standard for one-step culture dishes in our country. How to choose a manufacturer as a user when purchasing medium plates?
      A: first, according to the [2008]535 classification of the national food and drug monitoring equipment, the medium products are classified and classified into the category of medical device management.
      Secondly, in November 26, 2013, the State General Administration of food and Drug Administration (State Food and Drug Administration) will train basic class products in "6840 extracorporeal diagnostic reagent classification subcatalogue (2013 Edition)" in "the notice of the food and Drug Administration" on the publication of the classification subcatalogue of the extracorporeal diagnostic reagents.
      In third and September 5, 2014, the State Administration of food and Drug Administration (State Food and Drug Administration) pointed out that "since January 1, 2018, all medical equipment production enterprises should meet the requirements of the quality management regulations of medical instruments" in the fourth point of the notice on the implementation of the standards for the management of the quality of medical instruments.
      Combining the above three points, it shows that China has made a clear regulation on the training of the base class production from the laws and regulations. So, when selecting the supplier of the base plate, we must first choose a legitimate production enterprise.
      2. is it necessary to pre culture the finished product medium?
      Answer: according to the 2015 edition of the Chinese Pharmacopoeia (China Pharmacopoeia), the Fourth Department of China Pharmacopoeia (China Pharmacopoeia), the quality management guiding principle of the fourth 9203 medicine microbiology laboratory: "the medium for environmental monitoring must be specially protected, the best to be double layer packing and terminal sterilization, if the terminal sterilization medium can not be used, then 100% pre culture should be carried out before use. The presence of foreign pollutants is brought to the environment and avoid false positive results. At present, disposable culture dish products in the market are final sterilization products, so no pre culture is required before use.
      3. if the three layer vacuum packed one-off medium plate is found in the use of the bag, can it continue to be used?
      Answer: suggestions should not be brought into the clean areas, especially the A and B zones, which will bring pollution risks to the clean area environment.
      4. why is there too much water vapor after sterilization of laboratory dishes?
      Answer: there are two reasons: first, the temperature of the medium is too high when the plate is poured, and the cover is covered without cooling to the room temperature. Second, the dry performance of the sterilizer is not good.
      5. why does the culture medium plate shrink after the sampling of a one-step culture dish?
      Answer: if the temperature and humidity of the clean area (temperature: 18 - 22 - C, relative humidity control in 45%~65%; wind speed less than 0.54m/s), there is no special requirement. The shrink plate after sampling bacteria may be a problem of the quality of disposable plate.
      If there is a special requirement for temperature and humidity in the clean area, the exposure time of the dish should be determined according to the verification.
      6. why is there a lot of condensed water before using a disposable medium?
      Answer: the water in the culture base plate accounts for about 95% of the water. Under normal conditions, the surface of agar is smooth and wetted, and the condensate is too much. The main reason is that the environment temperature difference is too large in the medium plate, especially because of the improper transportation environment or storage, so it is necessary to ensure that the temperature difference in the environment is not too large.
      7. do R2A dishes for water system monitoring have to be pre cultured before use?
      Answer: if sterile medium can not be used, 100% pre culture should be carried out before use.
      8. in the environmental monitoring of settling bacteria sampling, in order to make the culture plate completely exposed, how can the lid of the dish be placed properly? Are there any differences between the top and the bottom of the lid if sampling is carried out under the 100 level laminar flow?
      A: in general, the lid is placed down, but it is also allowed to be placed upwards. It is equivalent to monitoring a flat dish with multiple samples, and the environmental monitoring is more stringent.
      9. does environmental monitoring result in the culture of settlement bacteria, with no bacteria growing at 0 or less than 1?
      A: it is suggested that the results should be expressed in 0, so as to facilitate future trend analysis.
      10. do biochemical equipment incubators, ultra clean worktables, sterilization pots and other equipment need IQ, PQ and OQ every year?
      Answer: the 2015 edition of the 2015 edition of China Pharmacopoeia (China Pharmacopoeia) requires the quality management guidelines of the Fourth Department of Pharmaceutical Microbiology of China Pharmacopoeia: the installation and replacement of biological safety cabinets, laminar super net worktables and high efficiency filters should be carried out by qualified personnel, and on-site biological and physical tests should be carried out in accordance with the confirmed methods, and revalidation should be carried out regularly. .
      The ventilation of laboratory biosafety cabinets and laminar clean workbench should meet the risk level of microorganism and meet the safety requirements. The biosafety cabinet and laminar clean workbench should be monitored regularly to ensure that their performance meets the relevant requirements. Laboratory shall keep inspection records and performance test results.
      The requirements for incubators in the GMP guide laboratory are: new equipment should be used only after IQ, PQ and OQ are qualified. Temperature monitoring should be carried out when using. It is recommended to confirm the temperature distribution at OQ, and to calibrate regularly and clean the equipment surface.
      The acknowledgement period is mandatory, generally once a year. If the extension period is necessary, the corresponding risk assessment is necessary.
      11. in the process of promoting growth test, it is often found that the recovery rate of individual experimental bacteria is less than 50%. What is the reason?
      Answer: the 2015 edition of China Pharmacopoeia 1105 stipulates that, according to table 1, the inoculation of bacterial liquid not more than 100cfu to soy sauce peptone liquid culture medium tube or cheese soybean Peptone Agar Medium plate or Shashi glucose agar medium plate, set table 1 under the conditions of culture. 2 tubes or 2 Petri dishes were prepared in parallel to each test strain. At the same time, the corresponding control medium was used instead of the tested medium.
      The ratio of the average number of colonies on the tested solid medium and the average number of the colony on the control medium should be within the range of 0.5 ~ 2, and the morphology of the colony should be the same as the colonies on the control medium.
      If the recovery is less than 50%, it is mainly caused by experimental error. In order to ensure that the concentration of each inoculation is equal, it is best to use the same bacteria liquid or quantitative strain.
      12. validation of microbiological methods must be done in 3 batches? If there is only one batch product, the next batch will wait 2 months later, then does this batch of product reports wait for the verification of the 3 batches?
      Answer: at present, the Pharmacopoeia does not have to do 3 batch products, so it is also allowed to do three times with a batch, and the present methodological applicability test is also considering the problem in order to make a better distinction from the concept of verification.
      13. is the finished product medium stored in the 2~8 C refrigerator, which has an effect on the quality of the dish?
      Answer: first, choose the appropriate temperature preservation according to the storage environment requirements of the culture base plate products purchased, and then ensure that the temperature distribution of the refrigerator you use is uniform, and it will not exceed the storage temperature range of the medium plate during the operation. If the temperature is below 0 C, the agar gel strength in the medium will be affected, and the quality of the medium will not meet the requirements.
      14. is there any need to monitor the floating bacteria and settling bacteria in class D clean area for the clean zone level specified in Appendix 1 of GMP?
      Answer: according to the risk level of each process pollution, enterprises can evaluate the dynamic microbial monitoring frequency of D level clean area. In general, monitoring of planktonic bacteria and settling bacteria is needed because of different sampling objects.
      15. GMP Appendix 1, "aseptic drugs", "note (2) individual settling disc exposure time can be less than 4 hours, the same location can be used multiple dissahers continuous monitoring and cumulative count." How do we describe the accumulation here?
      Answer: add all the results of this point, divide the actual hours into 4 hours. The monitoring time should cover the production time, but if the actual production time is shorter than 4 hours, the monitoring time does not need to reach 4 hours. For example, if the total operation time of a process is less than 4 hours, such as 2 hours, the total number of 5cfu (colony number) is detected. The conversion method is: 5/2*4=10cfu/4h.
      16. in Appendix I, the D level microbiological detection method is one of the three methods. Or do you need to do it all?
      Answer: all three methods need to be done. Because the three monitoring principles of settling bacteria, planktonic bacteria and surface microbes are different. They can not be completely replaced by different microorganisms. Different enterprises should set up monitoring plan and frequency according to the evaluation results of plant, product characteristics, process and equipment, such as exposure area and time.
      17. before the production of batch non terminal sterilization preparations, the monitoring of environmental microbes is carried out in two or one.
      Answer: when detecting microorganisms, planktonic bacteria and settling bacteria should be done. It should be noted that the clean air cleaning system for all sterile drugs should be kept running continuously and the corresponding cleanliness level should be maintained. The air purification system should be re opened for any reason. The necessary tests should be carried out to confirm that the required cleanliness level can be achieved.
      What is the limit of personnel density in the 18. clean area?
      Answer: "heating and ventilation and air conditioning design code" GB50019 - 2003 in section 3.1.9 second: industrial buildings should ensure that each person not less than 30m3/h of the new air volume; "pharmaceutical industry clean factory design code" GB50457 - 2008, section 9.1.3 second: clean room per person fresh air volume should not be less than 40m3/h. Mainly considering the average fresh air volume per person, so 4~6 square meters / person.
      19. is aseptic drug medium simulation test necessary?
      Answer: aseptic raw material should be managed by aseptic preparation according to the management of aseptic preparations.
      1., for open systems, enterprises have to choose segmented simulation schemes to help find sources of pollution.
      2., we should pay close attention to the bacteriostatic solvents and bacteriostasis conditions used in the simulation.
      3. the acceptance criteria should be established according to the maximum sub dose of the final product.
      20. what are the suitability tests for culture media used in the media simulation experiments?
      Answer: a part of the following experiments or the following experiments should be taken into consideration: aseptic test, growth promotion test, sterilization adaptability, fluidity experiment, solubility experiment and observation applicability.
      21. what is the general way of introducing sterile plates into the culture medium?
      Answer: most of them choose outsourcing by layer by layer, before the start of production.
      Method: the equipment that passes to the clean area, one is to need strict protection facilities, such as the Petri dish with special closed non embroidered steel barrel or breathing bag to fill the culture dish; two is to have a set of reasonable sterilization treatment, such as wiping (the sporicidal agent), irradiation (ozone or ultraviolet), vaporizing hydrogen peroxide and so on;Three, the effect is verified, which can be introduced into the clean area from the common area.
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