培養(yǎng)基平皿應(yīng)用中常見問題和解答
發(fā)布時(shí)間：
2022-12-24
作者：
培養(yǎng)基平皿應(yīng)用中常見問題和解答
1. 目前我國(guó)尚未有一次性平皿統(tǒng)一的質(zhì)量標(biāo)準(zhǔn)，作為用戶在購(gòu)置時(shí)該如何選擇廠家？
答：首先，根據(jù)國(guó)食藥監(jiān)械[2008]535號(hào)文件摘選中培養(yǎng)基產(chǎn)品分類界定，平板被劃分在醫(yī)療器械管理范疇。
其次、2013年11月26日，國(guó)家食品藥品監(jiān)督總局在《食品藥品監(jiān)管總局關(guān)于印發(fā)體外診斷試劑分類子目錄的通知》中將培養(yǎng)基類產(chǎn)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(guó)家食品藥品監(jiān)督總局在《關(guān)于醫(yī)療器械生產(chǎn)質(zhì)量管理規(guī)范執(zhí)行有關(guān)事宜的通告》第四點(diǎn)中指出“自2018年1月1日起，所有醫(yī)療器械生產(chǎn)企業(yè)應(yīng)當(dāng)符合醫(yī)療器械生產(chǎn)質(zhì)量管理規(guī)范的要求”。
綜合以上三點(diǎn)，說明我國(guó)已從法規(guī)上對(duì)這類生產(chǎn)作出明確規(guī)定，所以在選擇一次性平皿的生產(chǎn)廠家供應(yīng)商時(shí)，首先要選擇合法合規(guī)的生產(chǎn)企業(yè)。
2. 成品預(yù)裝培養(yǎng)基平皿是否需要預(yù)培養(yǎng)？
答：根據(jù)2015版《中國(guó)藥典》第四部9203藥品微生物實(shí)驗(yàn)室質(zhì)量管理指導(dǎo)原則中要求：“用于環(huán)境監(jiān)控的培養(yǎng)基須特別防護(hù)，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(yǎng)基，那么在使用前應(yīng)進(jìn)行100%的預(yù)培養(yǎng)，以防止外來的污染物帶到環(huán)境中及避免出現(xiàn)假陽(yáng)性結(jié)果”。目前市場(chǎng)上的一次性培養(yǎng)基平皿產(chǎn)品都是最終滅菌產(chǎn)品，所以使用前不需要預(yù)培養(yǎng)。
3. 若三層真空包裝的一次性培養(yǎng)基平皿在使用過程中發(fā)現(xiàn)漲袋，能否繼續(xù)使用？
答：建議不要帶進(jìn)潔凈區(qū)使用，特別是A、B級(jí)區(qū)，會(huì)給潔凈區(qū)環(huán)境帶來污染風(fēng)險(xiǎn)。
4. 實(shí)驗(yàn)室自制培養(yǎng)皿滅菌后為何會(huì)出現(xiàn)水汽太多的現(xiàn)象？
答：原因有兩個(gè)：第一、澆注平皿時(shí)培養(yǎng)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(yǎng)基平皿沉降菌采樣后為何會(huì)出現(xiàn)培養(yǎng)基縮板的現(xiàn)象？
答：如果潔凈區(qū)的溫濕度（溫度：18℃～22℃；相對(duì)濕度控制在45%~65%；風(fēng)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(zhì)量問題；
如果潔凈區(qū)的溫濕度有特殊要求，則要根據(jù)驗(yàn)證來確定在沉降菌采樣時(shí)平皿的暴露時(shí)間。
6. 使用前為何會(huì)出現(xiàn)冷凝水很多的現(xiàn)象？
答：培養(yǎng)基平皿中水分占95%左右。正常情況下瓊脂表面是光滑、潤(rùn)濕的，冷凝水太多，其原因主要是培養(yǎng)基平皿所處的環(huán)境溫差太大，特別是由于運(yùn)輸環(huán)境或庫(kù)房保存不當(dāng)造成的，所以必須確保平皿所處的環(huán)境溫差不要太大。
7. 用于水系統(tǒng)監(jiān)控的R2A平皿是否在使用前必須進(jìn)行預(yù)培養(yǎng)？
答：如果不能采用終端滅菌的培養(yǎng)基，那么在使用前應(yīng)進(jìn)行100%的預(yù)培養(yǎng)。
8. 在環(huán)境監(jiān)測(cè)沉降菌采樣時(shí)，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(jí)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(dāng)于多取樣監(jiān)測(cè)一塊平皿，環(huán)境監(jiān)測(cè)更嚴(yán)格一些。
9. 環(huán)境監(jiān)測(cè)沉降菌的培養(yǎng)結(jié)果沒有長(zhǎng)菌是以0還是以小于1表示結(jié)果？
答：建議用0表示結(jié)果，以便于今后的趨勢(shì)分析。
10. 生化培養(yǎng)箱、超凈工作臺(tái)、滅菌鍋等設(shè)備每年都需要做IQ、PQ、OQ嗎？
答：2015版《中國(guó)藥典》第四部9203藥品微生物實(shí)驗(yàn)室質(zhì)量管理指導(dǎo)原則中要求：應(yīng)由有資質(zhì)的人員進(jìn)行生物安全柜、層流超凈工作臺(tái)及高效過濾器的安裝與更換，要按照確認(rèn)的方法進(jìn)行現(xiàn)場(chǎng)生物和物理的檢測(cè)，并定期進(jìn)行再驗(yàn)證。
實(shí)驗(yàn)室生物安全柜和層流超凈工作臺(tái)的通風(fēng)應(yīng)符合微生物風(fēng)險(xiǎn)級(jí)別及符合安全要求。應(yīng)定期對(duì)生物安全柜、層流超凈工作臺(tái)進(jìn)行監(jiān)測(cè)，以確保其性能符合相關(guān)要求。實(shí)驗(yàn)室應(yīng)保存檢查記錄和性能測(cè)試結(jié)果。
《GMP指南-實(shí)驗(yàn)室》中對(duì)培養(yǎng)箱要求是：新設(shè)備應(yīng)該在進(jìn)行IQ、PQ、OQ合格之后方可使用。使用時(shí)應(yīng)進(jìn)行溫度監(jiān)測(cè)，建議在OQ時(shí)進(jìn)行多點(diǎn)溫度分布的確認(rèn)，并應(yīng)定期進(jìn)行校正和設(shè)備的表面清潔。
確認(rèn)周期均為硬性要求，一般一年一次，如果延長(zhǎng)周期需要進(jìn)行相應(yīng)的風(fēng)險(xiǎn)評(píng)估。
11. 在做培養(yǎng)平皿促生長(zhǎng)實(shí)驗(yàn)時(shí)，經(jīng)常會(huì)發(fā)現(xiàn)個(gè)別實(shí)驗(yàn)菌回收率小于50%，是什么原因？
答：2015版《中國(guó)藥典》1105中規(guī)定：按表1規(guī)定，接種不大于100cfu的菌液至胰酪大豆胨液體培養(yǎng)基管或胰酪大豆胨瓊脂培養(yǎng)基平板或沙氏葡萄糖瓊脂培養(yǎng)基平板，
置表1規(guī)定條件下培養(yǎng)。每一試驗(yàn)菌株平行制備2管或2個(gè)平皿。同時(shí)，用相應(yīng)的對(duì)照培養(yǎng)基替代被檢培養(yǎng)基進(jìn)行上述試驗(yàn)。
被檢固體培養(yǎng)基上的菌落平均數(shù)與對(duì)照培養(yǎng)基上的菌落平均數(shù)的比值應(yīng)在0.5～2范圍內(nèi)，且菌落形態(tài)大小應(yīng)與對(duì)照培養(yǎng)基上的菌落一致；
如果回收率小于50%，主要是實(shí)驗(yàn)誤差導(dǎo)致，為了確保每次接種量的濃度相當(dāng)，最好采用同一支試管的菌液或定量菌株。
12. 微生物方法驗(yàn)證必須做3個(gè)批次嗎？如果只有一個(gè)批次產(chǎn)品，下一個(gè)批次產(chǎn)品要等2個(gè)月以后，那現(xiàn)在這個(gè)批號(hào)的產(chǎn)品報(bào)告書是否等3個(gè)批次的驗(yàn)證完之后才能出呢？
答：目前藥典沒有規(guī)定必須做3個(gè)批次產(chǎn)品，所以用一個(gè)批次產(chǎn)品做三次也是允許的，而且現(xiàn)在的方法學(xué)適用性試驗(yàn)也在考慮這個(gè)問題，目的是為了與驗(yàn)證的概念有更好的區(qū)分。
13. 成品預(yù)裝培養(yǎng)基平皿儲(chǔ)存在2~8℃冰箱內(nèi)，對(duì)平皿質(zhì)量有影響嗎？
答：首先根據(jù)所購(gòu)置的培養(yǎng)基平皿產(chǎn)品的儲(chǔ)存環(huán)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(yùn)行過程中不會(huì)超出接觸碟儲(chǔ)存溫度范圍。如果溫度低于0℃，會(huì)使其中的瓊脂凝膠強(qiáng)度受到影響而導(dǎo)致平板質(zhì)量不符合要求。
14. 對(duì)于GMP附錄一《無(wú)菌藥品》中規(guī)定的潔凈區(qū)級(jí)別中，D 級(jí)潔凈區(qū)的浮游菌和沉降菌是否都需要進(jìn)行監(jiān)測(cè)？
答：企業(yè)可根據(jù)各工序污染的風(fēng)險(xiǎn)高低，評(píng)估決定D級(jí)潔凈區(qū)的動(dòng)態(tài)微生物監(jiān)測(cè)頻次。一般情況下，由于采樣的對(duì)象不同，浮游菌和沉降菌監(jiān)測(cè)都是需要的。
15. GMP附錄一《無(wú)菌藥品》中“注（2）單個(gè)沉降碟的暴露時(shí)間可以少于 4 小時(shí)，同一位置可使用多個(gè)沉降碟連續(xù)進(jìn)行監(jiān)測(cè)并累積計(jì)數(shù)。”這里描述的是如何累積的？
答：用這個(gè)點(diǎn)的所有結(jié)果相加，并除以實(shí)際小時(shí)數(shù)，再乘上4小時(shí)。監(jiān)測(cè)時(shí)間應(yīng)涵蓋生產(chǎn)時(shí)間，但如果實(shí)際生產(chǎn)時(shí)間短于4小時(shí)，監(jiān)測(cè)時(shí)間就沒有必要達(dá)到4小時(shí)。例如，如果某工序操作總時(shí)間不足4 小時(shí)，如2小時(shí)，累計(jì)共檢出5cfu（菌落數(shù)），換算方法是：5/2*4=10cfu/4h。
16. 附錄一 中 D 級(jí)微生物檢測(cè)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?yàn)槌两稻?、浮游菌和表面微生物三種監(jiān)測(cè)原理是不同的，針對(duì)不同的微生物，不能完全相互替代。不同企業(yè)應(yīng)根據(jù)廠房、產(chǎn)品特性、工藝流程及設(shè)備的特點(diǎn)（例如暴露區(qū)域及時(shí)間）評(píng)估結(jié)果制定監(jiān)測(cè)方案與頻次。
17. 間歇式非最終滅菌制劑生產(chǎn)前，對(duì)環(huán)境微生物情況進(jìn)行監(jiān)測(cè)，是浮游菌、沉降菌二選一還是都必須做？
答：檢測(cè)微生物時(shí)，浮游菌、沉降菌都應(yīng)該做。應(yīng)注意的是，所有無(wú)菌藥品生產(chǎn)的潔凈區(qū)空氣凈化系統(tǒng)應(yīng)當(dāng)保持連續(xù)運(yùn)行，維持相應(yīng)的潔凈度級(jí)別。因故停機(jī)再次開啟空氣凈化系統(tǒng)，應(yīng)當(dāng)進(jìn)行必要的測(cè)試以確認(rèn)仍能達(dá)到規(guī)定的潔凈度級(jí)別要求。
18. 潔凈區(qū)的人員密度限度為多少？
答：《采暖通風(fēng)與空氣調(diào)節(jié)設(shè)計(jì)規(guī)范》GB50019—2003中第3.1.9 條第2款：工業(yè)建筑應(yīng)保證每人不小于30m3/h的新風(fēng)量；《醫(yī)藥工業(yè)潔凈廠房設(shè)計(jì)規(guī)范》GB50457—2008中第9.1.3 條第2款：潔凈室內(nèi)每人新鮮空氣量不應(yīng)小于40m3/h。主要考慮每個(gè)人的平均新風(fēng)量，所以4～6平方米/人。
19. 無(wú)菌原料藥培養(yǎng)基模擬試驗(yàn)是否必須做？
答：無(wú)菌原料藥參照無(wú)菌制劑管理，應(yīng)該進(jìn)行培養(yǎng)基模擬灌裝驗(yàn)證，并應(yīng)注意以下問題：
1.對(duì)于開放系統(tǒng)，企業(yè)最好選擇分段式模擬方案，以利于尋找污染來源；
2.應(yīng)密切關(guān)注模擬過程中使用的抑菌溶劑和抑菌工藝條件；
3.應(yīng)根據(jù)最終成品的最大分裝劑量制定驗(yàn)證可接受標(biāo)準(zhǔn)。
20. 模擬實(shí)驗(yàn)中用到的培養(yǎng)基，要做哪些適用性檢查？
答：應(yīng)考慮以下實(shí)驗(yàn)或以下實(shí)驗(yàn)的一部分：無(wú)菌性試驗(yàn)，促生長(zhǎng)試驗(yàn)，滅菌適應(yīng)性、流動(dòng)性實(shí)驗(yàn)、溶解性實(shí)驗(yàn)和觀察適用性等。
21. 培養(yǎng)基平皿傳入無(wú)菌區(qū)，一般采用什么方式？
答：大多選擇逐層脫外包的方法，在生產(chǎn)開始前進(jìn)行。
方法：傳到潔凈區(qū)的設(shè)備，一是需要嚴(yán)格的保護(hù)設(shè)施，如培養(yǎng)皿用專用密閉的不繡鋼桶或呼吸袋盛裝培養(yǎng)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(jīng)驗(yàn)證，是可以從普通區(qū)傳入潔凈區(qū)。
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.