培养基平皿应用中常见问题和解答
发布时间：
2022-11-16
作者：
传统批培养培养基供应商
培养基平皿应用中常见问题和解答
1. 目前我国尚未有一次性平皿统一的质量标准，作为用户在购置时该如何选择厂家？
答：首先，根据国食药监械[2008]535号文件摘选中培养基产品分类界定，平板被划分在医疗器械管理范畴。
其次、2013年11月26日，国家食品药品监督总局在《食品药品监管总局关于印发体外诊断试剂分类子目录的通知》中将培养基类产品划分在《6840体外诊断试剂分类子目录（2013版）》。
第三、2014年9月5日，国家食品药品监督总局在《关于医疗器械生产质量管理规范执行有关事宜的通告》第四点中指出“自2018年1月1日起，所有医疗器械生产企业应当符合医疗器械生产质量管理规范的要求”。
2. 成品预装培养基平皿是否需要预培养？
答：根据2015版《中国药典》第四部9203药品微生物实验室质量管理指导原则中要求：“用于环境监控的培养基须特别防护，最好要双层包装和终端灭菌，如果不能采用终端灭菌的培养基，那么在使用前应进行100%的预培养，以防止外来的污染物带到环境中及避免出现假阳性结果”。目前市场上的一次性培养基平皿产品都是最终灭菌产品，所以使用前不需要预培养。
3. 若三层真空包装的一次性培养基平皿在使用过程中发现涨袋，能否继续使用？
答：建议不要带进洁净区使用，特别是A、B级区，会给洁净区环境带来污染风险。
4. 实验室自制培养皿灭菌后为何会出现水汽太多的现象？
答：原因有两个：一、浇注平皿时培养基温度太高，而浇注后没有冷却至室温就盖上盖子；第二、灭菌柜的干燥性能不佳造成。
5.一次性培养基平皿沉降菌采样后为何会出现培养基缩板的现象？
答：如果洁净区的温湿度（温度：18℃～22℃；相对湿度控制在45%~65%；风速小于0.54m/s）没有特殊要求，沉降菌采样后缩板，可能是一次性平皿的质量问题；
如果洁净区的温湿度有特殊要求，则要根据验证来确定在沉降菌采样时平皿的暴露时间。
6. 使用前为何会出现冷凝水很多的现象？
答：培养基平皿中水分占95%左右。正常情况下琼脂表面是光滑、润湿的，冷凝水太多，其原因主要是培养基平皿所处的环境温差太大，特别是由于运输环境或库房保存不当造成的，所以必须确保平皿所处的环境温差不要太大。
7. 用于水系统监控的R2A平皿是否在使用前必须进行预培养？
答：如果不能采用终端灭菌的培养基，那么在使用前应进行100%的预培养。
8. 在环境监测沉降菌采样时，为了使平皿完全暴露，平皿盖子如何放置比较合适？如果在百级层流下取样，盖子的面向上或向下有差异吗？
答：一般情况向下放置皿盖，但也允许向上放置，相当于多取样监测一块平皿，环境监测更严格一些。
9. 环境监测沉降菌的培养结果没有长菌是以0还是以小于1表示结果？
答：建议用0表示结果，以便于今后的趋势分析。
10. 生化培养箱、超净工作台、灭菌锅等设备每年都需要做IQ、PQ、OQ吗？
答：2015版《中国药典》第四部9203药品微生物实验室质量管理指导原则中要求：应由有资质的人员进行生物安全柜、层流超净工作台及高效过滤器的安装与更换，要按照确认的方法进行现场生物和物理的检测，并定期进行再验证。
实验室生物安全柜和层流超净工作台的通风应符合微生物风险级别及符合安全要求。应定期对生物安全柜、层流超净工作台进行监测，以确保其性能符合相关要求。实验室应保存检查记录和性能测试结果。
《GMP指南-实验室》中对培养箱要求是：新设备应该在进行IQ、PQ、OQ合格之后方可使用。使用时应进行温度监测，建议在OQ时进行多点温度分布的确认，并应定期进行校正和设备的表面清洁。
确认周期均为硬性要求，一般一年一次，如果延长周期需要进行相应的风险评估。
11. 在做培养平皿促生长实验时，经常会发现个别实验菌回收率小于50%，是什么原因？
答：2015版《中国药典》1105中规定：按表1规定，接种不大于100cfu的菌液至胰酪大豆胨液体培养基管或胰酪大豆胨琼脂培养基平板或沙氏葡萄糖琼脂培养基平板，
置表1规定条件下培养。每一试验菌株平行制备2管或2个平皿。同时，用相应的对照培养基替代被检培养基进行上述试验。
被检固体培养基上的菌落平均数与对照培养基上的菌落平均数的比值应在0.5～2范围内，且菌落形态大小应与对照培养基上的菌落一致；
如果回收率小于50%，主要是实验误差导致，为了确保每次接种量的浓度相当，最好采用同一支试管的菌液或定量菌株。
12. 微生物方法验证必须做3个批次吗？如果只有一个批次产品，下一个批次产品要等2个月以后，那现在这个批号的产品报告书是否等3个批次的验证完之后才能出呢？
答：目前药典没有规定必须做3个批次产品，所以用一个批次产品做三次也是允许的，而且现在的方法学适用性试验也在考虑这个问题，目的是为了与验证的概念有更好的区分。
13. 成品预装培养基平皿储存在2~8℃冰箱内，对平皿质量有影响吗？
答：首先根据所购置的培养基平皿产品的储存环境要求选择合适的温度保存，其次要保证您所用的冰箱温度分布均匀，在运行过程中不会超出接触碟储存温度范围。如果温度低于0℃，会使其中的琼脂凝胶强度受到影响而导致平板质量不符合要求。
14. 对于GMP附录一《无菌药品》中规定的洁净区级别中，D 级洁净区的浮游菌和沉降菌是否都需要进行监测？
答：企业可根据各工序污染的风险高低，评估决定D级洁净区的动态微生物监测频次。一般情况下，由于采样的对象不同，浮游菌和沉降菌监测都是需要的。
15. GMP附录一《无菌药品》中“注（2）单个沉降碟的暴露时间可以少于 4 小时，同一位置可使用多个沉降碟连续进行监测并累积计数。”这里描述的是如何累积的？
答：用这个点的所有结果相加，并除以实际小时数，再乘上4小时。监测时间应涵盖生产时间，但如果实际生产时间短于4小时，监测时间就没有必要达到4小时。例如，如果某工序操作总时间不足4 小时，如2小时，累计共检出5cfu（菌落数），换算方法是：5/2*4=10cfu/4h。
16. 附录一 中 D 级微生物检测方法，是三种方法任选一种呢？还是都需要做呢？
答：三种方法都需要做。因为沉降菌、浮游菌和表面微生物三种监测原理是不同的，针对不同的微生物，不能完全相互替代。不同企业应根据厂房、产品特性、工艺流程及设备的特点（例如暴露区域及时间）评估结果制定监测方案与频次。
17. 间歇式非最终灭菌制剂生产前，对环境微生物情况进行监测，是浮游菌、沉降菌二选一还是都必须做？
答：检测微生物时，浮游菌、沉降菌都应该做。应注意的是，所有无菌药品生产的洁净区空气净化系统应当保持连续运行，维持相应的洁净度级别。因故停机再次开启空气净化系统，应当进行必要的测试以确认仍能达到规定的洁净度级别要求。
18. 洁净区的人员密度限度为多少？
答：《采暖通风与空气调节设计规范》GB50019—2003中第3.1.9 条第2款：工业建筑应保证每人不小于30m3/h的新风量；《医药工业洁净厂房设计规范》GB50457—2008中第9.1.3 条第2款：洁净室内每人新鲜空气量不应小于40m3/h。主要考虑每个人的平均新风量，所以4～6平方米/人。
19. 无菌原料药培养基模拟试验是否必须做？
答：无菌原料药参照无菌制剂管理，应该进行培养基模拟灌装验证，并应注意以下问题：
1.对于开放系统，企业最好选择分段式模拟方案，以利于寻找污染来源；
2.应密切关注模拟过程中使用的抑菌溶剂和抑菌工艺条件；
3.应根据最终成品的最da分装剂量制定验证可接受标准。
20. 模拟实验中用到的培养基，要做哪些适用性检查？
答：应考虑以下实验或以下实验的一部分：无菌性试验，促生长试验，灭菌适应性、流动性实验、溶解性实验和观察适用性等。
21. 培养基平皿传入无菌区，一般采用什么方式？
答：大多选择逐层脱外包的方法，在生产开始前进行。
方法：传到洁净区的设备，一是需要严格的保护设施，如培养皿用专用密闭的不绣钢桶或呼吸袋盛装培养皿；二是要有一套合理的灭菌处理方式，如擦拭（杀孢子剂）、照射（臭氧或紫外线）、汽化双氧水等；三是效果经验证，是可以从普通区传入洁净区。
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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