The appropriate medium preparation method will affect
The microbial contamination in the production workshop and laboratory clean area of pharmaceutical enterprises is closely related to the source of pollution. Bacteria, fungi, viral contamination, etc. are more common, mainly caused by improper management of clean environment and facilities, irregular behavior and cross-contamination of personnel, leakage of operating strains, mixing of raw materials and auxiliary materials, contamination of equipment and containers, etc. Therefore, choosing a reliable microbial detection method in clean areas can effectively monitor the environment of clean areas, control the risk of microbial contamination, ensure the reliability and accuracy of microbial monitoring results, and reflect the true level of environmental microbial control in clean areas.
1. Culture medium
mediumIt is the basis of microbial test and directly affects the results of microbial test. Appropriate medium preparation methods, storage conditions and quality control tests are the guarantees for providing high-quality medium.
In microbiological testing, we should pay great attention to whether the media used (including the purchase of different batches of finished pre-packaged media, different batches of dehydrated media dry powder, and the use of different batches of raw materials to prepare the media according to the prescription), preparation procedures (including water quality control, preparation methods, sterilization procedures, etc.), storage conditions (temperature, humidity, time, and container conditions for containing the media, etc.) meet the requirements for microbiological testing.
Whether it is for clean area sedimentation bacteria, planktonic bacteria, or surface microorganisms, the culture medium flat dishes used must be tested for growth promotion before use. This is also the performance test of the culture medium and the inherent Quality Standard of the culture medium. It uses known standard strains to determine whether the performance of the medium meets the requirements according to the procedures and standards.
In this experiment, we can not ignore a link is the need to use control media. Regarding the control medium, in the "Chinese Pharmacopoeia", learn from the European and American Pharmacopoeia, and introduce the applicability test of the medium to ensure the accuracy and reliability of the microbial limit test results.
However, there is a lot of controversy over the adoption of the "previously verified culture medium" in the European and American Pharmacopoeia, which focuses on two points. First, the drug microbiology laboratories in our country are small and scattered, and thousands of enterprises and grassroots drug laboratories are difficult to effectively carry out the verification and evaluation of the control medium; second, in the supervision and inspection, it is difficult to effectively evaluate the verification of the control medium by the enterprise microbiology laboratory, and conduct the applicability test of the control medium with a unified, fully evaluated and verified control medium.
Therefore, when using the control medium, 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 suitability test.
The alternative use of different prescription media cannot be simply determined by the suitability test of the medium, and the medium should be used according to the standard.
It is not recommended to use other media that have passed the suitability test of the control medium as the "control medium" for laboratory use.
Unless otherwise specified, the medium can be prepared with distilled or purified water, but testing should be carried out.
The dry powder medium or the deterioration of the raw materials of the medium should not be reused; the finished medium should not be reused if discoloration, bacteria growth, severe dehydration, etc. are found during storage.
2. Clean area microbial testing
When evaluating the microbial status of the production clean environment, pharmaceutical companies need to conduct dynamic or static monitoring of the clean environment, and use sedimentation bacteria flat dishes, air-floating bacteria flat dishes and surface contact dishes to detect microorganisms in the clean area. Among them, the detection of planktonic bacteria and sedimentation bacteria is carried out according to GB16293-2010 "Test method for planktonic bacteria in the clean room (area) of the pharmaceutical industry" and GB16294-2010 "Test method for sedimentation bacteria in the clean room (area) of the pharmaceutical industry". Surface microbial detection methods include wiping method and surface contact plate method.
2.1 Sampling of sedimentary bacteria
The detection method of sedimentation bacteria adopts the sedimentation method, that is, through the principle of natural sedimentation, the biological particles in the air are collected in the culture medium flat dish, and after a certain period of time, they are allowed to multiply to visible colonies under suitable conditions for counting. The number of colonies in the flat plate culture dish is used to determine the number of live microorganisms in the crystalline environment, and the cleanliness of the clean area is evaluated.
Sampling method: Environmental monitoring personnel disinfect their hands with disinfectant, take out the TSA flat dishes, place them one by one according to the sampling point arrangement diagram, then open the petri dish lids one by one from the inside to the outside, and place the inside of the lids down next to the petri dish base to expose the surface of the petri medium to the air. In order to avoid contaminating the surface of the petri medium, do not pass over the petri dish with the open lid.
2.2 Sampling of plankton
The sampling of planktonic bacteria is to collect biological particles suspended in the air through an air sampler in a special medium, and make them visible after a certain period of time and suitable growth conditionscolonyAnd count to determine the microbial concentration in the clean area.
The planktonic bacteria sampler generally adopts the impact method mechanism and can be divided into slit sampler, centrifugal sampler or pinhole sampler.
The slit-type sampler sucks the airflow by the internal fan, and through the slit-type plate of the sampler, the collected air is sprayed and impacted on the surface of the slowly rotating plate culture medium, and the attached live microbial particles are cultured to form colonies.
Due to the high-speed rotation of the internal fan of the centrifugal sampler, the air flow is sucked in from the front of the sampler and flows out from the rear. Under the action of centrifugal force, the live microbial particles in the air have enough time to hit the dedicated solid culture strip, and the attached live microbial particles are cultured to form colonies.
The pinhole sampler is a gas stream sucked through a metal cover, which is densely machined with special holes. The fine air stream collected by the fan directly impacts the surface of the plate culture medium, and the attached live microbial particles are cultured to form colonies.
At present, most pharmaceutical companies use pinhole samplers to sample planktonic bacteria, so the sampling methods are introduced.
Sampling method (pinhole sampler):
Environmental monitoring personnel should first determine that the air sampler is within the scope of qualification.
Before sampling, wipe the outer surface of the air sampler with a sterile cloth soaked in disinfectant and let it dry naturally.
Prepare a sterilized sampler cover that can only be used once.
The environmental monitoring personnel sterilized their hands with disinfectant, took out the TSA flat dish, placed the petri dish on the top support surface, opened the lid and placed it aside, opened the sterilization bag, took out the porous collection cover, and covered it on the sampler to start collecting air samples.
Each sampling point, with a TSA medium dish, the sampling capacity of 1000 liters;
After sampling, mark the relevant information on the culture medium plate, such as room number, test item, level and shift, sampling date, etc.
Attention: During the sampling process, do not place any objects (including the operator's hands and head) over the culture dish and avoid moving around the sampling point.
2.3 Sampling of surface microorganisms
There are two methods for sampling surface microorganisms, namely, the wiping method and the surface contact plate method.
The wiping method is a commonly used surface microbial sampling method. Before the surface contact plate method, the surface microbial sampling in the clean area almost all adopts this method. It is often used for surface sampling of equipment. The advantage is that it can directly sample the curved surface. The contamination status of equipment is evaluated by examining the microbial level of representative parts, and it is also often used in the cleaning verification of equipment. However, the wiping sampling method has its limitations. Its microbial collection rate is usually affected by factors such as sampling tools, dissolved solvents, sampling personnel, and testing methods.
Specific operation method: Press the cotton swab on the sampling surface, bend it hard, wipe the sampling surface smoothly and slowly, and move it from side to side while moving forward. The wiping process should cover the entire surface, flip the cotton swab, and let the other side of the cotton swab also wipe, but perpendicular to the direction of the previous wiping movement, at least 25cm2 of area should be wiped.
The surface contact dish method is developed on the basis of the wiping method. A contact dish with a diameter of 55mm is used to contact the surface to be sampled. The flat dish should be filled with a volume-controlled agar medium (selected according to the target microorganism), specially made for surface sampling. The agar should form a raised meniscus on the petri dish. That is, after the inspector opens the flat dish, gently contact the surface of the sampling point with the surface of the petri medium. Be careful not to rotate. Immediately after sampling, cover the flat dish lid. The complete contact process is about 5s. After sampling, wipe the sampling point with a sterile clean cloth soaked in disinfectant.
3. Precautions
For the unidirectional flow clean area or feeding trend, the sampling port of the sampler should be facing the direction of the airflow; for the non-unidirectional flow clean area, the sampling port should be upward.
When arranging sampling points, at least try to avoid the trend of dust particles.
When sampling, environmental monitoring personnel should stand on the downwind side of the sampling port and move as little as possible.
All measures should be taken to prevent contamination during the sampling process and other possible contamination of the sample.
When a petri dish is used for testing, in order to avoid the influence of the petri dish transportation or handling process, a negative control test should be carried out at the same time. A control dish should be taken each time or in each area, and it should be operated in the same way as the sampling dish, but the sampling does not need to be exposed. Then, together with the sampled petri dish (TSA, SDA), it should be placed in the incubator for culture, and the result should be free of colony growth.
