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1. Purpose
Establish the standard operating procedures for cleanliness (sedimentation bacteria) inspection, and provide the correct standard operating methods for sedimentation bacteria inspectors.
2. Scope
Applicable to the whole process of cleanliness (sedimentation bacteria) inspection of our company.
3. Responsibility
QA is responsible for the supervision and inspection of the effective implementation of this procedure, and QC is responsible for the implementation of this procedure.
4. Procedures
4.1 Overview
This standard stipulates the rooms or areas that need environmental control for dust particles and microbial contamination. Its building structure, equipment and its use have the function of reducing the intervention, generation and retention of pollution sources in the area.
4.2 Test method
a. Method summary: This standard is based on the "Test Method for Sedimentation Bacteria in Clean Rooms (Areas) in Pharmaceutical Industry" issued by the State Administration of Technical Supervision. The sedimentation method is used, that is, the biological particles in the air are collected on the culture medium plate by the principle of natural sedimentation. After a period of time, under suitable conditions, they are allowed to multiply to visible colonies for counting. The number of colonies in the plate culture plate is used to determine the number of live microorganisms in the clean environment, and the cleanliness of the clean room (area) is evaluated on this basis.
b. Instruments
Instruments include: culture dishes, culture media, constant temperature incubators, and high-pressure steam sterilizers.
Culture dishes:Generally, culture dishes with a specification of f90mmx15mm are used.
Culture media:Soybean casein agar culture media (TSA) or Sabouraud culture media (SDA) or culture media approved and verified by the user.
4.3 Rules before testing
a. Test status:
Before the sedimentation bacteria test, the temperature and humidity of the tested Clean Room (area) must meet the specified requirements, and the static pressure difference, ventilation times, and air flow rate must be controlled within the specified values. The temperature and Relative humidity of the clean room (area) should be consistent with its production and process requirements (if there are no special requirements, the temperature is between 18℃~26℃ and the relative humidity is between 45%~65%), and it should also meet the use range of the test instrument.
Before the sedimentation bacteria test, the clean room (area) to be tested has been disinfected.
There are two test states: static and dynamic. The choice of test state must meet the production requirements and indicate the test state in the report.
During static testing, the exposure time of the culture dish is more than 30 minutes; during dynamic testing, the exposure time of the culture dish is no more than 4 hours.
b. Testers:
Testers must wear work clothes that meet the environmental cleanliness level.
During static testing, there shall be no more than 2 testers in the room.
c. Test time:
For unidirectional flow, such as Class 100 clean rooms and laminar flow workbenches, the test should start after the purification Air conditioning system is operating normally for no less than 10 minutes.
For non-unidirectional flow, such as Class B and Class b Clean Rooms, the test should start after the purification air conditioning system is operating normally for no less than 30 minutes.
d. Notes:
Test equipment should be sterilized to ensure the reliability and correctness of the test
Take all measures to prevent human contamination of samples.
Keep detailed records of culture medium, culture conditions and other parameters
Due to the wide variety of bacteria and their great differences, when counting, it is generally necessary to use transmitted light to carefully observe the back or front of the culture dish, and do not miss the colonies growing on the edge of the culture dish. Attention should be paid to the difference between bacterial colonies and culture medium sediments, and use a microscope for identification when necessary.
Before sampling, the quality of each culture dish should be carefully checked. If it is found to be deteriorated, damaged or contaminated, it should be discarded.
For unidirectional clean rooms (areas) or air outlets, the sampling port of the sampler should face the direction of the airflow; for non-unidirectional clean rooms (areas), the sampling port should face upward.
When arranging sampling points, at least try to avoid the return air outlet where dust particles are concentrated; when sampling, the tester should stand on the downwind side of the sampling port and walk as little as possible.
When the culture dish is used for testing, in order to avoid the impact caused by the transportation or moving process of the culture dish, it is advisable to carry out a control test at the same time. Take one control dish each time or in each area, operate it in the same way as the sampling dish but do not need to be exposed for sampling, and then put it into the incubator together with the sampled culture dish (TSA or SDA) for incubation. The result should be no colony growth.
4.4 Test steps
a. Sampling method:
4.4.1.1 Instruments and utensils: culture dish:
4.4.1.2 Method:
Sampling points are generally evenly arranged on a horizontal plane 0.8m above the ground,
When there are more than 5 sampling points, they can also be arranged in layers in the area 0.8m~1.5m above the ground, but each layer should not be less than 5 points.
Open the lid of the culture dish, expose the surface of the culture medium for 0.5h, then cover the culture dish and turn it upside down.
Minimum number of sampling points
| aera(Square meter) | Cleanliness level | aera(Square meter) | Cleanliness level | ||||
| A | B | B | A | B | B | ||
| <10 | 2~3 | 2 | 2 | ≥200~<400 | 80 | 20 | 6 |
| ≥10~<20 | 4 | 2 | 2 | ≥400~<1000 | 160 | 40 | 13 |
| ≥20~<40 | 8 | 2 | 2 | ≥1000~<2000 | 400 | 100 | 32 |
| ≥40~<100 | 16 | 4 | 2 | 2000 | 800 | 200 | 63 |
| ≥100~<200 | 40 | 10 | 3 | ||||
| Note: The area in the table refers to the air outlet area for unidirectional clean rooms, and the area of the room for non-unidirectional clean rooms. | |||||||
Minimum number of culture dishes
| Cleanliness level | Required p90mm culture blood number (0.5h sedimentation) |
| A | 14 |
| B | 2 |
| C | 2 |
| D | 2 |
b. Culture:
①Instruments and utensils: incubator, balance (1/10,000), weighing paper, conical flask (1000ml, 150ml), measuring cylinder (1000ml), high-pressure steam sterilizer.
②Culture medium:
Soybean casein agar culture medium (TSA):a) Ingredients: Casein pancreatic digest 15g Soy flour papain digest 5g Sodium hydroxide 5g Agar 15g Purified water 1000mlb) Preparation method: Take the above ingredients except agar, mix, dissolve with slight heat, adjust the H value to 7.3±0.2 after sterilization, add agar, heat and melt, divide, sterilize, cool to about 60℃, and pour about 20ml into a sterile plate (f90mm) under aseptic operation requirements. Cover and leave at room temperature until solidified.
Sabouraud medium (SDA):
a) Glucose 40g Casein pancreatic digest, animal tissue gastric digest 10g agar 15g Purified water 1000ml.
b) Preparation: Take the above ingredients except agar, mix, dissolve with slight heat, adjust the pH value to 5.6±0.2 after sterilization, add agar, heat and dissolve, divide, sterilize, cool to about 60℃, and pour about 20ml into a sterile plate (f90mm) under aseptic operation requirements. Cover and place at room temperature until solid.
③Method: After all sampling is completed, the culture dish is inverted and cultured in a constant temperature incubator. The culture dish prepared with soybean casein agar medium (TSA) is cultured in a 30~35℃ incubator for no less than 2 days; the culture dish prepared with Sabouraud medium (SDA) is cultured in a 20~25℃ incubator for no less than 5 days. Each batch of culture medium should have a control test to check whether the culture medium itself is contaminated. Three culture dishes can be selected for control culture in each batch.
c. Colony count:
Count directly with the naked eye, mark or count on the colony counter, and then check with a 5~10x magnifying glass to see if there are any omissions.
If there are 2 or more colonies overlapping on culture plate 4, they should still be counted as 2 or more colonies when they can be distinguished.
d. Result calculation:
Use the counting method to obtain the number of colonies in each culture dish.
Calculation of average colony count, see formula.
Where M-average colony count;
M1-number of colonies in culture dish No. 1:
M2-number of colonies in culture dish No. 2:
Mn-number of colonies in culture dish No. n:
n-total number of culture dishes.
e. Result evaluation: Use the average colony count to determine the microorganisms in the clean room (area) air.
The average colony count in the clean room (area) must be lower than the selected evaluation standard.
During the static test, if the average colony count of sedimentation bacteria in A Clean Room (area) exceeds the evaluation standard, the area must be disinfected first, and then resampled twice, and the test results must be qualified.
