Cleanroom sedimentation bacteria detection standard operating procedures

1. Purpose

To establish a standard operating procedure for sedimentation detection to standardize the Detection operation of employees.

2. Scope

Applicable to the detection of sedimentation bacteria in the company's internal production clean area.

3. Responsibility

The production department, production workshop, quality management department, QC inspection room director, and inspectors are responsible for the implementation of this procedure.

4. Testing basis

YY/T0188.6-1995 Drug Inspection Operation Procedure, Part 6, Drug Bioassay.

5. Content

5.1 Equipment: High pressure sterilizer, constant temperature incubator

5.2 Culture medium and plate preparation

• Culture dish: Generally use 490mmx15mm borosilicate glass culture dish.

• The culture medium generally uses ordinary broth agar culture medium or other culture medium approved by the pharmacopoeia.

• Place the culture dish in 121℃℃ wet heat sterilization for 20 minutes or dry heat sterilization at 180℃℃ for 2 hours.

• Heat the culture medium to melt, and when it cools to 45℃, inject the culture medium into the culture dish under the requirements of sterile operation, about 15ml per dish.

• After the agar is dissolved, invert the culture medium plane in a constant temperature incubator at 30~35℃ for 48 hours. If there is no colony growth on the culture medium plane, it can be used for sampling.

• The prepared culture medium plane should be stored in an environment of 2~8℃.

5.3 Sampling points

• The arrangement of sampling points should be uniform to avoid excessive concentration of sampling points in a certain local area. According to the size of the clean area, the minimum number of sampling points should be formulated. While meeting the minimum number of measurement points, it is also advisable to meet the minimum number of culture dishes.

• The location of the sampling point in the working area is about 0.8m~1.5m from the ground (slightly higher than the working surface). And add sampling points at key equipment or key work activity range.

• Test Standards

The static standards (a) for clean area microbial monitoring are as follows:

The dynamic standards (a) for clean area microbial monitoring are as follows:

5.4 Test Rules

a. Test Status

• Before the sedimentation bacteria test, the temperature and humidity of the clean room (area) to be tested must meet the specified requirements, and the static pressure difference, ventilation frequency, and air flow rate must be controlled within the specified values.

• The clean room (area) to be tested has been disinfected before the sedimentation bacteria test.

• There are two test states: static and dynamic. The choice of test state must meet the production requirements, and the test state must be indicated in the report.

• During the static test, the clean room purification and Air conditioning system is in normal operation.

During the dynamic test, the clean room is in normal production.

b. Testers

• Testers must wear work clothes that meet the environmental cleanliness level

• During the static test, all production equipment has been installed and ready, but not running, and there shall be no more than two testers in the room. Test time: For unidirectional flow, such as Class 100 Clean rooms and laminar flow workbenches, the test should start at least 10 minutes after the Clean air conditioning system has been operating normally; for non-unidirectional flow, such as Class 10000 and Class 100000 clean rooms, the test should start at least 30 minutes after the clean air conditioning system has been operating normally.

• During dynamic testing, all production equipment operates according to the predetermined process mode and a specified number of operators are on site.

c. Test steps

• Sampling method: Place the prepared culture dish according to the layout requirements of the sampling point, open the culture dish cover, expose the culture dish surface for 0.5 hours, then cover the culture dish cover and turn it upside down.

• Culture: After all sampling is completed, place the culture dish upside down in a constant temperature incubator and culture it at 30~35℃ for at least 48 hours.

• Colony counting: Count directly with the naked eye, mark or dot on the colony counter, and then check with a 5~10x magnifying glass to see if there are any omissions. If there are more than two colonies overlapping on the culture dish, count more than two colonies if they can be distinguished.

5.5 Notes

• The test equipment must be sterilized to ensure the reliability and correctness of the test.

• Take all measures to prevent human contamination of the sample.

• Make detailed records of the culture medium, culture conditions and other parameters.

• When counting, generally use transmitted light to carefully observe the back or front of the culture dish. Do not miss the colonies growing on the edge of the culture dish, and pay attention to the difference between fine colonies and culture medium sediments. If necessary, use a microscope for identification.

• The quality of each culture dish should be carefully inspected before sampling. If it is found to be deteriorated, damaged or contaminated, it should be discarded.

5.6 Result calculation

Use the counting method to obtain the number of colonies in each culture dish, and calculate according to the following formula:

Where: M--average colony count

M1--colony count of culture dish No. 1

M2--colony count of culture dish No. 2

Mn--colony count of culture dish No. n

n--total number of culture dishes

5.7 Result evaluation: Use the average colony count to judge the microorganisms in the air of the clean room (area).

• The average colony count in the clean room (area) must be lower than the selected evaluation standard

• If the average colony count 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.

5.8 Records

• The room temperature, relative humidity, pressure difference and test status should be recorded in the test report.

• The record book should be kept for two years after the last record.

• The archive location must be indicated in the record book.