Standard operating procedures for sedimentation bacteria testing in pharmaceutical purification workshops

Colony

A bacterial colony formed by the reproduction of one or several bacteria after bacterial culture, referred to as CFU. Usually expressed in number.

Test method

Sedimentation method, that is, the biological particles in the air are collected on the culture medium plate by the principle of natural sedimentation, and after more than 48 hours of culture, they are allowed to reproduce to a visible colony number under suitable conditions to assess the number of live microorganisms in the clean environment, and thus the cleanliness of the purification workshop is assessed.

Instruments and equipment used

• High pressure sterilizer: refer to the document GZ-ZL-SOP-066-00 for operation when using.

• Constant temperature incubator: The thermometer of the incubator must be calibrated regularly.

• Culture dish: Generally, a 90mmx15mm borosilicate glass culture dish is used. Place the culture dish at 121℃ for 20 minutes of wet heat sterilization before use. 

Culture medium

Ordinary nutrient agar culture medium. Heat the culture medium to melt, cool to about 45℃, and inject the culture medium into the culture dish under aseptic operation conditions, about 15ml per dish. After the agar medium solidifies, place the culture medium plate in a 30~35℃ constant temperature incubator for several hours. If there is no colony growth on the culture medium plate, it can be used for sampling. The prepared culture medium plate should be stored in an environment of 2~8℃.

Test steps

• Sampling method: Place the prepared culture plate at the predetermined sampling point, open the culture plate cover, expose the culture medium surface for 0.5 hours, and then cover the culture plate and turn it upside down.

• Cultivate for no less than 48 hours. Each batch of culture medium should have a control test to check whether the culture medium itself is contaminated. Three culture plates can be selected for control culture in each batch. 

• Colony count: Count directly with the naked eye, 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 the culture plate, they should still be counted as 2 or more colonies when they can be distinguished.

Notes

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

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

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

• Due to the large 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. Do not miss the colonies growing on the edge of the culture dish, and pay attention to the difference between bacterial colonies and culture medium sediments. Use a microscope to monitor if 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.

Test rules

a.Test status

• Before the sedimentation bacteria test: the temperature and humidity of the tested CleanRoom (area) must meet the specified requirements, and the static pressure difference must be controlled within the specified value. The tested cleanroom (area) has been disinfected.

• There are two test states: static and dynamic. The test state is noted in the report.

• Test personnel: The test personnel must wear work clothes that meet the environmental cleanliness level. During static testing, there shall be no more than 2 test personnel in the room.

b.Test time

• For unidirectional flow, such as in a Class 100 cleanroom and a laminar flow workbench, the test should start after the cleanroom air conditioning system has been operating normally for at least 10 minutes

• For non-unidirectional flow, such as cleanrooms of Class 10,000 and above, the test should start after the cleanroom air conditioning system has been operating normally for at least 30 minutes.

Settling bacteria count

a.Minimum number of sampling points: can be determined according to Table 1. While meeting the minimum number of measurement points, the minimum number of culture dishes should also be met.

b.Arrangement of sampling points: The test point in the work area is about 0.8~1.5m above the ground (slightly higher than the work surface). Measurement points can be added at key equipment or key work activity ranges. The arrangement of sampling points should be as uniform as possible to avoid sampling points being too concentrated in a certain local area and too sparse in a certain local area

c.Records: The room temperature, Relative humidity, pressure difference, test status and test data should be recorded in the test report.

d.Result calculation: The number of colonies in each culture dish is obtained by counting method.

The calculation of the average colony count is as follows: m1+m2+…tmn

Average colony count m=n

Where: m is the average colony count; m1 is the colony count of culture dish No. 1; m2 is the colony count of culture dish No. 2; mn is the colony count of culture dish No. n; n is the total number of culture dishes.

e.Result evaluation: Use the average colony count to judge the microorganisms in the air of the purification workshop.

f.The average colony count in the purification workshop (area) must be lower than the selected evaluation standard.

g.If the average colony count of a purification workshop (area) exceeds the evaluation standard, the area must be disinfected first, and then retested twice, and the test results must be qualified.