GB/T 16294-2010Test method for settling microbe in clean room(zone) of the pharmaceutical industry (English PDF)

3.1 Settling microbe

Visible number of colonies that are colonized using living microbial particles in the air collected by the method mentioned in this Standard, in special culture medium, under suitable growth conditions.

3.2 Settling microbe plate count

Number of setting microbe in the air that are collected in each plate petri dish in the specified period of time, expressed in cells/dish.

4.1 Method summary

This test method adopts the sedimentation method, that is, collect biological particles in the air on the culture medium plate through natural sedimentation principle, after some time, let it grow to visible colonies under appropriate conditions and count, determine the number of living microorganisms in a clean environment by counting the number of colonies in the plate petri dish, so as to assess the cleanliness of the clean room (zone).

4.2 Personnel responsibility and training

The test personnel of the clean room (zone) shall be trained in this specialty and obtain relevant qualifications before they can perform their duties in the clean room (zone) test, which includes hygienic knowledge and basic knowledge of the microorganisms involved.

4.3 Instruments

The instrument shall include.

4.3.1 Petri dish

It generally uses phi 90 mm × 15 mm petri dishes.

4.3.2 Culture medium

Soybean casein agar medium (TSA) or sabouraud medium (SDA) or user-approved and validated medium. The preparation method is shown in Annex B.

4.3.3 Constant temperature incubator

The constant temperature incubator must be checked regularly.

4.4 Test procedure

4.4.1 Before test, the surface of the petri dish must be strictly sterilized.

4.4.2 PLACE the prepared petri dishes one by one according to the sampling point layout, and then OPEN the lid of the petri dishes one by one from inside to outside so that the surface of the culture medium is exposed to air.

4.4.3 In the static state test, the exposure time of the petri dishes is more than 30 min; in the dynamic state test, the exposure time of the petri dishes is not more than 4 h.

4.4.4 After all sampling is complete, place the petri dishes in the constant temperature incubator.

4.4.5 The petri dish prepared by soybean casein agar medium (TSA) are sampled and cultured in the incubator at 30 °C to 35 °C for no less than 2 d.

The petri dish prepared by sabouraud medium (SDA) are sampled and cultured in the incubator at 20 °C ~ 25 °C for no less than 5 d.

4.4.6 Each batch of culture medium shall have a control test to test whether the culture medium itself is contaminated. It may select three petri dishes for control culture in each batch.

4.5 Colony count

4.5.1 Directly count and mark all the colonies on the petri dish with the naked eye or count on the colony counter, and then check with a magnifying glass 5 to 10 times for omission.

4.5.2 If there are 2 or more colonies overlapping on the plate, it will still count as 2 or more colonies when they are distinguishable.

4.6 Precautions

4.6.1 The test instruments shall be sterilized to ensure the reliability and correctness of the test.

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

4.6.3 Make a detailed record of the culture medium, culture conditions and other parameters.

5.1 Test conditions

Before test, the clean room (zone)-related parameters shall be pre-tested, which will provide environmental conditions for testing setting microbe. For example, the pre-test may include.

5.2 Test states

Both static and dynamic states can be tested.

In the static state test, the number of indoor testing personnel shall not be more than 2.

Before the settling microbe test, is decided by the user whether or not the clean room (zone) to be tested needs to be disinfected in advance.

The test report shall indicate the state used and the number of indoor testing personnel during the test.

5.3 Test time

5.3.1 In the empty state or static state a test, for unidirectional-flow clean rooms (zones), the test should be started after the clean air conditioning system is normally operating for not less than 10 min. For non-unidirectional-flow clean rooms (zones), the test should start after the clean air conditioning system is normally operating for not less than 30 min. In the static b test, for unidirectional-flow clean rooms (zones), the test should be started after the production operators are evacuated from the site and after 10 min self-cleaning; for non-unidirectional-flow clean rooms (zones), the test should be started after the production operators are evacuated from the site and after 20 min self-cleaning.

5.3.2 In the dynamic state test, it shall record the production start time and test time.

5.4 Calculation of settling microbe plate count

5.4.1 Number of sampling points and their layout

5.4.1.1 Minimum number of sampling points

The minimum number of sampling points for the settling microbe test may refer to GB/T 16292-2010.

5.4.1.2 Position of sampling points

The position of sampling points for setting microbe may refer to GB/T 16292-2010.

5.4.2 Minimum number of petri dishes

While satisfying the minimum number of sampling points, it should satisfy the minimum number of petri dishes, as shown in Table 1.

5.4.3 Number of sampling

Each sampling point is generally sampled once.

5.4.4 Precautions of sampling

5.4.4.1 For unidirectional-flow clean rooms (zones) or air supply ports, the sampling port of the sampler shall face the airflow direction; for non-unidirectional-flow clean rooms (zones), the sampling port shall be upward.

5.5 Records

The test report shall contain the following. […]

5.6 Result calculation

5.6.1 Count the number of colonies in each petri dish.

5.6.2 For the calculation of the average colony count of setting microbe at each test point, see equation (1).

5.7 Result evaluation

5.7.1 The average colony number of setting microbe at each test point must be lower than the limit in the selected evaluation criteria.

5.7.2 In the static state test, if the average colony number of setting microbe at a certain test point exceeds the evaluation criteria, it shall re-sample twice. It is determined qualified only when the two test results are qualified.

5.8 Daily monitoring

For monitoring of setting microbe, it should set correcting limits and warning limits, to ensure that the microbial concentration in the clean room (zone) is controlled. It shall carry out periodic test to check the microbial load and the effectiveness of the disinfectant to perform propensity analysis. Both static and dynamic monitoring can use this method.

For the sampling frequency of setting microbe, it shall be considered for modification if the following conditions occur. After evaluating the following conditions, it shall also determine the test frequency of other items.

- continuously exceeding correcting limits and warning limits;

- the duration of downtime is longer than expected;

- the presence of contamination in key areas;

- during the production, the air purification system performs any major maintenance;