Scope: This standard specifies the testing conditions and methods for planktonic bacteria in sterile rooms and clean areas of the pharmaceutical industry.

This standard is applicable to the testing and environmental validation of planktonic bacteria in sterile rooms and clean areas, sterile rooms or local air purification areas (including clean workbenches) in the pharmaceutical industry.

2. Reference standard: GB/T 16293? two thousand and ten

Appendix to the Drug Production Management Standards (1998 Revision).

3 standards

cleanliness	Number of live microorganisms/m3
Level 100	5
Level 10000	100
100000 level	500

4 Testing Methods

4.1 Method Summary

The method used for this specimen is the concentration counting method. That is, by collecting suspended biological particles in the air in a specialized culture medium (selecting a medium that can prove its ability to support microbial growth), and allowing them to reproduce under suitable growth conditions for a certain period of time until visible colony count is reached, in order to determine the microbial concentration in the cleanroom.

4.2 Responsibilities and Training of Personnel

The testing personnel in the sterile room should undergo this training and obtain the corresponding qualifications before they can fulfill their responsibilities for sterile room testing, including hygiene knowledge and basic microbiological knowledge.

The testing personnel in the sterile room should choose a wearing style that is suitable for the air cleanliness requirements of the production operation, and external clothing should not be brought into areas above level 100000.

4.3 Instruments, auxiliary equipment, and culture medium

Choose a suitable planktonic bacteria sampler, including using an oil-free suction pump, lower airflow velocity, and larger sampling flow rate, to ensure that the water distribution on the surface of the culture medium is dried.

This test requires the following instruments, auxiliary equipment, and culture medium:

a) Floating bacteria sampler;

b) Culture dish;

c) High pressure disinfection pot;

d) Culture medium;

e) Constant temperature incubator;

4.4 Principle of Floating Bacteria Sampler

Floating bacteria samplers generally use the impact method mechanism and can be divided into crevice samplers, centrifugal or pinhole samplers. The crevice sampler draws in airflow through an internal fan and sprays the collected air onto the slow surface of the agar plate through the crevice plate of the sampler. The attached live microbial particles form colonies after cultivation. Due to the high-speed rotation of the internal fan, the airflow of the centrifugal sampler enters from the front and flows out from the rear. Under the action of centrifugal force, the live microbial particles in the air have enough time to collide with the solid culture strip, and the attached live microbial particles form colonies after cultivation. A pinhole sampler is a device where air is drawn in through a metal cover, which is covered with densely machined small holes. The collected fine air is then directed onto the surface of a flat culture medium by a fan, and the attached live microbial particles are cultured and shaped into colonies.

4.5 Key points of testing

4.5.1 Testing instruments must be regularly calibrated according to their calibration cycle. Qualified instruments with valid validity period should be used.

When the testing instrument has not entered the tested area, if necessary, clean the surface first or prepare and store it in the corresponding sterile room (using a protective cover or other appropriate outer cover to protect the instrument).

When using paper in a Class 100 sterile room, a transparent, dust-free cover should be placed on top. Pencil erasers should not be used in Class 100 sterile rooms.

4.5.4 When using testing instruments, strictly follow the instructions.

4.5.4.1 After the instrument is turned on and preheated to stability, it can be calibrated according to the instructions. At the same time, the sampling flow rate should be checked, and the time should be set according to the sampling volume.

4.5.4.2 The sampling port must be made of materials that are easy to disinfect and have stable chemical properties.

4.5.4.3 The sampling tube must not leak, and the inner wall should be smooth.

4.5.4.4 The length of the sampling tube should be determined according to the height of the measuring point, and bending should be minimized as much as possible.

4.6 Culture dish

4.6.1 Generally, culture dishes with a diameter of 90mm × 15mm are used. Suitable culture dishes can be selected based on the selected sampler.

4.6.2 Solid culture strips used in centrifugal samplers

4.7 Culture medium

Soy casein agar medium (TSA) or Sabouraud medium (SDA) or other user approved and validated media.

4.8 Constant temperature incubator

Regular validation of the constant temperature incubator is necessary

4.9 Testing steps;

Before testing, the surfaces of instruments and culture dishes must be strictly disinfected.

4.9.1.1 Before entering the tested room, sterilize the sampler with disinfectant from the disinfection room, which is used for samplers in Class 100 sterile rooms

It should be placed in the tested room in advance.

Clean the outer surface of the culture dish with disinfectant.

Before sampling, disinfect the top cover, turntable, and inside and outside of the cover of the sampler with disinfectant. After sampling is complete, gently spray the inner wall of the cover and turntable with disinfectant.

4.9.1.4 The sampling port and sampling tube must be sterilized at high temperature before use. When disinfecting the outer and inner walls of the sampling tube with disinfectant, the residual liquid in the tube should be poured out and dried.

4.9.1.5 The sampler should wear work clothes corresponding to the clean area being tested. Before placing or exchanging the culture dish on the turntable, disinfect both hands with disinfectant or operate with sterile gloves.

4.9.1.6 After disinfection, the sampling instrument should not be placed in a culture dish. Instead, turn on the planktonic bacteria sampler to evaporate the residual disinfectant in the instrument for at least 5 minutes. Check the flow rate and adjust the set time according to the sampling volume.

4.9.1.7 Close the floating sampler, place it in a culture dish, and cover it with a lid.

4.9.1.8 Place the sampling port at the sampling point and turn on the planktonic bacteria sampler for sampling.

4.10 Cultivation

After all sampling is completed, invert the culture dish and incubate it in a constant temperature incubator.

4.10.2 The culture prepared with soybean casein agar medium (TSA) shall be cultured in a 30 ℃ to 35 ℃ incubator for no less than 2 days after sampling; the culture dish prepared with Sabouraud medium (SDA) shall be cultured in a 20 ℃ to 25 ℃ incubator for no less than 5 days after sampling.

4.10.3 Each batch of culture medium should have a control test to verify whether the culture substrate is contaminated. Three culture dishes can be selected for control culture in each batch.

4.11 Colony Count

4.11.1 Count and mark all colonies on the culture dish directly with the naked eye or count them on the colony counter, and then check with a 5-10 times magnifying glass for any omissions.

4.11.2 If there are 2 or more overlapping colonies on the plate, count them as 2 or more colonies when distinguishable.

4.12 Precautions

Before use, carefully check the quality of each culture dish. If the culture medium or dish is spoiled, damaged, or contaminated, it cannot be used.

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

4.12.3 Due to the wide variety and significant differences in bacterial species, when counting, it is generally observed carefully with transmitted light on the surface or true surface of the culture dish, without missing any colonies growing at the edge of the dish. It is necessary to distinguish between bacterial colonies or sediment in the culture medium, and if necessary, use a microscope for identification.

5 Testing Rules

5.1 Testing conditions

Before testing, it is necessary to conduct pre testing on the relevant parameters of the sterile room, which will test the environmental conditions of suspended particles. For example, this pre testing may include:

a) Testing of temperature and relative humidity. The temperature and relative humidity of the sterile room should be suitable for its production and process requirements

When there are no special requirements, it is advisable to control the temperature between 18 ℃ and 26 ℃ and the relative humidity between 45% and 65%. Simultaneously full

The scope of use of foot testing instruments;

b) Testing of indoor air supply volume or wind speed, or testing of pressure difference;

c) Leakage test of filter

5.1 Test Status

Both static and dynamic states can be tested.

During static testing, no more than 2 indoor testers are allowed

Before testing for planktonic bacteria, the user decides whether pre disinfection is necessary in the sterile room (area) being tested

During the testing of planktonic bacteria, the state used and the number of indoor testing personnel should be indicated.

5.3 Testing Time

5.3.1 During empty or static testing, for a unidirectional flow sterile room, the test should start no less than 10 minutes after the normal operation time of the purified air conditioning system. For non unidirectional sterile rooms, testing should begin at least 30 minutes after the purification system has been operating normally. During static b testing, for unidirectional flow cleanliness, it is recommended to start testing after production operators have evacuated the site and undergone 10 minutes of self-cleaning;; For non unidirectional sterile rooms, testing should begin after production operators have evacuated the site and undergone 20 minutes of self purification.

5.3.2 During dynamic testing, the start time of production and the testing time must be recorded.

5.4. Calculation of planktonic bacterial concentration

5.4.1 Number and arrangement of sampling points

5.4.1.1 Fewer sampling points

The sampling points for planktonic bacteria can refer to GB/T16292-2010

a) The measuring point in the work area is located about 0.8-1.5m above the ground (slightly higher than the working face).

b) The measuring point of the air supply outlet is located about 30cm away from the air supply surface.

c) Measurement points can be added at critical equipment or critical work activity areas.

5.4.2 Small sample size

The small sample size of planktonic bacteria is shown in Table 1.

Table 1 Small Sample Size

clean	sample size L/time
Level 100	1000
Level 10000	500
100000 level	100
300000 level	100

5.4.3 Sampling frequency

Each sampling point is usually sampled once.

5.4.4 Sampling precautions

5.4.4.1 For unidirectional flow sterile rooms or air supply ports, the sampling port of the sampler should face directly in the direction of the airflow; For non unidirectional sterile rooms, the sampling port should be facing upwards.

5.4.4.2 When arranging sampling points, at least try to avoid the return air outlet where dust particles are concentrated.

During sampling, the tester should stand on the downwind side of the sampling port and try to walk as little as possible.

5.4.4.4 Take all measures to prevent contamination during the sampling process and other possible contamination of the sample.

5.4.4.5 When the culture dish is used for inspection, in order to avoid the impact caused by the transportation or handling of the culture dish, negative control tests should be conducted simultaneously. One control dish should be taken from each time or area, and operated in the same way as the sampling dish but without exposing it. Then, it should be placed in the culture box together with the sampled culture dish (TSA or SDA) for cultivation, and the results should be sterile.

5.5 Records

The test report should include the following content

a) Name and address of the tester, testing date;

b) Test basis

c) Test the plane position of the sterile room (mark the plane position of adjacent areas if necessary);

d) Description of testing instruments and their testing methods: including testing environmental conditions, number and layout of sampling points, number of tests, sampling flow rate, or possible changes in testing methods, calibration certificates for testing instruments, etc; If it is a dynamic test, the number and location of on-site operators, as well as the number and location of on-site operating equipment, should also be recorded;

e) Result: Includes all statistical calculations.

5.6 Result Calculation

5.6.1 Use counting methods to determine the bacterial count of each culture dish.

The calculation of the average concentration of planktonic bacteria at each measuring point is shown in equation (1).

Average concentration (units/m3)=colony count/sampling volume??????????????? (1)

Example 1: If the sampling volume of a certain measuring point is 400L and the bacterial count is 1, then:

Average concentration=1/0.4=2.5 cells/m3

Example 2: If the sampling volume of a certain measuring point is 2m3 and the bacterial count is 3, then:

Average concentration=3/2=1.5 cells/m3

5.7 Result evaluation

5.7.1 The average concentration of planktonic bacteria at each measuring point must be lower than the limit of bacterial concentration in the selected evaluation criteria.

5.7.2 During static testing, if the average concentration of planktonic bacteria at a certain measuring point exceeds the evaluation criteria, it should be resampled twice. Only when both test results are qualified can it be judged as compliant.

5.8 Daily Monitoring

For the monitoring of planktonic bacteria, it is advisable to set correction limits and warning limits for the airflow on a section perpendicular to the airflow direction and parallel to the streamline in a single direction, to ensure that the microbial concentration in the sterile room is controlled. Regular checks on microbial load and disinfectant efficacy should be conducted, rather than propensity analysis. This method can be used for both static and dynamic monitoring.

For the sampling frequency of planktonic bacteria, if the following situations occur, it should be considered to be modified. In evaluating the following situations, the testing frequency of other items should also be determined:

---------------Continuously passing the correction and warning limits;

---------------The downtime is longer than expected;

--------------During production, any major maintenance of the air purification system is required;

--------------Regularly operating records to project biased data;

--------------Changes in disinfection procedures;

--------------Biological pollution, etc;

--------------When there are major repairs or additions to production equipment;

--------------When there are significant changes in the distribution of sterile room structural areas.

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