Sterile room floating bacteria test operating procedures

1.Scope

This standard specifies the test conditions and test methods for floating bacteria in sterile rooms and clean areas of the Pharmaceutical industry. This standard applies to the testing of floating bacteria and environmental verification in sterile rooms and clean areas of the pharmaceutical industry, sterile rooms or local air purification areas (including clean workbenches).

2.Reference standard: GB/T 16293.2010

Appendix to the Drug Production Management Standard (Revised in 1998).

3.Standard

4.Test method

4.1 Method summary

The method used in this specimen is the counting concentration method. That is, by collecting biological particles suspended in the air in a special culture medium (selecting a culture medium that can be proven to support the growth of microorganisms), after a certain period of time and under suitable growth conditions, let them multiply to visible colony counts to determine the microbial concentration of the clean room.

4.2 Personnel responsibilities and training

The test personnel of the sterile room should be trained in this profession and obtain the corresponding qualifications before they can perform the duties of Sterile room testing, which includes the relevant hygiene knowledge and basic microbiological knowledge.

The testers in the sterile room should choose a dressing style that is compatible with the air cleanliness level requirements of the production operation. Outside clothes cannot be brought into areas above Class 100,000.

4.3 Instruments, auxiliary equipment and culture medium

Select a suitable floating bacteria sampler, including an oil-free vacuum pump, a lower air flow rate and a larger sampling flow to ensure that the water distribution on the surface of the culture medium is blown dry. This test requires the following instruments, auxiliary equipment and culture medium:

• floating bacteria sampler;

• culture dish;

• high pressure sterilizer;

• culture medium;

• constant temperature incubator;

4.4 Principle of floating bacteria sampler

Plank bacteria samplers generally use the impact method mechanism and can be divided into slit samplers, centrifugal samplers or pinhole samplers. The slit sampler is inhaled by the internal fan, and the air is sprayed through the slit plate of the sampler to hit the surface of the slow plate culture medium. The attached live microbial particles form colonies after cultivation. Due to the high-speed rotation of the internal fan of the centrifugal sampler, the airflow enters from the front of the sampler and flows out from the back. Under the action of centrifugal force, the live microbial particles in the air have enough time to hit the special solid culture strip, and the attached live microbial particles form colonies after cultivation. The pinhole sampler is an airflow inhaled through a metal cover with dense and specially machined small holes on the cover. The fan directly hits the collected fine air on the surface of the flat culture medium, and the attached live microbial particles form colonies after cultivation.

4.5 Test points

a.The test instrument must be calibrated regularly according to the instrument calibration cycle. The instrument that has passed the calibration and is within the validity period should be used.

b.When the test instrument does not enter the test area, if necessary, clean the surface first, or prepare and store it in the corresponding sterile room (use a protective device or other appropriate external protective instrument.

c.When using paper in a Class 100 sterile room, it should be covered with a transparent dust-free cover. Do not use pencil erasers in a Class 100 sterile room.

d.When using the test instrument, operate it strictly in accordance with the instructions.

• After the instrument is turned on and preheated to stability, it can be calibrated according to the instructions. At the same time, check the sampling flow and set the time according to the sampling flow.

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

• The sampling tube must not be oozing and the inner wall should be smooth.

• The length of the sampling tube should be determined according to the height of the measuring point, and the bending should be minimized.

4.6 Generally, a 90mmx15mm culture dish is used. The appropriate culture dish can be selected according to the selected sampler.

The centrifugal sampler uses a special solid culture strip

Culture medium

4.7 Soybean casein agar (TSA) or Sabouraud culture medium (SDA) or other user-approved and verified culture medium.

4.8 Constant temperature incubator

The constant temperature incubator must be regularly tested

4.9 Test steps

The surface of the instrument and culture dish must be strictly disinfected before testing.

• The sampler should be sterilized with disinfectant in the disinfection room before entering the room to be tested. The sampler used in the 100-level sterile room should be placed in the room to be tested in advance.

• Wipe the outer surface of the culture dish with disinfectant.

• Before sampling, disinfect the top cover, turntable and the inside and outside of the sampler with disinfectant. After sampling, gently spray the inner wall of the turntable and the sampling port and sampling tube with disinfectant. They must be sterilized at high temperature before use.

• If disinfectants are used to disinfect the outer and inner walls of the sampling tube, the residual liquid in the tube should be poured out and dried.

• The sampler should wear work clothes corresponding to the clean area to be tested. Before placing or replacing the culture dish on the turntable, disinfect both hands with disinfectants or wear sterile gloves.

• After the sampling instrument is disinfected, do not put the culture dish in it first. Turn on the floating bacteria sampler to allow the residual disinfectant in the instrument to evaporate for no less than 5 minutes, check the flow and adjust the set time according to the sampling volume.

• Close the floating sampler, put in the culture dish, and cover it.

• After placing the sampling port at the sampling point, open the floating bacteria sampler for sampling.

4.10 Culture

After all sampling is completed, the culture dish is inverted and placed in a constant temperature incubator for culture. The culture dish prepared with soybean casein agar medium (TSA) is cultured in a 30℃~35℃ incubator after sampling for not less than 2 days; the culture dish prepared with Sabouraud culture medium (SDA) is cultured in a 20℃~25℃ incubator after sampling for not 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.

4.11 Colony count

• Directly count, mark or count all colonies on the culture dish with the naked eye 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 the plate, they should still be counted as 2 or more colonies when they can be distinguished.

4.12 Notes

• Before use, carefully check the quality of each culture dish. Culture medium and culture dish that are deteriorated, damaged or contaminated cannot be used.

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

• Since there are many types of bacteria and they vary greatly, when counting, it is generally necessary to observe carefully on the surface or true surface of the culture dish with transmitted light. Do not miss the colonies growing on the edge of the culture dish and distinguish the colonies or culture medium sediments. Use a microscope for identification when necessary.

5. Test rules

5.1 Test conditions

Before testing, the relevant parameters of the sterile room should be pre-tested. Such tests will provide the environmental conditions for the test county floating particles, for example: This pre-test may include

• Temperature and Relative humidity test. The temperature and relative humidity of the sterile room should be consistent with its production and process requirements (if there are no special requirements, the temperature should be controlled at 18℃~26℃, and the relative humidity should be controlled between 45%~65%. At the same time, it should meet the use range of the test instrument;

• Test of indoor air supply volume or Wind speed, or pressure difference test.

• Leakage test of high-efficiency filter.

5.2 Test state

Both static and dynamic states can be tested.

During static test, there should be no more than 2 testers in the room

Before the test of floating bacteria, the user decides whether the sterile room (area) to be tested needs to be disinfected in advance. When testing for floating bacteria, the state used during the test and the number of testers in the room should be indicated.

The minimum sampling volume of floating bacteria each time is shown in the table

5.3 Test time

• In the empty state or static test, for the unidirectional flow sterile room, the test should start at least 10 minutes after the purification Air conditioning system has been operating normally. For non-unidirectional flow sterile rooms, the test should start at least 30 minutes after the purification system has been operating normally. In the static b test, for the unidirectional clean room (area), the test should start after the production operators evacuate the site and self-clean for 10 minutes; for the non-unidirectional sterile room, the test should start after the production operators evacuate the site and self-clean for 20 minutes.

• In the dynamic test, the time of production start and the test time must be recorded.

5.4 Calculation of floating bacteria concentration

1.Number of sampling points and their arrangement

2.Minimum number of sampling points

The location of the sampling points for floating bacteria can refer to GB/T16292-2010

• The measuring point in the working area is about 0.8~1.5m from the ground (slightly higher than the working surface).

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

• Additional measuring points can be added at key equipment or key work activity range.

3.Number of sampling times

Each sampling point is generally sampled once.

4.Sampling precautions

• For unidirectional sterile rooms or air outlets, the sampling port of the sampler should face the direction of the airflow; for non-unidirectional sterile rooms, the sampling port should face upward.

• When arranging the 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 move as little as possible.

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

• When the culture dish is used for inspection, in order to avoid the impact of the transportation or moving process of the culture dish, it is advisable to carry out a negative 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 expose the sampling dish, and then put it into the incubator together with the sampled culture dish (TSA or SDA) for culture. The result should be no colony growth.

5.5 Records

The test report should include the following:

• Name and address of the tester, test date;

• Test basis

• Plane location of the test sterile room (mark the plane location of adjacent areas if necessary):

• Description of the test instrument and its test method: including test environment conditions, number of sampling points and layout diagram, number of tests, sampling flow, or possible changes in test methods, test instrument calibration certificate, etc.; if it is a dynamic test, the number and location of on-site operators, on-site operating equipment and number and location should also be recorded;

• Results: including all statistical calculation data.

5.6 Result evaluation

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

During static testing, if the average concentration of planktonic bacteria at a measuring point exceeds the evaluation standard, the sample should be resampled twice, and the results of both tests must be qualified to be judged as compliant.

5.7 Daily monitoring

For the monitoring of floating bacteria, it is advisable to set the correction limit and warning limit of the airflow speed on the section parallel to the streamline and perpendicular to the airflow direction in a single direction to ensure that the microbial concentration in the humid room is controlled. The microbial load and the effectiveness of the disinfectant should be checked regularly, not for trend analysis. This method can be used for both static and dynamic monitoring.