Sterile Room Airborne Bacteria Test Standard Operating Procedure

Purpose

Establish standard operating procedures for the test of airborne bacteria in sterile rooms and standardize the detection of airborne bacteria in sterile rooms.

Scope

Applicable to the test and environmental verification of airborne bacteria in sterile rooms (including clean workbenches).

Responsibilities

OAQC personnel shall comply with the implementation.

Contents

1.Reference standard: GBIT16293-2010

2.Test method

This test method is a counting concentration method, which is to collect biological particles floating in the air and place them in a special culture medium (select a culture medium that can prove that it can support the growth of microorganisms) for a period of time and under suitable growth conditions to allow them to grow to a visible colony count to determine the microbial concentration of the sterile room. Basic],

3.Test instruments, auxiliary equipment and culture medium

Floating bacteria sampler, culture dish (culture dish suitable for the instrument), culture medium, constant temperature incubator, high pressure steam sterilizer 4.4 Test state and test time Both static and dynamic states can be tested. During static testing, there shall be no more than 2 testers in the room.

4.During static a test, for unidirectional flow sterile room (area), the test shall start after the purification air conditioning system has been operating normally for at least 10 minutes; for non-unidirectional flow sterile room (area), the test shall start after the purification Air conditioning system has been operating normally for at least 30 minutes. During static b test, for unidirectional flow sterile room (area), the test shall start after the production operators have evacuated the site and self-cleaned for 10 minutes; for non-unidirectional flow sterile room (area), the test shall start after the production operators have evacuated the site and self-cleaned for 20 minutes.

5.Determination of sampling points
Minimum number of sampling points

The sampling points should be arranged evenly to avoid too sparse sampling points in local areas. The sampling points should be arranged in accordance with the requirements of the layout diagram of the workshop cleanliness monitoring sampling points in the Appendix xxx of the "Clean area environmental monitoring management Regulations". Each sampling point is generally sampled once.

The sampling point in the work area is about 0.8m~1.5m above the ground (the road is higher than the working surface), and the air outlet measurement point is about 30cm away from the air supply surface. Additional measurement points can be added at key equipment or key work activities.
Minimum sampling volume

6.Test preparation

Clean the surface of the sampler before entering the tested area, then disinfect it with 75% alcohol or store it in the tested area and disinfect it together with the tested area (the sampler used in the Class A sterile room must be placed in the tested room in advance).

Disposable culture dishes are used in the A/B grade area. Other areas shall prepare culture dishes in advance on the clean workbench in the inspection room according to the requirements of aseptic operation (dissolve the sterilized culture medium in a water bath, cool to about 50-60℃, pour about 20ml of culture medium into each 90mm culture dish, and pour about 30ml of culture medium into each 150mm culture dish). The culture dishes are covered and placed at room temperature until solidified. The homemade culture dishes shall not be stored in the sterile room for more than 48 hours, and the culture dishes stored at 2~8℃ shall generally not exceed 1 week. Disposable culture dishes shall be used within the marked validity period and shall not be used after the expiration date.

The culture dishes used in the A/B grade area shall be brought in once before the regional disinfection, disinfected together with the environment, temporarily stored on the shelf in the disinfectant taking room, and used within the validity period. If temporary introduction is required, it must be brought into the B grade area through the C grade and B grade transfer window before the ozone disinfection the day before, and used the next day. The surface must also be wiped with 75% alcohol before introduction. The culture dishes used in other areas shall be brought in and used on the same day according to the requirements for the introduction of items.

Before sampling, clean the top cover, turntable and the inside and outside of the cover of the sampler with 75% alcohol (the sampling head of the FKC type floating air dust sampler is cleaned with alcohol).

For specific test operations, see "JQ-Ⅱ type floating bacteria sampler standard operating procedures or FKC-1 type floating air dust sampler standard operating procedures"

After sampling, cover the culture dish lid, mark all information on the culture dish (including product batch number, placement, etc.), tie the culture dish in an appropriate way, and invert it in a 30~35℃ constant temperature incubator for incubation for no less than 2 days.

After the incubation, check the culture dishes one by one, count and mark all the colonies on the culture dishes 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 dish, they are still counted as 2 or more colonies when they can be distinguished. 47 The test record should include the following contents

7.Name of the tested area, test date, test basis, name of the tester, description of the test instrument and test method, the state used during the test and the number of indoor testers, number of sampling points, number of tests, sampling flow, test results (including all statistical calculation data), etc.

If it is a dynamic test, the number of on-site operators and the operation of on-site equipment should also be recorded.

8.Result calculation

• The number of colonies in each culture dish is obtained by counting method.

• The average concentration of planktonic bacteria at each measuring point is calculated according to the following formula

9.Result evaluation

• The average concentration of planktonic bacteria at each measuring point must be lower than the limit 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.

• If the specified value is exceeded, the production department must be contacted in time to disinfect or otherwise treat the area, and then retest until it is qualified.

10.Daily monitoring

Set correction limits and warning limits for daily monitoring of floating bacteria (see the "clean area Environmental Monitoring Management Regulations" for the limit range and sampling frequency) to ensure that the microbial concentration in the sterile room (area) is controlled, and regular testing is performed to check the microbial load and the effectiveness of the disinfectant, and to perform trend analysis. This method can be used for both static and dynamic monitoring.

For the sampling frequency of floating bacteria, if the following situations occur, modification should be considered. After evaluating the following situations, it should also be determined that the detection frequency of other items exceeds the correction limit and warning limit continuously;

• The downtime is longer than expected

• Contamination is found in the key area

• Any major maintenance of the air purification system during production

• Daily operation records reflect trending data

• Changes in disinfection procedures

• Accidents causing biological contamination, etc.

• When production equipment has major maintenance or equipment is added

• When there are major changes in the structure or regional distribution of the sterile room (area)

11.Precautions

• After disinfection, do not put the culture dish into the JYQ-Ⅱ floating bacteria sampler first, open the floating bacteria sampler, and allow the residual disinfectant in the instrument to evaporate for no less than 5 minutes, check the flow rate and adjust the set sampling time according to the sampling volume.

• For unidirectional flow sterile rooms (areas) or air outlets, the sampling port of the sampler should face the direction of the airflow. For non-unidirectional flow sterile rooms (areas), the sampling port should face upward.

• During the test, sterile clothing that meets the cleanliness level requirements of the tested area and has been sterilized by high pressure must be worn.

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

• After sampling, 75% alcohol is required to spray the inner wall and turntable of the sampler cover (FKC type floating air dust sampler spray sampling head and protective cover).

• All measures should be taken to prevent contamination during the sampling process and other possible contamination of the sample.

• 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 each batch as control culture. When the culture dishes are used for testing, in order to avoid the impact caused by the transportation or moving process of the culture dishes, it is advisable to carry out a 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 need to expose the sample, and then put it in the incubator together with the sampled culture dish for culture. The result should be no colony growth.

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

• Due to the wide variety of bacteria and great differences, when counting, it is generally necessary to use transmitted light to observe carefully on 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 or culture medium sediments. Use a microscope to monitor if necessary.

• The constant temperature incubator must be calibrated regularly.