File name: Standard operating procedures for clean room airborne bacteria detection					Document number:ZL/SOP/ZK/02300
Prepared by:			date: _{Year Month Day}		Document Type: Work Standard
Reviewed by:			date: _{Year Month Day}		_{Edition: First Edition}
Approved by:			date: _{Year Month Day}		Print quantity: 5 copies
Effective Date:			Year Month Day		Issuing Department: General Office
Distributed to: Vice President of Quality, Quality Management Department, QC Inspection Room, General Office
Change Record	Revision No.	Reviser	Approval Date	Effective Date	Reason and Purpose

1. Purpose

To establish a standard operating procedure for the detection of floating bacteria to standardize the detection operation of employees.

2. Scope

Applicable to the detection of floating bacteria in the company's internal production clean area.

3. Responsibilities

The production department, production workshop, quality management department, QC inspection room director, and inspectors are responsible for the implementation of this procedure.

4. Testing basis

GB/T16293-1996 (Test method for floating bacteria in clean rooms of pharmaceutical industry)

5. Contents

5.1 Instruments and materials

Floating bacteria sampler
Vacuum pump
Petri dish
Ordinary broth culture medium
Constant temperature incubator

5.2 Test steps

a. Before testing, the surface of the instrument and petri dish must be strictly disinfected.

The sample should be sterilized with the disinfectant in the disinfection room before entering the test room. The sampler used in the Class 100 Clean room should always be placed in the test room.
Wipe the outer surface of the culture dish with disinfectant.
Before sampling, disinfect the top cover, turntable and the inside and outside of the cover of the sampler with disinfectant. After sampling, gently spray the inner wall of the cover and the turntable with disinfectant.
The sampling port and sampling tube must be sterilized at high temperature before use. If the outer and inner walls of the sampling tube are disinfected with disinfectant, 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 disinfectant.

b. Sampling procedure

After the instrument is disinfected, do not put the culture dish in it first. Start the vacuum pump to exhaust air to evaporate the residual disinfectant in the instrument for no less than 5 minutes, and adjust the flow rate and turntable speed.
Turn off the vacuum pump, put in the culture dish, cover the lid and adjust the height of the sampler gap. 5.2.2.3 After placing the sampling port at the sampling point, turn on the sampler, vacuum pump, and rotating timer in turn, and set the sampling time according to the sampling volume.

c. Cultivation

After all sampling is completed, place the culture dish upside down in a constant temperature incubator for cultivation.
Cultivate in a 30~35℃ incubator 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 dishes can be selected for control culture in each batch.

d. Colony counting

Count directly with the naked eye, mark or count 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 are still counted as 2 or more colonies when they can be distinguished.

5.3 Precautions

The sampling port of the sampler must be made of materials that are easy to disinfect and have stable chemical properties.
The sampling tube is strictly prohibited from leaking, 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.
The quality of each culture dish should be carefully checked before use. Culture medium and culture dish that are deteriorated, damaged or contaminated cannot be used.
During operation, the contamination of the sampling tube and other human contamination of the sample should be prevented.
Detailed records of culture medium, culture conditions and other parameters should be made.
Since there are many types of bacteria and they are very different, when counting, transmitted light is generally used to carefully observe the back or bottom 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 for identification when necessary.

5.4 Test rules

a. Test status

Before the floating bacteria test, the temperature and humidity of the clean room (area) to be tested must meet the specified requirements, and the static pressure difference, ventilation frequency, and air flow rate must be controlled within the specified values.
Before the floating bacteria test, the clean room (area) to be tested has been disinfected.
There are two test states: static and dynamic. The choice of test state must meet the production requirements and the test state must be noted in the report.

b. Testers

Testers must wear work clothes that meet the environmental level.
During static testing, there must be no more than 2 testers in the room.

c. Test time

For unidirectional flow, such as Class 100 clean rooms and laminar flow workbenches, the test should start after the clean Air conditioning system has been operating normally for at least 10 minutes.
For non-unidirectional flow, such as Class 10000 and above clean rooms, the test should start after the Clean air conditioning system has been operating normally for at least 30 minutes.

d. Calculation of floating bacteria concentration

1.Number of sampling points and their arrangement

①Minimum number of sampling points: The minimum number of sampling points for floating Bacteria testing is divided into two situations: daily monitoring and environmental verification. See the table below for details.

aera	Cleanliness level
	100		10000		100000
	verify	monitor	verify	monitor	verify	monitor
<10	2~3	1	2	1	2	_
≥10<20	4	2	2	1	2	_
≥20<40	8	3	2	1	2	_
≥40<100	16	4	4	1	2	_
≥100<200	40	_	10	_	3	_
≥200<400	80	_	20	_	6	_
400	160	_	40	_	13	_
Note: (1) The area in the table refers to the surface area of the air outlet for Class 100 unidirectional cleanrooms (including laminar flow workbenches); for Class 10000 and 100000 non-unidirectional cleanrooms, it refers to the room area. (2) The number of sampling points for daily monitoring is determined by the key operating points of the production process.

②Location of sampling points

For each Class 100 clean operation area (such as laminar flow hood, laminar flow workbench), a measurement point can be set 30cm away from the drug opening.
For each Class 10000 clean work area (such as the drug opening work area), a measurement point can be set at the work surface.
The air outlet measurement point is located about 30cm away from the air supply surface.
Measuring points can be added at key equipment or key work activity ranges.

2.Minimum sampling volume

The sampling volume is determined according to daily testing and environmental verification. The minimum sampling volume for each time is shown in the table below.

Cleanliness level	Sampling volume, L
Cleanliness level	Daily monitoring	Environmental Verification
100level	600	1000
10000level	400	500
100000level	50	100

3.Number of sampling times: Each sampling point is generally sampled once.

e. Sampling precautions

For unidirectional air supply outlets, the sampling tube mouth of the sampler should face the airflow direction; for non-unidirectional air flow, the sampling tube mouth is upward.
When arranging the sampling points, they should be at least 1m away from the return air outlet where dust particles are concentrated.
When sampling, the tester should stand on the downwind side of the sampling port.