Cleanroom Microbial Testing Methods

1 Scope of application



Suitable for microbiological testing of settling bacteria, planktonic bacteria workbench, and worker hands in dust-free workshops.



2 Definition



2.1 Planktonic bacteria: Live microbial particles suspended in the air, propagated to visible bacterial activity using specialized instruments and specialized culture media under suitable growth conditions.



2.2 Settling bacteria: Live microbial particles in the air that propagate to visible bacterial activity under suitable growth conditions through specialized culture media.



2.3CFU: Colony forming unit.



3 Testing methods



3.1 Preparation of reagents/culture media



3.1.1 According to the sampling quantity, prepare 0.9% physiological saline and soy casein agar medium according to the specified requirements. Divide the prepared medium into triangular bottles, seal them with rubber stoppers, and wrap them in white paper; Transfer 10ml of 0.9% physiological saline into a test tube using a pipette, seal it with a stopper, and wrap it in white paper. Wrap the corresponding number of pipettes and cotton swabs in place; Put the petri dish into a stainless steel disinfection tube, and sterilize all items with high-pressure steam at 121 ℃ for 15 minutes;



3.1.2 After sterilization, part of the culture medium will be poured into a petri dish to detect planktonic and settling bacteria. If not used on the same day, it should be stored at a low temperature of 2-8 ℃. Place a portion of the culture medium in a constant temperature water bath at 45 ± 5 ℃, and allow the physiological saline tube to cool down before sampling in a dust-free workshop;



3.2 Sampling steps for settling bacteria are as follows:



3.2.1 Mark the solidified culture dish according to the sampling position;



When arranging sampling points, efforts should be made to avoid return air vents with concentrated dust particles;



3.2.3 Place the culture dish on a workbench about 0.8-1.5m above the ground, remove the lid of the dish, and expose the culture medium to the air for sampling for 30 minutes;



3.2.4 After all sampling is completed, cover the culture dish and invert it.



3.2.5 When using culture dishes for testing, in order to avoid the impact of transportation or handling of culture dishes, negative control tests should be conducted simultaneously. One control dish should be taken from each time or area, operated in the same way as the sampling dish but without exposing it for sampling. Then, the culture dish should be placed together with the sampled culture dish in the culture box for cultivation, and the results should be sterile.



3.3 Sampling steps for planktonic bacteria are as follows:



Before sampling, use disinfectant to disinfect the top cover, turntable, and inner and outer sides of the sampler;



3.3.2 Turn on the planktonic bacteria sampler, evaporate the residual disinfectant in the instrument for no less than 5 minutes, check the flow rate, and adjust the sampling parameters according to the sampling amount. Each sampling point should sample no less than 500L;



When arranging sampling points, the sampling point position should be about 0.8-1.5m above the ground, and the measuring point position of the air supply outlet should be about 30cm away from the air supply surface;



3.3.4 After disinfecting the hands with disinfectant, place the culture dish into a turntable, cover it with a porous sampling head cover, remove the protective cover, and open the planktonic bacteria sampler for sampling;



3.3.5 After the sampling is completed, mark the culture dishes that have been sampled;



3.3.6 After all sampling is completed, gently spray disinfectant on the inner wall of the instrument cover and the turntable;



When using culture dishes for testing, in order to avoid the impact of transportation or handling of culture dishes, negative control tests should be conducted simultaneously. One control dish should be taken from each time or area, operated in the same way as the sampling dish but without exposing the sample. Then, the culture dish should be placed together with the sampled culture dish in the culture box for cultivation, and the results should be sterile and grow.



3.4 Microbial sampling of workers' hands shall be carried out according to the following steps:



3.4.1 Mark the sterilized physiological saline tube;



3.4.2 Disinfect the hand with disinfectant, remove the sterilized cotton swab, open the sterilized physiological saline tube plug, immerse the cotton swab in the sterilized physiological saline, wait for the cotton swab to be wet, then gently press on the test tube wall several times to remove excess water;



3.4.3 Require the tested person to hold their five fingers together and apply a cotton swab soaked in disinfectant physiological saline back and forth on their hands 10 times, covering the entire palm area;



3.4.4 Remove the part of the cotton swab that comes into contact with the hand, drop the head of the cotton swab into a sterile saline tube, and send it for testing;



3.4.5 After sampling each sampling point, disinfect the hand scissors and hands with disinfectant.



After the sampling is completed, take another cotton swab and remove the part that comes into contact with the hand. The head of the cotton swab should be dropped into a sterile saline tube as a negative control.



3.5 Microbial sampling on the workbench should be carried out according to the following steps:



3.5.1 First, mark the sterilization physiological saline tube with a marking number corresponding to the worker's hand being taken;



3.5.2 The experimenter should wear sterile gloves, disinfect their hands and positioning frame with disinfectant, and if necessary, burn the positioning frame with an alcohol lamp to accelerate the evaporation of alcohol;



3.5.3 Select the sampling position and fix the positioning bracket;



3.5.4 Take out the sterilized cotton swab, open the sterilized physiological saline tube plug, immerse the cotton swab in the sterilized physiological saline, wait for the cotton swab to be soaked, and gently press on the test tube wall several times to remove excess water;



3.5.5 Use a cotton swab to apply back and forth in the positioning frame 10 times;



3.5.6 Remove the part of the cotton swab that comes into contact with the hand, drop the head of the cotton swab into a sterile saline tube, and send it for testing;



3.5.7 After sampling at each sampling point, disinfectants should be applied to the hands, positioning brackets, and scissors for disinfection.



After the sampling is completed, take another cotton swab and remove the part that comes into contact with the hand. The head of the cotton swab should be dropped into a sterile saline tube as a negative control.



3.6 Microbial testing after sampling



3.6.1 For sampling in a culture dish, invert the dish and incubate at 34 ± 1 ℃ for 24-48 hours, then count;



For sampling with a sampling tube, sonicate the tube in an ultrasonic cleaning machine for 5 minutes, mix well, and then dilute the diluted solution to a concentration of 10 times on a clean workbench; Take 1ml of diluted solution from a sterilized pipette and inject it into a sterile petri dish. Immediately pour the culture medium, mix well, and solidify. Pour 2 plates into each dilution, invert at 34 ± 1 ° C for 24-48 hours, and count;



3.7 Result determination



3.7.1 The bacterial count on the workbench should be ≤ 10cfu/cm2 (i.e. ≤ 250cfu/25cm2);



3.7.2 The hand bacterial count of workers should be ≤ 300cfu/hand;



3.7.3 The number of settling bacteria at 100000 levels should be ≤ 10 per dish;



The concentration of planktonic bacteria at 100000 levels should be ≤ 500 cells/m3 (≤ 125 cells/250L);