In the process of microbial sampling, cross contamination may arise from equipment residue, personnel operation, environmental microorganisms, or mutual contamination between samples, and strict pollution prevention measures need to be implemented throughout the entire process. The following are standardized operation guidelines and technical points for different scenarios:
1、 Sterilization of equipment and consumables before sampling
1. Hardware sterilization specifications
Reusable devices:
Metal sampling head (such as impact sampler): Sterilize with high-pressure steam at 121 ℃ for 20 minutes (GB/T38512-2020), or burn with 70% ethanol and flame (suitable for on-site emergency handling).
Plastic components (such as sampling pump tubing): Soak in 0.1% peracetic acid for 30 minutes, rinse with sterile water three times, air dry, and then irradiate with ultraviolet light (254nm, ≥ 30 minutes).
Disposable consumables:
Cultivation dishes, sampling bags, etc. should be sterilized by irradiation (cobalt-60 irradiation, dose ≥ 25kGy), and the packaging integrity should be checked before opening (ISO11607-1 standard).
2. Validation of sterilization effect
Perform biological monitoring after each sterilization: Place thermophilic Bacillus subtilis ATCC7953 spore strips, culture (56 ℃, 48 hours) after sterilization, and the colony count should be 0 (GB8599-2021).
2、 Anti pollution system for sampling site operations
1. Personnel aseptic operation guidelines
Level 3 protection requirements:
Normal environment: Wear disposable lab coats and medical gloves (such as nitrile gloves), and wipe the surface of the gloves with 75% ethanol before sampling.
High risk environment (such as clinical samples): Sterile isolation gowns, goggles, and double-layer gloves should be worn, and the outer gloves should be changed every time a sample is taken.
Action standards:
When sampling, avoid crossing over the sampling port with your arms, keep the sampling head at least 30cm away from your body, and keep your breathing direction at a 90 ° angle to the sampling port.
It is prohibited to tidy up hair or touch the face within the sampling area. If equipment adjustment is required, gloves must be replaced or hands disinfected first.
2. Space partitioning and airflow control
Sampling area division:
Set up a sterile operation area of 50cm x 50cm, spray the tabletop with 75% ethanol to form a disinfection belt, and place the sampler in the center of the area.
High cleanliness scenarios (such as ISO5 cleanrooms) require operation in laminar flow workbenches (wind speeds of 0.36-0.54m/s) or biosafety cabinets (BSC-II level).
Airflow interference control:
Close doors and windows before sampling, stop the air conditioning system from running for 10 minutes (to avoid external microorganisms being carried by the airflow), and keep personnel walking within 1 meter.
3、 Isolation measures during sample collection process
1. Batch sampling anti cross scheme
Batch interval disinfection:
After completing one sample sampling, wipe the sampler housing, sampling port, and operating table with 75% ethanol for 5 minutes before proceeding to the next sample collection.
If collecting different types of samples (such as environmental samples and clinical samples), it is necessary to replace the entire set of sampling heads and pipelines to avoid confusion of residual microorganisms.
2. Isolation techniques for special samples
High concentration sample processing:
When dealing with high load samples such as sewage and feces, use a sampler with negative pressure filtration (such as MilliporeEZ Pak), seal the filter element immediately after sampling to prevent aerosol diffusion.
Sampling of pathogenic bacteria:
Using a biosafety sampling head (such as a closed sampler with HEPA filtration), the sample is placed in a leak proof metal canister (lined with absorbent paper and 0.5% sodium hypochlorite solution) after sampling.