1、 Sample Transfer: Avoiding Pollution and Loss

When transferring samples from the sampler to processing containers (such as culture dishes, centrifuge tubes), it is necessary to simultaneously prevent external contamination and loss of target microorganisms:

Aseptic environment operation:

The transfer needs to be carried out in a biosafety cabinet. Before operation, disinfect the sampling head, container surface, and operator's hands with 75% alcohol; Use sterile straws and pipettes (with sterilized tips) to avoid contact with non sterile areas (such as the outer wall of the container).

Misconception tip: If operated in an open environment, miscellaneous bacteria in the air may mix into the sample, leading to an artificially high bacterial count, especially for low concentration samples.

Adequate elution of sampling solution:

For impact and sieve samplers, after sampling, the sampling head should be repeatedly rinsed with sterile eluent (such as 0.85% sterile physiological saline, phosphate buffered saline PBS) at least 3 times to ensure that the attached microorganisms completely enter the eluent; Vortex oscillation for 1-2 minutes (rotation speed of 2000-3000rpm) to break down microbial clusters and improve dispersion.

2、 Sample dilution: ensuring the accuracy of dilution multiples

When the concentration of microorganisms in the sample is high, gradient dilution is required to obtain a countable number of colonies (30-300CFU/dish), and the error in the dilution process will directly affect the accuracy of the results:

Dilution solution and container: Use sterilized gradient dilution tubes (such as 10mL capacity screw cap test tubes), pre install 9mL sterile dilution solution (with an error margin of ≤± 0.1mL); Clearly mark the dilution factor (10 ⁻¹, 10 ⁻², etc.) to avoid confusion.

Operating standards:

Ensure that the tip of the pipette/gun is in contact with 1-2cm below the liquid surface during pipetting to avoid inhaling air bubbles;

When diluting from high concentration to low concentration, each step needs to be mixed evenly (rub the test tube 10-15 times by hand) to ensure even dilution;

Make 2-3 parallel samples at the same dilution ratio to reduce random errors.

3、 Cultivation and Counting: Control Cultivation Conditions, Standardize Counting Standards

The growth of microorganisms is greatly influenced by cultivation conditions and requires strict standardization of parameters

Selection and preparation of culture medium:

Select the culture medium based on the target microbial type (such as nutrient agar for detecting bacteria and potato glucose agar PDA for fungi). The culture medium should be thoroughly sterilized (sterilized with high-pressure steam at 121 ℃ for 20 minutes) and have a pH value that meets the requirements (bacterial pH 7.2~7.4, fungal pH 5.6~5.8) to avoid microbial growth failure due to nutritional deficiencies or acid-base discomfort.

Cultivation condition control:

Temperature: Bacteria 36 ± 1 ℃, fungi 28 ± 1 ℃. The constant temperature incubator needs to be calibrated daily (error ≤ ± 0.5 ℃);

Time: Bacterial culture for 48 ± 2 hours, fungal culture for 72 ± 2 hours, to avoid colony counting due to too short culture or overlapping colonies due to too long culture.

Counting rules:

Select a plate count with a colony count of 30-300 CFU (below 30 results in large errors, above 300 results in easy overlap of colonies);

If the difference in bacterial count between parallel plates of the same dilution exceeds 1 times (such as 20 and 50 for plates of 10 ⁻ ³ dilution), retesting is required;

When counting, it is necessary to distinguish between target microorganisms and contaminating bacteria (which can be determined by colony morphology and staining microscopy).

4、 Quality control: Verify reliability throughout the entire process

Blank control experiment:

When processing each batch of samples, set the sampling blank (the sampler does not collect air, only washes the sampling head with eluent and cultivates it) and the operation blank (directly inoculates the sterile eluent for cultivation). If the number of blank colonies is greater than 5CFU, it indicates contamination and requires retesting.

Positive control and recovery verification:

Regularly simulate sampling with standard bacterial solutions of known concentrations (such as Escherichia coli and Staphylococcus aureus) and calculate the recovery rate (the recovery rate should be between 70% and 130%, otherwise the efficiency of the sampler or processing flow needs to be checked).

Instrument calibration:

The sampler needs to calibrate the flow rate regularly (with an error of ≤± 5%) to avoid sampling volume deviation caused by inaccurate flow rate;

Equipment such as pipettes and incubators must undergo annual metrological certification to ensure accurate parameters.