The general procedure for performing the microbial limit test (MLT) is as follows:
1. Prepare the sample by diluting, grinding, or filtering as necessary to obtain a suitable test suspension.
2. Perform the microbial enumeration test by inoculating a known volume of the test suspension onto the appropriate culture media, such as:
- soybean casein digest agar (SCDA) for bacteria.
- sabouraud dextrose agar (SDA) for fungi.
3. Incubate the plates at 30-35°C for bacteria and 20-25°C for fungi for 3-5 days.
4. Count the number of colonies and calculate the number of colony forming units (CFU) per gram or milliliter of the sample.
5. Perform the test for specified microorganisms by inoculating a known volume of the test suspension onto the selective and differential media for the target microorganisms, such as:
- MacConkey agar for E. coli
- Xylose lysine deoxycholate agar (XLD) for Salmonella
- Mannitol salt agar (MSA) for S. aureus
- Cetrimide agar for P. aeruginosa
- Chromogenic candida agar (CCA) for C. albicans
6. Incubate the plates at 30-35°C for bacteria and 20-25°C for fungi for 18-48 hours.
7. Observe the plates for the characteristic growth and color of the target microorganisms and confirm their identity by biochemical or molecular tests if necessary.
8. Compare the results of the MLT with the acceptance criteria for the product, which are based on:
- The intended use
- The route of administration
- The potential hazard to the user
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