SOP for Culture Dilution Preparation

1.0 Purpose:

To lay down a procedure for culture dilution preparation of selected microorganisms to obtain a known number of colony forming unit.

2.0 Scope:

This procedure is applicable for culture dilution preparation used for the positive control and growth promotion test in microbiology Laboratory.

3.0 Responsibilities:

Microbiologist : To perform and maintain the procedure as per SOP.

Head-QC : To review and implementation and ensure compliance of SOP.

Head-QA : To approve, implement and ensure compliance of SOP.

4.0 Procedure:

4.1 Prepare 24 hour old broth culture for bacterial culture in soybean casein digest medium or nutrient broth and 48 hours old culture of yeast or fungi in soybean casein digest medium or sabouraud dextrose broth by transferring a loopful of desired microorganism from validated slant.

4.2 Incubate tubes at the specified temperature (for bacteria 30-35°C and for yeast or fungi 20-25°C) and time. Check the growth in the test tube of the culture.

4.3 After completion of incubation take out a preincubated set of sterile sodium chloride peptone buffer solution pH 7.0 containing 9 ml of volume.

4.4 Transfer the culture and tubes in testing area & make a serial dilution of each culture under experiment from 10 –1 to 10-10. (Transfer 1ml of culture in to 9ml buffer tube.)

4.5 Transfer 1 ml of serially diluted cultures to petriplates in duplicate. (Microbiologist depending upon experience selects the dilution for pour plating). Select three or more dilutions to get measurable count on a plate.

4.6 Add 15-20 ml of sterile melted Soyabean casein digest agar in bacterial Cultures & Sabouraud Dextrose agar in fungal cultures.

4.7 Keep the negative control to test the sterility of the medium.

4.8 Allow the plates to solidify & incubate the plates at 30-35°C for 24 to 72 hours for bacterial cultures and 20-25°C for 72-120 days for fungal cultures.

4.9 Transfer the set of diluted culture to refrigerator at 2-8°C immediately after addition of cultures to petriplates to inhibit the further multiplication.

4.10 After completion of incubation period count the number of colonies.

4.11 For calculation select the plate of corresponding dilution and showing the number of colonies NMT 100. Record the observation.

4.12 Preserve the dilutions of each culture from which 10-100 CFU/ml is obtain.

4.13 The culture should be used within 15 day from the date of preparation.

4.14 Discard the remaining unwanted diluted culture.

4.15 Acceptance Criteria: NMT 100 CFU/ml.

4.16 Label selected culture suspension with name of culture, Generation No., prepared by and valid up to date.

4.17 Selected suspension shall be preserved up to 15 days for routine use in microbial testing.

4.18 Selected suspension should not kept outside of refrigerator for prolong period.

4.19 Precautions:

Avoid exposure of culture suspension to UV light.

Dispose the old culture suspension after decontamination.