SOP for Preparation of Culture Dilutions for Analysis

1.0 OBJECTIVE

To lay down the procedure for preparation of Culture dilutions.

2.0 SCOPE

This SOP shall be applicable to Quality Control Dept.

3.0 RESPONSIBILITY

Microbiologist.

4.0 ACCOUNTABILITY

Head Quality Control Department.

5.0 PROCEDURE

5.1 Serial Dilution

5.1.1 For the purpose of preparing appropriate microorganism from the weekly- transferred working culture slant, inoculate a loopful of culture to 10ml of SCDB for Bacteria & Fungi.
5.1.2 Incubate the bacterial suspension at 32.5 ± 2.5°C for 24 to 48 hours & fungal suspension at 22.5 ± 2.5°C for 24 to 72 hours for enrichment.
5.1.3 Serially dilute the enriched suspension in 0.9% sterile saline solution in a ratio of 1: 10 dilution by transferring & thoroughly mixing each dilution to yield count of 10 – 100 CFU/ml.
5.1.4 Check the no. of CFU/ml from the above-prepared dilutions by transferring 1 ml of the inoculum on Soybean casein digest agar plate for Bacteria and on Sabouraud dextrose agar plate for Fungi by pour plate method. Incubate the plates.
5.1.5 On observation of visible microbial growth on each plate, count & record the average number of CFU in Preparation of culture suspension register.
5.1.6 Calculate the amount of inoculums or the appropriate dilution containing colony forming units in the range of 10 – 100 CFU/ ml.

5.2 Storage & Usage

5.2.1 Store the appropriate dilution of 10 – 100 CFU/ ml of culture suspensions in refrigerator, use it for one week & discard (from date of preparation) as per SOP for disposal of media and record in the format.
5.2.2 Take 1 ml of the above prepared culture suspension for positive control, (for environmental monitoring, MLT and fertility test).

6.0 ABBREVIATIONS

6.1 CFU: Colony Forming Unit
6.2 SCDA: Soybean Casein Digest Agar
6.3 SDA: Sabouraud Dextrose Agar
6.4 SCDB: Soybean Casein Digest Broth
6.5 MLT: Microbial Limit Test
6.6 NaCl: Sodium Chloride
6.7 QC: Quality Control
6.8 SOP: Standard Operating Procedure

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