1.0 OBJECTIVE:
To lay down the procedure for preparation of culture dilutions.
2.0 SCOPE:
This SOP is applicable to preparation of culture dilutions in the microbiology laboratory.
3.0 RESPONSIBILITY:
Microbiologist: To perform the procedure as per SOP.
Head – Quality Control: To ensure the compliance of the SOP.
4.0 PROCEDURE:
4.1 Serial dilution:
4.1.1 From the working culture slant , take a loopful of organism and inoculate into 100ml of Soya bean casein digest medium / Fluid lactose medium and incubate for 24 hrs. Transfer 1 ml of the 24 hrs old broth culture into 10 ml of 0.9% sterile saline solution.
4.1.2 Serially dilute above inoculated saline solution (10 fold serial dilution) to yield of 10 – 100 cfu/ml. Negative control should be maintained separately.
4.1.3Check the no. of cfu/ml from the above prepared dilutions by transferring 1.0 ml of the inoculum into 20ml of molten Soyabean casein digest Agar plate for Bacteria and on Sabouraud dextrose agar/ Sabouraud chloramphenicol agar for Fungi. Incubate the Soyabean casein digest Agar plates at 32.5 ? 2.5°C for 24 to 48 hours & Sabouraud dextrose agar plate at 22.5 ? 2.5°C for 48 to 72 hours. Negative control of both the media should be maintained.
4.1.4 On observation of visible microbial growth on each plate, count & record the average number of CFU in “ Preparation of culture dilution record.
4.1.5 Calculate the amount of inoculums or the appropriate dilution containing colony forming units in range of 10 – 100 CFU/ ml.
4.2 Storage & usage:
4.2.1 Store the appropriate dilution of 10 – 100 CFU / ml of culture dilutions in refrigerator, use it for one week & discard (from date of preparation) as per procedure for disposal of media.
4.2.2 Take one loop full of inoculum of the above prepared culture dilution for positive control of selective medias & one ml of inoculum for growth promotion test.
Saline solution, microbiology, promotion test
