| Preparation and Storage of Microbial Culture Inoculums suspension |
1.0 OBJECTIVE To lay down a Procedure for the preparation and storage of microbial culture inoculums suspension. 2.0 SCOPE This procedure is applicable for preparation and storage of microbial culture inoculums suspension in the Quality Control Department of Microbiology Area. 3.0 RESPONSIBILITY • Microbiologist : is responsible for preparation, storage and maintain record as per SOP. • Manager microbiology : is responsible for ensuring that the SOP is followed correctly. 4.0 ACCOUNTABILITY • Department Head • Head Quality / QA 5.0 PROCEDURE 5.1 Safety considerations: 5.1.1 Microbiologist is aware of handling the microbial culture and microbial culture media carefully. 5.1.2 Microbiologist shall take all the necessary safety precaution during preparation of microbial inoculums. To Read this SOP click here OP for Entry and Exit in Microbiology Testing Area. 5.2 Preparation of Inoculums suspension 5.2.1 Take working culture slant/tubes of required microorganisms and transfer in bio-safety cabinet. 5.2.2 Take the required number of media plates or tubes and transfer in bio-safety cabinet. 5.2.3 Label the media tubes with name of microorganisms and date with the help of permanent marker. 5.2.4 Take sterile loop and inoculate a loop full of inoculums from working culture of into appropriaterly labeled SCDM media tubes and incubate tubes at 30-35°c for 24-48 hours. 5.2.5 For anaerobic culture, use cooked meat medium, instead of Soyabean Casine Digest medium. Incubate the tubes at 30-35°c for 24-48 hours for anaerobic bacteria culture 5.2.6 Incubate 20-25°c for 48-72 hours for 72-120hours for yeast and molds. 5.2.7 After completion of incubation, check the growth of microbial culture. 5.2.8 Assume that the broth culture 108 microorganisms per ml in case of aerobic bacteria and 10.6 microorganisms per ml in case of anaerobic and fungal. 5.2.9 Prepare set of 8dilution tubes (or up to appropriate dilution to get 10-100 cfu/ml) with 9ml 0.9% saline water for each culture and label them culture name and dilution name in the following order 10-1 to 10-8. 5.2.10 Take 1ml from stock to 9ml saline solution to make 10-1 dilution. Replace the tube with cap and mix the suspension. 5.2.11 Take 1ml from stock to 9ml saline solution to make 10-2, replace the cap and mix the suspension, further prepare the dilution up to 108. 5.2.12 By using pour plate technique, transfer 1ml from the dilution 10-4 to 10-8 plates out in delicate, SCDA used for bacterial culture and SCA/SDA for fungal culture. 5.2.13 Incubate the media plates at 30-35°c for bacteria culture 24-48 hrs. and 20-25°c for fungal culture for 48-72hrs. 5.2.14 After completion of incubation, count the colonies in plates and select the dilution with 10-100cfu/ml an its previous dilution (10-100cfu/0.1ml and store at 2-8°c. 5.2.15 If the dilution stock of 10-100cfu is exhausted, then prepare fresh stock from previous dilution stored by adding 1ml in 9ml of sterile 0.9% saline water. If required 0.1ml of previous dilution can be directly used to inoculate 10-100cfu/ml. 5.2.16 The culture suspension shall be stored at 2-8°c for 10days or as per validation data. 5.2.17 Record the details of inoculums preparation in format no. XXX. |
