SOP for Sub-culturing and Maintenance of Microbial Cultures

1.0 OBJECTIVE

To lay down the procedure for sub-culturing and maintenance of Microbial Cultures.

2.0 SCOPE

This SOP is applicable to the Quality Control Dept.

3.0 RESPONSIBILITY

Microbiologist

4.0 ACCOUNTABILITY

Head Quality Control Department.

5.0 PROCEDURE

5.1 Source: Organisms used for Microbiological analysis should be procured from a National or International recognized institute.
5.2 Approved Vendor: NCIM, Pune, India.
5.3 Mode of Supply: Freeze dried cultures are supplied in vacuum-sealed glass ampoules or sub-cultured on slants.

5.4 For Reviving Cultures

5.4.1 Prepare the specified growth medium as per the procedure is given in the SOP for Preparation of Microbiological Culture Media.
5.4.2 Transfer the Culture slants to the Microbiology testing area as per the procedure is given in the SOP for Entry and Exit Procedure into Microbiology Section.
5.4.3 Disinfect the culture slant surface with 70% isopropyl alcohol.
5.4.4 Remove the cotton plug and flame the neck of the tube carefully.
5.4.5 Transfer a loopful of freeze-dried culture in 10 ml of 0.9% sterile saline solution.
5.4.6 Again flame the neck of the tube to avoid the contamination and replace the cotton plug properly.
5.4.7 Mix the inoculated tube well to disperse the cells and incubate at the required temperature for 18-24 hrs.

5.5 Master Slant Preparation

5.5.1 Prepare the required Culture media slants for different microorganisms as per the procedure is given in the SOP for Preparation of Microbial Culture Media and mention the name of the media on each slant.
5.5.2 Take the slants and the incubated culture suspension into the microbiology testing area as per the procedure is given in the SOP for Entry and Exit Procedure to Microbiology Section.
5.5.3 Streak a loopful of culture form the 24 hours old culture suspension on to the respective media slants as described above and incubate at a specified temperature. Hence one will be Master culture, second will be stock culture and third will be working culture, incubate at specified condition mentioned.
5.5.4 Label the slants
5.5.5 Preserve the grown culture in the refrigerator at 2 to 6°C.
5.5.6 Prepare four master culture slants, one stock culture slant and one working culture slant from the 10 ml of culture suspension.
5.5.7 Sub-culturing in the pattern to get one slant per month.
5.5.8 Check the purity of mother culture at the time of subculturing. Stock culture purity is also checked monthly by the same manner by colony morphology and gram staining.

6.0 ABBREVIATIONS

6.1 NCIM: National Collection of Industrial Microorganisms
6.2 QC: Quality Control
6.3 Dept.: Department
6.4 SOP: Standard Operating Procedure

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