To lay down the procedure for preservation & maintenance of microbial cultures.
1.0 OBJECTIVE
To lay down the procedure for preservation & maintenance of microbial cultures.
2.0 SCOPE:
This SOP is applicable to the microbial cultures in Microbiology Laboratory.
? To maintain the authenticity, viability & purity of test cultures, which are used as positive controls in different microbiological testing.
? To minimize the risk of phenotype changes, which may occur due to prolonged storage of culture & thereby resulting in failure of organism, to grow in relevant medium.
3.0 RESPONSIBILITY:
Microbiologist : To perform the procedure as per SOP.
Head – Quality control: To ensure the compliance of SOP.
4.0 PROCEDURE:
4.1 Source:
Organisms used for Microbiological analysis should be from recognized institute.
4.2 Source for Microbial cultures:
? MTCC, Chandigarh, India.
? NCIM, Pune, India.
4.3 Mode of supply:
Freeze dried culture are supplied in vacuum-sealed glass ampoule or
Sub-cultured on slants.
4.4 For Reviving Freeze – dried cultures,
4.4.1 Disinfect the culture ampoule surface with sterile 70% isopropyl alcohol.
4.4.2 Cut the middle portion of the ampoule with an ampoule cutter.
4.4.3 Pour 2 ml of soybean casein digest medium into the ampoules containing lyophilized culture.. Mix well to disperse the cells or as per the recommendation of collection center where we procure the cultures. This is considered as a Mother culture
4.5 Master culture Slant preparation:
4.5.1 Prepare the soybean casein digest agar and sabourdox dextrose agar slants and preincubate the slants for 48 hrs.
4.5.2 After preincubation, revive the cultures as mentioned above and make a streak on the slants.
4.5.3 Prepare Master culture A1, Master culture B1, Master culture C1, Master culture D1 and Back up cultureslants from Mother culture.
4.5.4 Incubate the bacterial slants at 32.5+ 2.5 for 24 to 48 hrs and fungal slants at 22.5+ 2.5 for 48 to 72 hrs
4.5.5 After incubation, do subculturing of four working cultures for four weeks from Master culture A1 as per Appendix-I and incubate the working cultures as mentioned above.
4.5.6 Then preserve the Master and Working cultures in refrigerator at 2 to 8°C.
4.5.7 For next month, do subculturing of Master culture A2 slant, four working cultures and one backup culture slant from Master culture A1.
4.5.8 Similarly, prepare next Master culture, working culture and backup culture slants as per the Appendix- I
4.5.9 For next 9 month, follow the same pattern of subculturing from Master culture B1, C1 and D1.
4.5.10 Backup cultures can be used if any contamination occur in Master and working culture slants.
4.5.11 Refer Appendix –I for generations.
4.5.12 Check the purity of Mother culture at the time of sub culturing. Master culture purity is also checked
4.5.13 Record the Maintenance of cultures
4.5.14 If slants are purchased instead of lyopholised cultures, consider the slants as a mother cultures and do the sub culturing as per the Appendix-1.
Cultures, microbiology, 70% isopropyl alcohol
