1.0 OBJECTIVE
To lay down the procedure for subculturing and maintenance of microbial cultures.
2.0 SCOPE
This SOP shall be applicable for subculturing and maintenance of microbial cultures in the Microbiology Lab.
3.0 RESPONSIBILITY
3.1 Microbiologist and above shall be responsible for the preparation and execution of this SOP
3.2 QC & QA Head shall be responsible for the checking and approval of this SOP.
3.3 HOD shall be responsible for the authorization of this SOP.
4.0 ACCOUNTABILITY
Head QC
5.0 PROCEDURE
5.1 RECEIPT AND DISTRIBUTION OF LYOPHILIZED CULTURE
5.1.1 The lyophilized cultures received are first verified for their intactness, labeling, and so on.
5.1.2 The cultures are checked for their name and ATCC No / MTCC No./NCIM No. shown in the paper and on vial/ ampoule received from the source.
5.1.3 Before handling the cultures, glassware are first shall be sterilized and dehydrogenated for storage of stock cultures.
5.1.4 The outer surface of the received lyophilized culture shall be disinfected with Sterilized 70% I.P.A.solution.
5.1.5 Carefully and slowly remove the cotton plugin in such a manner that the fine particles of lyophilized culture do not release under L.A.F.
5.1.6 Suspend the contents in 100 ml of Soybean- Casein Digest Medium (SCDM) and vortex for about 15 seconds.
5.1.7 Bacterial culture shall be incubated at 32.5 ±2.5ºC for 48 hours and fungal culture shall be incubated at 22.5±2.5ºC for five days. (other media and incubation conditions may be necessary for some organisms).
5.1.8 For bacteria, a loopful of the 24-hour culture shall be streak on to Nutrient Agar slant(s) and incubate at 30 to 35ºC for 48 hours and for fungi inoculate a loopful of the 48 hours – 5 days old culture on to Sabouraud’s Dextrose Agar (SDA) slant (s) and shall be incubated at 20 to 25ºC for 5 days.
5.1.9 At the same time a loopful of the culture shall be streak onto a plate containing the appropriate medium and incubate.
5.1.10 Identify the mother culture 1 and mother culture 2 with its ATCC, MTCC, or NCIM and date of subculturing with the signature of the microbiologist.
5.1.11 The vials obtained shall be stored at a temperature of 2-8ºC in the refrigerator.
5.2 REVIVAL OF LYOPHILIZED CULTURE
5.2.1 From each mother culture ‘M1 and M2’ the subculturing shall be done on monthly basis for up to five generations.
5.2.2 From mother culture 1st seven slants shall be made for the six months and one as spare and this shall be designated as M1A to M1F
5.2.3 At the end of the fifth month make another seven slants from the second mother culture for the next six months and one as spare and this shall be designated as M2G to M2L.
5.2.4 The culture received in slants/stabs shall be directly revived by aseptically transferring loop full to the respective medium as shown in Annexure-1.
5.2.5 The inoculated medium is incubated at the temperature mentioned in Annexure-I
5.2.6 From these monthly cultures weekly / daily use cultures shall be prepare which shall be used on daily basis or weekly basis but not more than a week i.e.W1, W2, W3, W4 & D1, D2, D3, D4, and so on.
5.2.7 All slants shall be label clearly with the name of the organism batch number and date of sub-culturing
5.2.8 The cultures shall be preserved at a refrigerated temperature at 2-8º C.
5.2.9 The old culture shall not discard before confirming the purity of the subculture.
5.2.10 If any culture is found contaminated, subculture from the other tube of the previous set, if both the tubes are contaminated new culture shall be procured.
5.2.11 Previous subculture tubes shall be discarded by steam sterilization.
6.0 REFERENCES
N.A
7.0 ANNEXURE
|
Annexure No. |
Title of the Annexure |
Format No. |
|
Annexure I |
Record of subculturing |
F |
8.0 ABBREVIATIONS :
SOP : Standard Operating Procedure
QCD : Quality Control Department
Dept. : Department
QC : Quality Control
QA : Quality Assurance
QCD : Quality Control Department
No. : Number
N.A : Not Applicable
Lab. : Laboratory
IPA : Iso Propyl Alcohol
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