sop for Handling and Sub culturing of Microbial cultures

1.0 OBJECTIVE
1.1 To lay down the procedure for Maintenance, Handling and Sub-culturing of Microbial cultures.

2.0 SCOPE
2.1 This procedure is applicable to the handling and maintenance of microbial cultures and to ensure that the standard Microbial

cultures used in Microbiology laboratory for testing purpose are in pure form and for long time use

3.0 RESPONSIBILITY
3.1 Microbiologist QC

4.0 ACCOUNTABILITY
4.1 Q.C. Manager

5.0 PROCEDURE

5.1 PRECAUTIONS
5.1.1 Take Soyabean casein Digest Agar slants that are free from any contamination.
5.1.2 Do not use slants containing moisture content.
5.1.3 Seed lot culture maintenance techniques should be employed so that the viable microorganisms used for inoculation are

not more than 4 passages removed from the ATCC/MTCC cultures.
5.1.4 Contaminated slants should be discarded.
5.1.5 Prepare bacterial &fungal culture slant at a different day, to avoid intermixing of tubes or label.

5.2 MATERIALS REQUIRED
Media (Soyabean Casein Digest Agar, Soyabean Casein Digest Medium, Sabouraud Dextrose Agar, Columbia Agar),

Mother microbial culture, Inoculating loop, test tubes, 70%IPA, Beaker, Sterile forceps.

5.3 PROCUREMENT OF CULTURES
Generally Microbial cultures must be procured from any source listed below-
National collection for Industrial Microorganism
Division of Biochemical Science
National Chemical Laboratory
Pune – 411 008
Or

Microbial Type Culture Collection Chandigarh
Sector 39 A Chandigarh- 160 036
Phone-(0172-690562, 690173)
Fax-(0172-690585, 690173)
Or
American Type Culture Collection
12301, Park Lawn Drive
Rockville, MD 20852
Or
Hi-media Laboratories
A- 516,Swastik Disha Business Park
LBS Marg, Mumbai- 400086 India
Phone- 022-61471919, Fax-022-61471920.

Following is the list of standard culture with ATCC Nos. and their equivalent number of other
Manufacturers:-

Name of Culture	ATCC	MTCC	NCIM	Hi-media
Escherichia coli	8739	1687	2065	0483P
Bacillus subtilis	6633	441	2063	0486P
Staphylococcus aureus	6538	737	2079	0485P
Staphylococcus epidermidis	12228	3615	2493	0371P
Salmonella enteric ser. A	NCTC 6017	3858	2257	0890P
Pseudomonas aeruginosa	9027	1688	2200	0484P
Clostridium sporogenes	19404	2684	5125	0317P
Candida albicans	10231	227	3471	0443P
Aspergillus brasiliensis	16404	1344	1196	0392P

After receipt check the detail match the strain no. with record, and all culture store at 2 -8 0C in refrigerator.

5.4 EQUIPMENTS REQUIRED
5.4.1 Horizontal Laminar Air Flow, Double Door autoclave, Refrigerator.

5.5 PROCEDURE
5.5.1 Preparation of slants
5.5.2 Prepare media and Dispense 10ml media in each test tube and plug them with non absorbent cotton, sterilized at 121˚C for 20 minutes.
5.5.3 Immediately after unloading, place the tubes under HLAF such that the base of the tube slopes
at an angle of about 30˚C with the horizontal surface.
5.5.4 Leave the tubes under HLAF until the agar media solidify. Incubate the tubes at 30˚C to 35˚C for 48 hours. Check the

status for contamination then transfer in refrigerator.
5.5.5 Before transferring the culture, bring the slants at room temperature.
5.5.6 With the help of sterile nichrome loop pick up a part of earlier culture and streak on slant surface.
5.5.7 Incubate at 30˚C to 35˚C for 24 to 48 hours 20˚C to 25˚c for 72 hours.
5.5.8 Transfer the tubes to refrigerator wrapped with aluminum foil.
5.6 Preparation of stabs
5.6.1 Prepare media Dispense about 5ml of media in each test tube and plug them with nonabsorbent cotton, sterilized at 121˚C for 20 minutes.
5.6.2 Immediately after unloading place the test tube stand under HLAF, Leave the test tubes on
HLAF until the agar media get solidify.
5.6.3 Incubate the test tubes at 30oC to 35oC for 48 hours. Check the status for contamination before transferring to refrigerator.
5.6.4 Before transferring the culture to stab bring the stab to room temperature.
5.6.5 With the help of sterile straight nichrome wire, pick the culture and stab in fresh stab.
5.6.6 Incubate at 30˚C to 35˚C for 24 to 48 hours.
5.6.7 Transfer the culture tubes to refrigerator wrapped with aluminum foil.
5.7 Maintenance of Mother Culture:
5.7.1 Procure fresh culture from an authentic supplier along with Certificate of purity of the culture.
5.7.2 Before use check the initial purity of the all receiving culture.
5.7.3 After ensuring the purity & physical characteristic of mother culture by staining.
5.7.4 Inoculate Soyabean Casein Digest medium with the new culture from ATCC/MTCC/NCIM/Hi-media
cultures and incubate at the temperature and time required by the organism as specified in

Certificate or follow the procedure as per by vender/manufacturer.
5.7.5 After the growth in Soyabean Casein Digest medium subculture on three fresh, pre-incubated slants.

Mark first as ”Master Culture MC I , second as MC II and third one as “Master Stock Culture MCS”
5.7.6 Incubate the slants at specified temperature for each culture.
5.7.7 After sure the sufficient growth is obtained label on these slants store in the refrigerator at 2-8oC for their specific period.
5.7.8 Use the prepared culture as per Schematic diagram specified in Annexure-I
5.7.9 For the 1st month, 2nd month, 3rd month, 4thmonth , 5th month, 6th month use M1, M2, M3, M4, M5, M6 respectively.

Use monthly culture for preparation of weekly cultures. If one of the above culture get contaminated use M7” Stand by culture”.
5.7.10After ensuring the purity of culture, transfer a loopful growth from monthly culture to a five
fresh slant and treat as a W1,W2,W3,W4,& W5.
5.7.11After incubation observe the growth and label the culture as per Annexure no. 1.
5.7.12Assign the batch number i.e. MC1/M1/W1, MC1/M1/W2, MC1/M1/W3, MC1/M1/W4 and
MC1/M1/W5 These slant shall be used for weekly purpose.
5.7.13 In case of Clostridium use Columbia agar for culture maintenance by following procedure from

step 5.7.2 to 5.7.12 with Columbia agar and incubating the culture in anaerobic condition.
5.7.14 After completion of work discard the culture slant or test tube by autoclaving at 1210C / 15 psi
Pressure for 30 minutes.
5.7.15 Check the purity of each monthly culture, before preparation of weekly culture, by colony
characteristics and Gram’s nature.

Specimen Label

Name of Organism:

Strain No.:

Batch No.:

Sub. Date:

Use before:

Storage temp.: 2 to 8⁰C

5.8 FREQUENCY
5.8.1 Whenever required as per procedure.

6.0 ABBREVIATIONS

Abbreviation used	Full form of abbreviation used
SOP	Standard Operating Procedure
IPA	Iso Propyl Alcohol
QA	Quality assurance
HEPA	High Efficiency Particulate Air
MLT	Microbial Limit Test
MTCC	Microbial Type Culture Collection
CFU	Colony forming unit
ATCC	American Type Culture Collection
NCIM	National collection for Industrial Microorganism