sop for Handling and Sub culturing of Microbial cultures

sop for Handling and Sub culturing of Microbial cultures


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

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

3.1 Microbiologist QC

4.1 Q.C. Manager


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.

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.

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

Microbial Type Culture Collection Chandigarh
Sector 39 A Chandigarh- 160 036
Phone-(0172-690562, 690173)
Fax-(0172-690585, 690173)
American Type Culture Collection
12301, Park Lawn Drive
Rockville, MD 20852
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


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.

5.4.1 Horizontal Laminar Air Flow, Double Door autoclave, Refrigerator.

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

5.8.1 Whenever required as per procedure.


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

sop for calibration and validation of micro autoclave

sop for Sterility failure investigation

cleaning and operation of discard autoclave

sop for operation of fogger machine

sop for Biological assay of lactic acid bacillus

sop for preparation of culture inoculum

STP for sterility testing of sterile gloves

sop for Operation and calibration of active air sampler

sop for transfer of material for testing and sampling in sterile area

entry & exit procedure in microbiology laboratory

Growth Promotion Test In Microbiology Laboratory

Operation of B.O.D in Microbiology Laboratory

Operation of Horizontal Laminar Air Flow in the microbiology laboratory

Operation and cleaning of Pass Box.

Operation and cleaning of air sampler

Cleaning and Sterilization of Glassware

Analysis of water for microbial load in microbiology lab

Operation and temperature monitoring of Refrigerator

Fumigation of Microbiology Laboratory.

Entry & Exit procedure In Sterility Area

SOP for Microbial analysis of Raw Material Finished Products

SOP for Operation & Calibration of pH meter in Micro Department

SOP for Operation & Calibration of pH meter in Micro Department

SOP Operation and calibration of Hot Air Oven In Microbiology

SOP for operation cleaning & calibration of Digital colony counter

SOP for Operation And Cleaning of Microscope

sop for Media Preparation and Consumption

sop for Receipt Storage and Usage of Culture Media

sop for Cleaning Sanitization And Disinfection In Microbiology

sop for Environmental monitoring of all the Classified area

sop for Handling and Sub culturing of Microbial cultures

sop for Media Growth Promotion Test and various Microbiological test

sop for BOD incubator operation and cleaning

sop sampling of water for microbiological analysis

sop for Disinfectant Efficacy Test

sop for for cleaning and operation of vortex mixture

sop for Temperature & Relative Humidity Monitoring

sop for Operation and Calibration of Heating Block

sop for Sterility Testing of Microbiology

sop for Disposal of Culture Media

sop for Drain point of Microbiology

sop for entry & exit procedure In Microbial limit test and Biosafety

sop for Gram Staining of Bacteria in Microbiology Laboratory

sop for Monitoring of Compressed Air/gases for microbiological analysis

sop for BET (Bacterial Endotoxin) test in Microbiology

sop for receipt storage and Determining the population of Biological indicators

sop for qualification of analyst microbiologist

sop for Bioburden test of Packing materials in Microbiology Laboratory

sop for microbiological assay of erythromycin antibiotic

sop for liquid particle counter

sop for operation and calibration of digital zone reader

sop for monitoring of ultraviolet efficiency LAF and pass box

microbiological assay of cyanocobalamin or vitamin B12

gowning procedure for microbiological testing area

swab testing of various surfaces for bioburden determination

sop for endotoxin challenge test

Hold time study protocol for sterilized media

sop for personnel Qualification protocol for aseptic area

sop for sampling and testing of drain water

Sop for Operation of Airborne Particle Counter

sop for Inoculum Preparation

sop for Validation protocol of steam sterilizer autoclave

sop for pathogen detection from drain point

Sop for Analysis of Raw water Purified water water for injection and pure steam water

sop for preservatives efficacy test

sop for collection and preservation of in house isolated microorganisms

sop for Operation Calibration and Maintenance of Micropipette

Sop for UV Efficacy Test

sop for gram staining

Sop for swab testing

sop for microbiological testing of water


sop for depyrogenation of apparatus

sop for media preparation

sop for fertility test growth promotion test of media

sop for Operation and cleaning of moist heat sterilizer

sop for monitoring by active air sampler

sop for swab sampling and testing for clean rooms in production area

sop for monitoring in microbiology laboratory

sop for Fumigation of aseptic area and microbiology lab

sop for monitoring of personnel in aseptic area

sop for maintenance of cultures

sop for Operation and cleaning of laminar bench

preparation of settle plates

sop for monitoring of pure steam

sop for entry and exit procedure to m.l.t and b.e.t room

sop for storage of and use of media

sop for disposal of microbiological media and cleaning of microbiological glassware

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