sop for preparation of culture inoculum



sop for preparation of culture inoculum



To describe the procedure for Preparation of Culture Inoculum (10 – 100 Cfu/ml).

It is the policy of Multani pharmaceuticals Limited (Analytical Division) that a written procedure shall be followed for Preparation of Culture Inoculum (10 – 100 Cfu/ml).

This SOP shall be applicable for preparation of culture inoculum (10-100 cfu/ml) in Microbiology Laboratory (Analytical Division).

Persons along with their responsibilities are given below:

S. No Designation Responsibility
01 Microbiologist Preparation & revision of SOP.
02 Officer-QA Distribution and retrieval of SOP.
03 Technical Manager Training of SOP.
04 Quality Manager Approval & Implementation of SOP.



5.1.1 Prepare soybean casein digest Agar medium for bacteria and sabouraud chloramphenicol agar for fungi or other selective media

required for specific bacteria or fungi in a conical flask as per Media Preparation SOP
5.1.2 Prepare 0.9 % w/v solution of Sodium chloride with distilled water in a conical flask in required quantity & transfer 9-9 ml to each

test tube, plug the tubes with cotton plug and sterilize in an autoclave at 121°c and 15psi for 20 minutes.
5.1.3 Sterilized Petri plates in autoclave at 121°C and 15psi for 30 minutes.
5.1.4 Perform the activity of serial dilution in the Bio Safety Cabinet.
5.1.5 Transfer 100 µl 24 hour old broth culture i.e. M1/W1(1st month/1st week) from SCDM to 9 ml sterile Normal Saline

(Stock Suspension). Homogenize the suspension by gentle shaking.
5.1.6 Prepare serial dilutions by diluting each time 1 ml of culture suspension with 9 ml of Normal Saline up to 10-8 dilution.
5.1.7 Preserve the original undiluted culture suspension & subsequent dilutions in a refrigerator between 2°C to 8°C.
5.1.8 Aseptically pour 1 ml each of 10-3 to 10-8 dilution of each organism in each of the sterilized petridishes.
5.1.9 Aseptically pour in each plate about 20-25 ml sterilized Soyabean Casein Digest agar medium for bacterial culture and

sterilized sabouraud chloramphenicol agar for fungal culture, previously liquefied and cooled to around 45°C, mix and allow

the medium to solidify. Incubate the Plates at 32.5 ± 2.5°C for 24 to 48 hours for bacterial culture and 22.5 ± 2.5°C for 48 to 72 hours for fungal culture.
5.1.10 At the end of incubation count the number of Colony Forming Units developed on each of the petridishes.
5.1.11 Select the culture suspension containing 100-1000 cfu/ml and preserve in refrigerator between 2°C to 8°C. Use 100 µl from this suspension for routine positive controls & GPT.

Each diluted culture suspension containing about 100-1000 cells per ml shall be preserved in a refrigerator between 2°C to 8°C.

5.3.1 The diluted culture suspension containing about 100-1000 cfu of each organism per ml shall be subjected to determination of

Total Viable Count of the same at the time of preparation.
5.3.2 The diluted culture suspension as prepared above and preserved in refrigerator shall not be used beyond 15 days after its date of preparation.
5.3.3 All the data including observations related to preparation of culture suspension and the data related to establish their shelf life

shall be recorded and preserved in the Annexure I


5.4.1 All inoculum preparation should be carried out under Biosafety cabinet.
5.4.2 Use nose mask and hand gloves while working with cultures.
5.4.3 After completion of work wash your hand with 70% IPA solution.
5.4.4 After the completion of work all micropipette tips, test tubes & other accessories which is contaminated by culture should be autoclaved.

5.5 FREQUENCY :15 days



QA Quality Assurance
SOP Standard Operating Procedure
BOD Biological Oxygen Demand
QM Quality Microbiology
LAF Laminar Air Flow
FR Format
cfu Colony Forming Unit
S. Serial
No. Number
SCDA Soybean Casein Digest Agar

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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