sop for preparation of culture inoculum

1.0 OBJECTIVE

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

2.0 PURPOSE
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).

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

4.0 RESPONSIBILITY
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.

5.0 PROCEDURE

5.1 METHODOLOGY
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.

5.2 STORAGE OF CULTURE SUSPENSION
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 ASSIGNING SHELF LIFE TO DILUTED CULTURE SUSPENSION
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 PRECAUTION
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

6.0 CROSS REFERENCES: SOP FOR SOP

7.0 ABBREVIATIONS & DEFINITION

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

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