SOP on Preparation of Culture Dilution

1.0 OBJECTIVE

The Purpose of this SOP to lay down the procedure for preparation of Culture dilutions.

2.0 SCOPE

This procedure is applicable for Culture Maintenance in the Microbiology Laboratory at Pharmaceutical Manufacturing Industry.

3.0 RESPONSIBILITY

Authorized designee of microbiology department shall be responsible for Preparation of culture Suspension procured from an authorized / recognized standard laboratory.

Authorized designee or Microbiologist shall be responsible for receipt, primary physical check of cultures and storage of culture as per Manufacturer requirement.

Head of Microbiology is responsible for effective implementation of the SOP. 

QA is responsible for review and approval of the SOP

5.0 PROCEDURE

5.1 Serial Dilution

 For the purpose of preparing appropriate microorganism from the weekly- transferred working culture slant, inoculate a loopful of culture to 10 ml of Nutrient Agar for Bacteria & Fungi.
 Incubate the bacterial suspension at 32.5 ± 2.5°C for 24 to 48 hours & fungal suspension at 22.5 ± 2.5°C for 24 to 72 hours for enrichment.
Serially dilute the enriched suspension in 0.9% sterile saline solution in a ratio of 1: 10 dilution by transferring as follows.

Transfer the culture suspension in a sterile test tube.

Collect the suspension in a sterile test tube.

Vortex the culture suspension to obtain a uniform suspension.

Carry out serial dilution so as to obtain a culture suspension of 10-100 cfu/ml by     following the steps given below.

                Transfer 1 ml of the suspension to 9 ml sterile normal saline solution – 101    Dilution.

1 ml of 101 Dilution to 9 ml sterile normal saline solution – 102 Dilution.

    1 ml of 102 Dilution to 9 ml sterile normal saline solution – 103 Dilution.

    1 ml of 103 Dilution to 9 ml sterile normal saline solution – 104 Dilution.

    1 ml of 104 Dilution to 9 ml sterile normal saline solution – 105 Dilution.

    1 ml of 105 Dilution to 9 ml sterile normal saline solution – 106 Dilution.

    1 ml of 105 Di Dilution to 9 ml sterile normal saline solution – 107 Dilution.

Check the CFU/ml from the above-prepared dilutions by transferring 1 ml of the inoculum on Nutrient agar plate for Bacteria & Fungi by pour plate method. Incubate the plates for 18 to 24 hours at respective Temperature as mentioned above.
On observation of visible microbial growth on each plate, count & record the average number of CFU in Preparation of culture suspension register. Count the CFU/ml from each Dilution & select the dilution tube which gives 10-100 CFU/ml. Store the same Tube for daily use of Culture for Positive Control. Record all Dilutions Result In annexure. Use it for one week & discard (from date of preparation) as per SOP for disposal of media and record in the format.
Take 1 ml of the above prepared culture suspension for Positive control for environmental monitoring, MLT and fertility test.

5.2 Abbreviations :

CFU: Colony Forming Unit
MLT: Microbial Limit Test
NaCl: Sodium Chloride

6.0 Training :

As per employee training SOP.

7.0 Annexure :

Annexure I: Culture suspension preparation Record

Annexure II : Culture Suspension Usage Record

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