STANDARD OPERATING PROCEDURE Preparation of Culture Suspension

	STANDARD OPERATING PROCEDURE
	Department: Quality Control	SOP No.: QC/MB/046-05
Preparation of Culture Suspension		Effective Date:
		Review Date:
		Page No.: 1 of 5

1.0            OBJECTIVE:

1.1                   To lay down a procedure for preparation of culture suspensions by serial dilution method.

2.0            SCOPE:

2.1                   This procedure is applicable to preparation culture suspension of all the cultures used for growth promotion test and validation of microbial limit test in microbiology laboratory at  Pharma Private Limited.

3.0            RESPONSIBILITY:

3.1                 Microbiologist – Responsible for execution of activity.

3.2                 Section In charge – Responsible for review of activity.

3.3                 QC Head – Responsible for implementing of procedure.

3.4                 QA Head – Overall Compliance.

4.0            PROCEDURE:

4.1                 Bacterial Organisms for Culture Suspension:

4.1.1             Staphylococcus aureus ATCC 6538

4.1.2             Pseudomonas aeruginosa ATCC 9027

4.1.3             Bacillus subtilis ATCC 6633

4.1.4             Escherichia coli ATCC 8739

4.1.5             Salmonella NCTC 6017

4.2                 Fungal Organisms for Culture Suspension:

4.2.1             Candida albicans ATCC 10231

4.2.2             Aspergillus brasiliensis ATCC 16404

4.3                   Prepare required quantity of desired media as per current version of media preparation SOP QC/MB/xxx

4.4                   Take out working cultures of above organisms from the refrigerator and place it under LAF to attain room temperature.

4.5                   Label the test tubes containing buffer solutions as Stock (10mL) and remaining test tubes (18mL) as serially from 10^-1 to 10^-8 for each microorganism.

4.6                   With the help of sterile loop, take a loop full of culture and inoculate in 10 mL of sterile buffer sodium chloride peptone solution (BPW) or 0.9 % w/v sterile saline solution (Stock suspension).

4.7                   For Aspergillus brasiliensis add 0.05% v/v polysorbate 80 to buffer sodium chloride peptone solution or saline solution to suspend the spores.

4.8                   Thoroughly mix the contents by using cyclo mixture to get uniform suspension.

4.9                   With the help of micro pipette take 2 mL from the stock suspension and inoculate into 18 mL of buffer sodium chloride peptone solution or 0.9 % w/v sterile saline solution (10^-1dilution).

4.10               Mix thoroughly by using cyclo mixture to get uniform suspension and perform the serial dilution in the same manner up to 10^-8.

4.11               Follow the flow chart for the preparation of serial dilutions of culture suspension as mentioned in annexure-II.

4.12               Perform the whole exercise with all the organisms mentioned in point no. 4.5 to 4.9.

4.13               The growth in higher dilutions shall be high, hence lower dilutions (preferably from 10^-4 dilution) shall be used for plating.

4.14               Pipette out 1.0 mL of the each selected dilutions (10^-5, 10^-6, 10^-7 &10^-8 for bacterial & Candida and 10^-4, 10^-5, 10^-6 &10^-7 for Aspergillus) into sterile petri plate with duplicate and pour 15 to 20mL of sterile molten SCDA media for bacterial suspension and SDA media for fungal suspension.

4.15               Incubate bacterial culture dilution plates at 30°C to 35°C for 72 hours and fungal culture dilution plates at 20°C to 25°C for 5 days.

4.16               Discard the left over dilutions by decontamination procedure as per current version of SOP QC/MB/035.

4.17               After the specified incubation period count the number of colonies and record the details in Annexure-I.

4.18               Identify the dilution which gives 10-100 cfu/mL, it is useful in growth promotion tests.

4.19               Label the above tested dilution tubes with the label mentioned in Annexure – III.

4.20               Store labelled culture suspension tubes in refrigerator for 15days from the date of preparation.

4.21               After completion of 15days discord the stored culture suspension tubes as per current version of SOP QC/MB/xxx

5.0            ABBREVIATIONS:

QC: Quality Control	SOP: Standard Operating Procedure
QA: Quality Assurance	BPW: Buffered Peptone Water
LAF: Laminar air flow	SCDA: Soyabean Casein Digest Agar
IPA: Isopropyl alcohol	SDA: Sabouraud Dextrose Agar
CFU: Colony Forming Unit	ATCC: American Type Culture Collection
mL: Milli Liter	NCTC: National Collection of Type Culture