SOP for Preparation of Culture of Microorganism

1.0 OBJECTIVE

To describe the procedure for preparation of microorganism.

2.0 SCOPE

This SOP is applicable for preparation of microorganism fertility test.

3.0 RESPONSIBILITY

Microbiologist - Quality Control

4.0 ACCOUNTABILITY

Manager – Quality control

5.0 PROCEDURE

5.1 Open a freeze-dried culture of the organism under a laminar airflow hood. Suspend the contents in 10 ml of Trypticase Soya Broth (TSB also called Soyabin-Casein Digest Broth - SCDB) for bacteria and Sabouraud Dextrose broth for yeasts and fungi, vortex for about 15 seconds. Incubate the bacterial culture at 32.5 ±2.5ºC and 22.5±2.5ºC for 48 hours and Five days respectively (media and incubation conditions may be different for some organisms).
5.2 For bacteria incubate a loopful of the 24 hours old culture on to Trypticase Soya Agar (TSA) slant /slope at 30 to 35ºC for 48 hours and for fungi inoculate a loopful of the 48 hours – 5 days old culture on to Sabouraud’s Dextrose Agar (SDA) slope, incubate at 20 to 25ºC for 5 days. At the same time streak a loopful of the culture on to a plate containing the appropriate medium and incubate. Refer to table 1 for further information in respect of medium, temperature and incubation requirements.
5.3 If the culture is not pure. Purify it by subculturing from a selected isolated colony and streak on to appropriate plate (refer to Table 1). Incubate as required. Check colony morphology and carry out gram reaction growth from a select colony to either TSA or SDA slope (s) repeat step 5.2.
5.4 Label all slopes clearly with the name of the organism batch number and date of subculture.
5.5 Store the slope at 2°C to 8°C and use it as seed culture/ mother culture.
5.6 From the mother culture three master slants i.e.M1, M2 and M3 should be prepared.
5.7 From M1 seven working cultures should be prepared for each month for first six months i.e. W1,W2,W3,W4,W5,W6 and W7.
5.8 After six months again these monthly cultures should be prepared from M2 similarly as point no 5.7.
5.9 From these monthly cultures weekly / daily use cultures should be prepared which should be used for daily basis or weekly basis but not more than a week i.e. D1,D2, D3, D4 and so on.
5.10 After use of these cultures that is after expiry discard these culture tubes.
5.11 Open a freeze-dried culture every year and discard old culture stock.
5.12 After six month of the opening of freeze dried ampoule check the identity of the organism on the current slope.

Table-1

Organism	ATCC # MTCC	Growth Medium	Growth temp.±1ºC	Incubation time -day	Condition
Bacillus subtilis	441	Trypticas Soya Broth (TSA)	37	1	Aerobic
Candida albicans	ATCC 19403	Sabouraud Dextrose Agar (SDA)	30	2	Aerobic
Clostrdium sporogenes	NCIM 10627	Horse Blood Agar (HBA)	32	2	Anaerobic
Escherichium coli		TSA	37	1	Aerobic
Pseudomonas aeruginosa		TSA	37	1	Aerobic
Staphylococcus aureus		TSA	37	1	Aerobic

6.0 ABBREVIATIONS

6.1 SOP - Standard Operating Procedure
6.2 ATCC - American Type Culture Collection
6.3 MTCC - Microbial Type Culture Collection
6.4 NCIM - National Collection of Industrial Microorganism
6.5 SDA - Sabouraud’s Dextrose Agar
6.6 TSA - Trypticase soya agar
6.7 HBA - Horse blood Agar
6.8 SCDB - Soybean casein digest broth

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