SOP for Quantification of Microbial Culture

1.0 OBJECTIVE

To describe a procedure for quantification of culture

2.0 SCOPE

This SOP is applicable for quantification of culture

3.0 RESPONSIBILITY

Microbiologist - Quality Control

4.0 ACCOUNTABILITY

Manager- Quality Control

5.0 PROCEDURE

5.1 To perform the experiments select 24 hours old bacterial culture, yeast culture and sporulated culture of fungi.
5.2 The cultures are to be selected i.e. environment isolates. Bacillus subtilis, Staphylococcus aureus, Salmonella sp, pseudomonas aeruginosa. E.coli, Candida albicans, Aspergillus niger.
5.3 Preparation of culture media
5.3.1 Prepare the culture media as per the SOP of media preparation and qualify the medium for the fertility test.

5.4 Quantification of culture

5.4.1 Take the bacterial culture 24 hours old slant, harvest the slant in peptone water aseptically in pre-sterilized 9 ml peptone water tube.
5.4.2 Dilute the harvested culture in pre-sterilized 9 ml peptone water containing test tube using sterile micropipette tips.
5.4.3 Quantification of fungal culture, use sterile saline and sterile glass beads for harvesting
5.4.4 Do the serial dilution of culture up to 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, 10-9, 10-10 dilution.
5.4.5 Do the pour plating of 1 ml culture suspension from all dilution in duplicate add SCDA medium for bacteria, SDA for fungi (temperature of media should be 40°C- 45°C.
5.4.6 Incubate the bacterial culture at 30°C- 35°C for 72 hours and fungal culture at 20°C- 25°C for 5 days.
5.4.7 Record the result after incubation as per Annexure – I
5.4.8 Select the dilution, which falls between 10 to 100 cfu /ml. Refrigerate all dilution at 2°-8°C.
Related: Maintenance of Microbial Cultures

6.0 ABBREVIATIONS

6.1 SOP - Standard Operating Procedure
6.2 SCDA - Soybean Casein Digest Agar
6.3 Cfu - Colony Formation Unit
6.4 SDA - Saboraud’s Dextrose Agar

ANNEXURE- I

CULTURE TITRATION RECORD (Culture Quantification)

Name of organisms	Date of sub culturing	B. No. of culture	Date of plating/ incubation	Incubation time & temp	Media lot No

Quantification Table

Dilution	Count obtained
	Name of organisms
10-1
10-2
10-3
10-4
10-5
10-6
10-7
10-8
10-9
10-10
Culture titre value
Dilution Vol. Suspension used
Date of completion

Remarks:

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