SOP for Growth Promotion Test and Calibration of Inoculum

1.0 OBJECTIVE

To lay down the procedure for testing each prepared Microbiological media lot for its growth promotion properties and its suitability for use in microbiological work.

2.0 SCOPE

This SOP shall be applicable to Quality Control Dept.

3.0 RESPONSIBILITY

Microbiologist

4.0 ACCOUNTABILITY

Sr. Manager Quality Assurance

5.0 PROCEDURE

5.1 Growth Promotion Test

5.1.1 Carry out growth promotion test for every lot of media prepared.
5.1.2 Every time a particular medium is used for analysis, prepare two extra tubes/ flasks/plates of the medium.
5.1.3 Inoculate one tube/flask/plate of the medium with approximately 100 viable organisms of the respective standard culture and incubate at the respective temperature.
5.1.4 Leave one tube/flask/plate uninoculated, to serve as a negative control.
5.1.5 Early and copious growth should be observed in the inoculated media. If delayed growth or scanty growth is observed, reject the media and prepare a fresh lot of media.
5.1.6 Carry out growth promotion test every time a medium is used for microbiological analysis.
5.1.7 If the test is negative for a particular medium, then the analysis performed using that particular medium shall be considered void and a fresh lot of media shall be prepared and the growth promotion test and analysis shall be repeated with fresh medium.

5.2 Calibration of Inoculum

5.2.1 Prepare 1% aqueous barium chloride and 1% aqueous sulfuric acid solutions. Add the amounts indicated in Table-1 to clean dry tubes.

Table - 1

Tube	Barium Chloride 1% (ml)	Sulfuric Acid 1% (ml)	Density of bacteria (million/ml)
1 2 3 4 5 6 7 8 9 10	0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0	9.9 9.8 9.7 9.6 9.5 9.4 9.3 9.2 9.1 9.0	300 600 900 1200 1500 1800 2100 2400 2700 3000

5.2.2 Prepare a series of 5 inoculum tubes, each tube containing varying amounts of inocula, from an active 24 hour old culture, in 0.1% Peptone water.
5.2.3 Compare the transmittance with the barium sulfate standards on a suitable spectrophotometer at 620 nm using 40.1% Peptone water as a blank.
5.2.4 Select one inoculum (A), which corresponds to the transmittance of tube no. 3 and one inocula (B), which corresponds to the transmittance of tube no. 4 i.e., 1200 (bacteria in million/ml).
5.2.5 Dilute the two inocula, aseptically, in 0.1% Peptone water, as per the dilution scheme is given below.
5.2.6 Calculate cfu/ml of Inocula C and D, by pour plate technique.
5.2.7 Select the inocula, which shows a count of 100 cfu/ml
5.2.8 Use this diluted culture for inoculation in growth promotion tests and for validation of media.

6.0 ABBREVIATIONS

6.1 SOP: Standard Operating Procedure
6.2 QA: Quality Assurance
6.3 QC: Quality Control
6.4 Dept.: Department

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