SOP for Enumeration of Spore Count in Biological Indicators : Pharmaceutical Guidelines

SOP for Enumeration of Spore Count in Biological Indicators

Standard operating procedure to enumerate the spores in Biological Indicator used in validation of steam sterilizer.

1.0 OBJECTIVE

To describe the procedure for the enumeration of spore count in Biological Indicators.

2.0 SCOPE

This SOP is applicable for the enumeration of spore count in Biological Indicators.

3.0 RESPONSIBILITY

Microbiologist - Quality Control

4.0 ACCOUNTABILITY

Manager - Quality Control

5.0 PROCEDURE

5.1 Aseptically transfer one specimen paper strip of biological indicator to 1 ml of chilled sterile distilled water in a sterile 20x150 mm screw-capped tube with 5 sterile glass beads of 4-5 mm diameter.
5.2 Pulp the paper strips in its components fiber by vortexing, using the moderate speed, for approximately 1 minute.
5.3 Dilute with an additional 9 ml of sterile distilled water and vortex again for approximately one minute. This is a 1:10 dilution of the spore strip. When pipetting from this suspension avoid drawing fiber masses into the pipette.
5.4 Heat shock the suspension of Bacillus stearothermophilus spore in boiling water bath at 95-100°C for 15 minutes, start the timer when the temperature reached 95 °C (with the help of stopwatch).
5.5 Cool the suspension rapidly in an ice water bath (0-4°C).
5.6 Transfer two; one ml aliquots to separate sterile test tube.
5.7 Prepare a series of tenfold dilution from each aliquots using chilled sterile distilled water as the diluent. The range of dilution should be selected to yield between 30 to 300 CFU/ml from one dilution series.
5.8 Transfer 1 ml from each dilution to duplicate, add 15 ml Trypticase Soya Agar into each plate (temperature 40-50°C). Let it solidify.
5.9 Incubate plates at 55-60°C and examine after 24 to 48 hour.
5.10 Record the number of colonies on each plate, enter the result in the biological indicator enumeration form.
5.11 Determine the average count using the appropriate dilution factor.
5.12 The number of spore per strip should be not less than 50 % and not more than 300% of the number of visible spore of the specified strain stated on the manufacturer label. Note: Repeat this procedure each time a new batch /lot number of spore is received.
Related: New Genetically Engineered Biological Indicators

6.0 ABBREVIATIONS

6.1 SOP - Standard Operating Procedure
6.2 CFU - Colony Forming Units

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Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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