Sterility Test Procedure for vial sample

Introduction :

The tests for sterility are intended for detecting the presence of viable forms of micro – organisms in preparations. The tests must be carried out under conditions designated to avoid accidental contamination of the product.

The following procedure is based on the procedure described in British Pharmacopoeia.

 

1. Thoroughly clean the laminar flow module.

2.  Switch on the U.V. tube and laminar flow at least 15 min.  

before commencing the work .

 3. Wear sterile apron  while carrying out the test.

4.  Before starting the work, check the U.V. tube is switched off.

 Precaution  While Testing :

Procedure :

1.  Equipments :

A) Sterilise clean membrane filtration assembly by placing    

them in butter papers and autoclave it at 121 ° C for 15 minutes.

B) Sterilise 47 mm x 0.45 micron membrane filters by placing them   in  a Peteridish enclosed with filter paper & autoclaving at 121°C   for 15 min.Also sterilised all other accessories like SS foreceps,glass peteridishes & other glassware as required by autoclaving  at 121^o C for 15 minutes.

2.  Media & Reagents :  Culture media will be prepared as directed by the manufacturer.  Where ever water is required for testing purpose, used casein peptone  water. The media should be sterilized by heating in a autoclave at 121° C for 15 minutes, unless otherwise indicated on the respective media  container.

 Sample : swab the vial by IPA.

3. Using membrane filtration 

i) First arranged the filter assembly  as per requirement & attach a vacuum pump to filter the sample solution.

 ii) Give washing to membrane filter paper with sterile water &

discard the washing .

iii) Take 20 vial . Withdraw 1 gm  sample from each vial &     

dissolve in  100 ml of sterile 0.9 & sodium chloride solution.

iv) Filter sample solution through membrane filter paper.

v) Then give washing with sterile water & dismantle the assembly

vi) Cut the membrane filter paper into two equal part with forceps.

vii) One part of  the paper insert in soyabean casein digest medium

 (for fungi)  & one part of the paper insert in fluid thioglycolate  medium  ( for   bacteria ).

viii) For 14 days incubate soyabean casein digest media at  20 to 25º  

C & fluid thioglycolate media at 30 to 35 º C  & observe the media every day upto 14 days .

 ix) There should not be any  growth observed in soyabean casein

digest medium & fluid thioglygolate medium for 14 days.

Negative controls are carried out as follows :

Incubate one 100 ml test tube of  Soyabean Casein Digest medium  at 20-25°C & observe every day upto 14 days.

Incubate one 100 ml test tube of  fluid thioglycolate  medium  at  30-35°C & observe every day upto 14 days.

 

Positive controls are carried out as follows:

Following culture should be insert in specified media & observed the growth for 14   days.

            For bacteria  - Bacillus subtilis ( ATCC 6633 )

For fungi -  Candida albicans ( ATCC 10231 )

Observation and interpretation of results :  

At intervals during the incubation period and at its conclusion, examine the media for  macroscopic evidence of microbial growth.

If the material being tested renders the  medium turbid so that the presence or absence of microbial growth cannot be readily determined by visual examination, 14 days after the beginning of incubation transfer  portions (each not less than 1 ml) of the medium to fresh vessels of the same  medium and then incubate the original and transfer vessels for not less than 4 days.

If no evidence of microbial growth is found, the product to be examined complies with  the test for sterility. If evidence of microbial growth is found the product to be  examined does not comply with the test for sterility.