Sterility Test

Sterility Test

Introduction:

Sterility test is one of the most important test to declare an injectable dosage form sterile Yet the first official compendial requirement of sterility testing of  Drugs administered by the parenteral route didn’t appear until 1932 in BP &  1936 USP.

Definition:

The sterility Test is designed  to reveal the presence of contamination with live microorganisms in preparation intended for parenteral administration  or for other sterile applications.

  
 

Test Condition:

1.  The test should be carried out under aseptic condition as free from contamination.

2.  Use disinfecting agents, germicidal lamps and laminar airflow

3.  Use sterilized dress, static- free clothing including head and foot wear,

4.      The air pressure in the sterility testing room should be greater than that of the exterior

      area.

5.      The performance of the laminar air flow and of the Sterility  testing room should be 

      monitored  by particulate count and  microbial count.

Apparatus used:

1.  Filling Funnel -250 ml

2.  Membrane Filter of Diameter 47mm. Pore size  0.20µm to 045µm. The filter unit

     must be autoclaved at 121°C &15 lb pressure  for 15 minutes.

Media Used:

1.  Fluid Thioglycolate  Medium(FTM)

2.  Tryptic Soy  Broth (TSB)

3.  Meat Peptone or Casein Peptone.

Media Preparation:

The media should  be prepared as per requirement of the test following the instruction of the manufacturer.

Methods:

The methods used for sterility testing are :

1.      Method-I: Membrane Filtration.

2.      Method-II : Direct transfer to test media.

Method-I: Membrane Filtration

In the case of SVPL

Transfer aseptically the contents of 20 container in the  filtering funnel. Filter the solution using 500 ml 0.1 % meat peptone or casein peptone as neutralizing agent. Upon completion of the filtration, divide the membrane filter into two approximately equal parts. Transfer one part of the membrane filter into one flask and the other part into another flask . Pour 100 ml fluid Thioglycolate Medium into one flask and the same  volume of Tryptic Soy Broth into another flask. Incubate the Fluid Thioglycolate Medium flask at 30-35°C and Tryptic Soy Broth flask at 20- 25°C  for 14 days.

In the Case of Powder for Injection (Soluble Solids):

Aseptically transfer the contents of 20 containers (total weight should be 300 mg – 6.0 g ) into a flask containing 200 ml of meat peptone  or casein  peptone ( 0.1%). Transfer the solution into a filtering funnel and filter the solution as in the case of SVPL.

Method-II : Direct Transfer

The Direct Transfer Method is the more traditional  sterility Test Method . Basically , the Direct Transfer Method involves  three steps.

1.      Aseptically Opening each sample container from a recently sterilized batch of  product.

2.      Using sterile syringe and needle to withdraw  the required volume of the sample for both media from the container.

3.      Injecting one-half of the required volume of sample into a flask containing the required volume of Fluid Thioglycolate  Medium and the other half volume of sample into a second flask containing the required  volume of Tryptic Soy Broth.

In the case of powder for Injection transfer aseptically an amount 300mg to 6.0 g of the product from 20 containers into a flask containing 100ml Fluid Thioglycolate  Medium and the same amount into another flask containing Tryptic Soy Broth of the same volume.

Incubate the flask containing Fluid Thioglycolate  Medium at 30- 35°c and Tryptic Soy Broth at 20-25°C  for 14 days and examine the media visually for growth at least as often as on the third or fourth or fifth day, on the seventh or eighth day, and on the last day of the test period .

  

Interpretation of the result:

If no evidence of growth is found is any of the culture vessels ,except in the positive control, the articles tested  pass the sterility test. Repeat the test if growth is found. If no growth is found is the repeat test the sample passes the sterility test. The sample   fails is growth is found in the repeat test. 

Positive Control;

The media used for sterility test is checked for its efficacy to support growth of different organism is done by adding 10-100 cfu of the microorganisms. This is known as positive Control. 

 

Negative Control: 

Negative controls consists of  culture media without addition of product sample or microbial challenge. The negative control is used to verify the sterility of the medium before , during and after the incubation period of the sterility test. If microbial growth is detected with a negative control either the medium was not sterilized properly, contamination was introduced accidentally during  the test procedure , or there exists an inefficiency in the  container or packaging system. If such microbial growth in a negative control occurs and in the absence of evidence from the environmental monitor , equipment or personnel of accidental contamination, it becomes a clear indication for retesting the product.

Conclusion:

The pharmacopoeial procedures for sterility test are not by themselves designed to ensure that a batch of product is sterile or has been sterilized. When evidence of microbial  contamination in the article is obtained by the appropriate pharmacopoeial  methods,  the result so obtained is conclusive evidence of failure of the article to meet the requirements of the test for sterility, even using an alternative procedure then different result is obtained.

Though there are many Limitation of the sterility test , but still it is the most accepted method of a batch to be declared sterile.

Challenge Test:

Challenge test is a standard test procedure , that establishes & validates  a particular test  e.g. Microbial Challenge test of a filter is the ability of the filter to retain microorganisms involving filtration of a standardized culture containing a large number of small microorganisms.