Sterility Test Validation Standard Operating Procedure

Sterility Test Validation Standard Operating Procedure is a crucial document that outlines the step-by-step process for validating the sterility testing procedures used in a pharmaceutical or biotechnology setting. This SOP ensures that the sterility testing methods are reliable, accurate, and consistent. Here’s an outline that you can use to create an SOP for Sterility Test Validation:


To assure that sterility test with direct inoculation will produce the same results when tested as per Sterility Test Validation Standard Operating Procedure. State the purpose of the SOP, which is to establish a standardized procedure for the validation of sterility testing methods.


This Sterility Test Validation Standard Operating Procedure is applicable for sterility of products. Define the scope of the SOP, specifying the types of products or processes to which it applies.


  • Microbiologist from QC departments prepared SOPs.
  • QC Manager reviewed and signed the SOPs.
  • QA Head approved the SOPs.
  • Director Operations authorized the SOPs for implementation.
  • Clearly outline the responsibilities of personnel involved in the sterility testing validation process, including roles such as Quality Assurance, Microbiologist, and Validation Specialist.


  • European Pharmacopoeia 10.0
  • Test for Sterility (Ph. Eur. method 2.6.1)
  • List any relevant documents, standards, or regulations that provide guidance on sterility testing validation.


SOPs: Standard Operating Procedures
FTM: Fluid Thioglycollate Medium
TSB: Soyabean Casein Digest Broth

Provide definitions for key terms used in the Sterility Test Validation Standard Operating Procedure to ensure clarity and consistency.

Sterility Test:

Sterility is the test in which we ensure that the batch of product is sterile or has been sterilized”. The test is applied to substance, preparations or article which, according to the pharmacopoeia, are required to be sterile. However, a satisfactory result only indicates that no contaminating microorganism has been found in the sample examined in the condition of test.



  • Sterile S.S Forceps
  • Sterile Surgical Blade No.22
  • Sterile Scissor
  • Sterile Syringes
  • Sterile Gloves
  • Sterile Glass Media Bottle
  • Isopropyl Alcohol 70%
  • Sterile Lint Free Clothes
  • Laminar Flow Hood
  • Sterile Suit
  • Fluid Thioglycollate Medium
  • Soyabean Casein Digest Broth
  • List and describe all equipment and materials required for sterility test validation, including sterilization methods, culture media, incubators, and testing vessels.

Validation Test:

  1. This method validation is performed, when the test for sterility to be carried out on a new product.
  2. Whenever there is a change in the experimental conditions of the test.
    This method of validation may be performed simultaneously with the test for sterility of the product to be examined.
  3. Carry out a test as describe below under test for sterility of the product to be examined using exactly the same methods, except for the modifications.

Direct Inoculation:

  1. After transferring the contents of the container or containers to be tested to the culture medium, add an inoculum for a small number of a viable microorganisms (NMT 100 cfu) of each of the strains listed in Table No:1(NMT 100cfu) to the medium.
  2. Perform a Growth Promotion Test as a positive control.
  3. Incubate all the containers containing medium for NMT 5 days.

Antimicrobial Activity:

If clearly visible growth of microorganisms is obtained after the incubation, visually comparable to that in the positive control container without product, either the product possesses no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated. This test for sterility may then be carried out without further modification.
If clearly visible growth is not obtained in the presence of the product to be tested, visually comparable to that in the positive control containers without product, the product possesses antimicrobial activity that has not be satisfactorily eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity and repeat the validation test.

Table-1 Test Microorganisms Suitable for Use in the Growth Promotion Test

Medium Microorganism Incubation (05 days)
Strain Temperature Conditions
Fluid Thioglycollate Staphylococcus aureus ATCC 6538 32.5±2.5°C Aerobic
Pseudomonas aeruginosa ATCC 9027 32.5±2.5°C Aerobic
Clostridium sporgenes ATCC 11437 32.5±2.5°C An aerobic
Soyabean Casein Digest Bacillus subtilis ATCC 6633 22.5±2.5°C Aerobic


To minimize the errors in validation test, the validation test must be carried out by the trained personnel.
All the materials, vessels and equipment’s that are used in sterility validation test sterilized by the dry heat oven or autoclave. Uniforms that are used by the personnel must be sterilized and germ free.

Remember to tailor this template to the specific requirements and processes of your organization. Additionally, ensure that the SOP complies with relevant regulatory guidelines and standards applicable to sterility testing in your industry.

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