SOP for investigation of OOS (out of specifications) for sterility test to find out the root cause is described in this post which you can follow in the section of microbiological lab.


This procedure has been established for finding out the root cause for failure of sterility test.


This procedure is applicable to microbiology lab.




Quality Control Manager.            


If microbial growth observed during incubation period then it means that the test for sterility test for respective batch does not comply with specifications.

Parameters to be Investigated

Investigate the results of microbiological monitoring reports of the sterile area (microbiology lab) and sterile manufacturing and filling of the suspected product. 

Investigate the cleaning and sanitization logbook record of both the microbiology testing area for sterility testing and sterile manufacturing area for product manufacturing.

Investigate the chemical or biological indicator record for sterilization cycle of the media, sterility set, uniform set, and sterilization parameters i.e. (Temperature/Pressure/Time). 

Investigate the logbook for dry heat sterilization process of glass vials, autoclavation of rubber stoppers and uniform set and machine parts.

Investigate and review the swab testing of the sterile manufacturing and testing area as well as the Finger DAB test of the operators and microbiologist.

Investigation of the sample showing growth

Preserve the  sample showing positive result along with positive and negative control to complete investigation. 

Take a loop full of culture from sample showing growth on two pre-incubated nutrient agar plates.

If the sample is taken from FTM, incubate the plates at 32oC±2.5oC for 48 hrs. 

If the sample is taken from TSB, incubate the plates at 22oC±2.5oC for 72 hrs. 

After Incubation, observe the cfu and identify the colony by staining techniques or other biochemical techniques. 

If colony observed on agar plate is same to the positive control then there are chances of contamination during testing.

Repetition of Sterility Test

The test must be repeated if microbial growth observed in the negative controls.

If microbial growth observed in the laminar airflow cabinet at the time of sterility testing or in the sterility test room that could be a cause of contamination i.e.(Reports of microbiological monitoring of the sterility test room and area shows a growth evidence).

The test protocols are not followed properly leading to contamination then repeat the test. 

if glassware are not depyrogenated or sterilized properly lead to the contamination then repeat the test.

incase of contaminated raw material/powder (decided after atleast three consecutive repetitive tests) discard the batch after 3 repetitions of the same results.

 CAPA(Corrective and Preventive Actions):

At the end of investigation of the whole manufacturing and testing procedures and reviewing the data compile all data and find out the root cause and give a comprehensive report to the relevant department suggesting them the corrective and preventive action report (CAPA) tat clearly indicate the solutions to avoid sterility testing failures in future.