What is the meaning of Sterility Test?
Meaning of sterility test:-
Sterility testing is set of activity to confirm that products are free from any Viable Microorganism. Sterility can be defined as absence of viable Microorganism.
PRECAUTIONS AGAINST MICROBIAL CONTAMINATION
The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any micro-organisms which are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls.
Strains of the test microorganisms suitable for use in the Growth promotion test and the Method suitability test(Ph. Eur 2.6.1)
Minimum Quantity To be used for each medium (Ph. Eur 2.6.1)
Minimum number of items to be tested(Ph. Eur 2.6.1)
Standerd Operation Procedure Of Sterility Testing :
- Sterile Sterility test filter holder unit (Manifold).
- Sterile membrane filter – 0.45 m Pore size, 47 mm Diameter
- Sterile Soyabean Casein Digest Medium (SCDM), 100 ml in each tube.
- Sterile Fluid Thioglycollate Medium (FTM), 100 ml in each tube.
- Sterile Scissors.
- Sterile Dusters.
- Sterile Forceps.
- Sterile gloves.
- 70% Isopropyl Alcohol (Filtered through 0.22 µm membrane filter)
- Laminar Air Flow Cabinet (LAF)
- Test Samples
- Sterile gown, mask and booties.
- Sterile Bacteriological peptone (0.1% w/v).
- Prepare media for sterility testing as per SOP.
- Use pre-incubated media for the sterility testing of injection.
- Before taking for the testing observe the media tubes for its sterility.
- Insure GPT of Prepared media is performed before use for testing.
- Take the required quantity of media tubes and disinfect the other surface of media tubes using filtered 70% v/v IPA.
- Sterilize all material required for performing sterility test i.e. manifold, scissors, forceps, cutters, membrane filters, filtration cones, dusters, in double door autoclave at 121ºC for 30 minutes.
- Keep ON U.V. lamp of laminar air flow bench before one hour of sterility testing.
- Clean outer surface of sample with filtered 70% IPA in case of raw material and transfer through pass box provided in sterility testing room.
- Incase of Finished product, take required quantity of samples as per pharmacopoeia. Dip the samples in filtered 70% IPA or spray with filtered 70% IPA and then transfer through pass box provided to sterility testing room.
- Enter in to the sterility testing room by following SOP of entry and exit procedure.
- Switch “OFF’ UV Light, if it is ON and switch “ON” Laminar Air Flow. Wipe the working bench of LAF with 70% v/v IPA with clean and sterile lint free duster.
- Take all the sterilized materials required for sterility test from required for sterility test from double door autoclave and place in the laminar air flow unit of sterility test from Laminar flow unit of sterility testing area.
- Arrange samples to be tested in the LAF unit with proper disinfection.
- Assemble the filtration cones on sterility test filter holder unit which is attached to be source of vacuum and put the sterilized membrane filter on to the filtration cone.
- Wet the filter paper with 100 ml of 0.1 % sterilized bacteriological peptone.
- In the case of glass ampule, cut the 20 number of ampule with cutter and transfer whole content of ampules to the filtration cone and allow to filter it completely.
- After completion of filtration wash the membrane with 3 X 100 ml of sterilized 0.1% bacteriological peptone and filter it.
- After completion of filtration, close the knob of each filtration assembly and the top of assembly shall be carefully removed. Filters are cut in to two halves with the help of sterilized forceps or scissor. Transfer one half in to Soyabean Casein Digest Medium tube and second half in to Fluid Thioglycollate tube for one sterility test.(Step X)
- In similar fashion, perform negative control (Blank) on every testing day for each FTM and SCDM by using sterile 0.1% bacteriological peptone.
In similar fashion, inoculate the bacterial and fungal culture of known population in particular media.
- Repeat the same procedure for every lot or load of sample to be analysed
- In case of powder, dissolve the powder in 100 ml sterilized 0.1% bacteriological peptone and filter through membrane filter.
- Wash the filter with 3 x100 ml of sterilized 0.1% bacteriological peptone and follow the (Step X)
- The inoculated sterility test tubes of Soyabean Casein Digest Medium are incubated at 20 to 25°C and Fluid Thioglycollate Medium are incubated at 30 to 35°C for 14 days.
- Examine the test tube on each working day for the evidence of microbial growth throughout the 14 days incubation period and record the results. If the 14 th day is weekly off or half day then observe the test tube on next working day.
- After completion of incubation dispose the media as per SOP
- Negative Control
- Expose 1 tube each of sterile Soyabean Casein Digest Medium and Sterile Fluid Thioglycollate Medium in LAF during sterility testing and incubate same tubes at 20 to 25°C and 30 to 35OC respectively for 14 days.
The positive control run separate in MLT area of used media for particular test
- For detail GPT of prepared media refer Annexure-I.
- For detail of minimum items to be tested and minimum quantity to be used for each medium refer the Annexure –II.
Observation and Interpretation of Results:
- Observe the incubated tubes daily and examine the media for macroscopic evidence of microbial growth.
- If the material being tested render the medium turbid so the the presence or absence of microbial growth can not be easily determined by visual examination, 14 days after the beginning of incubation, transfer portion (each not less than 1 ml) of the medium to fresh vessel of the same medium and then incubate the original and transfer vessels for not less the 4 days.
- If No evidence of Microbial growth is found, the preparation under examination complies with the test for sterility, provided that the negative controls does not showing evidence of growth.
- If evidence of microbial growth is found, the preparation being examined does not comply with the test for sterility. Do not repeat the test unless it can be clearly shown that the test was invalid for causes unrelated to the preparation under examination. The test may be considered invalid only when one or more of the following conditions are fulfilled.
- Microbial growth is found in negative control.
- Data on microbial monitoring of the sterility facility show a fault.
- A review of the testing procedure used for the test in question reveals a fault.
- After identifying the microorganism isolated from the container showing microbial growth the growth may be ascribed without any doubt to faults with respect to the materials and/ or the technique used in conducting the test procedure.
- If the test is declared to be invalid it is repeated with the same number of units as in the original test.
- If no evidence of microbial growth is found in the repeat test the product examined complies with the test for sterility. If microbial growth is found in the repeat test and confirmed microscopically, the preparation under examination does not comply with the test for sterility and the product is rejected.
After luxurious growth shown in Positive control discard it after five days from incubation.
- Record the results of the test in annexure –III.
Frequency: Every Manufacturing g Batch
- Annexure-I : Growth Promotion Test of Media (Organism Used, Temperature,
- Annexure-II : Minimum Number of Items to be Tested and Minimum Quantity to Be
- Annexure-III: Sterility Test Report (Membrane Filteration Method).