sop for Sterility Testing of Microbiology

1.0 OBJECTIVE

1.1 To lay down the Procedure for Sterility Testing of Microbiology Laboratory.

2.0 SCOPE

2.1 This procedure applicable for Sterility Testing of Microbiology Laboratory

3.0 RESPONSIBILITY

3.1 Microbiologist QC

4.0 ACCOUNTABILITY

4.1 QC Manager

5.0 PROCEDURE

5.1 Requirement
5.1.1 Manifold holder assembly
5.1.2 Vacuum pump Sterile FTM
5.1.3 Sterile SCDM
5.1.4 Sterile 0.1% w/v Peptone water.
5.1.5 70% filtered IPA Solution.
5.2 Methodology
MEMBRANE FILTRATION METHOD
5.2.1 Wipe the sample article individually with 70% IPA solution and keep in a clean S.S trays marked with Product Name,

Batch No. and Lot No, and then transfer the samples to the sterility room through clean pass box for performing sterility.
5.2.2 Prepare the media tubes (FTM and SCDM) as per the SOP for preparation of culture media. Dispense 100 ml

quantity for membrane filtration and for Direct inoculation Method. Sterilize both the media at 1210C and 15 psi pressure

for 20 minutes as per SOP for Media Sterilization by Autoclaving.
5.2.3 After autoclave Label the tubes with Name of Media, Media Batch No. and pre-incubate the media tubes

at appropriate temperature i.e. SCDM tubes at 20 to 250C whereas FTM tubes at 30 to 350C for 24 – 48 hrs before subjecting them for sterility operations.
5.2.4 Autoclave Dress, Scissors, forceps and Filtration unit in a S.S Container at 1210C temperature and 15psi pressure for 30 minutes.
5.2.5 After Sterilization cool the contents and aseptically transfer in a S.S. container to cleaned Pass box.
5.2.6 Transfer the pre incubated sterile media tubes, SCDA plates, sterile swabs, sterilized Filtration unit,

and 0.1% w/v peptone water to the sterility test room through pass box.
5.2.7 Enter in sterility room as per the Entry / Exit procedure for Sterility Room.
5.2.8 Start the LAF as per SOP.
5.2.9 Wipe out all samples to be tested for sterility with 70% filtered IPA solution.
5.2.10 Before starting sterility test, expose the SCDA plates as specified locations throughout the testing.
5.2.11 Connect the Glass filtration cup holder with the Filtration flask properly with pipe under laminar airflow unit.
5.2.12 Check the Manometer reading of working LAF and check the temperature as well as humidity of the sterility room.
5.2.13 Switch ON the vacuum pump with the help of switch, present on the wall. Now place 0.45 sterile membrane filters

between filtration cup and receptacle with the help of sterilized forceps and clamped it properly.
5.2.14 Wet the membrane filter by adding approx.. 15 ml of sterilized Fluid A (0.1% peptone water) to filter holder,

and filter the fluid by employing vacuum.
5.2.15 Cut the tip of bottle/vial or ampoule with sterile SS blade in front of the gas burner and immediately

transfer the contents of sample to membrane filter. For dry powder or lyophilized container, add the sterile

water 0.1% peptone dissolve and then collect it in sterile flask and immediately transfer the contents of sample to membrane filter.
5.2.16 Immediately filter the solution with the aid of vacuum and wash the membrane three times with 100 ml of sterilized fluid A (0.1% peptone water).
5.2.17 After complete filtration, stop the vacuum pump.
5.2.18 Lift the membrane carefully with the help of sterile forceps, aseptically cut the membrane filter into

two halves with sterile SS scissor and transfer one half to FTM and one half to SCDM tubes by unplugging in front of gas burner only.
5.2.19 Label both the tubes with Product name, B. No, Report No.., date of testing, Completion date & Tested by.
5.2.20 Simultaneously prepare a negative control by filtering 100 ml of 0.1% peptone water
5.2.21 Instead of product sample, cut the membrane into two halves with sterile SS scissor and transfer one

half to FTM and one half to SCDM and label both the tubes as Negative control.
5.2.22 For media negative control, keep one – one tube of each autoclaved lot of FTM & SCDM un-inoculated.
5.2.23 After completion of work transfer all inoculated media through hatch box and then transfers all the

equipment and exposed plates to microbiology analysis section.
5.2.24 Incubate the FTM tubes at 300C – 350C and SCDM tubes at 200C – 250C. Incubation period for terminally

sterilized products are 7 days and 14 days for aseptically filled products as per IP. If the Product is as

per USP, BP, incubation period is 14 days for both terminally sterilized as well as for aseptically filled products.
5.2.25 Start the LAF of Biosafety cabinet as per SOP and prepare three positive Control tubes by inoculating

aseptically not more than 100 cfu in FTM tubes with S. aureus, P. aeruginosa and Clostridium sporogenes.

Similarly prepare three SCDM positive control by inoculating not more than 100 cfu separately

with C. albicans, A. niger, and Bacillus subtilis. Incubate FTM positive control tubes at 30 – 35ºC & SCDM

positive control tubes at 20 – 25ºC. For bacterial positive controls, incubation period is not more

than 3 days & for fungal positive control, incubation period is not more than 5 days
5.3 DIRECT INOCULATION METHOD:
5.3.1 For sampling and sterility test preparation follow the same procedure as specified in Membrane filtration method.
5.3.2 Take sufficient number of sample, aseptically cut with the help of sterile SS blade and transfer

approx.. 2 ml from each vial to a each 10 tubes containing 100 ml of Fluid Thioglycollate medium

and 10 tubes of 100 ml Soybean Casein Digest Medium. Mix the liquid with medium but do not aerate excessively.
5.3.3 Simultaneously prepare a negative control by inoculating 2 ml of sterile fluid A into tubes containing 100 ml FTM and SCDM.
5.3.4 Incubate the inoculated media for not less than 14 days at 30 to 350C for fluid Thioglycollate

medium and at 20 to 250C for Soyabean-casein digest medium.
5.4 Observation and Interpretation of Results
5.4.1 Visually examine the media tubes daily to its conclusion for macroscopic Evidence of Microbial growth.
5.4.2 If no evidence of growth observed in any of the tube the product to be examined for the

test complies with the test for sterility. The test is not valid unless the Negative control shows negative till
6.0 ABBREVIATIONS