sop for Sterility Testing of Microbiology

sop for Sterility Testing of Microbiology



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


2.1 This procedure applicable for Sterility Testing of Microbiology Laboratory


3.1 Microbiologist QC


4.1 QC Manager


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
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
5.3.1 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

Sr. No. Abbreviation used Extended Form
1.0 SOP Standard Operating Procedure
2.0 0C Degree Celsius
3.0 % Percentage

sop for calibration and validation of micro autoclave

sop for Sterility failure investigation

cleaning and operation of discard autoclave

sop for operation of fogger machine

sop for Biological assay of lactic acid bacillus

sop for preparation of culture inoculum

STP for sterility testing of sterile gloves

sop for Operation and calibration of active air sampler

sop for transfer of material for testing and sampling in sterile area

entry & exit procedure in microbiology laboratory

Growth Promotion Test In Microbiology Laboratory

Operation of B.O.D in Microbiology Laboratory

Operation of Horizontal Laminar Air Flow in the microbiology laboratory

Operation and cleaning of Pass Box.

Operation and cleaning of air sampler

Cleaning and Sterilization of Glassware

Analysis of water for microbial load in microbiology lab

Operation and temperature monitoring of Refrigerator

Fumigation of Microbiology Laboratory.

Entry & Exit procedure In Sterility Area

SOP for Microbial analysis of Raw Material Finished Products

SOP for Operation & Calibration of pH meter in Micro Department

SOP for Operation & Calibration of pH meter in Micro Department

SOP Operation and calibration of Hot Air Oven In Microbiology

SOP for operation cleaning & calibration of Digital colony counter

SOP for Operation And Cleaning of Microscope

sop for Media Preparation and Consumption

sop for Receipt Storage and Usage of Culture Media

sop for Cleaning Sanitization And Disinfection In Microbiology

sop for Environmental monitoring of all the Classified area

sop for Handling and Sub culturing of Microbial cultures

sop for Media Growth Promotion Test and various Microbiological test

sop for BOD incubator operation and cleaning

sop sampling of water for microbiological analysis

sop for Disinfectant Efficacy Test

sop for for cleaning and operation of vortex mixture

sop for Temperature & Relative Humidity Monitoring

sop for Operation and Calibration of Heating Block

sop for Sterility Testing of Microbiology

sop for Disposal of Culture Media

sop for Drain point of Microbiology

sop for entry & exit procedure In Microbial limit test and Biosafety

sop for Gram Staining of Bacteria in Microbiology Laboratory

sop for Monitoring of Compressed Air/gases for microbiological analysis

sop for BET (Bacterial Endotoxin) test in Microbiology

sop for receipt storage and Determining the population of Biological indicators

sop for qualification of analyst microbiologist

sop for Bioburden test of Packing materials in Microbiology Laboratory

sop for microbiological assay of erythromycin antibiotic

sop for liquid particle counter

sop for operation and calibration of digital zone reader

sop for monitoring of ultraviolet efficiency LAF and pass box

microbiological assay of cyanocobalamin or vitamin B12

gowning procedure for microbiological testing area

swab testing of various surfaces for bioburden determination

sop for endotoxin challenge test

Hold time study protocol for sterilized media

sop for personnel Qualification protocol for aseptic area

sop for sampling and testing of drain water

Sop for Operation of Airborne Particle Counter

sop for Inoculum Preparation

sop for Validation protocol of steam sterilizer autoclave

sop for pathogen detection from drain point

Sop for Analysis of Raw water Purified water water for injection and pure steam water

sop for preservatives efficacy test

sop for collection and preservation of in house isolated microorganisms

sop for Operation Calibration and Maintenance of Micropipette

Sop for UV Efficacy Test

sop for gram staining

Sop for swab testing

sop for microbiological testing of water


sop for depyrogenation of apparatus

sop for media preparation

sop for fertility test growth promotion test of media

sop for Operation and cleaning of moist heat sterilizer

sop for monitoring by active air sampler

sop for swab sampling and testing for clean rooms in production area

sop for monitoring in microbiology laboratory

sop for Fumigation of aseptic area and microbiology lab

sop for monitoring of personnel in aseptic area

sop for maintenance of cultures

sop for Operation and cleaning of laminar bench

preparation of settle plates

sop for monitoring of pure steam

sop for entry and exit procedure to m.l.t and b.e.t room

sop for storage of and use of media

sop for disposal of microbiological media and cleaning of microbiological glassware

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