SOP for Sterility Testing Using Manifold Filtration Method : Pharmaguideline

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SOP for Sterility Testing Using Manifold Filtration Method

Standard operating procedure to test the samples for sterility using filtration manifold.


To lay down the procedure for sterility testing of samples by membrane filtration method using filtration manifold.


Applicable to sterility testing of dilution study and stability study samples at Quality Control Microbiology Laboratory.


Trained Quality Control/Microbiology


Head of Department


5.1 General Instructions and Precautions

5.1.1 Follow SOP for entry and exit to Microbiological testing and Sterility Testing areas.
5.1.2 Ensure that all the sterile or sterilized items are within their expiry/use before the date and the packets are integral. If any packs are found damaged or integrity is compromised, then do not use such materials.
5.1.3 Ensure that the all the media and diluents to be used are pre-incubated. Check the media for contamination or physical damages. In case of Fluid Thioglycollate Medium, if the more than upper one-third of the medium has acquired pink color (resazurin ring), then do not use such media containers.
5.1.4 Check the physical parameters (Temperature, Humidity and Differential Pressure) of the area and ensure they are within limits before starting the activities.
5.1.5 During each sterility test session, perform environmental monitoring as per SOP.

5.2 Transfer of Materials to Sterility Testing Area and cRABS

5.2.1 Materials such as media, rinsing fluid or diluents, test accessories viz., forceps, scissors, equinox drain tray with tubing, drain collection container, etc required for sterility testing shall be sterilized in an autoclave, unloaded and stored in the sterile side (buffer room).
5.2.2 All other materials shall be transferred as follows: Keep all the required materials related to sterility testing such as samples and other sterile accessories in pass box in Incubator room opening to LAL test room. From the LAL room unload the materials and keep in LAF. Remove the flip-off seals of the product vials and disinfect the outer surface of vials using filtered disinfectant solution. Disinfect the outer surface of the sterile accessories packs using filtered disinfectant solution. Transfer all the materials into dynamic pass box opening to buffer room. Ensure the dynamic pass box is in ON condition. From the buffer room, unload the materials and transfer to sterility testing room.
5.2.3 Operate cRABS as per SOP
5.2.4 Take all the required materials like media containers, sterilized accessories like the manifold, filtration funnels, forceps, scissors, etc., filtered disinfectant solution & 70% IPA and product samples to sterility testing room.
5.2.5 Keep all materials except for product samples in cRABS pass box.
5.2.6 Close the cRABS pass box and disinfect the outer surface of the material with filtered 70% IPA by wiping. Turn on the UV light and expose the materials for not less than 15 minutes.
5.2.7 After minimum 15 minutes, transfer the materials from the cRABS pass box to cRABS through the channel provided.
5.2.8 In case of samples, keep them in cRABS pass box, close the pass box and disinfectant the outer surface of the samples with filtered 70% IPA by wiping. Do not turn ON the UV light. Allow the disinfectant to dry.
5.2.9 Transfer the samples from the cRABS pass box to cRABS through the channel provided.
5.2.10 Before the test, ensure that the disinfectant on each article is dried properly and the packs are integral.
5.2.11 Connect the drain collection container to cRABS drain port securely.

5.3 Sample Requirements

5.3.1 Number of Containers: As per respective protocols
5.3.2 Volume of Sample to be tested
Stability Samples:
Content of Container
Minimum Quantity to be tested for each medium
Less than 1 ml
Total Content
1 to 40 ml
Half the contents but not less than 1 ml
More than 40 ml but less than 100 ml
20 ml
100 ml or more
10% of contents, but not less than 20 ml
Note: In case fill volume greater than 10 ml then consider the leftover sample from 3 vials for bacterial endotoxin test (To be pooled before the test)
Dilution Study Sample: As per respective protocol but not less than 20 ml

5.4 Sterility Testing By Open Funnel Method Using Manifold

5.4.1 Place the sterilized manifold inside the cRABS and connect to vacuum pump through drain collection container or flask.
5.4.2 Place the sterilized membrane filtration funnel onto the holder of the manifold. Place sterile 0.45µ membrane filter on the filter holder of the filtration funnel.
5.4.3 Open the cap of the filtration assembly and wet the membrane with 100 ml of rinsing fluid and filter by turning on the vacuum pump.
5.4.4 Aseptically open the product container seal and transfer the contents of each product container to the filtration assembly. Alternatively aseptically withdraw the contents using sterile syringe and needle and transfer. In case of lyophilized products first, reconstitute the sample with required volume sterile diluent and then transfer the contents.
5.4.6 Turn on the vacuum pump and filter the contents.
5.4.7 After filtration of the sample, add 100 ml of rinsing fluid and filter. Repeat this until 3 x 100 ml of rinsing fluid filters through the membranes.
Note: The rinsing fluid and volume of rinsing fluid shall be changed as per validation if different from above.
5.4.8 After rinsing, aseptically cut the membrane into two half with sterilized forceps and scissors. Transfer one half of the membrane to 100 ml of Soybean Casein Digest Medium and the other part to 100 ml Fluid Thioglycollate Medium tubes.
5.4.9 Label the tubes with the Product name, Batch No/A.R No, Media Lot No, Date and Analyst initials.

5.5 Negative Product Control Test

5.5.1 Perform one negative control test during each sterility test session as a control for media and diluents sterility and conditions during the test session.
5.5.2 Perform the testing by considering 100 ml of 0.1% sterile peptone water as product and performing maximum manipulations performed during the session as follows: If the maximum manipulations performed during the test session are for the lyophilized product, then all steps involved in lyophilized product testing shall be performed for negative product control. If the maximum manipulations performed during the test session are for the liquid product then all steps involved in the liquid product shall be performed for negative product control.

5.6 Transfer and Disposal of Material

5.6.1 After completion of the test, transfer all accessories, spent sample containers, empty media diluents containers to autoclave area through pass box from Buffer Room to Autoclave Room.
5.6.2 Transfer the canisters and media tubes for incubation through dynamic pass box from buffer room to LAL Test Room and then through pass box from LAL Test Room to Incubator Room.
5.6.3 Clean the cRABS with the filtered disinfectant agent. Disinfect the port and tubing for drain collection by transferring sufficient volume of disinfectant solution through the port and tubing such that all the surface comes in contact with the disinfectant solution.
5.6.4 Close the valve if the drain collection tubing, disconnect the stainless steel container by opening the TC clamp. Transfer the container to autoclave through pass box from buffer room.
5.6.5 Deactivate the solution in the container by addition of appropriate deactivating agent and dispose of as per SOP.

5.7 Incubation and Observation

5.7.1 Incubate Soybean Casein Digest Medium (SCM) sterility test tubes at 22.5 ± 2.5°C and Fluid Thioglycollate Medium (FTG) sterility test tubes at 32.5 ± 2.5°C for 14 days.
5.7.2 Examine on each working day for the evidence of microbial growth throughout the 14 days incubation period and record the results. If the 14th day is weekly off or holiday then observe on next working day.
5.7.3 During observation, any evidence of clear growth in terms turbidity, cloudiness, precipitate, cotton ball type growth, clear medium with twister raising from the bottom when swirled, then remove such canisters/tubes and proceed for identification/investigation as per SOP on the day of such observation.
5.7.4 After completion of incubation, dispose of the media as per SOP for disposal of media.

5.8 Acceptance Criteria

5.8.1 If there is no evidence of microbial growth in any of the sterility test media at the conclusion of the incubation period, the sample/ product under test complies with the requirements for sterility.
5.8.2 If evidence of microbial growth is found in either of the media, the sample/ product being tested does not comply with the test for sterility unless it can be clearly demonstrated that the test was invalid for causes unrelated to the sample/ product being tested. The test may be considered invalid only if one or more of the following conditions apply: The microbiological monitoring of the sterility testing area shows a fault. A review of the testing procedure used during the test in question reveals a fault. Microbial growth is found in the negative controls. After determination of the identity of the microorganisms isolated from the test, the growth of this species (or these species) may be ascribed unequivocally to faults with respect to the material and or the technique used in conducting the sterility test procedure.
5.8.5 If the test is declared invalid, the test must be repeated with the same number of units as in the original test.
5.8.6 If no evidence of microbial growth is found in the repeat test, the product examined complies with the test for sterility.
5.8.7 If microbial growth is found in the repeat test, the product examined does not comply with the test for sterility.
Related: Incubation Conditions for Common Media used for Fungus and Bacteria


6.1 SOP - Standard operating procedure
6.2 cRABs - Closed Restricted Access Barrier System
6.3 % - Percent
6.4 UV - Ultraviolet
6.5 °C - Degree centigrade
6.6 ml - Milliliter
6.7 SCA - Soybean Casein Digest Agar
6.8 SCM - Soybean Casein Digest Medium
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