MICLAB-060 Micro Laboratory Procedure for Sterility Testing

All Microbiology Laboratory colleagues

1. Obtaining of Samples

1.1.         Sampling

Production personnel conduct all sampling in the following areas.  Details of these sampling procedures are outlined in MICLAB 095.

Terminally Sterilized Products

Samples are to be selected randomly from throughout the autoclave load.

Aseptically Filled Products

Samples are to be selected from the beginning, middle and end of each batch, plus the first units filled after any prolonged downtime (greater than one hour).  Samples are to be of saleable standard, not rejects.

1.2.         Sample Size required for Initial Sterility Testing

Determine the sample size of the product type and batch size.

2. Setting up a Testing Session

2.1.         Checking the testing requirements for each product code

Every product sampled must be documented properly in details when setting up a test session.
This will indicate what tests are required for each product code.  Always cross-check the BPN on the sample container against the sample ID sheet.

2.1.1.     Parametric Release

Not all terminally sterilized products require sterility testing.  Check for Parametric Release status of any product code.

2.1.2.     Sterility Test

These products require sterility testing.  A standard sterility testing session is comprised of 4 products and a sterile control to be membrane filtered and any number of direct inoculations along with a suitable sterile control.

2.1.3.     Bacterial Endotoxin

Check if a product requires Bacterial Endotoxin testing and also by which test method, either gel clot or KCA.  If the product requires Bacterial Endotoxin testing, separate these samples from the sterility samples.  Ensure that these samples are labelled with the Product Name & strength, batch, code and place these in the receptacle for the type of Endotoxin test required.

2.1.4.     Biological Assay

If a biological assay is required separate the samples from the Sterility samples.  Ensure these samples are labelled with the Product Name, batch and code and are given or communicated to the technician responsible for Assay testing.

2.2.         Known Sterile Controls

A known sterile control is to be included in each test session appropriate for each type of testing carried out. i.e. for testing by Membrane Filtration use 20 units of Water for Injection that have been autoclaved twice.

For testing by Direct Inoculation use a 100 mL bottle of Paraffin Oil that has been dry heat sterilized in the Micro. Lab. depyrogenation oven at 180°C for at least three hours.
Approximately 2 mL from the bottle of Paraffin Oil is to be poured into the appropriate media to simulate the squeezing of jelly from a tube.  The type of media that is used for testing of the product is used for testing of the Direct Inoculation Known Sterile Control (i.e. Sterile Paraffin Oil).

2.3.         Equipment Sterilization

The equipment necessary for a sterility test session includes the following; a stainless steel waste bucket, a number of stainless steel trays (usually 6), scissors, forceps, drain tray and tube, 10L plastic waste bottle, 2 plastic swabbing tubs, a pre-rinsed with distilled water 10L plastic bottle for preparation of 250 ppm Chlorine solution and 250mL glass Schott bottle for 70% IPA.  The items are to be autoclaved before placing in the transfer hatch.

NOTE   Equipment is to be suitably wrapped where necessary in Sterilope bags, Kraft paper or Dacron Bags and taped with autoclave tape.  The Date of Autoclaving should be written on the tape.

Always check that the indicator stripes on the autoclave tape have altered in color to a dark Grey/Black and that the chart recorder shows that both the temperature and pressure for the cycle have conformed to standards.

2.4.         Chlorine Disinfection

Prepare 10L of 250ppm Chlorine Solution in a pre-sterilized 10L plastic bottle (which has been pre rinsed with distilled water before autoclaving) using Haz-Tabs (Sodium Dichloroisocyanurate Dihydrate) or its equivalent as per manufacturer’s instructions.

Manufacturer’s Instructions for the preparation of 250ppm of Chlorine Solution using HAZ–TABS are to dissolve 1 tablet in 10L of Distilled Water with gentle agitation.  Ensure the tablet is fully dissolved before solution is used.

The solution has an expiry of 7 days after the date of preparation.  Log the preparation details into the sterility test room logbook, include date, batch number of Haz-Tab, expiry date of the Haz-Tabs and name of person preparing.

2.5.         Swabbing Down

Prior to being placed into the Sterility Transfer Hatch, all items are to be decontaminated by swabbing down with 250ppm Chlorine Solution in the Transfer Hatch, using sterile wipes stored in the Transfer Hatch.  The exception being for Pre-sterilized gamma irradiated Agar plates that can be transferred straight from their packaging to the transfer cabinet (remove one of the outer layers of irradiated wrapping) and air sampler, which should be decontaminated using 70% IPA.

Fill approx. 200mL of sterile filtered 70% IPA into a pre-sterilized 250mL sterile Schott bottle and swab down to take into the sterility test suite.

Swabbing takes place in autoclaved sterile tubs in the following order.

The length of time for submersion should be no less than 2 minutes, to ensure the removal of any surface contamination from the items being taken into the Sterility Test Suites.

Ensure there is no excess Chlorine solution on all articles that are disinfected, as this solution is corrosive on metals.

Heavier items and testing equipment, i.e. media, waste bucket and waste bottle may be placed on the bottom shelf.

Products are to be clearly segregated to ensure that no mix up of batches takes place.

2.6.         Recording of the Session

The contents of the testing session including sterile controls, media and diluents used are to be recorded in the log book.

Add into the Batches form for the correct BPN if major stoppage samples have been tested for sterility.

Add into the ‘Comments’ section in the sterility test log book if the sterility test session was swabbed down by a technician other than the one performing the testing, also record the Steritest lot number from the Steritest, Sterility Testing device Certificate of Analysis.  Equipment sterilization details for the session should also be recorded in the comments section, including the autoclave used, the cycle number and the date of sterilization.

After completing the sterility test session enter into the sterility test log book and any information that might impact on the test results.

3. Sterility Testing Procedures

3.1.         Procedure –Always check batch number before starting test.

3.1.1.     Aqueous Solutions

All aqueous injection solutions could be tested using the Steritest II canister (brand) Membrane Filtration system employing 0.45-micron filters.  Products are to be sterility tested individually and each Autoclave Load is to be tested separately. However machines that run in tandem from the same Bulk Solution can be pooled.

The entire contents of the above mentioned number of containers or 20mL from each, whichever is least, is to be filtered through the sterile needle by piecing through the softest part of the container, this will vary depending on the container type.

The Steritest canisters and the filters are then washed 3 times with 100mL of Sterile Peptone Water.  These washes should be individual.  After the final wash the plugs are to be placed in the bottom of the individual canisters.

One canister is to be clamped and filled with 100mL of sterile Fluid Thioglycollate Medium (FTM) + 0.5% v/v Tween 80 and the other canister is to be clamped and filled with 50 mL of sterile Trypticase Soy Broth (TSB) + 0.5% v/v Tween 80.  These are then sealed for incubation.

3.1.2.     Non-Injectable, Non‑Filterable Products

Example- 1 jelly form

Aseptically transfer 1mL from each unit into a 600mL Wheaten bottle containing 250mL of sterile Trypticase Soy Broth + 0.1% w/v Lecithin + 0.7% w/v Polysorbate 80.

Repeat dispensing from the remaining units into a second, third and fourth Wheaten bottle.

Aseptically transfer 1mL (about 3cm) from each of the units into a 600mL Wheaten bottle containing 500mL of sterile Fluid Thioglycollate Medium + 0.1% w/v Lecithin + 0.7% Polysorbate 80.

Repeat dispensing from the remaining units into a second Wheaten bottle.

Example- 2 suspension form

Aseptically insert a sterile air filter into a 600 mL bottle containing 600 mL sterile Fluid Thioglycollate Medium + 0.5% v/v Tween 80 (FTM).

Using a Liquid Transfer Kit, (e.g. Millipore TEA000010), push the “thick” needle through the rubber stopper of the bottle containing the first media type.

Push the “thin” needle through the bottom of the plastic container and transfer the whole contents of all containers into the FTM bottle.

Repeat the procedure above with a 600mL bottle containing 600mL sterile TSB+ 0.5% v/v Tween 80 (TSB).

Incubate the TSB and FTM bottles as described below in point 3.6 – ‘Incubation’.

Secondary Transfer Process

After not less than 14 days of incubation and working within the sterility test LAF, aseptically transfer the following:

– 1 mL of the initial FTM media into a new bottle of 100mL FTM, and,
– 1 mL of the initial TSB media into a new bottle of 100 mL TSB.

Continue the incubation of the initial media and transfer media bottles as follows:

FTM – Not less than 4 days @ 32±2°C.

TSB – Not less than 4 days @ 22±2°C.

Examine the media for growth at intervals during the incubation period. If no evidence of growth is found the batch meets the requirements for the Test of Sterility.

3.2.         Agar Plates

One irradiated Letheen Agar plate and one irradiated Nutrient Agar plate are to be taken into the Sterility Test room for every sterility test session.

The Nutrient Agar plate is to be left exposed on the Laminar Flow bench (sterile Environmental) throughout the entire time of the sterility test session.

The Letheen Agar plate is to be used to record the Operator’s finger print impressions during the test session or at its conclusion.  If a needle stick injury occurs, before leaving the sterility test room to change gloves, conduct a finger print impression.  Use an additional Letheen Agar plate to monitor the finger impressions at the conclusion of the session.

Four (4) contact plates are used for personnel monitoring of the hood, sleeve, chest and leg after all testing has been completed, on exiting the change room.

NOTE: If a double consecutive session is being tested, perform personnel monitoring after the second session only.

Record the Name, Date and Session details on the plates.  Incubate all plates according to MICLAB 045.  Record results in the “Monitoring Results for Sterility Test Sessions Finger, Fallout, Environmental and Personnel Results” File.
NOTE: After recording the finger print impression, the Operator must don a new pair of sterile gloves in the change room and disinfect their hands with 70% IPA prior to resumption of testing.  See MICLAB 045 for limits.

3.3.         Environmental Monitoring

Environmental monitoring must be performed with each Sterility Testing Session and in each room and transfer hatch in use, on a daily basis.  See the Table below for details:

Area	Environmental Monitoring Required	Frequency
Laminar Flow Cabinet	1000L Air Sample 1 x Contact Plate	Each Session Each Session
Change Room	200L Air Sample 1 x Contact Plate (Floor)	Daily, when in use Daily, when in use
Transfer Hatch	200L Air Sample 1 x Contact Plate	Daily, when in use Daily, when in use
Sterility test Room	200L Air Sample 1 x Contact Plate (Floor) 1x Contact Plate (Trolley)	Daily, when in use Daily, when in use Daily, when in use

The environmental Agar plates and contact plates are to be labelled with all details and incubated according to MICLAB 045.  Results from the Laminar Flow monitoring are to be entered in the “Monitoring Results for Sterility Test Sessions Finger, Fallout, Environmental and Personnel Results” File.  Results from ALL environmental samples are to be entered in the “Environmental Results Air / Surfaces Non-Sterile File”.  After the total count is recorded, re-incubate the samples according to MICLAB 045.  Following incubation, record the number of molds present in the relevant file, as mentioned above.  If any colonies are present, keep the samples and do not discard until time of batch release.

3.4.         Completion of Sterility Test Session

On completion of each sterility test session the sterility test bench, Millipore Steritest unit, trolleys and seat are to be cleaned with 70% IPA.  The test suite and change room floors are then to be mopped with the appropriate disinfectant after leaving the sterility test session.
If an incident has occurred during the session that could impact on the outcome of the test result, (i.e. stabbing you finger with the Steritest testing needle) raise a Deviation Report, notify the Microbiology Manager or senior technician and invalidate the test at this point, have the batch re-sampled and repeat the test.

3.5.         Transfer Hatch

When emptying out the Transfer Hatch after completing a Sterility session, all equipment is to be rinsed and left to dry.  The test canisters are to be placed in the correct incubators (refer to 3.6).  The Transfer Hatch is to be wiped down with filtered 70% IPA.

3.6.         Incubation

The FTM is incubated at 32°⁺2°C for 15 days and the TSB at 23°⁺2°C for 15 days, or the results can be read after 14 full days.  When placing canisters into the incubators the trays are to be labelled with the date of the test and the time of incubation.

4. Observation

Examine all Steritest canisters or bottles of media daily on working days for 15 days, (or 14 full days), recording results in the work books.

4.1.         Suspected Contamination

If any turbidity is apparent in the medium prior to carrying out the sterility test due to the Lecithin additive or the nature of the product disregard the Medium, by destroying in a ‘Contaminated’ autoclave cycle.

Do NOT use any media that is not sterile.

After full incubation time 15 days, if the material being tested renders the medium turbid so that the presence or absence of microbial growth cannot be determined by visual examination, transfer 2 ml of the medium to fresh bottles of the same medium and then incubate the original and the transfer bottles for not less than 4 days. Re inspect for growth.

4.2.         Contamination

4.2.1.     If contamination, i.e. turbidity, a pellicle or any abnormal odour is suspected in the FTM, plate onto:

1. a)A Nutrient Agar plate for aerobes and incubate at 32±2°C.  Examine after 24 and 48 hours.
2. b)A Reinforced Clostridial Agar plate or Schaedler Agar plate for anaerobes and incubate at 32±2°C in an anaerobic jar.  Examine the plate after 48 hours.

4.2.2.     If contamination is evident in the TSB, plate onto a Nutrient Agar plate and incubate at 25±2°C.  Examine after 24 and 48 hours.  Continue incubation and daily examination, for 5 days for fungi.  If no growth occurs on the Nutrient Agar plate, repeat the above procedure using a Sabouraud Dextrose Agar plate.

4.2.3.     Identify the contaminant of the sterility test to at least Genus level (see MICLAB 070).  And identify to genus any associated growth on environmental plates and 70% IPA.  If not able to grow the contaminant, record the organism as a viable non-culturable microorganism.

4.2.4.     Record of Results:

The Microbiology Manager or senior technician can record the results for the 7 day and 15 day checks, (or 14 full days), into the work book.

If either media is found to be contaminated, the following data should be recorded:

Media involved

Microscopic and morphological appearance of the organism/s

Number of days of incubation

Identity of the contaminant to at least Genus level.

4.2.5.     Any contaminant/s found in an initial sterility test are to be retained in the refrigerator.  The contaminants should be retained for a minimum of 6 months on a TSA slope.  If the test is found to be invalid one repeat test may be performed.

5. Interpretation of Results

5.1.         The product meets requirements for sterility if no growth is evident in either the FTM or the TSB media after 14 full days incubation.  This interpretation applies even if growth occurs in the control canisters.

5.2.         If contamination has occurred in the initial sterility test, The Sterility Test Out Of Specification Result Investigation form, (Form 680) is to be filled out, a Deviation Report is to be raised and an Incident / investigation started, including a meeting held with all relevant parties.  The Form 680 is to be used as a minimum guide and does not limit the extent of the investigation.  From the incident investigation the Microbiology Manager is to decide if the product is to be Repeat sterility tested or rejected depending on the out come of the incident investigation findings. The decision is to be based on the current compendial guidelines, Observation and Interpretation of Results.

See Appendix 1 (Flowchart of Sterility Test Results – Interpretation and Repeat Test), can be used as a guideline.

5.3.         In the event of an initial sterility test failure the test may be considered invalid only when one or more of the following conditions are fulfilled:

The data of the microbiological monitoring of the sterility testing facility 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 maybe ascribed unequivocally to faults with respect to the material and/or the technique used in conducting the sterility test procedure.

5.4.         If the test is found NOT to be invalid, then the product is to be rejected.

6. Repeat Testing of Sterile Products

6.1.         Sample Size

If the above review justifies that a repeat sterility test can be conducted on a product, the sample size and the test method used are to be as for an initial sterility test, (Section 1).

6.2.         Sampling

Samples must be collected at random by trained sampling staff.  As the product requiring sampling may be at various stages of production, consultation with the Microbiology Manager will be necessary to decide the sampling plan to be conducted.

6.3.         Repeat test Procedure

The product is to be regarded as a separate session with a known sterile control.  No other product is to be tested during this procedure.

a) Non‑injectable, Non‑filterable products

For non‑injectable, non‑filterable products follow the same procedure as for the initial test. The known Sterile Control is Paraffin Oil in this test session.

b) Aqueous Solutions

For aqueous solutions the same quantity of product is filtered.

Include a known sterile control of X units of Water for Injection that has been autoclaved at least twice in the test session.

Wash the canister filters with 3 x 100 mL of Sterile Peptone Water.  These 3 washes should be individual.  Fill the canisters with the appropriate medium, seal and incubate as for an initial sterility test.

6.4.         Recording of the session and results

Details of the test sessions and the results are to be recorded in the log book.

6.5.         Interpretation of sterility repeat test results

6.5.1.     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, the product examined does not comply with the test for sterility.

7. Media Check after Sterility Testing (Stasis Test)

7.1.         Membrane Filtration and Direct Inoculation

Once every 12 months media containers from a product tested by Membrane Filtration and also if possible those from a product tested by Direct Inoculation must be subjected to a challenge by a low number of microorganisms after the sterility test has been completed.  This is to ensure that any inhibitory effects present in the products have been successfully inactivated by the dilution effect of the testing procedure, or the washing procedure as appropriate to the Test Method, and hence any microorganisms that may have been present in the product would have been able to grow in the media when the product was tested.  It is important that on an annual basis, the range of sterile products are to be subjected to a Stasis Test.

A Stasis Check on Sterility Tests Form must be filled out on an annual basis detailing the product type and date the Stasis test was conducted.  NOTE: The product types listed are not exhaustive; they represent the range of products manufactured by the company and list only the specific products most likely to be inhibitory to the Sterility test.

7.2.         See MICLAB 090 for the procedure of Stasis tests and to determine the method of preparation of Stock Suspensions of micro organisms used for Stasis work and media checks.

8. Validation and Revalidation of Sterility Test Methods

8.1.         General Requirements

8.1.1       All products, which require sterility testing, must have a corresponding validated method.

8.1.2       Validation is required for each new product formulation, changes in product formulation, changes in containers / closure types and any other changes that impact the status of a validated system, process or equipment.

8.1.3       Re-validations of existing methods are to be performed once every 5 years.

8.1.4       A revalidation of the sterility test system, using at least one product is to be conducted on an annual basis.

8.2.         Documentation

8.2.1      Documentation must be completed for each product undergoing initial validation or revalidation.

8.2.2      Following the entry of test results, the completed validation form must be reviewed and authorized by Microbiology Manager, prior to the method being used in routine testing.

8.3.         Test Methods

8.3.1.     Test Articles

8.3.1.1. Aqueous Solutions

For aqueous solution validations the process is identical to routine product sterility testing except for the following two conditions:

1)      The product volume filtered is 3 (three) times the routine test volume.

2)      The final 100mL peptone water rinse must contain 10-100cfu of a viable challenge organism.  The peptone water is to be inoculated immediately prior to the validation procedure.  The required challenge organisms should be listed and must be used to validate each method.

8.3.1.2. Non-Injectable, Non‑Filterable Products

For non-injectable, non-filterable product validations the process is identical to routine product sterility testing except for the following condition;

1)      The final containers prior to incubation are inoculated with 10-100cfu of a viable challenge organism.  The required challenge organisms should be listed and must be used to validate each method.

8.3.2.     Control Articles

8.3.2.1. Positive controls must be performed each day validations are performed.  The preparation of positive control units is identical to test units, however, no test product is filtered, (liquids) or added directly to the media, (non-filterables).

8.3.2.2. A minimum of one (1) positive control canister must be prepared for each organism used in the validation.  The required organism is introduced into the test either by inoculating the final rinse (liquids), or direct inoculation (non-filterables).

8.3.2.3. Control canisters are incubated at the same time and under the same conditions as test canisters.

8.4.         Incubation and Inspection

8.4.1.     All test and control units are to be incubated and inspected as per the following table:

Canister Media & Organism	Incubation conditions	Inspection interval
FTM + Staph aureus	32±2°C	Not more than 5 days
FTM + Ps aeruginosa	32±2°C	Not more than 5 days
FTM + Cl. Sporogenes	32±2°C	Not more than 5 days
TSB + B. subtilis	22±2°C	Not more than 5 days
TSM + C. albicans	22±2°C	Not more than 5 days
TSB + A. niger	22±2°C	Not more than 5 days

8.4.2.     All units are to be inspected by an experienced analyst at the interval indicated above and confirmed by either the Microbiology Manager. If clearly visible growth is obtained after the incubation, visually comparable to that in the control vessel without product, either the product contains no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated.

8.4.3.     All results from validations are to be recorded on the relevant forms:

8.5.         Interpretation of Results

8.5.1.     If the results from the validation test show comparable growth recovery for all required organisms the test method is deemed valid and following completion of the relevant documentation, is placed into routine use.

8.5.2.     If the growth of any test organism is not observed in the test canisters, is not visually comparable to that in the positive control, or is poor, it is considered that the product possesses antimicrobial activity that has not been adequately neutralized under the conditions of the test.  Under these circumstances the test method must be modified either by increasing the rinse volume, changing rinse type or by increasing the amount of medium used in the test, in order to eliminate the antimicrobial effect of the product.  It is only necessary to repeat the test for specific organisms that failed to grow, unless the modified test requires a significant method change, e.g. change in rinse fluid type, or test equipment change.

9. Appendix 1 – Flowchart of Sterility Test results – Interpretation and Retests

10. Summary of Changes

Version #	Revision History
MICLAB 060	New