MicroFunnel™ ST Filter Funnel for Sterility Testing Applications Presence/Absence and Recovery Testing Protocol

1.0 Purpose

This testing is to validate that MicroFunnel ST filter funnel products are suitable for use in sterility testing applications and will recover the pharmacopeial organisms defined in this protocol.

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2.0 Scope

To perform presence/absence (direct transfer) and microbial recovery studies in MicroFunnel filter funnels using organisms listed in USP-24 and EP-2000. Three lots of MicroFunnel ST filter funnels will be tested in triplicate for each organism.

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3.0 References

3.1 USP24 <71> Sterility Tests

3.2 USP24 <1227> Validation of Microbial Recovery From Pharmacopeial Articles

3.3 EP 2000 2.6.1. Sterility

3.4 Standard Methods for the Examination of Water and Wastewater, 19th Edition,1995


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4.0 Sample Requirements

150 MicroFunnel ST filter funnels from each of three individual lots. Each lot of MicroFunnel ST filter funnels will be evaluated in triplicate for each test organism.

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5.0 Materials

5.1 Test Organisms

Escherichia coli, ATCC 8739

Pseudomonas aeruginosa, ATCC 9027

Staphylococcus aureus, ATCC 6538P

Bacillus subtilus, ATCC 6633

Clostridium sporogenes, ATCC 19404 (or Clostridium sporogenes, ATCC 11437)

Candida albicans, ATCC 10231

Aspergillus niger, ATCC 16404

5.2 Equipment

Vacuum manifold

Vortex Mixer

Incubators, 32.5 ± 2.5 °C and 22.5 ± 2.5 °C

Water bath at 45 ± 1°C

Quebec colony counter

Petri plates, 15 X 100 mm and 15 X 150 mm

Pipettes, 1 mL and 10 mL, sterile

Pipettor, 1000 lambda

Ice water bath

Anaerobe jars or pouches

Stereomicroscope

5.3 Media and Reagents

Soybean Casein Digest (SCD) Agar

Anaerobic Agar

Soybean Casein Digest Broth, 200 mL bottles, sterile

Fluid Thioglycollate Medium (FTM),

200 mL bottles, sterile

Saline (0.9% NaCl), sterile

Sterile 0.1% Peptone Water

Sterile saline TS containing

0.05% polysorbate 80

Sterile 0.1% Peptone Water with 0.05 - 0.1% Tween 80.


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6.0 Procedure

6.1 Organism Preparation

6.1.1 Propagate growth of pharmacopeial organisms according to standard laboratory practices.

6.1.2 Dilute each organism suspension to obtain a density of less than 100 colony forming units per mL. Use sterile saline for non-spore-forming, aerobic organisms and sterile saline TS containing 0.05% polysorbate 80 for spore-forming bacteria and fungal spore suspensions.

6.2 Funnel Analysis

6.2.1 Open three MicroFunnel™ ST filter funnels from one lot to be tested and place unit directly onto the manifold. Blue adapters with #8 stoppers may be used as an alternative.

6.2.2 Dispense 100 mL of sterile rinse fluid into each MicroFunnel ST filter funnel. Use sterile 0.1% peptone water for non-spore-forming, aerobic organisms and sterile 0.1% peptone water containing 0.1% Tween 80 for spore-forming bacteria and fungal spore suspensions. Replace the filter funnel lids, apply vacuum, allow fluid to drain and turn off vacuum.

6.2.3 Repeat step 6.2.2.

6.2.4 Dispense 100 mL of sterile rinse fluid into each MicroFunnel ST filter funnel. Use sterile 0.1% peptone water for non-spore-forming, aerobic organisms and sterile 0.1% peptone water containing 0.1% Tween 80 for spore-forming bacteria and fungal spore suspensions.

6.2.5 Using a 1.0 mL pipette or equivalent, dispense 1.0 mL of a test organism suspension into each filter unit.

6.2.6 Replace the filter funnel lids, apply vacuum, allow fluid to drain and turn off vacuum.

6.2.7 Retrieve the membrane from the first MicroFunnel ST test funnel by removing the lid and gently squeezing the funnel cylinder until it disengages from the base and using alcohol flamed forceps gently lift the edge of the membrane, being careful not to touch the effective filtration area.

6.2.8 Plate the membrane right side up onto the surface of a pre-poured agar plate using a rolling motion to avoid any bubbles being trapped beneath the membrane. Use an SCD agar plate for aerobic organisms and an Anaerobic Agar plate for Clostridium sp.

6.2.9 Retrieve the membrane from the second MicroFunnel ST test funnel as described in 6.2.7 and using aseptic technique immerse the membrane into a 200 mL bottle of FTM.

6.2.10 Retrieve the membrane from the third MicroFunnel ST test funnel as described in 6.2.7 and using aseptic technique immerse the membrane into a 200 mL bottle of SCD broth. It is not necessary to perform immersion in SCD broth for Clostridium sp.

6.3 Replicate testing within a MicroFunnel ST filter funnel lot

6.3.1 Three replicates are performed for each lot of MicroFunnel ST filter funnel by both the recovery and the presence/absence method.

6.3.2 Three lots of MicroFunnel ST filter funnels are evaluated for performance by both recovery and presence/absence method.

6.3.3 Repeat steps listed in 6.2 until all replicates for each of three lots of MicroFunnel ST filter funnels have been examined.

6.3.4 Repeat steps listed in 6.2 until all organisms have been evaluated in triplicate for each of three lots of MicroFunnel ST filter funnels.

6.4 Controls

6.4.1 Inoculum Count: Prepare five to ten density control plates for each organism suspension prepared in step 6.1.2 by dispensing 1.0 mL into separate sterile petri plate. Add about 20 mL of molten agar cooled to about 45 - 48 °C and mix by carefully swirling several times in both directions then allow agar to solidify. Use Anaerobic agar for Clostridium sp. and SCD agar for all other organisms.

6.4.2 Growth Promotion Controls: Directly inoculate each lot of every medium (broth and agar) used with the applicable organism or both as used in the test. Incubate as indicated in section 6.5.

6.4.3 Media Sterility Controls: Incubate a minimum of two uninoculated replicates of each lot of every agar and broth medium used in the evaluation.

6.4.4 Rinse Fluid/Membrane Negative Controls: Filter 300 mL of each lot of rinse fluid through quadruplicate MicroFunnel filter funnels. Plate one filter on Anaerobic Agar, plate one on SCD agar, immerse one in SCD broth, and immerse the remaining filter in FTM. Incubate as in section 6.5. Repeat these controls for each lot of filter or MicroFunnel used.

6.4.5 Rinse Fluid/Membrane Positive Controls: Filter 300 mL of each lot of Peptone water used as a rinse fluid through duplicate MicroFunnel ST filter funnels and immerse one in SCD broth and the other in FTM then inoculuate with test organism suspension. Repeat for each organism used.

6.5 Incubation

6.5.1 Incubate Anaerobic Agar plates under anaerobic conditions at 32.5 ± 2.5 °C for a maximum of 7 days. Check for growth periodically and perform colony count when colonies are clearly visible. Stereomicroscope may be used to aid in visualizing colonies on membranes, or a Quebec counter for pour plates.

6.5.2 Incubate B. subtilus, C. albicans, and A. niger organisms cultured on SCD agar plates at 22.5 ± 2.5 °C for a maximum of 7 days. Check for growth periodically and perform colony count when colonies are clearly visible. A Stereomicroscope may be used to aid in visualizing colonies on membranes, or a Quebec counter for pour plates.

6.5.3 Incubate E. coli, P. aeruginosa, and S. aureus organisms cultured on SCD agar plates at 32.5 ± 2.5 °C for a maximum of 7 days. Check for growth periodically and perform colony count when colonies are clearly visible. A Stereomicroscope may be used to aid in visualizing colonies on membranes, or a Quebec counter for pour plates.

6.5.4 SCD broth

6.5.4.1 Incubate SCD broth inoculated with E. coli, P. aeruginosa, and S.aureus organisms at 32.5 ± 2.5 °C for a maximum of 3 days. Check for turbidity periodically.

6.5.4.2 Incubate SCD broth inoculated with B. subtilus, C. albicans, and A. niger at 22.5 ± 2.5 °C for a maximum of 5 days. Check for turbidity periodically.

6.5.5 FTM

6.5.5.1 Incubate FTM inoculated with E. coli, P. aeruginosa, C. sporogenes, and S. aureus at 32.5 ± 2.5 °C for a maximum of 3 days. Check for turbidity periodically.

6.5.5.2 Incubate FTM broth inoculated with B. subtilus, C. albicans, and A. niger at 22.5 ± 2.5 °C for a maximum of 7 days. Check for turbidity periodically.


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7.0 Results

7.1 Record colony count and morphological characteristics of colonies obtained from membrane filters.

7.2 Count colonies from pour plates using the Quebec counter. Reference Standard Methods for the Examination of Water and Wastewater, current edition, section 9215 A.8 for counting and recording.

7.3 Check FTM and SCD broth for turbidity and record results. Comment on whether growth in test samples is equivalent to controls.

7.4 Calculations

7.4.1 Calculate the mean count for each organism and lot of MicroFunnel™ ST test samples and control membrane.

7.4.2 Calculate the standard deviation, σ, and cV for each mean calculated.
cV = ( σ ÷ x ) ( 100 )

7.4.3 Calculate the mean and standard deviation for the SCD pour plate density controls (inoculum counts).

7.4.4 Calculate the percent recovery of the MicroFunnel samples and control membranes:

% Recovery =   Mean of membrane    x 100

Mean of inoculum count

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8.0 Limits and Conclusions

8.1 The percent recovery of each lot of MicroFunnel ST test funnels must be ≥ 70% of the density control in order to be considered suitable for use as a sterility test funnel.

8.2 If the cV for a group of samples is > 20%, the test for that group, on that media is considered invalid and must be repeated.

8.3 If the cV for the media control group is > 20%, the entire test (all test funnels and controls) on that media may be considered invalid.

8.4 If the cV for a group of density plates is > 20%, the entire test that used that specific organism suspension may be considered invalid.

8.5 For presence/absence tests, the growth in the test broths must be equivalent to the growth obtained in the control broths. The method can be considered validated if all of the test samples show copious growth for all microorganisms.

8.6 Turbidity should be observed in all inoculated blanks of SCD broth and FTM for each lot of MicroFunnel ST test samples in order to be considered suitable for use as a sterility test funnel.

8.7 Failure of direct inoculum of test suspension into SCD broth or FTM to produce a turbid result will render testing invalid and must be repeated.

8.8 All other Negative and Positive Controls should yield the expected response. Report any discrepancies.


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Ordering Information

MicroFunnel™ ST Filter Funnels
Product No. Description Pore Size
4750 MicroFunnel ST with Supor¨ 450 membrane, plain, sterile, 100 mL capacity 0.45 µm
4751 MicroFunnel 300 ST with Supor 450 membrane,plain, sterile, 300 mL capacity 0.45 µm
4811 MicroFunnel ST with GN-6 Metricel¨ membrane, grid, sterile, 100 mL capacity 0.45 µm
4812 MicroFunnel 300 ST with GN-6 Metricel membrane, plain, sterile 300 mL capacity 0.45 µm
Packaging Note:

100 mL funnels (40 units total per box): 10 individually bagged funnels within an overpack bag, 4 overpack bags per box.

300 mL funnels (20 units total per box): 5 individually bagged funnels within an overpack bag, 4 overpack bags per box


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