Table I

The Development of Consensus Test Parameters for Rating a 0.1 μm Rated Filter

Initial PreparationPhase OnePhase TwoPhase Three
TaskIdentify relevant consensus test parametersScreen candidate culture media with regard to mycoplasma cell growth and confirm the ability of the mycoplasma cells to penetrate a randomly chosen 0.2 μm rated penetration control filter

  1. Choose challenge fluid by Task Force Committee member consensus

  2. Evaluate five candidate culture media with regard to mycoplasma cell growth and ability to penetrate a randomly chosen 0.2 μm rated penetration control filter in 5 different laboratories

  3. Choose culture media

Confirm that the chosen media will generate test cells that will penetrate three different 0.2 μm rated filters, thereby ensuring a robust test of a 0.1 μm rated filter.
When testing a 0.1 μm rated filter, the 0.2 μm rated filter will serve as a penetration control, similar to the role of a 0.45 μm rated penetration control filter when testing a 0.2 μm rated filter.
Supporting ActivityIdentify what test parameters will require testing to reach a consensus.Testing was performed in a single independent (volunteer) laboratory.Testing was performed in five different filter manufacturer laboratories.Testing was performed in a single contract laboratory.
Final OutcomeConsensus Test parameters to be evaluated:

  1. The test organism

  2. Challenge level

  3. The test pressure

  4. The challenge volume for a 47 mm disc

  5. The challenge fluid (selected in Phase 2)

  6. The positive control (penetration control): penetration of a 0.2 μm rated filter

  7. Culture media for the generation of test cells (this test parameter required testing to reach a consensus)

All candidate media, except one, supported good growth of A. laidlawii and generated cells capable of penetrating a 0.2 μm rated penetration control filter.A challenge fluid was chosen by consensus of the Task Force Committee: phosphate buffer
A culture media was chosen (designated Mycoplasma Task Force Media) as a result of the testing and consists of

  1. 20 g/L mycoplasma broth base (Beef heart infusion broth)

  2. 25 g/L yeast extract

  3. 100 mL/L of heat-inactivated horse serum (held at 55 °C for a minimum of 30 min)

The test cells penetrated all three different 0.2 μm rated penetration control filters.
Summary of final consensus test parameters:

  1. The test organism: A. laidlawii ATCC® 23206™ (NC 10116; Strain PG8)

  2. Challenge level (≥1 × 107 CFU/cm2 of test filter area)

  3. The test pressure: 30 psid (207 kPa)

  4. The challenge volume for a 47 mm disc: 200 mL

  5. The challenge fluid: phosphate buffer

  6. The positive control (penetration control): penetration of a 0.2 μm rated filter

  7. Culture media: Mycoplasma Task Force Media