What is a microbial aerosol challenge testing?

Microbial aerosol challenge testing evaluates a finished product’s final sterile barrier system’s package integrity by exposing the terminally sterilized packaged product to aerosolized Bacillus atrophaeus bacteria. After the package exterior has been exposed to Bacillus atrophaeus, the package contents are tested for bacterial contamination.

Aerosolized bacteria challenge testing begins with the preparation of Trypticase Soy Broth (TSB) media that will be used as a negative control (without bacteria) and positive control (with bacteria). For now, the TSB is without bacteria. All microbial aerosol challenge methods are performed in an ISO Class 5 laminar flow hood to maintain sterility during the assessment. All positive controls, negative controls, and samples will have adapters for syringe use. For the preconditioning process, ten samples (terminally sterilized vials containing product) and all controls are held statically in an ISO Class 7 controlled environment for 28 days. Five of the samples will be laid down on their side, and five will be left standing on their base during the 28 days of storage. Two dose removals will be performed from the TSB vial at times of 0, 7, 14, 21, and 28 days. After 28 days of sample conditioning, the vial adapter/syringe assembly will undergo the microbial aerosol challenge test.

A Bacillus atrophaeus suspension is prepared for the microbial challenge so that the aerosol chamber receives a minimum of 1.0 x 10⁷ spores when the bacterial is aerosolized. The final suspension concentration is verified using the spread plate method. Once the aerosol chamber with Bacillus atrophaeus suspension has been prepared, the ten test media sample vials with attached syringe-adapter assemblies are loaded, equally spaced and upright, into the aerosol chamber along with the positive controls. Positive controls are created by piercing the vial septum with a sterile 23 gauge needle and leaving the needle in place during the microbial challenge. Negative control vials are not loaded into the chamber. Once all positive controls and sample vials are loaded into the aerosol chamber, the doors are sealed, and the chamber fan is turned on. The prepared Bacillus atrophaeus suspension is loaded to the chamber’s nebulizers, and the suspension is aerosolized until the nebulizers are empty. Samples are left in the chamber for an hour after the suspension has been fully aerosolized. Next, samples are taken out of the chamber, and the exterior is thoroughly disinfected. Finally, all positive control, negative control, and test vials are incubated upright for seven days.

After seven days, all control and sample vials are checked for microbial growth. The level of microbial growth is assessed by streaking the samples onto TSA agar and incubating them. Any growth on the agar plates is compared to Bacillus atrophaeus by Gram staining and direct microscopic observation. Negative TSB vial sterility results indicate that the sterile barrier vial system acted as an effective microbial barrier for the product under the microbial aerosol challenge testing conditions. Positive TSB media vial results indicate that the sample closures did not act as an effective microbial barrier.