A previous article discussed the selection of challenge microorganisms for bacterial retention validation (for review, see: Must Bacterial Retention Validation Use Brevundimonas diminuta?). Taking Brevundimonas diminuta ATCC® 19146™ as an example, the strain is typically obtained in lyophilized form from the American Type Culture Collection (ATCC®). After reviving the microorganism according to ATCC® procedures, it may be stored under refrigeration or frozen in a suitable culture medium in accordance with standard microbiological practices. Storage conditions for process isolates intended for challenge studies must be established.
Two standard techniques are recognized as suitable for the preparation and maintenance of Brevundimonas diminuta for bacterial challenge testing: the Saline Lactose Broth (SLB) method and the Frozen Cell Paste (FCP) method. Both methods can effectively produce a suitable suspension of Brevundimonas diminuta with dimensions of approximately 0.3-0.4 μm in diameter and 0.6-1.0 μm in length.
Alternative culture media and cultivation methods may be equally effective, provided they yield single, dispersed cells of appropriate size capable of passing through a 0.45 μm pore-size membrane filter. Alternative cultivation methods must be validated. The aggregation status of stock bacterial challenge cultures can be examined using optical microscopy. If aggregation is observed, consider placing the stored culture in cold water within an ultrasonic cleaning bath for 10 minutes to disperse the aggregates. Cavitation generated by the water bath can disperse bacterial cells without compromising cell viability. The effectiveness of dispersion should be confirmed using optical microscopy, viable cell counting, and downstream recovery through a 0.45 μm pore-size control filter.
The concentration for the bacterial challenge test must ensure uniformity throughout the intended process duration, with a challenge level of at least 10⁷ CFU/cm² based on the filter surface area. When calculating the bacterial challenge concentration, operational parameters such as flow rate, duration, and pressure drop must be comprehensively considered.
The viability and titer of the Brevundimonas diminuta suspension should be confirmed using an appropriate recovery medium, such as Soybean-Casein Digest Broth or Miller-Hinton Agar. During filter challenge testing, the viability titer of the bacterial challenge suspension must be determined immediately before and after the challenge. The upstream bacterial titer should be verified using validated microbiological testing methods. The same medium must be used to confirm any Brevundimonas diminuta recovered downstream.