Lyopholized Pellet Use In Testing LIF-Based RMMs

New Rapid Microbiological Method (RMM) instruments permitting the instantaneous detection of microbes in water are emerging for use in the assessment of pharmaceutical water quality. Online and offline systems provide an excellent risk management tool, permitting the routine analysis of pharmaceutical grade waters throughout distribution loops, storage tanks, and at points of use via conventional point sampling or continuous monitoring. These Laser Induced Fluorescence (LIF)-based RMM instruments utilize the simultaneous detection of Mie scatter for particle presence, and intrinsic fluorescence for biologic status, to provide results in real time, without the need for staining, reagents or sample manipulation.

Customer assessment of system performance and appropriate methods for system validation are an important topic for new technologies. Commercial products such as bioMérieux’s BioBall® and Microbiologics EZ-Accu Shot™ offer very precise and easy-to-use microbial inoculations in the form of a lyophilized pellet. These products are excellent options for use in growth promotion testing, sterility assurance testing, antimicrobial effectiveness testing, and other applications where a precise microbial concentration is desired. Yet, these products are not well suited for use in the validation or assessment of LIF-based RMM instruments due to significant background particulate and fluorescence present in the lyophilized pellet matrix.

Off-the-shelf lyophilized pellets contain the microorganism of interest in addition to other materials required for the freeze-drying process, such as cryoprotectants and lyoprotectants like serumi. Such materials cause background particulate interference when analyzed using a flow cytometer and also fluoresce at excitation wavelengths, such as 405nm, used with LIF-based RMM systems for wateri. If water-for-injection (WFI) or ultrapure water (UPW) is utilized to rehydrate these pellets (as opposed to the manufacturer’s rehydration fluid), background particulate and fluorescence may be reduced but the pellet matrix itself still has a significant particulate load.