Development of precise microbiological reference materials

Quality Control (QC) reference materials are widely used in microbiology to demonstrate the efficacy of testing methods and culture media. The current method for preparation of QC materials is by serial dilution of a microbial broth culture to obtain a suspension that contains an estimated number of colony forming units (cfu). Commercial reference material products are available with dried microbial cells, however, the numbers of cells are variable between batches as the production processes are reliant on cell suspensions of estimated cell number.This study developed a method to produce precise microbial reference materials with a accurate number of viable cells. Flow cytometry was used to count and dispense precise numbers of cells into a single droplet of fluid. The droplets were then mixed with a lyoprotectant solution and subjected to freeze-drying. The resultant freeze-dried pellets showed consistent average cfu counts between 28-33 cfu with a standard deviation 90% of the cells to survive freeze-drying and remain viable for one year at a storage temperature below -18°C.The methodology for the production of freeze-dried pellet was applied to a range of genera including, different E. coli strains, Gram positive bacteria such as Listeria and Staphylococcus, the yeast Candida albicans and a spore-producing Bacillus cereus. The precision of cell numbers was comparable between different microbial genera and strains and a consistent standard deviation below 3 cfu was achieved. The same freeze-dried pellet method was used for the different micro-organisms, except for changes to preparation of cell suspensions. Different methods of broth culture were developed to ensure freeze-dried cell survival. A measurement of method reproducibility was obtained when 99 batches of pellets were produced, and within batch and between batch variation was determined