Application of flow cytometry for enumerating individual bacterial cultures from a mixed culture system : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
Cultured dairy products are often made with more than one microbial culture. Yoghurt requires the cultivation of several bacterial species for its production and the level of each is important for different reasons. Differential plate count methods to enumerate the separate species in yoghurt are not ideal because many of the bacteria used have similar growth profiles and plate counts take several days to produce a result. A fast specific method for enumerating each culture would be beneficial because quick results would enable tighter control of processing or experimental conditions and the ability to track individual species amongst a background of similar bacteria. Flow cytometry combined with fluorescent in-situ hybridisation (FLOW-FISH) was investigated as a potential solution and successful enumeration was achieved within 1 day for a yoghurt microorganism, Streptococcus thermophilus (ST55), grown in M17 medium. This method may be improved to increase the signal-to-noise ratio and to reduce the assay time. The chemical propidium monoazide enabled a closer match to plate counts for flow cytometry results using a total viable count assay and may be useful combined with the FLOW-FISH assay for removing non-viable or viable, but non-culturable, cells from the results. An enzyme and/or detergent pre-treatment may achieve successful FLOW-FISH enumeration of cells grown in reconstituted skim milk – a similar matrix to yoghurt.