<i>In situ</i> examination of <i>Lactobacillus brevis</i> after exposure to an oxidizing disinfectant

Beer is a hostile environment for most microorganisms, but some lactic acid bacteria can grow in this environment. This is primarily because these organisms have developed the ability to grow in the presence of hops. It has been speculated that hop resistance is inversely correlated to resistance against oxidation, and this would have great impact on the use of various disinfectants in the brewing industry. In this study, we cultivated bacteria under aerobic and anaerobic conditions, and then investigated the in situ outgrowth of individual cells into microcolonies on MRS agar after exposure to the oxidizing agent peracetic acid (PAA). An automated microscope stage allowed us to analyse a much larger number of cells over extended periods of incubation. After PAA treatment, the lag time increased markedly, and extensive variation in morphology, µmax as well as stress resistance was observed between and within the tested L. brevis strains. The results suggest that aerobic cultivation increased the oxidative stress tolerance in L. brevis. The results also show that dead cells are randomly distributed in a microcolony and the majority of non-growing individual cells do not stain with a membrane impermanent dye (Propidium iodide), which indicates that PAA may not destroy the plasma membrane. In conclusion, the developed microscopic analysis of individual cells on MRS agar can provides faster results and more details of cell physiology compared to the traditional CFU method

Heterogeneity between and within Strains of <em>Lactobacillus brevis</em> Exposed to Beer Compounds

This study attempted to investigate the physiological response of six Lactobacillus brevis strains to hop stress, with and without the addition of Mn(2+) or ethanol. Based on the use of different fluorescent probes, cell viability and intracellular pH (pHi) were assessed by fluorescence microscopy combined with flow cytometry, at the single cell level. The combined approach was faster than the traditional colony based method, but also provided additional information about population heterogeneity with regard to membrane damage and cell size reduction, when exposed to hop compounds. Different physiological subpopulations were detected under hop stress in both hop tolerant and sensitive strains. A large proportion of cells were killed in all the tested strains, but a small subpopulation from the hop tolerant strains eventually recovered as revealed by pHi measurements. Furthermore, a short term protection against hop compounds was obtained for both hop tolerant and sensitive strains, by addition of high concentration of Mn(2+). Addition of ethanol in combination with hop compounds caused an additional short term increase in damaged subpopulation, but the subsequent growth suggested that the presence of ethanol provides a slight cross resistance toward hop compounds