Changes

Viable but nonculturable ('''VBNC''') is a newly identified state for bacteria that are not able to grow or form colonies on typical growth media, but remain viable (alive) and retain a limited level of metabolic activity (reduced nutrient transport, respiration, and synthesis of compounds) while sometimes being able to regain their population when returned to a more ideal environment. The cells often exhibit dwarfing, and they can remain in this state without dying for 4-12+ months depending on the species. An appropriate viability test for a given species can be performed to show that the cells are not dead, even though they don't grow on typical growth media (for example, intracellular hydrolysisof hydrolysis of CTC or reduction of INT as an indication of metabolic activity, by establishing the presence of an intact cytoplasmic membrane via BacLight® or propidium iodide, or by multi-parameter flow cytometry). Cells enter this state as a way to survive some sort of stress in their environment (for example, osmotic stress, too much oxygen exposure, exposure to white light, etc.). Treatments such as pasteurization in milk and chlorination of wastewater have also been shown to induce VBNC. A number of species have been found to be able to enter the VBNC state, including ''E. coli'', ''Lactobacillus plantarum'', ''L. lactis'', ''L. linderi'', ''L. casei'', ''L. plantarum'', ''L. paracollinocides'', ''L. acetotolerans'', and several species of ''Salmonella''. Early studies on VBNC microbes were not able to fully show that the resuscitation was truly from VBNC cells rather than a very small number of culturable cells, but later studies were able to show that some bacteria can be resuscitated from a VBNC state, although most bacteria that enter a VBNC state have not been shown to be able to be resuscitated <ref>[https://pdfs.semanticscholar.org/e661/934dca6bb0dbb31a8781f3193232b7b5a8a4.pdf The Viable but Nonculturable State in Bacteria. James D. Oliver. The Journal of Microbiology. 2005.]</ref><ref name="Liu_2018">[https://www.frontiersin.org/articles/10.3389/fmicb.2018.02076/full#B28 Junyan Liu, Yang Deng, Thanapop Soteyome, Yanyan Li, Jianyu Su, Lin Li, Bing Li, Mark E. Shirtliff, Zhenbo Xu, and Brian M. Peters. Front. Microbiol. 2018. DOI: https://doi.org/10.3389/fmicb.2018.02076.]</ref>.

A couple of published studies have reported inducing the VBNC state in bacteria strains that were isolated from contaminated beer. Liu et al. (2017) were able to induce a VBNC state (meaning that they were not able to grow on growth media for up to 14 days) in a strain of ''Lactobacillus lindneri'', a species that is responsible for 15-25% of spoiled beer reports, and determined VBNC via a Live/Dead BacLight® bacterial viability kit. They induced this state by storing the cells in beer at 0°C without shaking for 190 days. They also found that storing the VBNC cells at -80°C in glycerol stocks was the best way to maintain the cells. They were able to resuscitate the cells by growing them on MRS media that had 500-1000-μL of the enzyme catalase spread onto them (trying higher temperatures did not work to resuscitate, nor did using higher concentrations of MRS), thus showing that brewers can use catalase to help grow VBNC state ''L. lindneri'' cells on MRS media (and perhaps other species of ''Lactobacillus'' as well). It took 3-4 days to begin showing signs of growth on the catalase supplemented MRS media. It was also demonstrated that VBNC cells could grow in beer after 30 days of incubation, and showed final cell counts similar to normal ''L. lindneri'' and resuscitated cells <ref>[https://www.sciencedirect.com/science/article/pii/S0882401017303030?via%3Dihub First study on the formation and resuscitation of viable but nonculturable state and beer spoilage capability of Lactobacillus lindneri. Junyan Liu, Lin Lia, Bing Li, Brian M. Peters, Yang Deng, Zhenbo Xua, Mark E. Shirtliff. Microbial Pathogenesis, Vol 107. 2017. DOI: https://doi.org/10.1016/j.micpath.2017.03.043.]</ref>. Lui et al. (2018) also found very similar reproduced these results with a hop tolerant strain of ''L. brevis'' <ref name="Liu_2018" />.