Changes

====Cell Counting====

[[File:Martyniak.JPG|thumb|300px|right|Bright-field and fluorescent images of ''B. clausenii'', ''B. bruxellensis'', and ''B. lambicus''. The fluorescent images show the counting of the yeasts with and without declustering the bud and pseudohyphae. Source: https://link.springer.com/article/10.1007/s10295-016-1861-4. Used with permission from Brian Martyniak.]]

One of the problems with cell counting for ''Brettanomyces'' is that they tend to form pseudohyphae (elongated cells that are attached to one another) during growth. This makes counting the cells difficult. Martyniak et al. (2017) has proposed a new way of counting cells for ''Brettanomyces'' by using the fluorescence capability of a [https://www.nexcelom.com/nexcelom-products/cellometer-fluorescent-viability-cell-counters/cellometer-x2-fluorescent-automated-viability-cell-counter/ Nexcelom X2 image cytometer] with stains created from acridine orange (AO) and propidium iodide (PI). The AO-PI stains only the nuclei of the individual cells, allowing them to be counted easily by the cytometer regardless of pseudohyphae formation. During their research, they found that a "clausenii" strain of ''B. anomalus'' formed more pseudohyphae than two strains of ''B. bruxellensis'' (one of which was refered to as a "lambicus" strain), and this corresponded with higher viability over time. It was therefore hyopthesized that pseudohyphae might play a role in the longer survival of some strains of ''Brettanomyces'' over others <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1362220137139530/ Brian Martyniak. Milk The Funk thread on counting ''Brettanomyces'' cells. 06/29/2016.]</ref><ref>[https://link.springer.com/article/10.1007/s10295-016-1861-4 A novel concentration and viability detection method for Brettanomyces using the Cellometer image cytometry. Brian Martyniak, Jason Bolton, Dmitry Kuksin, Suzanne M. Shahin, Leo Li-Ying Chan. 2017.]</ref><ref>[https://www.mbaa.com/meetings/archive/2017/proceedings/Pages/91.aspx 91. A novel concentration and viability detection method for Brettanomyces using image cytometry. Leo Chan. MBAA Poster. Retrieved 07/19/2018.]</ref>. It has been observed that pseudohyphae might be less common in ''Brettanomyces'' cultures that have been grown in wort versus lab media, making them easier to count <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1362220137139530/?comment_id=1932020246826180&comment_tracking=%7B%22tn%22%3A%22R%22%7D Caroline Whalen Taggart. Milk The Funk thread on Brettanomyces cell counting. 12/26/2017.]</ref>, but this method makes it easy to count cells which are attached via pseudohyphae. This method has been criticized for its relatively high cost (both for the fluorescent microscope and the dyes) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2201177583243777/ Justin Amaral, Jimmy Healy, Shawn Savuto, Matt Jumbard. MTF Facebook thread on the cost of using fluorescent micscopes and dyes for easier counting of ''Brettanomyces'' cells. 07/27/2018.]</ref>.