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WINEMAKING Secondly, once a barrel has been identified as possibly containing Brettanomyces, samples need be carefully removed for analysis. Initially, many winemakers opt for microscopic evaluation using a phase contrast microscope because the method is rapid. In general, Brettanomyces exhibits cellular morphologies described as being boat-shaped, gothic arches or ogival. While cell appearance is useful for identification, caution must be applied given variable cell morphology depending on age, culture medium and environmental stress. In fact, some cells can appear elongated, rectangular or other have shapes.1 Using a microscope, populations of more than 5,000 cells per milliliter are sometimes needed to be able to see a single cell. If cells suspected of being Brettanomyces are observed, other methods such as culturing the yeast on specific microbiological agar media (e.g., WLC) or application of genetic protocols such as Scorpions may be necessary to confirm its presence.2 Culturing microbes in-winery is actually a pretty easy task since a number of companies offer agar media and supplies. Finally, large wineries may routinely measure volatile phenols (i.e., 4-EP and 4-EG), as few non-Brettanomyces microorganisms synthesize these compounds. These analyses require very expensive equipment, however, so smaller wineries would need to send samples elsewhere for analysis. In the past, a widely used method of control has been the use of SO2. In general, many winemakers maintain 0.4-0.6 milligrams per liter (mg/L) molecular SO2 as a means to limit growth of Brettanomyces. Therefore, lowering wine pH will also affect Brettanomyces by increasing the concentrations of molecular SO2 present. However, there is growing evidence that the yeast may become "viable but not culturable" upon exposure to sulfites. As the name implies, the yeast remains alive but cannot be cultured on standard microbiological media for unknown reasons. Some researchers believe that the so-called "VBNC" is really a form of microbial injury; some disagree, citing that injured cells should grow on non-selective (general) media where VBNC cells allegedly do not. Whatever the case, there is little doubt that SO2 affects Brettanomyces such that the cells are not necessarily dead (injured or VBNC), and culturability and synthesis of volatile phenols can eventually return if precautions are not taken. From a more holistic point of view, perhaps the best method to minimize Brettanomyces infections is implementation of "hurdle technology" methods. Applied in the food industry, the concept is to put in place as many "hurdles" as feasibly possible to prevent infections and spoilage. As examples, a good hurdle program would not only include frequent monitoring with additions of SO2 but also implementation of 1) comprehensive sanitation programs; 2) maintaining low cellar temperatures (<15°C); 3) use of other antimicrobials such as Velcorin, chitosan or ozone, and/or other procedures. A large advantage in applying this technology is that many times the sum ef- Some wineries have greatly reduced average 4-ethyl phenol levels in only a couple of vintages even though the yeast can still be found in the cellar. Where does Brettanomyces come from? Two important sources of contamination involve the importation of infected wines and/or barrels. If wine is purchased from other establishments, it is critical to filter prior to introduction into the winery (use membranes with at least 0.8 µm absolute porosity). Furthermore, avoid purchasing used barrels, as these can harbor yeast. Once established in wood cooperage, complete sanitization is generally unattainable because of the deep cracks and crevices within barrels. While there have been promising reports describing use of high-powered ultrasound and ozone as means to disinfect barrels, more research is needed before those methods can be uniformly accepted. There are other vectors by which Brettanomyces can enter a winery. Early evidence from the 1950s pointed out that Brettanomyces could be transported on insects (fruit flies), although this has not been confirmed using modern techniques. More recently, it was reported that Brettanomyces could be moved within a building through air currents. But perhaps the most controversial potential vector has been whether grapes could be a habitat for Brettanomyces. While some scientific papers have claimed the yeast could be found on grapes, the scientific validity of some of these reports has been questioned. If the yeast is found to be conclusively present on grapes, populations would likely be quite low, given the number of past failed attempts to find Brettanomyces ("needle in a haystack"). Clearly, additional research is needed to determine if the yeast is found on grapes with any regularity. If Brettanomyces is found, should the winery be burned down? Twenty or so years ago, some winemakers believed that burning down the winery was an option to exterminate Brettanomyces. Since that time, many wineries have altered their approach from "complete eradication" to more of "management" using various means. In fact, some wineries have greatly reduced average 4-EP levels in only a couple of vintages even though the yeast can still be found in the cellar through rigorous monitoring and implementation of specific protocols. 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