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:''Special thanks goes out to Bryan Heit from Sui Generis blog and Jeff Mello from Bootleg Biology for providing the information in this article.'' ==Isolating Pure Cultures=====Preparation===* [http://suigenerisbrewing.blogspot.ca/p/yeast-wrangling.html Sui Generis Blog] video guides on setting up a clean workspace, building and using an alcohol lamp, and aseptic techniques.* [http://bootlegbiology.com/diy/creating-plates/ Bootleg Biology's] guide on creating agar plates using wort, agar powder, and yeast nutrient.* [http://suigenerisbrewing.blogspot.ca/2015/03/new-video-casting-agar-plates.html Sui Generis Blog] guide to more types of agar plates, and video tutorial.* [https://teach.genetics.utah.edu/content/microbiology/liquids/ All growth media should be sterilized in an autoclave or a pressure cooker if possible, and boiled if not.] ===Catching===* [http://bootlegbiology.com/diy/capturing-yeast/ Bootleg Biology] defines three methods of catching a wild yeast.* [http://suigenerisbrewing.blogspot.ca/2014/12/two-new-videos.html Sui Generis Blog] video tutorial for catching wild yeast from fruit or from the air.* David Thornton of [[SouthYeast Labs]] describes his favorite method of catching wild yeast/bacteria: "Most of our cultures are taken from over-ripened fruit macerated in its own juice and left to ferment 24 hours at the location in a small Erlenmeyer with a perforated cap. Always grows something, and almost always get an alcohol fermenting strain, and of the alcohol fermenting strains id say 10% are worth brewing with. I like this method because I can stir my loop and do a streak on regular UBA aerobically, then pull favorable looking colonies for yeast and ''Lactobacillus'' and do a quadrant streak on selective media for isolation." <ref name="Thornton">[https://www.facebook.com/groups/MilkTheFunk/permalink/1141930175835195/?comment_id=1142286619132884&reply_comment_id=1142394019122144&total_comments=4&comment_tracking=%7B%22tn%22%3A%22R6%22%7D Conversation with David Thornton on MTF. 09/06/2015.]</ref>. See also the [https://www.milkthefunk.live/podcast/2018/6/28/episode-005-bio-prospecting-with-david-thornton-and-keston-helfrich-of-southyeast-labs-and-carolina-bauernhaus MTF 'The podcast' episode with David Thornton].* Monitor for mold growth. If mold begins to grow on the growth media, scrape it out and continue on with the isolation process. This should be safe since isolating the yeast will separate it from any potential mycotoxins that developed during the initial catching. Do not consume any growth media that has had mold grow on it (see [[Mold]]). One suggested method is to remove any materials (fruit, leaves, etc.) that were used as an inoculant as soon as visible fermentation happens; this can help avoid mold growth by removing floating material <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1812527172108822/?comment_id=1812566615438211&comment_tracking=%7B%22tn%22%3A%22R%22%7D Caroline Whallen Taggart on removing fruit to prevent mold. Milk The Funk Facebook group. 09/05/2017.]</ref>.* In general, summer and spring are more successful times to catch wild yeast, however any time of year can be successful. Simply choose fruits or flowers that bloom/grow during that particular season. For example, in spring, flowers and fruit blossoms are a good source. In summer, wild summer fruit can be a good source (raspberries, strawberries, cherries, etc.). In the fall, apples and grapes can be a good source. In winter, juniper berries <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1683379841690223/?comment_id=1683799054981635&comment_tracking=%7B%22tn%22%3A%22R%22%7D Milk The Funk thread on the best seasons for bioprospecting. 05/11/2017.]</ref>.* A new study showed that wild yeast actually exists more so in leaf matter on the ground than it does on bark or fruit. See the [[Wild_Yeast_Isolation#Videos|"Where (Do) the Wild Yeast Roam" video by Bryan from Sui Generis blog]] below and the below studies. Unless isolating microbes, this method could be dangerous unless the starter medium pH is lowered to be under 4.5 and enough ethanol (vodka) is added to reach 3-5% ABV to kill potential pathogens. Purging the flask with CO2 might help prevent mold growth.** [http://onlinelibrary.wiley.com/doi/10.1111/1758-2229.12446/full A systematic forest survey showing an association of Saccharomyces paradoxus with oak leaf litter.]** [http://onlinelibrary.wiley.com/doi/10.1111/mec.13120/abstract The interaction of Saccharomyces paradoxus with its natural competitors on oak bark.]** [http://macau.uni-kiel.de/receive/dissertation_diss_00018537?lang=en The natural ecology of Saccharomyces yeasts.]** [https://www.facebook.com/groups/MilkTheFunk/permalink/1910439962317542/ Associated MTF thread.]** [https://www.facebook.com/groups/MilkTheFunk/permalink/1916797218348483/ Matt Spaanem's attempt at isolating yeast from leaf litter.]* [https://www.facebook.com/groups/MilkTheFunk/permalink/1034954619866085/ David Thornton's use of foam as stoppers.] * Bryan of [https://suigenerisbrewing.blogspot.com/ Sui Generis Blog] instructions for using a jar of wort and cheesecloth to catch microbes in the air: <blockquote>"1) Prepare wort with an OG of 1.040, with enough lactic acid added to reduce the pH to 4.5. I'll often split this off of a normal brew day, prior to my hop addition, or you can make it from DME/LME. I am usually filling a 1L wide-mouthed pickel jar with the wort. I fill the jar to just below the threads. 2) I place the jar, covered with sanitised cheesecloth, in a place where I want to collect <nowiki>[microbes]</nowiki> from - usually I aim for an area with either growing plants (garden, etc), or in an area with lots of deciduous trees, as I've had the best success in these areas. I put the jars out in the evening, once the afternoon heat has passed, and gather them the next morning. I don't worry about having the wort hot and letting it "cool" to mimic a cool-ship as I've found this to not matter. In fact, I often pre-can (using a pressure canner) a few of these, and take them with me camping or to the cottage, to gather the local yeasts. You could even "guerrilla capture" yeast by sneaking these jars into local orchards/farms/etc...not that I'd ever do that ;-) 3) After collecting the (hopefully inoculated) jars, I remove the cheesecloth and replace it with the jar's lid + an airlock fitted into a hole I've drilled. I then place the jar in a dark warm place and let it sit for 3-4 weeks before I sniff test it. If it smells OK I will typically brew with it within the next 2 weeks. A few other pieces of advice: 1) This works best in the spring and fall - e.g. when nighttime temperatures are getting below ~10C/50F. When its warmer I've found my success rate drops from >80% to ~50%. This doesn't work well if the temperature is going below freezing, as the small volume of wort will inevitably freeze. 2) A small amount of "pure" alcohol can be added to improve your chances - e.g. add enough vodka or other unflavoured cheap booze (whiskey, unflavoured rum, etc) to bring your starters alcohol content up to 3-5%; this will suppress a lot of the unwanted <nowiki>[microbes]</nowiki>. For example, in my case I typically have ~900ml of wort in my 1L jar. To get 5% ABV I would make up 790 ml of wort (at ~1.045), to which I would add 110 ml of 40% ABV vodka. This will give ~5% alcohol, and keep the gravity above 1.035. Obviously, you add the alcohol after the wort has been boiled/canned/etc as you won't want to boil it off. 3) Hoped wort can be used as well, although my success at getting yeast + lacto with hopped wort is poor...I usually only get yeast, even with a meagre 3-5 IBU. 4) Raccoons, possums, rats, squirrels, deer, and pretty much every other fur-bearing animal (and some of the feather-bearing kinds as well) love unfermented wort, so try to place your jar somewhere they cannot get at it - while still maintaining airflow. We've got wild turkeys in the back, and those f***ers can fly (editor's note: language, Bryan!), so its a real challenge to keep them out." <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1817522898275916/?comment_id=1817911281570411&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Bryan of Sui Generis blog. Milk The Funk Facebook group post on catching wild yeast. 09/10/2017.]</ref></blockquote> ===Isolating===* [http://bootlegbiology.com/diy/isolating-yeast/ Bootleg Biology's] guide to streaking agar plates to isolate the captured yeast cultures.* [http://suigenerisbrewing.blogspot.ca/2014/08/your-home-yeast-lab-made-easy-streak.html Sui Generis Blog] video guide on how to streak agar plates.* [http://suigenerisbrewing.blogspot.ca/2014/12/two-new-videos.html Sui Generis Blog] video guide on streaking plates and further isolating yeast.* [http://basementbrewlab.com/lab/lab-media/sla/ Selecting for and isolating for ''Lactobacillus'' using Rogosa SL Agar.]* [https://www.facebook.com/groups/MilkTheFunk/permalink/1713165188711688/ MTF thread with tips on isolating and growing from a single colony.]* [https://drive.google.com/open?id=17wqLEBinSVCx6zKEoXpsUI9hMPN_MAUHgZ22TD552r0 MTF thread with tips on removing bacteria from yeast colonies using selective media and antibiotics.]* [https://www.facebook.com/groups/MilkTheFunk/permalink/3298177256877132/ Ruth Barry details her process for isolating individual strains in a mixed culture with the goal of characterizing each strain for a more controlled mixed fermentation.]* [https://www.youtube.com/watch?v=dQzbeE5_ehc Dr. Matt Winans from Imperial Yeast on "Chop n Brew" demonstrates that incubating agar plats at different temperatures can result in widely different growth success rates (~11:00 minutes in).] See also [https://www.facebook.com/groups/MilkTheFunk/posts/6025908170770680/ this MTF thread exploring this approach]. ===Identifying===* [http://suigenerisbrewing.com/index.php/2019/10/30/a-primer-on-yeast-morphology "Basic Yeast Morphology" by Dr. Bryan Heit of Sui Generis Blog.]* [http://suigenerisbrewing.blogspot.ca/2014/12/new-video.html Sui Generis Blog] video guide on how to visually identify usable yeasts versus potential pathogens and molds on agar plates.* Microbes cannot be accurately identified based on what their pellicle looks like. See [[Pellicle#Pellicle_Appearance_as_a_Microbe_Identifying_Indicator|Pellicle]] for more information.* Species is difficult to establish from fermentation characteristics or morphology <ref>[https://suigenerisbrewing.blogspot.com/2013/04/old-school-identification-of-wild.html "Old School Identification of Wild Yeasts & Bacteria". Sui Generis blog. 04/10/2013. Retrieved 02/14/2018.]</ref>. DNA sequencing is the only way to be completely sure of the species identification. ** See also [https://www.facebook.com/groups/MilkTheFunk/permalink/1989236037771267/ instructions submitting cultures to Genewiz for Sanger Sequencing ($18-20 US).]** [https://www.facebook.com/groups/MilkTheFunk/permalink/1989236037771267/?comment_id=1989247777770093&reply_comment_id=1989250314436506&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Nick Impellitteri's offer to ID species at a low cost and when time permits (contact Nick at The Yeast Bay).]** [https://www.facebook.com/groups/MilkTheFunk/permalink/1989236037771267/?comment_id=1989499621078242&comment_tracking=%7B%22tn%22%3A%22R%22%7D Sleight Beer Lab will ID at slightly higher price, but it includes the BLAST report done for you.] BLAST can be done with a cheap software called [http://www.mbio.ncsu.edu/bioedit/bioedit.html BioEdit] and [https://www.megasoftware.net/home MEGA] (see also their [https://www.youtube.com/user/rsingh1980/videos instructional videos]), or [http://biologylabs.utah.edu/jorgensen/wayned/ape/ ApE].* Shawn Savuto recommends [http://my.asbcnet.org/ItemDetail?iProductCode=52206 "Illustrated Guide to Microbes and Sediments in Wine, Beer and Juice" by Charles G. Edwards] <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2056634557698081/?comment_id=2056742781020592&comment_tracking=%7B%22tn%22%3A%22R%22%7D Shawn Savuto. Milk The Funk Facebook thread on books on identifying microbes. 04/11/2018.]</ref>.* [[Books#Science_Textbooks|Yeast Textbooks]] for help with identification. ===Growing and Testing==Culturing=* [http://suigenerisbrewing.blogspot.ca/2012/11/yeast-banking-iii-stepping-up-to.html Sui Generis Blog] guide to stepping up yeast to pitchable amounts. * [https://www.youtube.com/watch?v=CAIS29Pj8AY Dr. Bryan Heit from Sui Generis Brewing blog] discusses and demonstrates his approach to trialing wild yeast isolates.* [http://braukaiser.com/wiki/index.php?title=Growing_Yeast_from_a_Plate Kai Troester's] guide to growing yeast from an agar plate.* [https://www.facebook.com/groups/MilkTheFunk/permalink/2089020794459457/?comment_id=2089022064459330&comment_tracking=Plate Streaking%7B%22tn%22%3A%22R0%22%7D Some wild yeasts might perform well at lager temperatures.] ===Storage===Slants are a good option for longer term storage (3 months to potentially 2+ years). For instructions on how to make slants at home capable of storing any microbe for potentially 2+ years, [http://suigenerisbrewing.blogspot.com/2015/11/easy-home-yeast-banking-and-video.html see Bryan's video on Sui Generis Brewing (requires a pressure cooker)]. ==David Thornton's "Yeast Bootcamp"==This method was developed by David Thornton of [[SouthYeast Labs]] and [https://carolinabauernhaus.com/ Carolina Bauernhaus] for collecting wild ''Saccharomyces'' yeast and lactic acid bacteria. David described his method in detail on [https://www.milkthefunk.live/podcast/2018/6/28/episode-005-bio-prospecting-with-david-thornton-and-keston-helfrich-of-southyeast-labs-and-carolina-bauernhaus the Milk The Funk "The Podcast" episode #005]. Thornton encourages people to alter this process to suit their required results and conditions; for example, developing a different approach that might allow this method to be better suited towards catching ''Brettanomyces''. The following is a summary of this process: # Start with 75 mL of 6° Plato wort with yeast nutrient in a 250 mL flask. Add the sample (fruit, flower, bee, etc). If it's fruit, squish it up. Set the flask on a shaker or stirplate at 80-90F with a [https://www.facebook.com/groups/MilkTheFunk/permalink/1034954619866085/ foam stopper] (the foam stoppers can be autoclaved/boiled and create an aerobic environment; aluminum foil should work as a substitute) for 12-48 hours. Look for frothing and as soon as that happens put an airlock on the flask, but keep on stirplate to assist fermentation. # After the 24-48 hours, decant and discard the liquid and leave the biomass at the bottom. Add 75 mL of new starter media to the flask (with the biomass still at the bottom of the flask) that is 8-10* Plato with yeast nutrient, and purge the flask with CO2. Put an airlock on it on a stirplate and grow until the sugars have fermented.# Again, decant and discard the liquid and leave the biomass at the bottom of the flask. Purge the flask (with the biomass still at the bottom) with CO2, and add 75 mL of 20° Plato wort with yeast nutrient and an airlock, and place on a stirplate or shaker. Look for the wort to ferment down to around 2 Plato or so. # After the 20° Plato wort has finished fermenting, samples from the liquid (the liquid contains the more active yeast versus the biomass at the bottom of the flask) are streaked onto media to isolate the strains that have survived. If agar plating is not an option, a less advanced approach can be taken by [[Wild_Yeast_Isolation#Growing_and_Testing_Without_Plating|growing and testing without plating]]. Agar that David uses (see also [[Laboratory_Techniques#Growth_Media|Growth Media]] on the wiki for other media types that might also be useful): * Wallenstein labs nutrient: serves as a catch-all.* HLP: isolate ''Lactobacillus'' and ''Pediococcus''.* LMDA: color indicators for acid production (then do a gram stain to see if it is positive or negative; ''Lactobacillus'' versus ''Acetobacter'').* Lin’s Cupric Sulfate Medium: selects for wild ''Saccharomyces''. Other notes:* Measure pH to make sure it gets down to under 4.5; this helps monitor for fermentation activity. * Generally this method selects for 1-3 strains of yeast with usually a lactic acid bacteria strain.* The anaerobic nature of this method selects against mold growth.* This method may not select for wild ''Brettanomyces'' since aerobic growth is limited. This method could possibly be altered to support the growth of ''Brettanomyces'' by allowing for small amounts of oxygen exposure after the initial aerobic step.* Agitation during growth helps both aerobic and anaerobic microbes grow by evenly distributing nutrients and waste. David has used something as simple as setting flasks on his washing machine to keep the growth media agitated. See also:* [https://loschicoshomebrewing.wordpress.com/2021/01/17/capturing-wild-yeasts-first-try Fernando Sánchez Leal's detailed blog post on trialing three different capturing methods, including the David Thornton "Bootcamp" method and links to many good resources.] ==Growing and Testing Without StreakingPlating==While using agar plates to isolate yeast colonies is the most effective way to culture wild yeast, it is not the only way. Wild yeast should first be caught using DME wort as outlined in [http://bootlegbiology.com/diy/capturing-yeast/ one of Bootleg Biology's methods] or [[Wild_Yeast_Isolation#David_Thornton.27s_.22Yeast_Bootcamp.22|David Thornton's Yeast Bootcamp method]]. Hopping the wort will help decrease bacteria if that is desired. The wort's pH should be lowered to 4.5 or lower with lactic acid to avoid bacteria as much as possible. Molds may still grow in the yeast starter even with the lower pH; wort that has grown mold should not be consumed because mycotoxins can contaminate the wort (see [[Mold]]). Keeping the foraged fruit, flowers, etc. under the surface of the wort will help reduce the chances of mold growth. Some brewers recommend submerging the fruit/flowers in the wort for a few minutes and then removing them to prevent them from attracting mold growth <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1659565197405021/?comment_id=1711116612249879&comment_tracking=%7B%22tn%22%3A%22R%22%7D Jeff Porn. Milk The Funk Facebook group. 06/06/2017.]</ref>. If the yeast cannot be separated from wort that mold grew on then it should be thrown out. Signs of a small krausen within 1-3 days is a good sign that viable wild yeast has been collected. After another few days, the yeast will start dropping to the bottom of the collection vessel <ref name="manoaction">[http://www.homebrewtalk.com/collecting-wild-yeast.html Collecting Wild Yeast. Manoaction. Homebrewtalk. Oct 30, 2012.]</ref>. Once the wort has fermented out (allowing 2 weeks total is a good rule of thumb), decant the beer and pitch the collected yeast into 500ml of starter wort, again lowering the pH of the starter wort to 4.5 with lactic acid. After the starter wort has been fermented, the yeast should have enough of a population to ferment out a 1 gallon batch of wort. Keep the recipe of the wort simple, and in the 1.050 gravity range. The yeast cell count should be high enough at this point to out-compete bacteria and lower the pH of the wort within a few days. The pH of the 1 gallon batch, therefore, does not need to be lowered, although the brewer may choose to do so anyway. For [[Wild_Yeast_Isolation#Safety|safety reasons]], test to make sure that the beer fully attenuated and has a pH of 4.5 or less. Allow for one month after fermenting the 1 gallon batch of beer before sampling. If it smells like feces or vomit, do not sample it and throw it away. If the beer smells ok, feel free to sample the beer to see if the wild yeast produced a good tasting beer. David Thornton from [[SouthYeast Labs]] estimates that only about 10% of yeast that can fully attenuate produces favorable results, so failures are to be expected (keep trying!) <ref name="Thornton"></ref>. Fermenting a few batches of beer at different temperatures is a useful method for identifying the ideal fermentation temperature range for the yeast. Once a wild culture has been determined to be something that the brewer wants to keep for future batches, yeast harvesting methods can be used to maintain the culture. See [http://www.wyeastlab.com/yeast-harvesting-re-pitching Wyeast's article on yeast harvesting] and [http://brulosophy.com/methods/yeast-harvesting/ harvesting from an over-built yeast starter]. See also:* [https://www.facebook.com/groups/MilkTheFunk/permalink/1676671082361099/ Advice from DeWayne Schaaf and others on bioprospecting techniques without isolating microbes on MTF]. * [[Mold]] for identifying mold and safety issues with mold. See this [https://www.facebook.com/groups/MilkTheFunk/permalink/2129463057081897/ MTF thread] for tips on avoiding mold when bioprospecting. * Microbes cannot be accurately identified based on what their pellicle looks like. See [[Pellicle#Pellicle_Appearance_as_a_Microbe_Identifying_Indicator|Pellicle]] for more information. * Dr. Bryan Heit of Sui Generis Blog demonstrates a simple way for [http://suigenerisbrewing.com/index.php/2019/02/12/wild-mead/ collecting yeast from bees using a vodka fortified starter (no plating or isolating)]:: <youtube width="300" height="200">6Fd-LVd0HoA</youtube> ===Notes on Fermentation Timeframes===Some wild captures might take a long time to ferment. Most wild caught, non-isolated cultures that can ferment the sugars in wort take 2-4 weeks to ferment, although some can take longer and some can take less time (even just a couple of days). This is impossible to predict due to the impossibility of predicting what microbes are in a non-isolated wild culture. For example, wild ''Brettanomyces'' might take a long time to finish fermenting (up to 8 months to reach a stable gravity, according to Matt Spaanem). In some experiences, krausen will form for a few days, and then drop, and then form again (presumably as another microbe starts fermenting). Others ferment long and slow over a month or so. Once re-pitched with higher cell counts, often the fermentation times will hasten, but other times they will not. The best piece of advice is to take gravity readings to determine when the fermentation is finished instead of relying on visual indicators. Even though there could be a krausen and signs of fermentation, if the fermentation is going slow then it is possible that the sugars in the wort are not being fermented effectively (this might improve upon re-pitching as this should help select for microbes that can withstand this environment; see reference for more details on MTF members' experiences) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1777108328984040/ DeWayne Schaaf, Christophe Boebion, Tobias Häberli, Justin Amaral, Bryan from Sui Generis blog, Matt Spaanem, Josh Regenold, Guy Greenhalgh, Jason Pallett, Caroline Whalen Taggart. Milk The Facebook group on how long fermetnation takes for wild caught cultures. 08/01/2017.]</ref>. ===Safety===An often asked question when it comes to tasting beer fermented from a wild culture that wasn't plated and isolated is, "When is it safe to drink?" To our knowledge, no studies have shown when a wild-caught beer is scientifically ~99.9% safe to drink. However, several studies can give us some guidance on this, as well as [http://suigenerisbrewing.blogspot.com/ Dr. Bryan Heit from Sui Generis Blog]. * The addition of hops in the starter wort will inhibit or kill some Gram-positive bacteria pathogens (''L. monocytogenes'' and ''S. aureus'') <ref name="Menz1">[http://www.ncbi.nlm.nih.gov/pubmed/22004814 Growth and survival of foodborne pathogens in beer. Menz G., Aldred P, Vriesekoop F . Oct 2011.]</ref><ref name="BryanMTF">[https://www.facebook.com/groups/MilkTheFunk/permalink/1104757552885791/?comment_id=1105118642849682&offset=0&total_comments=15&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Bryan of Sui Generis Blog on MTF regarding when it is safe to taste wild beer. 7/6/2015.]</ref>. The use of [https://en.wikipedia.org/wiki/Lysozyme lysozyme], which kills Gram-positive bacteria, might also be a good way to limit the growth of bacteria (including lactic acid bacteria).* A low pH is not the only requirement for making beer safe. A combination of alcohol, low pH, and hops will ensure that pathogens cannot grow (survival is another matter) <ref name="Bryan">[http://suigenerisbrewing.blogspot.ca/2014/02/fact-or-fiction-can-pathogens-survive.html ''Fact or Fiction? Can Pathogens Survive in Beer?'' Sui Generis Blog.]</ref><ref>[http://www.ncbi.nlm.nih.gov/pubmed/20207437 The growth and survival of food-borne pathogens in sweet and fermenting brewers' wort. Menz G1, Vriesekoop F, Zarei M, Zhu B, Aldred P. May 2010.]</ref>. * Two studies have shown that ''E. coli'' can survive in moderate strength beer (~5% ABV, 4.3 pH) for at least 28 days (more than 30 days in one study) if the beer is stored cold (39-41°F or 4-5°C) <ref name="Menz1"></ref><ref>[http://www.ncbi.nlm.nih.gov/pubmed/24674433 Survival of foodborne pathogenic bacteria (Bacillus cereus, Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Listeria monocytogenes) and Bacillus cereus spores in fermented alcoholic beverages (beer and refined rice wine). Kim SA1, Kim NH, Lee SH, Hwang IG, Rhee MS. March 2014.]</ref>. Therefore, the wild culture should be stored at room temperature for a least a month before tasting. '''Additionally, the pH of the beer should be less than 4.6, and alcohol should be present for at least one month before tasting. The smell of feces or vomit indicates that bad bacteria may be present; if so dump it out ''' <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1104757552885791/?comment_id=1104851526209727&offset=0&total_comments=8&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Conversation with Bryan of Sui Generis Blog on MTF regarding when it is safe to taste wild beer. 7/5/2015.]</ref>.* In unfermented wort, ''E. coli O157:H7'' and ''Salmonella typhimurium'' were shown to grow at a pH of 4.3, but stopped growing at a pH of 4.0 <ref name="Menz1"></ref>. However, some of these food poisoning bacteria can survive the low pH of the stomach (~2.0) long enough to cause illness, so relying on a low pH alone is not adequate. Bryan of Sui Generis Blog recommends a pH of 4.5 or less, the use of well sanitized equipment, and monitoring the starter for the rapid onset of fermentation without putrid smells. Some oxidative yeasts that are the first "barriers" to bacterial growth may be inhibited at 4.0 pH <ref name="BryanMTF"></ref>.* The risk of botulism toxin is extremely low, but not zero. Generally, ''C. botulism'' is not able to grow and produce toxin in low protein (non-meats) substances at a pH lower than 4.6 <ref>[http://www.ingentaconnect.com/content/iafp/jfp/1982/00000045/00000003/art00005 Toxin Production by Clostridium Botulinum in Media at pH Lower Than 4.6. Nobumasa, Tanaka. Journal of Food Protection®, Number 3, February 1982, pp. 214-284, pp. 234-237(4)]</ref><ref>[http://edis.ifas.ufl.edu/fs104 Preventing Foodborne Illness: Clostridium botulinum. University of Florida IFAS Extension. Retrieved 7/5/2015.]</ref>. However, proteins in the wort may allow growth at a lower pH than 4.6 (studies have not been done on wort, only on meats) <ref name="BryanMTF"></ref>. The presence of more than 5% oxygen in the wort will also reduce the risk <ref>[http://www.ncbi.nlm.nih.gov/pubmed/15895734 Growth and toxin production by Clostridium botulinum in steamed rice aseptically packed under modified atmosphere. Kasai Y, Kimura B, Kawasaki S, Fukaya T, Sakuma K, Fujii T. May 2005.]</ref>.* To our knowledge, there have been no documented cases of food poisoning from beer <ref name="Bryan"></ref>. However: "It is well established that at least a dozen human pathogens can survive in fully fermented beer; moreover, sour-brewing (and home brewing in general) offers an additional opportunity for pathogens to gain a foothold or produce toxins which persist into the final product. Reality is that the source of most food poisonings is never established. A few industries (milk, meat, canning, etc) are under close scrutiny, and as such we have firm numbers for rates of food-borne illnesses from those industries. Neither commercial nor homebrewing is subject to that monitoring, so any cases of disease arising from contaminated products from either source would likely go unidentified." - Bryan of Sui Generis Blog on MTF <ref name="BryanMTF"></ref>. For more information on the potential for pathogens surviving in beer, see this [http://suigenerisbrewing.blogspot.com/2014/02/fact-or-fiction-can-pathogens-survive.html Sui Generis Blog article.].* In the words of Bryan from [http://suigenerisbrewing.blogspot.ca/2014/02/fact-or-fiction-can-pathogens-survive.html Sui Generis Blog]: "If you have an immunodeficiency or are immunosuppressed: Obviously, don't take medical advice off the internet. Talk to your medical doctor to see if you are at risk of infection - especially if you're condition/treatment increases your risk of fungal infections. If so, or if you are concerned, stick to beers that are fermented using commercial strains of Saccharomyces yeast." <ref name="Bryan"></ref>* See also [https://www.facebook.com/groups/MilkTheFunk/permalink/1283377785023766/ this thread on MTF].* See also [https://www.facebook.com/groups/MilkTheFunk/permalink/1447181778643365/?match=Ym90dWxpc20%3D this MTF thread on the mechanisms of botulism poisoning, and how it applies to different brewing processes] and this [http://suigenerisbrewing.com/index.php/2020/01/29/botulism-in-beer/ blog post on Sui Generis Brewing blog by Dr. Bryan Heit].* [https://suigenerisbrewing.blogspot.com/2017/01/fact-of-fiction-can-pathogens-survive.html Further explanation of what makes microbes "pathogenic" by Bryan from Sui Generis Blog] and a related [https://www.facebook.com/groups/MilkTheFunk/permalink/1246624848699060 MTF discussion].* If mold grows on growth media and the yeast can be isolated cleanly, then this is not an issue, however, do not consume beer or wort that has had mold growing on it. If the yeast is not being isolated on agar plates, then throw out the capture sample and do not use it. See [[Mold]] for identifying mold and safety issues with mold.* [https://www.facebook.com/groups/MilkTheFunk/permalink/2054548517906685/ An example of identifying a pathogenic species of ''Candida'' (''C. lusitaniae'') in a wild capture.]* Do not taste wort soured with malted grain; grain soured wort has been found to grow pathogens. Wait until fermentation with yeast is finished. See [[Grain#Malt_Inoculated_Wort|Grain]] for more information. ==Domestication==It has been demonstrated that wild yeast can adapt to brewing conditions if they are exposed to a repeated and consistent environment in as little as 200 generations. The average beer fermentation produces 3-5 generations, and the average starter for growth produces around 10 generations. For more information, see this [https://www.facebook.com/groups/MilkTheFunk/permalink/4374738789220968/ MTF thread] by Dr. Bryan Heit for a summary of [https://elifesciences.org/articles/63910 "Phenotypic and molecular evolution across 10,000 generations in laboratory budding yeast populations"]. See also:* [[Saccharomyces#History_of_Domestication|History of Yeast Domestication]] ==Wild ''Brettanomyces''==''Brettanomyces'' is notoriously difficult to bioprospect from the wild. For example, an ongoing survey of wild yeasts in most of the US which isolated nearly 2,000 isolates with 262 unique species has not yet found a single occurrence of ''Brettanomyces'' in the wild (so far they have only surveyed non-human inhabited wild areas of the US) <ref>[https://www.biorxiv.org/content/10.1101/2021.07.13.452236v1 Substrate, temperature, and geographical patterns among nearly 2,000 natural yeast isolates. William J. Spurley, Kaitlin J. Fisher, Quinn K. Langdon, Kelly V. Buh, Martin Jarzyna, Max A. B. Haase, Kayla Sylvester, Ryan V. Moriarty, Daniel Rodriguez, Angela Sheddan, Sarah Wright, Lisa Sorlie, Amanda Beth Hulfachor, Dana A. Opulente, Chris Todd Hittinger. bioRxiv 2021.07.13.452236; doi: https://doi.org/10.1101/2021.07.13.452236.]</ref>. While it has been alleged by many that the natural environment for ''Brettanomyces'' is the skins of fruit or the bodies of insects, it wasn't until 2007 that science was able to show that ''Brettanomyces'' survives on the skins of grapes, although it does so in a very low population and a possibly [[Quality_Assurance#Viable_But_Nonculturable|"viable but not culturable"]] state. Renouf et al. (2007) and Comitini et al. (2019) demonstrated that an "enrichment ''Brettanomyces bruxellensis''" media called EBB is more efficient at first growing up ''Brettanomyces'' before trying to culture it on DBDM. ''Brettanomyces'' was allowed to grow for 80 days in the EBB media, and then streaked onto DBDM for selection for ''Brettanomyces'' (other wild yeast such as ''Hanseniaspora'' and ''Pichia'' grew much more readily than ''Brettanomyces'' that was cultured from wine grapes). Using EBB, Comitini et al. (2019) found that after 80 days of incubation in the EBB medium, half of grapes samples from a vineyard with several varieties of grapes had wild ''Brettanomyces'' on their skins. The researchers also tested the air, the trailers, and the winery where the grapes were processed. They found no ''Brettanomyces'' in the air or the trailers, but did find a couple of valves that were used to transfer must have ''Brettanomyces''. Out of the four varieties of grapes sampled, Merlot grapes did not have any ''Brettanomyces'' growing on them, indicating that the variety of the grape might play a role in the ability of ''Brettanomyces'' to survive on grape skins. The researchers found a wide range of strains that matched the strains found in the winery, indicating that the ''Brettanomyces'' strains did originate from the grape skins <ref>[https://www.sciencedirect.com/science/article/pii/S0944501306000231?via%3Dihub Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries. Vincent Renouf, Aline Lonvaud-Funel. 2007. DOI: https://doi.org/10.1016/j.micres.2006.02.006.]</ref><ref>[https://www.frontiersin.org/articles/10.3389/fmicb.2019.00415/abstract Occurrence of Brettanomyces bruxellensis on grape berries and in related winemaking cellar. Francesca Comitini1, Lucia Oro, Laura Canonico, Valentina Marinelli, Maurizio Ciani. 2019. DOI: 10.3389/fmicb.2019.00415.]</ref>. More recent information indicates that the natural habitat of ''Brettanomyces'' might be the root systems of certain plants, known as the [https://www.nature.com/scitable/knowledge/library/the-rhizosphere-roots-soil-and-67500617/ "rhizosphere"]. See [[Brettanomyces#Environment_and_Survival|''Brettanomyces'' Environment and Survival]] and the links below for more information. See also: * [https://suigenerisbrewing.com/index.php/2022/10/06/this-is-not-the-yeast-your-are-looking-for/ Dr. Bryan Heit's first attempt to find wild ''Brettanomyces'' in the rhyzosphere of plants.]** [https://suigenerisbrewing.com/index.php/2022/11/02/this-is-the-wild-brettanomyces-your-are-looking-for/ Second attempt which may have been successful (pending genetic testing).] *** A follow up [https://www.facebook.com/groups/MilkTheFunk/posts/6294804440547717/?comment_id=6294865953874899&reply_comment_id=6297362253625269 thread in MTF] reports Dr. Heit finding a strain of ''B. nanus'' and (likely) ''B. custersianus''.*** [https://suigenerisbrewing.com/index.php/2023/03/24/wild-brettanomyces-fermentation-test/ A blog update on the flavor and aroma results from fermenting with rhyzosphere-isolated ''Brettanomyces''.]* [[Laboratory_Techniques#Brettanomyces|Agar recipes for ''Brettanomyces'']].* [https://www.facebook.com/groups/MilkTheFunk/permalink/1620419974652877/ MTF discussion on the rare but possible isolation of wild ''Brettanomyces''.]* [[Brettanomyces#Environment_and_Survival|''Brettanomyces'' natural environment.]] ==Misc==* [https://www.facebook.com/groups/MilkTheFunk/permalink/2859721020722760/?comment_id=2859768487384680&reply_comment_id=2860162564011939&comment_tracking=%7B%22tn%22%3A%22R%22%7D Reports of wild caught yeast that produce EPS (ropiness).] * Escarpment Labs presentation on catching wild yeast:: <youtube width="300" height="200">hvgyVrMpa4k</youtube> * Two recent studies have found a very high occurrence of ''Lactobacillus acetotolerans'' in many mixed fermentation sour beers (see [https://www.facebook.com/groups/MilkTheFunk/posts/4652629078098603 this associated MTF thread for discussions]:** [https://www.sciencedirect.com/science/article/pii/S0740002020302471 Characteristics of bacterial and yeast microbiomes in spontaneous and mixed-fermentation beer and cider; Tyakht et al. (2021).]** [https://www.biorxiv.org/content/10.1101/2021.07.21.453094v1 Mixed culture metagenomics of the microbes making sour beer; Bochman et al. (2021).] ===MTF Wild Capture Swap===DeWayne Schaaf organizes an occasional swap of wild caught microbes on MTF. * Fall 2017 swap: currently under way. See [https://www.facebook.com/groups/MilkTheFunk/permalink/1810727062288833/ this thread] to sign up.
==See Also==
===Additional Articles on MTF Wiki===* [[Mixed Laboratory Techniques]]* [[Microscope]]* [[Spontaneous Fermentation]]* [[Alternative Bacteria Sources]]* [[Bootleg Biology]]* [[Brettanomyces]]* [[Saccharomyces]]* [[Lactobacillus]]
===External Resources=======Links====* [httphttps://bootlegbiology.com/diy/capturinglocal-yeast-project/ Bootleg Biology's local yeast project to collect yeast from every zip code in the US.]* [https://onlinelibrary.wiley.com/doi/10.1002/yea.3380 "Bioprospecting for brewers: Exploiting natural diversity for naturally diverse beers", scientific review on bioprospecting (2019).]* [https://www.youtubefacebook.com/groups/MilkTheFunk/permalink/1489822481045961/ James Howat from Black Project shares his simple setup on MTF for continuous CO2 purging in order to discourage mold in wild starters.]* [http://www.brewingscience.com/identification_key.htm Brewing Science Bacteria on LMDA (visual identification of various bacteria species on LMDA plates).]* [http://bootlegbiology.com Bootleg Biology provides a full toolkit and materials for yeast isolation.] They also provide a [http://bootlegbiology.com/community/ community yeast bank].* [http://suigenerisbrewing.blogspot.ca Sui Generis Blog.]* [https://bkyeast.wordpress.com/2012/12/17/how-to-build-a-yeast-ranch/ BKYeast, "How to Build a Yeast Ranch".]* [http://www.themadfermentationist.com/playlist?list=PLZ3Z2428mCTqahdOz2011/04/ambient-spontaneous-tVJyeast-pGKfg9sTblk Hunting Wild starters.html "Ambient Yeast videos Starters" by Bryan Heit of 'Michael Tonsmeire.]* [https://dcylab.wordpress.com/2012/04/16/things-ive-learned/ "Things I've Learned", DC Yeast Lab.]* [https://eurekabrewing.wordpress.com/yeast-cultivation/ "Yeast Cultivation Basics", Eureka Brewing.]* [https://eurekabrewing.wordpress.com/2012/08/24/yeast-banking-2-agar-plates/ "Yeast Banking - #2 Agar Plates", Eureka Brewing.]* [http://www.dhbrewingconsulting.com/culturing-instructions/ Culturing Instructions by Double Helix Brewing Consulting.]* [http://suigenerisbrewing.blogspot.com/2014/02/fact-or-fiction-can-pathogens-survive.html "Fact or Fiction? Can Pathogens Survive in Beer?", by Sui Generis'' Blog.]* [http://www.alesoftheriverwards.com/2016/04/forest-and-main-brewing-co-exploring.html "Forest and Main Brewing Co.: Exploring their indigenous culture", By Ed Coffey on Ale of the Riverwards Blog.]* [http://www.craftcommander.com/brewing/2016/7/14/brewing-beer-with-a-sense-of-place-foraging-yeast-for-beginners "Brewing Beer With a Sense of Place - Foraging Yeast for Beginners" by Andrew "Gus" Addkison on the Craft Commander blog.]* [http://brouwerij-chugach.com/?p=1171 "Yeast Wrangling Adventure", Brian Hall of Brouwerij Chugach blogshares wild yeast hunting techniques in the Alaskan wilderness.]* [https://www.youtube.com/channel/UC8_kozGERzHnjVIizhnh43g/videos "Brewing Viking" home yeast lab videos on YouTube.]* [https://www.mbaa.com/publications/tq/tqPastIssues/2022/Pages/TQ-59-3-1204-01.aspx "Methods for Harvest, Isolation, Selection, and Utilization of Saccharomyces and Non-Saccharomyces Yeast Strains for Commercial Brewing," by Tim Faith and Alex Nham, MBAA Technical Quarterly, 2022.] See also their interview on [http://sites.libsyn.com/315398/episode-114-yeast-foraging-harvesting-isolating-and-selection-w-tim-faith-alex-nham Bru Lab podcast]. ====Books====* [http://www.amazon.com/Yeast-Practical-Fermentation-Brewing-Elements/dp/0937381969 Yeast: The Practical Guide to Beer Fermentation (Brewing Elements)], by Chris White and Jamil Zainasheff.* [http://dunham.gs.washington.edu/DunhamLabMinistats.pdf Dunham Lab Ministat Manual.] ====Videos====<youtube height="200" width="300">-tmbnPXZcFQ</youtube><youtube height="200" width="300">y43x7nLTEPo</youtube><youtube height="200" width="300">SwDjJKkuT74</youtube><youtube height="200" width="300">viXPMtkTfk8</youtube><youtube height="200" width="300">ChHcR9qaxj0</youtube><youtube height="200" width="300">g9mwpnXeZmc</youtube>
==References==
<references/>
[[Category:Techniques]]