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[[File:Thornton Wild Capture.jpg|thumb|[https://www.facebook.com/groups/MilkTheFunk/permalink/1034954619866085/ Picture of the first step to catching wild yeast by David Thornton of SouthYeast Labs and Carolina Bauernhaus Ales.]]]
'''Wild yeast isolation''', sometimes known as "yeast wrangling" or more formally [https://en.wikipedia.org/wiki/Bioprospecting "bioprospecting"], refers to the process of catching wild, native yeast ([https://www.merriam-webster.com/dictionary/autochthonous autochthonous] yeast), and isolating it with agar plates as a pure culture. This article will also contain contains information on growing up a ''captured'' mixed culture in wort rather than isolating single strain yeast cultures on agar plates. Culturing wild yeast and bacteria should not be confused with [[Spontaneous Fermentation]] because many of the microbes that might make a flavor impact during spontaneous fermentation are killed off during the bioprospecting processes. For general lab techniques, see [[Laboratory Techniques]]. For more information on nonconventional microbes, see [[Nonconventional Yeasts and Bacteria]].
:''Special thanks goes out to Bryan Heit from Sui Generis blog and Jeff Mello from Bootleg Biology for providing the information in this article.''
* [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===
* [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.
** [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===
* [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=%7B%22tn%22%3A%22R0%22%7D Some wild yeasts might perform well at lager temperatures.]
* 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 Plating==
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 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>.
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|EBB recipeAgar 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===
===Additional Articles on MTF Wiki===
* [[Laboratory Techniques]]
* [[Microscope]]
* [[Spontaneous Fermentation]]
* [[Alternative Bacteria Sources]]
* [http://brouwerij-chugach.com/?p=1171 "Yeast Wrangling Adventure", Brian Hall of Brouwerij Chugach blog shares 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].
* [https://surradelupulo.com.br/projeto-manipueira-resultados-cientificos/ "Manipueira, wild beers and scientific results" by Nana Ottoni's podcast; interviewing Brazilian scientists Estela Virgílio and Ana Carolina about microbes identified during spontaneous fermentation in Brazilian beer (English translation available).]
====Books====