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Wild Yeast Isolation

2,682 bytes added, 22:47, 21 March 2017
added a section for wild Brettanomyces isolation
'''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 yeast, and isolating it with agar plates as a pure culture. This article will also contain information on growing up a ''captured'' culture in wort rather than isolating yeast cultures on agar plates.
:''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 Bryan Heit'sSui 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 Bryan Heit'sSui Generis Blog] guide to more types of agar plates, and video tutorial.
===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 Bryan Heit'sSui 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 erlynmeyer 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 lacto 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>
===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 Bryan Heit'sSui Generis Blog] video guide on how to streak agar plates.* [http://suigenerisbrewing.blogspot.ca/2014/12/two-new-videos.html Bryan Heit'sSui 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.]
===Identifying===
* [http://suigenerisbrewing.blogspot.ca/2014/12/new-video.html Bryan Heit'sSui Generis Blog] video guide on how to visually identify usable yeasts versus potential pathogens and molds on agar plates.
===Growing and Testing===
* [http://suigenerisbrewing.blogspot.ca/2012/11/yeast-banking-iii-stepping-up-to.html Bryan Heit'sSui Generis Blog] guide to stepping up yeast to pitchable amounts.
* [http://braukaiser.com/wiki/index.php?title=Growing_Yeast_from_a_Plate Kai Troester's] guide to growing yeast from an agar plate.
 
===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)].
==Growing and Testing Without Plating==
===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/ Bryan 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>.* 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 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/ Drgroups/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 Heit form of Sui Generis Blogrecommends 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].* [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].
* The addition of hops in the starter wort will inhibit or kill some gram positive bacteria pathogens (==Wild ''L. monocytogenes'' and ''S. aureusBrettanomyces'') <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 Heit on MTF regarding when it is safe to taste wild beer. 7/6/2015.]</ref>.* 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, by Bryan Heit.]</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 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/11047575528857911620419974652877/?comment_id=1104851526209727&offset=0&total_comments=8&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Conversation with Bryan Heit 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 discussion on a low pH alone is not adequate. Bryan Heit recommends a pH of 4.5 or less, the use rare but possible isolation 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 non-zero. Generally, wild ''C. botulismBrettanomyces'' 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 Heit on MTF <ref name="BryanMTF"></ref>. For more information on the potential for pathogens surviving in beer, see [http://suigenerisbrewing.blogspot.com/2014/02/fact-or-fiction-can-pathogens-survive.html Bryan Heit's Blog article.].* In the words of Bryan Heit 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 MD 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==
* [[Alternative Bacteria Sources]]
* [[Bootleg Biology]]
* [[Brettanomyces]]
* [[Saccharomyces]]
* [[Lactobacillus]]
===External Resources===
====Links====
* [https://www.facebook.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 by Bryan HeitBlog.]* [https://bkyeast.wordpress.com/2012/12/17/how-to-build-a-yeast-ranch/ BKYeast, ''"How to Build a Yeast Ranch''".]* [http://www.themadfermentationist.com/2011/04/ambient-spontaneous-yeast-starters.html ''"Ambient Yeast Starters'' " by 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 Bryan Heit of 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 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.]
====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>-tmbnPXZcFQ</youtube>
<youtube>y43x7nLTEPo</youtube>
<youtube>SwDjJKkuT74</youtube>
<youtube>viXPMtkTfk8</youtube>
==References==
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