13,691
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→Propagating Yeast
==Equipment==
===General (links to other pages)===
* [[Nonconventional Yeasts and Bacteria]]
* [[Quality Assurance]]
* [[Microscope]]
* [[Microscope#Using_a_Microscope|How To Use a Microscope]]
* [[PH_Meter|pH Meter]]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/2365023236859210/ MTF thread on advice for what kind of pipette to get for cell counting.]
* [http://www.terifahrendorf.com/Lab-Manual.pdf Teri Fahrendorf’s "Small Brewery Lab Procedures Manual"]
* [https://coloradobeer.org/tech-safety-post/so-you-want-to-add-a-brewing-lab/ Colorado Brewers Guild "So You Want to Add a Brewing Lab?"]
===Bunsen Burner===
https://www.facebook.com/groups/MilkTheFunk/permalink/2069735766387960/
====Diastatic strains of ''S. cerevisiae''==== * [https://www.facebook.com/groups/MilkTheFunk/permalink/1888017211226484/?comment_id=1907916755903196&comment_tracking=%7B%22tn%22%3A%22R9%22%7D MTF thread on identifying diastatic strains of ''Saccharomyces cerevisiae'' var. ''diastaticus'' using "Instagene".] Richard Preiss warns of [https://www.facebook.com/groups/MilkTheFunk/permalink/2149139905114212/?comment_id=2150233945004808&reply_comment_id=2150769964951206&comment_tracking=%7B%22tn%22%3A%22R1%22%7D false positives] when using STA1 PCR.** See also: [[Saccharomyces#Saccharomyces_cerevisiae_var._diastaticusDiastatic_strains_of_Saccharomyces_cerevisiae|diastatic strains of ''S. cerevisiae'' var. ''diastaticus'']].
===Titratable Acidity Meter===
==Growth Media==
===General Notes===
* MRS media and some other media types are not food grade, and should only be used for initial propagation from frozen stocks (and afterwards decanted or pelleted) or for making agar for isolation. MRS media should not be used for large propagation such as starters or yeast propagators at breweries <ref>Nick Impellitteri. Private correspondence with Dan Pixley. 03/23/2020.</ref>.
* Richard Preiss from [[Escarpment Laboratories]] reported better agar results when limiting autoclave times to 15 minutes for recipes that include copper-containing ingredients <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2874530432575152/?comment_id=2874872599207602&reply_comment_id=2874886849206177&comment_tracking=%7B%22tn%22%3A%22R%22%7D Richard Preiss. Milk The Funk Facebook group thread about making agar. 08/27/2019.]</ref>.
* Cycloheximide will inhibit ''Saccharomyces'' and ''Brettanomyces'' at 100µg/ml <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/4744159795612197/?comment_id=4744240825604094 Christophe Pichon. Milk The Funk Facebook post regarding acetic acid bacteria isolation. 06/30/2021.]</ref>.
See also:
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1697319680296239/ MTF tips on autoclaving wort extract based media in such a way that prevents precipitation and darkening.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1320512184643659/ MTF tips on what media to use for wild yeast isolation.]
* For techniques on separating yeast strains from themselves and from bacteria, see [[Laboratory_Techniques#Yeast.2FBacteria_Isolation|Yeast/Bacteria Isolation]].
* To detect ''[[Brettanomyces]]'' in a ''[[Saccharomyces]]'' culture, WLN (or WLN along with DBDM) or even something as cheap as YPD can be used because ''Saccharomyces'' will show significant colony growth at 3 days, while ''Brettanomyces'' colonies will grow at 5-10 days <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1957870547574483/ Richard Preiss, Justin Amaral, and Lance Shaner. Milk The Funk Facebook thread on detecting Brettanomyces in a Saccharomyces culture. 01/18/2018.]</ref>.
Reference on DBDM: https://www.facebook.com/groups/MilkTheFunk/permalink/1805210829507123/?comment_id=1805397166155156&comment_tracking=%7B%22tn%22%3A%22R%22%7D
===''Lactobacillus/Pediococcus''===
Catalase enzyme can be spread onto MRS media to assist with culturing so-called "viable but non-culturable" (VBNC) bacteria cells; see [[Quality_Assurance#Viable_But_Nonculturable|the Quality Assurance page for details]].
See also: [https://www.facebook.com/groups/MilkTheFunk/posts/5310978085597029 Zach Taggart offers QC tips on testing ''Lactobacillus'' growth using modified MRS agar plating and recording morphology].
'''NBB Media'''
|-
|}
'''[[The Yeast Bay]] Media'''
Nick Impellitteri from [[The Yeast Bay]] shared his formulation for a 1 liter formulation <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3360639773964213/?comment_id=3360874993940691 Nick Impellitteri. Milk The Funk Facebook group post on his Lactobacillus growth media formulation. 03/20/2020.]</ref>.
{| class="wikitable"
|-
! Chemical !! Usage Amount
|-
| Dextrose || 25 grams
|-
| Fermaid O nutrient || 10 grams
|-
| CaCO3 || 2.5 grams
|-
| Apple Juice (no preservatives) || 100 milliliters
|-
| Tomato Juice || 20 milliliters
|-
| Tween 80 || 1 milliliter
|-
| Distilled Water || 1 liter
|-
| Final pH || 6.2-6.3
|-
|}
'''HLP Media'''
Developed by the ASBC and BA. Hsu’s Lactobacillus and Pediococcus (HLP) media can be employed to screen for common beer spoilers, while selecting against other organisms, such as yeast, gram negative bacteria and aerobic microbes. This video outlines the ASBC Methods of Analysis technique for utilizing HLP media in a brewery. The benefit of this media is that it doesn't need to be autoclaved or incubated anaerobically, so it is a good option for small breweries with limited lab equipment.
<youtube>BNzOzDC1oWo</youtube>
See also: [https://www.brewersassociation.org/educational-publications/asbc-methods-of-analysis-training-videos/ ASBC Methods of Analysis Training Videos] and [https://www.masterbrewerspodcast.com/045 MBAA Podcast episode with Eric Jorgenson].
====Misc====
See also [[Lactobacillus#Samuel_Aeschlimann.27s_Starter_Procedures|''Lactobacillus'' starters]] for food safe, brewery-friendly formulations.
===''Saccharomyces''===
A wide variety of media can be used for ''Saccharomyces''. Bromocresol Green can also be added to these media, as in the commercial WLN formulation. Most ''Saccharomyces'' cannot metabolize this dye, causing the colonies to stain green. Chloramphenicol can also be added to eliminate bacterial growth<ref name="Cubillos_2019">[https://onlinelibrary.wiley.com/doi/10.1002/yea.3380 Bioprospecting for brewers: Exploiting natural diversity for naturally diverse beers. F.A. Cubillos, B. Gibson, N. Grijalva‐Vallejos, K. Krogerus, J. Nikulin. 2019. DOI: https://doi.org/10.1002/yea.3380.]</ref>. Although not all of these media are specifically for your average brewers ''Saccharomyces'', most strains should have no issues growing.
'''YPD Media'''
|}
'''Wild Yeast Medium from [https://academic.oup.com/femsyr/article/15/3/fov002/544271?login=true Sylvester et al. 2015]'''
{| class="wikitable"
|-
| Ammonium sulfate || 5 grams
|-
| Synthetic Complete Dropout mix (US Biological) || 1 gram
|-
| Yeast Nitrogen Base without amino acids, Carbohydrate & w/o AS (US Biological) || 1.72 grams
|-
| Ampicillin || 0.1 grams
|-
| Chloramphenicol || 0.03 grams
|-
| Glucose || 8% or 0.8%
|-
|}
'''LCYM /LCSM Media'''(for wild yeast and diastatic strains of [[Saccharomyces#Diastatic_strains_of_Saccharomyces_cerevisiae|''S. cerevisiae'']]) <ref>[http://himedialabs.com/TD/M2027.pdf Himedia LCSM technical Data Sheet. Retrieved 01/04/2019.]</ref>
{| class="wikitable"
|-
| Yeast Extract || 4 grams
|-
| Agar || 17 20 grams|-| Distilled/De-ionized Water || Fill to 1000 mL|} '''Omega-optimized LCYM Media''' (Autoclaved for no longer than 15 minutes to preserve copper-containing ingredients. This media is specifically for detecting [[Saccharomyces#Diastatic_strains_of_Saccharomyces_cerevisiae|diastatic strains of ''S. cerevisiae'']]; reported more reliable detection than regular LCSM and Weber diastaticus media; see [https://www.facebook.com/groups/MilkTheFunk/permalink/2874530432575152/ this MTF thread] by Laura Burns from [[Omega Yeast Labs]] and the [https://omegayeast.com/news/improved-functional-assays-and-risk-assessment-for-sta-strains-of-saccharomyces-cerevisiae associated write up]) {| class="wikitable"|-! Chemical !! Usage Amount |-| Ammonium sulfate || 0.5 grams|-| Dipotassium ortho phosphate || 1 gram|-| Cupric sulphate (anhydrous) || 0.6 grams|-| Dextrose || 10 grams|-| DME || 2 grams|-| Peptone || 2 grams|-| Yeast Extract || 4 grams|-| Agar || 20 grams|-| Distilled/De-ionized Water || Fill to 1000 mL|} '''Escarpment CSSM''' (A modified version of the Omega LCYM Media. This media is specifically for detecting [[Saccharomyces#Diastatic_strains_of_Saccharomyces_cerevisiae|diastatic strains of ''S. cerevisiae'']]; Also reported to culture beer strains of ''Brettanomyces'', ''Pichia'', and other wild yeasts<ref>[https://www.facebook.com/groups/MilkTheFunk/posts/5844925622202270/?comment_id=5852798014748364 Shawn Savuto. Milk The Funk Facebook group post on Escarpment Labs CSSM. 05/05/2022.]</ref> - see [https://cdn.shopify.com/s/files/1/0323/8825/1780/files/TQ-58-2-0611-01_1.pdf?v=1631327125 this MBAA paper].) {| class="wikitable"|-! Chemical !! Usage Amount |-| Ammonium sulfate || 0.5 grams|-| potassium phosphate dibasic || 0.5 gram|-| Cupric sulphate (anhydrous) || 0.6 grams|-| Maltose || 0.5 grams|-| potato starch (Alfa Aesar no.213400) || 20 grams|-| Peptone || 2 grams|-| Liquid malt extract (Briess Ultralight) || 2 grams|-| Yeast extract || 4 grams|-| Agar || 20 grams
|-
| Distilled/De-ionized Water || Fill to 1000 mL
===''Brettanomyces''===
A few different media can be used to isolate ''[[Brettanomyces]]'' but DBDM medium is commonly used. WLD with additions of cycloheximide can also be used. A simple Malt agar with cycloheximide has been shown to grow ''Brettanomyces'' that has been adapted to the brewing environment more efficiently than Dekkera medium (recommended by the European Brewing Convention, but not listed here) and [https://foodsafety.neogen.com/pdf/acumedia_pi/7574_pi.pdf universal beer medium] (recommended by the Brewery Convention of Japan; also not listed here) <ref>[https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-2008-0917-01 Effects of Beer Adaptation on Culturability of Beer-Spoilage Dekkera/Brettanomyces Yeasts. Koji Suzuki, Shizuka Asano, Kazumaru Iijima, Tomoo Ogata, Yasushi Kitagawa & Tsunehiro Ikeda. 2018.]</ref>. DBDM may not allow all strains of ''Brettanomyces'' to grow <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2538786349482897/?comment_id=2538810702813795&reply_comment_id=2538942496133949&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Richard Preiss. Milk The Funk Facebook group thread on DBDM medium. 03/01/2019.]</ref>. The [https://cdn.shopify.com/s/files/1/0323/8825/1780/files/TQ-58-2-0611-01_1.pdf?v=1631327125 Escarpment CSSM media] for diastatic ''S. cerevisiae'' is reported to also be a good growth medium for beer strains of ''Brettanomyces'' (see [[Laboratory_Techniques#Saccharomyces|''Saccharomyces'']] above). 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) <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>. Due to the slow growth of ''Brettanomyces'' on traditional growth media, a new technique called ''indirect impedance'', which utilizes electrical currents to measure the impedance of the electricity caused by microorganisms, has been proposed as a way to detect ''Brettanomyces'' in wine <ref>[https://www.mdpi.com/2673-8007/1/2/24 van Wyk, S.; Silva, F.V.M. Enumeration of Brettanomyces in Wine Using Impedance. Appl. Microbiol. 2021, 1, 352-360. https://doi.org/10.3390/applmicrobiol1020024.]</ref>. See also: [[Wild_Yeast_Isolation#Wild_Brettanomyces|Wild Isolation of ''Brettanomyces'']]. '''YPD''' See [https://suigenerisbrewing.com/index.php/2022/11/02/this-is-the-wild-brettanomyces-your-are-looking-for/ this article] by Dr. Bryan Heit on using a modified YPD media that is cheaper and easier to prepare than WLN, along with techniques to help improve the chances of culturing wild ''Brettanomyces'' while mostly eliminating other yeasts and mold. {| class="wikitable"|-! Chemical !! Usage Amount |-| Yeast Extract || 10 grams|-| Peptone || 20 grams|-| Dextrose || 20 grams |-| Agar(optional) || 15 grams|-| Distilled Water || Fill to 1000 ML|} '''EBB Media Recipe'''{| class="wikitable"|-! Chemical !! Usage Amount |-| Red grape juice (possibly replaceable with DME?) || 200 mL/L|-| Ethanol || 40 mL/L|-| Malt extract || 1.5 g/L|-| Yeast extract || 1.5 g/L|-| (NH4)2 SO4 (sourced from Sigma-Aldrich) || 0.5 g/L|-| MgSO4 || 0.2 g/L|-| Tween 80 || 0.5 mL/L|-| Biphenyl (limits mold) || 0.2 g/L (w/v)|-| Chloramphenicol (limits bacteria) || 0.05 g/L (w/v)|-| Adjust pH to 5.0 with sodium hydroxide || |-|} Notes: gentle agitation during growth (120 rpm).
'''DBDM Media Recipe'''
|}
==Storage==
<youtube height="200" width="300">pdHdfCBTzj4</youtube>
Growing cultures from slants or plates:
<youtube height="200" width="300">76xnhjkt5bA</youtube>
===Liquid Slurry===
* [https://www.masterbrewerspodcast.com/189 MBAA Podcast episode 189 "Long Term Yeast Storage" with Matt Couch; storing yeast slurries with monopotassium phosphate and sunflower oil.]
==Propagators==
<youtube height="200" width="300">g0GE3oTMZrY</youtube>
<youtube height="200" width="300">mCOtWztCObY</youtube>
<youtube height="200" width="300">RUOePCX0DKY</youtube>
<youtube height="200" width="300">Kh_McZnTHg4</youtube>
===Making Agar Plates===
<youtube height="200" width="300">iqLuTNUZa2E</youtube>
* Making a starter from slants or plates:
<youtube height="200" width="300">76xnhjkt5bA</youtube>
===Yeast/Bacteria Isolation===
* [https://www.escarpmentlabs.com/single-post/2019/03/18/agar-plating-guide Escarpment Labs agar plating guide.]
* [https://www.mbaa.com/districts/EasternCanada/events/Documents/2012-11-21Goineau.pdf Siebel presentation on detecting brewery contamination with agar plating.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1341845122510365/ Mark Trent details his method for isolating ''Brettanomyces'' from ''Saccharomyces'' from dregs or other mixed cultures.]
* [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.facebook.com/groups/MilkTheFunk/permalink/3754592574568929/ Cory Widmayer's guide to yeast isolation and media growth techniques, with an example of ''S. pombe'' and tips for ''Brettanomyces'', ''Z. bailii'', and others.]
* See [[Wild_Yeast_Isolation#Isolating|Wild Yeast Isolation]].
* See [[Lactobacillus#Selecting_for_on_Agar|isolating ''Lactobacillus'']].
===Cell Counting===
* Cell counting:: <youtube height="200" width="300">EcYhAegVewU</youtube>* Cell Density Equation:: <youtube height="200" width="300">One0wc95kk8</youtube>* Semi-Automated Cell Counting:: <youtube height="200" width="300">f6TwzVAKMZQ</youtube>
Various other tips:
* [https://www.escarpmentlabs.com/single-post/2018/10/30/Crop-pray-count-yeast-counting-guide Escarpment Labs guide to yeast counting.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1139453072749572/ MTF thread with tips on using "cheap Chinese" haemocytometers hemocytometers for cell counting.]* [http://masterbrewerspodcast.com/113-reducing-error-in-cell-counts MBAA podcast episode 113 with Bill Maca on how to properly prepare a yeast sample for counting.]
===Gram Staining===
===Propagating Yeast===
* Propagation techniques:: <youtube height="200" width="300">YMDw6xj1uvo</youtube>* [https://www.masterbrewerspodcast.com/239 "Yeast Propagation at Urban Chestnut" interview with Florian Kuplent of Urban Chestnut on MBAA Podcast episode #239.]* [https://www.themadfermentationist.com/2022/11/small-craft-brewery-yeast-management.html "Small Craft Brewery Yeast Management," by Michael Tonsmeire.]
Various other tips:
* When propagating yeast in an Erlenmeyer flask, use a flask that allows for plenty of headspace. This allows for a thinner layer of foam and thus more oxygen diffusion. Set the stir plate or shaker to the highest speed that doesn't produce a lot of foam. A good general rule of thumb is to use a flask that allows for 20-33% starter volume and 66-80% headspace (ideally 20% starter volume and 80% headspace). For example, for a 1L starter, use a 3L, 4L, or 5L flask (ideally 5L). More volume can be used with a Fernbach or a baffled flask, but these require a shaker table. When propagating yeast for use in beer, use wort and a magnesium and zinc nutrient for best yeast propagation practices. Cover the flask with foil (or [https://www.facebook.com/groups/MilkTheFunk/permalink/1034954619866085/ foam stoppers] if fruit flies are a problem) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1986378521390352/ Various MTF members. Milk The Funk Facebook thread started by Jon Stanley on Erlenmeyer flask usage for yeast starters. 02/12/2018.]</ref>.
* [https://suigenerisbrewing.com/index.php/2024/06/14/diy-culture-rotator/ Dr. Bryan Heit's DIY Culture Rotator.]
===Semi-Anaerobic Containers for Incubating Plates===
===Identification===
* [http://suigenerisbrewing.com/index.php/2019/10/30/a-primer-on-yeast-morphology "Basic Yeast Morphology" by Dr. Bryan Heit of Sui Generis Blog.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/2284645151563686/?comment_id=2285158518179016&comment_tracking=%7B%22tn%22%3A%22R%22%7D Richard Preiss explains the differences between ITS sequencing, PCR fingerprinting, Low coverage whole genome short read sequencing, Higher coverage whole genome short read sequencing, and Long read sequencing (PacBio), as well as why some methods have a high cost.]
* [https://www.facebook.com/groups/592560317438853?view=permalink&id=2820993514595511 MTF thread on methods for identifying strain level differences.]
* [[Wild_Yeast_Isolation#Identifying|Identifying Wild Yeast]].
<youtube height="200" width="300">9LXEAZbei_8</youtube>
===Hybridization and Modification===
* [https://www.facebook.com/groups/592560317438853/?multi_permalinks=5776258545735645 Kristoffer Krogerus explains his published technique of "return-to-growth" to improve brewing yeast genetics.]
==Shipping Cultures==
==Laboratory Information Management Systems==
* [http://brewerypi.com/ Brewery Pi] (open source, but still in development; sponsored by Deschutes).
==Chemistry==
* [https://www.facebook.com/groups/MilkTheFunk/posts/5384596774901826/ Ehren Schmidt shares examples of using paper chromatography to test for malic acid.]
==Historical==
* [http://www.garshol.priv.no/blog/376.html "Pasteur and the beer of national revenge", by Lars Garshol (how Louis Pasteur proved that microbes were alive in 1857, and other discoveries).]
* [http://www.garshol.priv.no/blog/377.html "Emil Chr. Hansen and the yeast revolution", by Lars Garshol (how yeast was first isolated from other strains in 1883).]
* [https://www.mdpi.com/2311-5637/6/1/9?fbclid=IwAR1Emz2IsfcPKGwGv2rccb6VMX190fUNEK7mZd-HxpT53_s0QqRdecPHs8o Characterization of Old Wine Yeasts Kept for Decades under a Zero-Emission Maintenance Regime.]
==See Also==
===External Resources===
* [https://www.youtube.com/watch?v=5SWOFBhdvrY ASBC Webinar: Green Chemistry & Beer Science by Dana Garves of Oregon BrewLab.] See also [https://www.masterbrewerspodcast.com/307 MBAA Podcast 307].
* [https://www.youtube.com/watch?v=FL5uE4y6Eog Microwaving Micro Media by Oregon BrewLab.] See also [https://www.facebook.com/groups/MilkTheFunk/posts/8489399607754845/ comments on this method in MTF].
* [https://journals.asm.org/doi/pdf/10.1128/jmbe.00336-21 Bootleg Biology: a Semester-Long CURE Using Wild Yeast to Brew Beer.]
* [https://www.brewerspublications.com/products/yeast-the-practical-guide-to-beer-fermentation "Yeast: The Practical Guide to Beer Fermentation," by: Chris White and Jamil Zainasheff.]
* [http://traffic.libsyn.com/basicbrewing/bbr07-27-17homelab01.mp3 BasicBrewing interview with Zack Taggart, lab manager at 42 North Brewing, on how to set up our own homebrew lab to analyze yeast health and count cell population (part 1)] and [http://traffic.libsyn.com/basicbrewing/bbr08-17-17homelab02.mp3 isolating, propagating, and storing yeast and bacteria (part 2)].
* [http://www.brewingscience.com/PDF/BSI_brewers_lab_handbook.pdf "Brewers' Laboratory Handbook: Brewing Without the Blindfold™", by Brewing Science Institute.]