Changes

See [[Lactobacillus#Selecting_for_on_Agar|Rogosa SL Agar]].

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.

'''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>

| 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

A few different medias media can be used to isolate ''[[Brettanomyces]]'' but DBDM media 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).

| Distilled Water || Fill to 1000 MLmL

| Distilled Water || Fill to 1000 MLmL|} '''Malt Agar Media Recipe''' [http://himedialabs.com/TD/M1964.pdf Click here] for directions on how to make this medium. The below recipe is using the [https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-2008-0917-01 Suzuki (2008)] recipe instead of the recipe in the linked directions. {| class="wikitable"|-| Malt extract || 30 grams|-| Cycloheximide || 0.01 grams|-| Agar || 15 grams|-| Distilled Water || Fill to 1000 mL|-

===Misc/Other==='''Yeast Growth Media''' * This is a simple non-synthetic yeast growth media shared by Cory Widmayer on Milk The Funk, and is based off of [https://wwwacademic.facebookoup.com/groupsfemsyr/MilkTheFunkarticle/permalink13/16973196802962391/ MTF tips on autoclaving wort extract based media in such a way that prevents precipitation and darkening.]* For techniques on separating yeast strains from themselves and from bacteria, see [[Laboratory_Techniques#Yeast.2FBacteria_Isolation|Yeast34/Bacteria Isolation]]544881 Leite et al.* To detect ''[[Brettanomyces]]'' in a ''[[Saccharomyces]]'' culture, WLN (or WLN along with DBDM2013) 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>]. See [https://www.facebook.com/groups/MilkTheFunk/permalink/19578705475744833328794017148789/ Richard PreissWidmayer's post on MTF] for directions on using this media (requires aeration; the batch feeding process may not be necessary, Justin Amaral, and Lance Shanersee comments by Dr. Bryan Heit). Milk The Funk Facebook thread on detecting This media and method are claimed to grow ''Brettanomyces in a Saccharomyces culture'' very quickly. 01/18/2018.]</ref>Widmayer also provides a synthetic version of the recipe on the MTF post. {| class="wikitable"|-| Glucose or another sugar type || 100 grams|-| Yeast Extract || 90 grams|-| Distilled Water || Fill to 1000 mL|-|}

Reference on DBDM: ====Misc====* [https://www.facebooksciencedirect.com/groupsscience/MilkTheFunkarticle/permalinkabs/1805210829507123pii/?comment_id=1805397166155156&comment_tracking=%7B%22tn%22%3A%22R%22%7DS0168160519303460 A simple procedure for detecting ''Brettanomyces bruxellensis'' in wine environment by RNA-FISH using a novel probe.]

* 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" 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.]

* 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.]

* 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. For When propagating yeast propagation for use in beer, use wort and a magnesium and zinc nutrient, and set the stir speed to the highest setting for best yeast propagation practices. Cover the flask with foil (or [https://www.facebook.com/groups/MilkTheFunk/permalink/1034954619866085/ foamstoppers] 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>.

[[File:Shipping cultures.jpg|thumbnail200px|right|[https://www.facebook.com/kveikworldorder/posts/1307377055994331 Example of DeWayne Schaaf's packaging for shipping cultures.]]]

# Place each liquid filled tube into its own snack sized baggie to minimize cross -contamination if they do happen to leak.

# Each of these smaller bags will be placed into in a quart Ziplock.

# My main shipping container are is Uline Poly bubble mailers. I don't tend to use ice packs as I've found them unnecessary when shipping in favorable temps (use ice packs during the summer).