Changes

The morphology of ''Brettanomyces'' can vary immensely from strain to strain (and species to species). Some strains can look similar in size and shape to ''S. cerevisiae'' under a microscopic image, while others are elongated or much smaller. This makes it difficult to identify ''Brettanomyces'' without DNA analysis (see [[Laboratory_Techniques#PCR.2FqPCR|PCR)]]. Morphologies of ''Brettanomyces'' grown on agar plates can also be different from strain to strain. For example, Devin Henry found that a sample of WLP648 that contained two closely related strains of ''B. bruxellensis'' grew completely differently on the same growth media. At first, larger, slightly off-white colonies grew on the plates (this was the first strain), and then a few days later the second strain grew as many smaller white-colored colonies. Other strains may appear as glossy or matted with jagged edges, etc. Morphology on agar plates can change depending on the type of growth media <ref>[http://web.archive.org/web/20240623073158/http://brettanomycesproject.com/dissertation/analysis-of-culturability-on-various-media-agar/morphological-traits/ Yakobson, Chad. "Morphological Trains". Masters Dissertation. 2011. Retrieved 05/12/2017.]</ref><ref name="bryan_vrai" /><ref>[https://eurekabrewing.wordpress.com/2012/03/27/brettanomyces-bruxellensis-microscopy-pictures/ Samuel Aeschlimann. "Brettanomyces bruxellensis microscopy pictures". Eureka Brewing blog. 03/12/2012. Retrieved 05/12/2017.]</ref>. While genetic (PCR) identification is required for any kind of confident identification of ''Brettanomyces'', specialized selective media can also help identify ''Brettanomyces''; see [[Laboratory_Techniques#Brettanomyces|Selective Media]].

* [http://web.archive.org/web/20240519145413/http://brettanomycesproject.com/2010/06/brettanomyces-yeast-cell-images/ ''Brettanomyces'' morphology examples from Remi Bonnart.]

''Brettanomyces'' has been thought to occur naturally on the skins of fruit such as apples and grapes. However, there are only a handful of reports of ''Brettanomyces'' being identified on the skins of fruit, and in some cases where ''Brettanomyces'' has been found, its abundance is extremely minimal <ref name="Aires_2025">[https://www.mdpi.com/2076-2607/13/3/538 Aires, C.; Maioto, R.; Inês, A.; Dias, A.A.; Rodrigues, P.; Egas, C.; Sampaio, A. Microbiome and Microbiota Within Wineries: A Review. Microorganisms 2025, 13, 538. https://doi.org/10.3390/microorganisms13030538.]</ref><ref>[https://onlinelibrary.wiley.com/doi/full/10.1002/jib.154 Lentz, M., Putzke, T., Hessler, R. and Luman, E. (2014), Genetic and physiological characterization of yeast isolated from ripe fruit and analysis of fermentation and brewing potential, J. Inst. Brew., 120: 559– 564. DOI: 10.1002/jib.154.]</ref><ref name="Comitini">[https://www.frontiersin.org/articles/10.3389/fmicb.2019.00415/abstract Occurrence of Brettanomyces bruxellensis on grape berries and in related winemaking cellar. Francesca Comitini, Lucia Oro, Laura Canonico, Valentina Marinelli, Maurizio Ciani. 2019. DOI: 10.3389/fmicb.2019.00415.]</ref><ref name="Renouf_2007">[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>. In contrast, there are also studies that indicate ''Brettanomyces'' only being found during or after food processing, which indicates that the processing equipment may be the primary source for the ''Brettanomyces''. In addition, ''Brettanomyces'' has been isolated in abundance from the surfaces of equipment/processed materials in wineries and breweries <ref name="Aires_2025" /><ref name="smith_divol_2016" /><ref name="Schifferdecker" /><ref name="Loureiro_2003">[https://www.ncbi.nlm.nih.gov/pubmed/12892920 Spoilage yeasts in the wine industry. Loureiro V, Malfeito-Ferreira M. 2003.]</ref><ref name="Steensels" /><ref name="Barata_2008">[https://www.ncbi.nlm.nih.gov/pubmed/18077036 Survival patterns of Dekkera bruxellensis in wines and inhibitory effect of sulphur dioxide. f Barata A, Caldeira J, Botelheiro R, Pagliara D, Malfeito-Ferreira M, Loureiro V. 2008.]</ref> (Table 1). For example, an ongoing survey of wild yeasts in different regions of United States wilderness areas 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 and Alaska; substrates sampled included leaves, soil, bark, moss, mushrooms, needles, pine cones, twigs/wood, and other plant matter) <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>. It is therefore unclear that ''Brettanomyces'' found on grape skins originated there or from the industrial processing where it is more abundant. It is also thought to disperse via fruit-flies (called "vectors" in the scientific literature), similar to how ''Saccharomyces'' travels, although direct evidence for this has been reported rarely and only on fruit-flies in wineries that are likely to come into contact with equipment/processed material that is already contaminated with ''Brettanomyces'' <ref>[https://youtu.be/G2nhUM5PIrg?t=309 Dr. Bryan Heit. BotB - Where (Do) The Wild Brettanomyces Roam?. ~5 mins in. Retrieved 07/10/2022.]</ref><ref name="Renouf_2007" /><ref name="Steensels">[http://www.sciencedirect.com/science/article/pii/S0168160515001865 Brettanomyces yeasts — From spoilage organisms to valuable contributors to industrial fermentations. Jan Steensels, Luk Daenen, Philippe Malcorps, Guy Derdelinckx, Hubert Verachtert, Kevin J. Verstrepen. International Journal of Food Microbiology Volume 206, 3 August 2015, Pages 24–38.]</ref><ref name="Barata_2008" /><ref name="Loureiro_2003" />. ''Brettanomyces'' is known to be difficult to grow in a lab due to slow growth, specific nutrient requirements, or perhaps because of a "VBNC" state (see [[Wild_Yeast_Isolation#Wild_Brettanomyces|Wild ''Brettanomyces'']] for more information), which may account for the lack of evidence for fruit being the primary natural habitat for ''Brettanomyces''. More recently, techniques have been invented to more easily isolate and grow ''Brettanomyces'' <ref name="Renouf_2007" /><ref name="Comitini" />. There is also significant evidence that the natural habitat of ''Brettanomyces'' might actually be the root systems of certain plants, known as the [https://www.nature.com/scitable/knowledge/library/the-rhizosphere-roots-soil-and-67500617/ "rhizosphere"]. The rhizosphere refers to the complex symbiotic community of microbe populations that live on and around the root system of plants. Wild strains of ''Brettanomyces'' have been found in the root systems of dill, common beans, sunflowers, maize, corn, jute, cassava, and grey mangroves found in the estuaries of Indonesia <ref>[https://onlinelibrary.wiley.com/doi/abs/10.1111/aab.12309 Weisany, W., Raei, Y., Salmasi, S., Sohrabi, Y. and Ghassemi-Golezani, K. (2016), Arbuscular mycorrhizal fungi induced changes in rhizosphere, essential oil and mineral nutrients uptake in dill/common bean intercropping system. Ann Appl Biol, 169: 384-397. https://doi.org/10.1111/aab.12309.]</ref><ref>[https://archive.aessweb.com/index.php/5003/article/view/3333 I.O, S. ., & G.P, O. . (2012). Diversity of Fungal Populations in Soils Cultivated With Cassava Cultivar TMS 98/0505. Journal of Asian Scientific Research, 2(3), 116–123. Retrieved from https://archive.aessweb.com/index.php/5003/article/view/3333.]</ref><ref>[https://www.ajol.info/index.php/swj/article/view/149513 Rhizosphere and non-rhizosphere soil mycoflora of Corchorus olitorius (Jute). G.S. Olahan, I.O. Sule, T Garuba, Y.A. Salawu. Science World Journal. 2016.]</ref><ref>[https://ojs.unud.ac.id/index.php/jbb/article/view/36023 NOERFITRYANI, Noerfitryani; HAMZAH, Hamzah. THE EXISTENCE OF ENTOMOPATHOGENIC FUNGI ON RICE PLANTS RHIZOSPHERE. International Journal of Biosciences and Biotechnology, p. 12-24, dec. 2017. ISSN 2655-9994. doi: https://doi.org/10.24843/IJBB.2017.v05.i01.p02.]</ref><ref>[https://www.sciencedirect.com/science/article/abs/pii/S2452219818300259 Marcela Sarabia, Saila Cazares, Antonio González-Rodríguez, Francisco Mora, Yazmín Carreón-Abud, John Larsen, Plant growth promotion traits of rhizosphere yeasts and their response to soil characteristics and crop cycle in maize agroecosystems, Rhizosphere, Volume 6, 2018, Pages 67-73, ISSN 2452-2198, https://doi.org/10.1016/j.rhisph.2018.04.002.]</ref><ref>[https://www.sciencedirect.com/science/article/abs/pii/S1049964419303238 Nivien A. Nafady, Mohamed Hashem, Elhagag A. Hassan, Hoda A.M. Ahmed, Saad A. Alamri. The combined effect of arbuscular mycorrhizae and plant-growth-promoting yeast improves sunflower defense against Macrophomina phaseolina diseases. Biological Control. Volume 138, 2019, 104049. ISSN 1049-9644, https://doi.org/10.1016/j.biocontrol.2019.104049.]</ref><ref>[http://ejurnal.its.ac.id/index.php/sains_seni/article/view/5613 Isolation and Characterization of Yeast from Rhizosphere Avicennia Marina Wonorejo. Sitatun Zunaidah, Nur Hidayatul Alami. 2014. DOI: 10.12962/j23373520.v3i1.5613.]</ref>. See Dr. Bryan Heit's video [https://www.youtube.com/watch?v=G2nhUM5PIrg "Where (Do) The Wild Brettanomyces Roam?"] and [https://www.facebook.com/groups/MilkTheFunk/posts/5940213029340195 his comments in Milk The Funk], as well as [https://www.youtube.com/watch?v=BrR7G_YyfmA "Philip Poole. Plant Control of the Rhizosphere Microbiome"]. For documented isolation attempts from plant rhizospheres, see [[Wild_Yeast_Isolation#Wild_Brettanomyces|Wild Yeast Isolation]].

''Brettanomyces'' has the ability to form a [[Quality_Assurance#Biofilms|biofilm]]. Biofilm formation is a survival mechanism induced by stress whereby the cells adhere to non-living surfaces such as plastic and stainless steel <ref>[https://ives-technicalreviews.eu/article/view/4544 "Brettanomyces bruxellensis biofilms: a mode of life to withstand environmental stresses?" Sandrine Rousseaux, Manon Lebleux, Hany Abdo, Louise Basmacyian, Chloé Roullier-Gall, Hervé Alexandre, Stéphanie Weidmann. 2020. DOI: https://doi.org/10.20870/IVES-TR.2020.4544.]</ref><ref>[https://academic.oup.com/femsle/advance-article/doi/10.1093/femsle/fnae105/7917616 Scott J Britton, Thijs Dingemans, Lisa Rogers, Jane S White, Dawn L Maskell, Excitation of filamentous growth in dekkera spp. By quorum sensing aromatic alcohols 2-phenylethanol and tryptophol, FEMS Microbiology Letters, 2024;, fnae105, https://doi.org/10.1093/femsle/fnae105.]</ref>. After adhesion to the surface, the cells produce a protective layer of proteins and polysaccharides that help protect the organism from cleaning and sanitizing agents.

Montagner et al. (2024) examined the biofilms of several strains of ''B. bruxellensis'' that were found at one of three different wineries in the Bordeaux region of France. They reported that phenol production occurred within the biofilm and hypothesized that it is possible that phenol contamination in wine could be a result of the wine coming into contact with the biofilm rather than solely from ''Brettanomyces'' growing in the wine. They also observed the detachment of ''B. bruxellensis'' cells from the biofilm and into the wine <ref name="Montagner_2024" />. 

There is some evidence that ''Brettanomyces'' can be sensitive to high levels of light. [https://www.frontiersin.org/articles/10.3389/fmicb.2021.747868/full Catrileo et al. (2021)] showed that under laboratory conditions, ''Brettanomyces bruxellensis'' was not able to grow when exposed to a 2500 lux and 4000 lux light source. For reference, the lux of indirect daylight is around 10,000 - 25,000 and the lux of office lighting is usually between 350 and 500 <ref>[https://en.wikipedia.org/wiki/Lux "Lux". Wikipedia. Retrieved 02/20/2022.]</ref>. However, when p-coumaric acid, a phenolic precursor that is present in plants and fruits (including malted barley and wheat), is present, certain genes are expressed during the growth of ''B. bruxellensis'' that allow it to adapt to the high light exposure conditions. While this study does not show at what level light begins to affect ''B. bruxellensis'' (the lowest light intensity that they tested was 2500 lux), [https://journals.asm.org/doi/abs/10.1128/jb.133.2.692-698.1978 Woodward et al. (1978)] demonstrated that ''Saccharomyces cerevisiae'' growth is unaffected by light until about 1,250 lux, at which point it begins to inhibit growth and the transfer of nutrients across the cell membrane <ref>[https://www.frontiersin.org/articles/10.3389/fmicb.2021.747868/full Catrileo D, Moreira S, Ganga MA and Godoy L (2021) Effect of Light and p-Coumaric Acid on the Growth and Expression of Genes Related to Oxidative Stress in Brettanomyces bruxellensis LAMAP2480. Front. Microbiol. 12:747868. doi: 10.3389/fmicb.2021.747868.]</ref><ref>[https://journals.asm.org/doi/abs/10.1128/jb.133.2.692-698.1978 J R Woodward, V P Cirillo, L N Edmunds, Jr. Light effects in yeast: inhibition by visible light of growth and transport in Saccharomyces cerevisiae grown at low temperatures. ASM Journals. Journal of Bacteriology. Vol. 133, No. 2. 1978. https://doi.org/10.1128/jb.133.2.692-698.1978.]</ref>. Grangeteau et al (2024) demonstrated that 10 minutes of ultra-high irradiance (UHI) blue light treatment resulted in the complete death of ''B. bruxellensis'' within a biofilm <ref>[https://www.sciencedirect.com/science/article/pii/S0023643824013215 C. Grangeteau, M. Lebleux, V. David, S. Rousseaux, H. Alexandre, L. Beney, S. Dupont. Ultra-high irradiance (UHI) blue light treatment: A promising method for inactivation of the wine spoilage yeast Brettanomyces bruxellensis. LWT, 2024, 117038. ISSN 0023-6438. https://doi.org/10.1016/j.lwt.2024.117038.]</ref>. Additionally, Cvetkova et al (2025) showed that UV inactivation of ''Brettanomyces bruxellensis'' in white wine was more efficient at 280 nm than at 254 nm, but at 280 NM, white wine flavor properties such as phenols were negatively affected <ref>[https://www.sciencedirect.com/science/article/abs/pii/S0956713525001197 Svetlana Cvetkova, Elke Herrmann, Jutta Keiser, Benedikt Woll, Mario Stahl, Maren Scharfenberger-Schmeer, Elke Richling, Dominik Durner. Comparing the effect of UV treatment at wavelengths 254 nm and 280 nm: inactivation of Brettanomyces bruxellensis and impact on chemical and sensory properties of white wine. Food Control, 2025, 111250. ISSN 0956-7135. https://doi.org/10.1016/j.foodcont.2025.111250.]</ref>.

BRETTANOMYCES BRUXELLENSIS. Jackeline M. Silva, Gilberto H. Teles, Ester Ribeiro & Will B. Pita. Department of Antibiotics, Federal University of Pernambuco, Recife, Pernambuco, Brazil. Aug 2024.]</ref>. Acetic acid, glycerol, succinic acid, and ethanol are only consumed if oxygen is present <ref name="smith_divol_2016"></ref>. The addition of H+ acceptors such as acetaldehyde, acetone, pyruvic acid, and other carbonyl compounds, stimulates anaerobic fermentation. Small amounts of oxygen also stimulate fermentation <ref name="yakobson_introduction">[http://web.archive.org/web/20240415090559/http://www.brettanomycesproject.com/dissertation/introduction/ Yakobson, Chad. The Brettanomyces Project. Introduction. Retrieved 8/11/2015.]</ref>. The presence of small amounts of oxygen can allow some strains of ''Brettanomyces'' to utilize certain carbon sources. For example, several strains of ''B. bruxellensis'' can consume ethanol, glycerol, and acetic acid as food sources only when at least a low amount of oxygen is present (semi-aerobic conditions) and no other sugar is available. Acetic acid and glycerol are used as food sources by some strains only under fully aerobic conditions, but not under semi-aerobic or anaerobic conditions. It has been hypothesized that acetic acid and glycerol are only consumed by ''Brettanomyces'' when ethanol and other food sources are no longer available <ref name="Smith_2018" />.

''Brettanomyces'' is capable of synthesizing several ethyl esters from ethanol and fatty acids, as well as other types of esters from various alcohol types (methanol, for example). Among the most prolific of these are ethyl acetate (synthesized from ethanol and acetic acid), ethyl lactate (synthesized from ethanol and lactic acid), phenethyl acetate, ethyl caproate, ethyl caprylate, ethyl deconoate <ref name="Tyrawa_2017" />, along with the hydrolysis (breakdown) of isoamyl acetate. Esters have been found to attract fruit flies and other flying insects, which help many species of yeast transfer from one food source to another (namely 2-phenyl-ethanol, 3-methyl-1-butanol, ethyl acetate, 2-methyl-1-butanol, and 3-methyl-3-butenol). Some of these esters are also released by blooming flowers and it is thought that the attraction to flowers by insects is also driven by these same esters <ref>[https://onlinelibrary.wiley.com/doi/epdf/10.1002/ece3.3905 Chemical signaling and insect attraction is a conserved trait in yeasts. Paul G. Becher, Arne Hagman, Vasiliki Verschut, Amrita Chakraborty, Elżbieta Rozpędowska, Sébastien Lebreton, Marie Bengtsson, Gerhard Flick, Peter Witzgall, Jure Piškur. 2018.]</ref>. During non-mixed fermentations where lactic acid is minimal to none, insignificant amounts of ethyl lactate esters are produced, whereas ethyl caprylate and ethyl caproate have a general increase. With the addition of lactic acid, ethyl lactate levels are greatly increased although may still not reach the flavor threshold level of 250 mg/L (strain dependent), and ethyl acetate is generally slightly increased. The amounts of esters produced vary widely based on species and strain <ref>[http://web.archive.org/web/20240415090559/http://www.brettanomycesproject.com/dissertation/introduction/ Yakobson, Chad]. Pure Culture Fermentation Characteristics of Brettanomyces Yeast Species and Their Use in the Brewing Industry. Production of Secondary Metabolites. 2011.</ref>. A similar but slower evolution of esters has been seen in a long-term study on examining how Belgian lambic from Cantillon ages in bottles. The study found that lactic acid (produced by lactic acid bacteria) and ethyl lactate increased as bottles aged, while ethyl decanoate and isoamyl acetate decreased, all presumably from ''Brettanomyces'' metabolism over time <ref>[http://horscategoriebrewing.blogspot.com/2016/02/thoughts-on-spitaels-and-van.html "Thoughts on Spitaels and Van Kerrebroeck et al, 2015." Dave Janssen. Hors Catégorie Blog. 02/20/2016. Retrieved 03/15/2016.]</ref>.

| Ethyl butyrate (pineapple, mango, tropical fruit <ref>[http://www.flavoractiv.com/products/ethyl-butyrate-beer-flavour-standards/ Ethyl Butyrate Beer Flavour Standard. FlavorActIV. Retrieved 6/20/2015.]</ref>, juicy fruit gum <ref>Private corrospondance with Richard Preiss by Dan Pixley. 12/1/2016.</ref>) || [[Butyric Acid]] and ethanol || 0.4ppm (flavor) <ref>[http://www.flavoractiv.com/products/ethyl-butyrate-beer-flavour-standards/ Flavoractiv. Ethyl butyrate. Retrieved 1/18/2015.]</ref> || C₆H₁₂O₂ <ref name="pubchem_ethylbutyrate">[http://pubchem.ncbi.nlm.nih.gov/compound/ethyl_butyrate PubChem. Ethyl Butyrate. Retrieved 08/15/2015.]</ref> || Low levels of production by some species of Brettanomyces; production decreases with higher acidity <ref name="yakobson1">[http://web.archive.org/web/20240623080847/http://www.brettanomycesproject.com/dissertation/pure-culture-fermentation/pure-culture-fermentation-discussion/ Yakobson, Chad. Pure Culture Fermentation Characteristics of Brettanomyces Yeast Species and Their Use in the Brewing Industry. Pure Culture Fermentation Discussion. 2011.]</ref>. Also known as ethyl butanoate <ref name="pubchem_ethylbutyrate"></ref>.

| Ethyl lactate (fruity, creamy, rum <ref>[http://www.aroma-chemical.com/ethyl-lactate/ Best Aroma website. Ethyl Lactate. Retrieved 08/15/2015.]</ref><ref>[https://books.google.com/books?id=avYMy82EBuAC&pg=PA384&lpg=PA384&dq=ethyl+lactate+flavor&source=bl&ots=AZufxA6Htu&sig=rTbNo4rOSBY_6kuhGDtW_JqQ5oA&hl=en&sa=X&sqi=2&ved=0CD0Q6AEwBWoVChMI35jXjuirxwIVyKOICh0klgDF#v=onepage&q=ethyl%20lactate%20flavor&f=false Dictionary of Flavors. Dolf De Rovira. John Wiley & Sons, Feb 28, 2008. Pg 384.]</ref>) || [[Lactic Acid]] and ethanol || 0.2 ppm-1.66 ppm (odor) <ref>[http://hazmap.nlm.nih.gov/category-details?id=1179&table=copytblagents Haz-Map, Ethyl Lactate odor threshold.]</ref> || C₅H₁₀O₃ <ref>[http://pubchem.ncbi.nlm.nih.gov/compound/7344 PubChem. Ethyl Lactate. Retrieved 08/15/2015.]</ref> || Increases production with higher amounts of Lactic Acid <ref>[http://web.archive.org/web/20240723012858/http://www.brettanomycesproject.com/dissertation/pure-culture-fermentation/impact-of-initial-concentration-of-lactic-acid/ Yakobson, Chad. The Brettanomyces Project. Impact of the Initial Concentration of Lactic Acid on Pure Culture Fermentation. Retrieved 6/16/2015.]</ref>

| Claussenii|| ''Dekkera anomala'' || ''Brettanomyces anomalus'' ||WLP645||Fruity, pineapple. Wine grape-like aroma, with light wood-like, floral, and citrus aromas. More fruit forward in the flavor, clean aftertaste with little to no "funk" <ref name="danpixley_mtf" />. || Approx. 500 million cells per mL; homebrew vials are approx. 17.5 billion cells at 35 mL <ref name="reddit_brett"></ref>. See also [https://www.facebook.com/groups/MilkTheFunk/permalink/1385144124847131/?match=YXR0ZW51YXRpb24sYXR0ZW51YXRlZCxjbGF1c3Nlbmlp this MTF thread] and [https://www.facebook.com/groups/MilkTheFunk/permalink/1309024112459133/?comment_id=1310955328932678&comment_tracking=%7B%22tn%22%3A%22R1%22%7D this MTF thread] which discuss the purity of this culture, and references [http://web.archive.org/web/20240623083404/http://brettanomycesproject.com/dissertation/pure-culture-fermentation/impact-of-pitching-rate/ Yakobson's data] that indicates that it does not attenuate wort efficiently when purely isolated.

There are generally two approaches to handling ''Brettanomyces'' starters. The first is to use a stir plate set to a medium-high RPM with tin foil on top of the flask for 7-8 days, cold crash for a few days, and then decant the beer before pitching the sedimented yeast. The second approach is to use an orbital shaker set to 80 RPM to create a ''semi-aerobic'' environment (this means that the oxygen levels are low, but also not non-existent) for 7-8 days as described in ''The Brettanomyces project'' <ref name="chad_rpm">[http://web.archive.org/web/20240623090139/http://www.brettanomycesproject.com/dissertation/propagation-and-batch-culture-growth/propagation-methods/ Yakobson, Chad. The Brettanomyces Project. Propagation and Batch Culture Methods. Retrieved 2/18/2015.]</ref>, cold crashing can be skipped, and the entire starter is pitched into the wort. An alternative to the second approach is to use a stir plate on a very low setting so that only a very small "dimple" of a vortex is formed <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/?comment_id=1174867645874781&reply_comment_id=1174924805869065&total_comments=1&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Mark Trent, Richard Preiss, and Roy Ventullo on MTF regarding creating a semi-aerobic starter without an orbital shaker. 11/06/2015]</ref>. If a stir plate is not available, give the starter an initial dosage of pure O2, and then cover it with foil so that oxygen can slowly diffuse into the starter, and gently agitate as often as possible <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1019859158042298/?comment_id=1020313737996840&offset=0&total_comments=24&comment_tracking=%7B%22tn%22%3A%22R6%22%7D Conversation with Nick Impellitteri on MTF in regards to semi-aerobic starters. 2/16/2015.]</ref>.

Current yeast pitching calculators for brewers are not adequate for determining ''Brettanomyces'' pitching rates based on starter volume size because the maximum cell density of ''Brettanomyces'' per mL of wort is 3 to 6 times the cell density of ''Saccharomyces''. For example, a given ''Saccharomyces'' strain may reach a cell density of 130 million cells per mL in a 1.040 wort (different ''Saccharomyces'' strains can have different cell densities as well, although they are a lot lower than ''Brettanomyces'' overall). Different ''Brettanomyces'' strain cell densities have been reported to be 600 to 885 million cells per mL in 1.040 wort depending on the species/strain <ref name="Yakobson_Propagation">[http://web.archive.org/web/20240623065612/http://www.brettanomycesproject.com/dissertation/propagation-and-batch-culture-growth/propagation-results/ Yakobson, Chad. The Brettanomyces Project. Propagation and Batch Culture Results. Retrieved 2/17/2015]</ref><ref name="MarkTrent">[https://www.facebook.com/groups/MilkTheFunk/permalink/1114254011936145/ Conversation with Mark Trent and Lance Shaner on MTF regarding Brett pitching rates. 07-21-2015.]</ref>. Since yeast calculators are based on ''S. cerevisiae'' or ''S. pastorianus'' cell density, using one of these tools for ''Brettanomyces'' starters will create an unexpectedly high cell count in reality. There is not currently enough publicly available data that the authors of this wiki are aware of to accurately determine starter volumes for ''Brettanomyces'', particularly because each strain and species have a different maximum cell density per mL of wort. However, pitching around 500-600 mL of a ''Brettanomyces'' starter for 5 gallons of 1.060 SG wort will achieve a pitching rate that is similar to lager yeast pitching rates, which has been recommended for [[Brettanomyces_Fermentation|100% Brettanomyces Fermentation]]. [[Omega Yeast Labs]] is currently working on a project to create a more accurate ''Brettanomyces'' pitching rate calculator (it will also contain pitching rate calculations for specific strains of ''Saccharomyces'', which is something that current yeast pitching calculators do not include) <ref name="MarkTrent"></ref>.

For yeast laboratories, "Malt Yeast Peptone Glucose" growth substrate has been shown to be a better substrate than wort for initially growing ''Brettanomyces'' from a plate or slant. When grown in wort, ''Brettanomyces'' will often go through a 24 hour lag phase, a growth phase, another lag phase, and a second growth phase (all within 7-8 days). When grown in MYPG substrate, there is only a single growth phase and no lag phase, which has been reported by Yakobson to produce a larger cell count in the same amount of time <ref>[http://web.archive.org/web/20240519142207/http://www.brettanomycesproject.com/2009/08/mypg-vs-wort-as-the-growth-substrate/ Yakobson, Chad. The Brettanomyces Project. MYPG Compared to Wort as a Growth Substrate. Retrieved 2/18/2015.]</ref>. Cells grown in MYPG also are better adapted to grow in wort <ref>[http://web.archive.org/web/20240623065300/http://www.brettanomycesproject.com/dissertation/propagation-and-batch-culture-growth/propagation-discussion/ Yakobson, Chad. The Brettanomyces Project. Propagation and Batch Culture Discussion. Paragraph 5. Retrieved 2/18/2015.]</ref>. Practical instructions for making this substrate can be found on Jason Rodriguez's blog, "[http://sciencebrewer.com/2011/04/29/wild-yeast-project-mypg-culture-media/ Brew Science - Homebrew Blog]". Unfortunately, growing ''Brettanomyces'' pitches in MYPG for breweries isn't very practical due to needing almost 4 times the amount of MYPG versus wort to get the same pitching rate. In a brewery or homebrewery, using wort for ''Brettanomyces'' starters is more practical <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1150169708344575/ Conversation with Mark Trent, Lance Shaner, and Richard Preiss on MTF. 09/18/2015.]</ref>.

* [http://web.archive.org/web/20240519133925/http://www.brettanomycesproject.com/ The Brettanomyces Project - Chad Yakobon's Brett research.]