{"id":2910,"date":"2022-11-02T08:31:24","date_gmt":"2022-11-02T13:31:24","guid":{"rendered":"https:\/\/suigenerisbrewing.com\/?p=2910"},"modified":"2025-03-21T06:57:37","modified_gmt":"2025-03-21T11:57:37","slug":"this-is-the-wild-brettanomyces-your-are-looking-for","status":"publish","type":"post","link":"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/","title":{"rendered":"These ARE The Yeast You Are Looking For"},"content":{"rendered":"<div class=\"wp-block-image\">\n<figure class=\"alignright size-large is-resized\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"606\" data-attachment-id=\"2900\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/10\/06\/this-is-not-the-yeast-your-are-looking-for\/notbrett\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/notBrett.jpg?fit=1178%2C893&amp;ssl=1\" data-orig-size=\"1178,893\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"notBrett\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/notBrett.jpg?fit=800%2C606&amp;ssl=1\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/notBrett.jpg?resize=800%2C606&#038;ssl=1\" alt=\"A WLN agar plate showing a failed attempt at isolating wild brettanomyces\" class=\"wp-image-2900\" style=\"width:256px;height:194px\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/notBrett.jpg?resize=1024%2C776&amp;ssl=1 1024w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/notBrett.jpg?resize=300%2C227&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/notBrett.jpg?resize=768%2C582&amp;ssl=1 768w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/notBrett.jpg?w=1178&amp;ssl=1 1178w\" sizes=\"auto, (max-width: 800px) 100vw, 800px\" \/><figcaption class=\"wp-element-caption\">My previous failed attempt<\/figcaption><\/figure>\n<\/div>\n\n\n<p><a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/10\/06\/this-is-not-the-yeast-your-are-looking-for\/\">A few weeks ago<\/a> I attempted to isolate wild <em>Brettanomyces<\/em> from the soil, <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/07\/07\/where-do-wild-brettanomyces-live\/\">based on some papers<\/a> which identified Brettanomyces as part of the rhizosphere (microorganisms associated with plant roots) of several plant species. This first attempt simply plated water which I had used to rinse the roots of bean plants onto agar selective for <em>Brettanomyces<\/em>. This attempt did not work, and instead I grew some pretty &#8211; but utterly useless for fermentation &#8211; filamentous fungi.<\/p>\n\n\n\n<p>I thought this would be it for the year &#8211; it was already well into fall, and most plants in our garden were on their way to dormancy or death. But, thanks to some late season warm weather, I was able to make a second attempt at it. This time using an enrichment approach prior to plating.<\/p>\n\n\n\n<p>So did it work?<\/p>\n\n\n\n<p>This is a monster post, so here&#8217;s a quick TOC for anyone who wants to jump around:<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li><a href=\"#enrich\">Enriching for <em>Wild Brettanomyces<\/em><\/a> &#8211; my strategy for this experiment<\/li>\n\n\n\n<li><a href=\"#EIP\">The Enrichment in Practice<\/a> &#8211; the details of the experiment itself<\/li>\n\n\n\n<li><a href=\"#results\">Results<\/a> &#8211; what I found<\/li>\n\n\n\n<li><a href=\"#cultures\">Overview of Isolated Cultures<\/a> &#8211; a summary of the 12 pure cultures I generated<\/li>\n\n\n\n<li><a href=\"#conclusions\">Conclusions<\/a> &#8211; what I found<\/li>\n\n\n\n<li><a href=\"#limitations\">Limitations<\/a> &#8211; caveat emperor<\/li>\n\n\n\n<li><a href=\"#next\">What&#8217;s next<\/a> &#8211; yes, there will be a Part III, IV and V to this series<\/li>\n<\/ul>\n\n\n\n<h5 class=\"wp-block-heading\"><strong>Other Parts to this Series:<\/strong><\/h5>\n\n\n\n<ul class=\"wp-block-list\">\n<li>Part 0:&nbsp;<a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/07\/07\/where-do-wild-brettanomyces-live\/\">Where do the Wild Brettanomyces Roam?<\/a><\/li>\n\n\n\n<li>Part I: <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/10\/06\/this-is-not-the-yeast-your-are-looking-for\/\">These Are Not The Yeast You Are Looking For<\/a> <\/li>\n\n\n\n<li>Part II: <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/\">These ARE The Yeast Your Are Looking For<\/a> (this post)<\/li>\n\n\n\n<li>Part III: <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/06\/wild-brettanomyces-needle\/\">Wild Brettanomyces &#8211; a Needle in a Haystack<\/a><\/li>\n\n\n\n<li>Part IV: <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2023\/03\/24\/wild-brettanomyces-fermentation-test\/\">Fermentation characteristics<\/a><\/li>\n\n\n\n<li>Part V:&nbsp;<a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2024\/02\/10\/wild-brettanomyces-2023\/\">Wild Brettanomyces \u2013 The 2023 Search<\/a><\/li>\n\n\n\n<li>Part VI:  <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2025\/03\/21\/wild-brettanomyces-project-2024-update\/\">Wild Brettanomyces \u2013 The 2024 Search<\/a><\/li>\n<\/ul>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"enrich\">Enriching for Wild <em>Brettanomyces<\/em><\/h3>\n\n\n\n<p>My plan was to &#8220;grow-out&#8221; any wild <em>Brettanomyces<\/em> that were present, while also suppressing the growth of unwanted yeast, fungi, and bacteria. Microbiologically speaking, this is a selective approach that relies on several properties to enrich for <em>Brettanomyces<\/em> while suppressing the unwanted organisms.<\/p>\n\n\n\n<p><strong>Growth Medium<\/strong>: I chose to use <a href=\"http:\/\/cshprotocols.cshlp.org\/content\/2017\/8\/pdb.rec090563.\">YPD<\/a> medium instead of the WLN medium I used last time. This is a simpler medium that is nutritionally rich and <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC368351\/\">supports the growth of <em>Brettanomyces<\/em><\/a>. It&#8217;s also much cheaper than WLN, and is much easier to prepare, which were the real reasons for the switch.<\/p>\n\n\n\n<p><strong>Inhibiting Bacteria:<\/strong> I added the antibiotics ampicillin and chloramphenicol, both at 50 mg\/L, to act as a broad-spectrum anti-bacterial agent.<\/p>\n\n\n\n<p><strong>Inhibiting Most Fungi and Yeast:<\/strong> I used the same &#8220;special sauce&#8221; as last time &#8211; 50 mg\/L cycloheximide* &#8211; to inhibit the growth of most yeast and fungi. <\/p>\n\n\n\n<p><em>*Cycloheximide is highly toxic. Please do not try to replicate this project unless you know how to work with it safely.<\/em><\/p>\n\n\n\n<p><strong>Selecting for wild <em>Brettanomyces:<\/em><\/strong> This is where my plan differs the most from last time. To help enrich for <em>Brettanomyces<\/em> I took advantage of two properties of <em>Brett<\/em> which non-yeast fungi lack. The first property is a relatively strong ability to grow in liquid culture compared to filamentous fungi. As a general rule (meaning there are exceptions), yeast will dominate in a liquid culture during early time-points, with filamentous fungi becoming dominant later on. To take advantage of this, I transfered a small amount of the cultures started from plant roots into fresh medium after 7 days (a process called &#8220;sub-culturing&#8221;). This inoculated the fresh medium at a time when <em>Brettanomyces<\/em> should be at or near peak growth, but before filamentous fungi become dominant. In theory, this should give <em>Brettanomyces<\/em> a good chance of dominating the sub-cultures.<\/p>\n\n\n\n<p>The second property is the one that should strongly select for wild <em>Brettanomyces<\/em> &#8211; the ability to grow in the absence of oxygen. I created an anaerobic (oxygen-free) environment by layering mineral oil over the liquid culture. All filamentous fungi are obligate aerobes, meaning they cannot survive in the absence of oxygen, while <em>Brettanomyces<\/em> has the ability to ferment and grow in the absence of oxygen. Combined with the cycloheximide, there is perhaps two or three genera of yeast which will grow under these anaerobic conditions&#8230;including <em>Brettanomyces<\/em>.<\/p>\n\n\n\n<p><strong>Detecting <em>Brettanomyces<\/em>:<\/strong> I used <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/10\/06\/this-is-not-the-yeast-your-are-looking-for\/\">the same approach as last time<\/a> to detect <em>Brettanomyces<\/em>. Two drops (~100 uL) of the cultures were plated on solid medium (this time YPD-agar) containing the same antibiotics and cycloheximide as the liquid medium used above, plus 22 mg\/L of bromocresol green. The antibiotics + cycloheximide suppress bacteria and most yeast\/fungi, while the bromocresol green allows for the identification of <em>Brettanomyces<\/em>. Yeasts will take up this dye, but cannot process it, leading to blue\/green colonies. <em>Brettanomyces<\/em> can break down the dye, producing white colonies. While this does not provide a definitive identification, it is a good enough of an identification to justify the cost and time that it takes to perform a full work-up of the putative <em>Brettanomyces<\/em>.<\/p>\n\n\n\n<p>From a microbiological perspective, these agar plates are termed &#8220;selective and differential media&#8221; (hence the meme in the header image). The medium is <em>selective<\/em> as it allows the preferential growth of a limited range of organisms, and it is <em>differential<\/em> as the colour-change of the medium allows you to differentiate between certain species.<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"EIP\">The Enrichment In Practice<\/h3>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"alignleft size-full is-resized\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"720\" height=\"827\" data-attachment-id=\"2912\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/fermplan-1\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/FermPlan-1.png?fit=720%2C827&amp;ssl=1\" data-orig-size=\"720,827\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"FermPlan-1\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/FermPlan-1.png?fit=720%2C827&amp;ssl=1\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/FermPlan-1.png?resize=720%2C827&#038;ssl=1\" alt=\"Visual flow-chart showing the process that will be used to enrich for wild brettanomyces\" class=\"wp-image-2912\" style=\"width:360px;height:414px\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/FermPlan-1.png?w=720&amp;ssl=1 720w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/FermPlan-1.png?resize=261%2C300&amp;ssl=1 261w\" sizes=\"auto, (max-width: 720px) 100vw, 720px\" \/><figcaption class=\"wp-element-caption\">A graphical overview of the enrichment plan<\/figcaption><\/figure>\n<\/div>\n\n\n<p>I&#8217;ve depicted the enrichment process in the diagram on the left. I collected a mixture of roots from the last of the living plants in my garden &#8211; dill, purple beans, chives, parsley, and a bit of crab grass. I then used the chopped roots to inoculate two tubes of highly oxygenated medium.<\/p>\n\n\n\n<p>48 hours later I mixed the contents of the two tubes to re-oxygenate and equalize the microbial contents. I then transferred a portion of the medium back to the emptied tube such that both tubes had the same volume. I placed 1 mL of mineral oil on top of one cultures to deprive it of oxygen (anaerobic culture). The other tube was left open to allow for continued air exchange (aerobic culture).<\/p>\n\n\n\n<p>Five days later (1 week after the initial inoculation) I inspected both cultures in my microscope, looking for yeast-like (and preferably <em>Brettanomyces<\/em>-like) cells. None were observed in the aerobic culture, but a few were found in the anaerobic culture. As such, I plated a small amount of the anaerobic culture.<\/p>\n\n\n\n<p>At this point I sub-cultured both the aerobic and anaerobic cultures, even though the former lacked any obvious <em>Brettanomyces<\/em>-like cells. The rationale for sub-culturing the aerobic culture was that <em>Brettanomyces<\/em> may still be present, but at too small a number to be identified by microscopy. I sub-cultured the aerobic culture by transferring 1 mL of this culture into two tubes of freshly made YPD + antibiotics + cycloheximide (e.g. 0.5 mL into each of the new tubes). I immediately topped one of the new tubes with mineral oil, and left the other exposed to air. I then did the same with the anaerobic culture. This gave me a total of four sub-cultures: anaerobic and aerobic sub-cultures of the initial aerobic culture, and anaerobic and aerobic sub-cultures of the initial anaerobic culture.<\/p>\n\n\n\n<p>For simplicity I&#8217;ll refer to the sub-cultures as &#8220;initial-subculture&#8221;, so the aerobic sub-culture of the initial anaerobic culture would be called &#8220;anaerobic-aerobic&#8221;.<\/p>\n\n\n\n<p>I allowed the sub-cultures to grow for another week. At this point I inspected them by microscopy and plated all four sub-cultures.<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"results\">Results<\/h3>\n\n\n\n<h5 class=\"wp-block-heading\">Initial cultures<\/h5>\n\n\n\n<figure class=\"wp-block-image size-large\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"199\" data-attachment-id=\"2920\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/montage\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Montage.png?fit=3012%2C750&amp;ssl=1\" data-orig-size=\"3012,750\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"Montage\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Montage.png?fit=800%2C199&amp;ssl=1\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Montage.png?resize=800%2C199&#038;ssl=1\" alt=\"Montage of the plants, roots, and liquid cultures used to start the wild brettanomyces project\" class=\"wp-image-2920\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Montage.png?resize=1024%2C255&amp;ssl=1 1024w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Montage.png?resize=300%2C75&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Montage.png?resize=768%2C191&amp;ssl=1 768w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Montage.png?resize=1536%2C382&amp;ssl=1 1536w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Montage.png?resize=2048%2C510&amp;ssl=1 2048w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Montage.png?w=1600&amp;ssl=1 1600w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Montage.png?w=2400&amp;ssl=1 2400w\" sizes=\"auto, (max-width: 800px) 100vw, 800px\" \/><figcaption class=\"wp-element-caption\">Setting up the initial cultures. Left \u2192 Right: The section of the garden I harvested roots from, the roots being cut up, the initial inoculation after two days showing some growth, and on the far-right, the tubes a few hours after blending and adding the mineral oil to one tube.<\/figcaption><\/figure>\n\n\n\n<p>To setup the initial cultures I harvested roots from a variety of plants which were still green and living from my garden. This included beans, dill, chives, parsley, and crab grass. The roots were finely chopped, mixed, and divided evenly between two well-oxygenated tubes of media. Two days later there was visible evidence of growth, in the form of a slight haze to the medium (see the third image from the left, above). At this point I mixed the contents of the two tubes together, in order to even out any microbial differences between the tubes, and then separated the mixture back into two separate tubes. I layered oil over one of these cultures to create an anaerobic environment and allowed the tubes to grow for an additional 5 days.<\/p>\n\n\n\n<p>On the final day placed a single drop of the aerobic and anaerobic cultures onto a slide and imaged the suspensions on my microscope. My intent here was two-fold. Firstly, I was looking for the presence of yeast-like cells. Secondly, if yeast-like cells were present I wanted to get a rough count of how many were present. The reason for the latter was to allow me to dilute the sample properly in order to plate it out on my selective and differential medium. <\/p>\n\n\n\n<figure class=\"wp-block-gallery has-nested-images columns-default is-cropped wp-block-gallery-1 is-layout-flex wp-block-gallery-is-layout-flex\">\n<figure class=\"wp-block-image size-large\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"800\" data-attachment-id=\"2926\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/oxygen\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oxygen.png?fit=931%2C931&amp;ssl=1\" data-orig-size=\"931,931\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"Oxygen\" data-image-description=\"\" data-image-caption=\"&lt;p&gt;Yeasts, Moulds, and Bacteria&lt;\/p&gt;\n\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oxygen.png?fit=800%2C800&amp;ssl=1\" data-id=\"2926\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oxygen.png?resize=800%2C800&#038;ssl=1\" alt=\"Fungal and bacterial cells in the aerobic culture.\" class=\"wp-image-2926\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oxygen.png?w=931&amp;ssl=1 931w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oxygen.png?resize=300%2C300&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oxygen.png?resize=150%2C150&amp;ssl=1 150w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oxygen.png?resize=768%2C768&amp;ssl=1 768w\" sizes=\"auto, (max-width: 800px) 100vw, 800px\" \/><figcaption class=\"wp-element-caption\">Field of view of the fungal cells and bacteria found in the aerobic culture. Scale bar is 5 \u03bcm<\/figcaption><\/figure>\n\n\n\n<figure class=\"wp-block-image size-large\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"800\" data-attachment-id=\"2925\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/oil\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oil.png?fit=1058%2C1058&amp;ssl=1\" data-orig-size=\"1058,1058\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"Oil\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oil.png?fit=800%2C800&amp;ssl=1\" data-id=\"2925\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oil.png?resize=800%2C800&#038;ssl=1\" alt=\"Wild brettanomyces like cells from the anaerobic ulture\" class=\"wp-image-2925\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oil.png?resize=1024%2C1024&amp;ssl=1 1024w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oil.png?resize=300%2C300&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oil.png?resize=150%2C150&amp;ssl=1 150w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oil.png?resize=768%2C768&amp;ssl=1 768w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/Oil.png?w=1058&amp;ssl=1 1058w\" sizes=\"auto, (max-width: 800px) 100vw, 800px\" \/><figcaption class=\"wp-element-caption\">Montage of yeast-like cells found in the anaerobic culture. Scale bar is 5 \u03bcm<\/figcaption><\/figure>\n<\/figure>\n\n\n\n<p>The aerobic culture (image to the left, above) had a thin layer of mould on the top of the tube, and unsurprisingly, under the microscope I found large numbers of highly elongated and vacuolated cells typical of moulds. Surprisingly, I also found a large number of bacteria. While some antibiotic resistant bacteria are to be expected, the numbers present were much higher than is reasonable. I suspect that there was some metabolism of the antibiotics by the moulds, thus allowing the bacteria to grow. Despite a lot of time spent searching, I found little evidence of yeast. Because of this I didn&#8217;t bother plating this sample.<\/p>\n\n\n\n<p>My anaerobic culture (image to the right, above) was, at first glance, a little dismaying. Unlike the aerobic culture, there was very little growth visible in the sample, with most of the objects in the sample appearing to be dust and other debris. However, after a lot of searching, I was able to find several clumps of cells that had the prototypical appearance of yeast cells. Several of these also had interlinked cells in a growth pattern similar to that of <em>Brettanomyces<\/em>. Because <em>Brettanomyces<\/em>-like cells were present &#8211; though-be-it at a low density &#8211; I plated out a few drops (~200 ul) of the undiluted culture onto an agar plate. <\/p>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"alignleft size-medium\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"300\" height=\"199\" data-attachment-id=\"2934\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/firstplate\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/firstPlate.jpg?fit=987%2C656&amp;ssl=1\" data-orig-size=\"987,656\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"firstPlate\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/firstPlate.jpg?fit=800%2C532&amp;ssl=1\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/firstPlate.jpg?resize=300%2C199&#038;ssl=1\" alt=\"agar plate showing many hundreds of (hopefully wild brettanomyces) colonies\" class=\"wp-image-2934\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/firstPlate.jpg?resize=300%2C199&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/firstPlate.jpg?resize=768%2C510&amp;ssl=1 768w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/firstPlate.jpg?w=987&amp;ssl=1 987w\" sizes=\"auto, (max-width: 300px) 100vw, 300px\" \/><figcaption class=\"wp-element-caption\">Lots of colonies from the initial anaerobic culture!<\/figcaption><\/figure>\n<\/div>\n\n\n<p>The plate was incubated at room temperature (currently around 19C) for 4 days, at which point hundreds of colonies were visible on the plate (image to left). Of these, none were green, consistent with the expected decolourization of the bromocresol green by <em>Brettanomyces<\/em>. That said, this wasn&#8217;t a total success as the unexpectedly high density of colonies essentially prevents me from picking pure colonies from the plate. As such, I will only pick colonies from the sub-cultures, which may limit the diversity of <em>Brettanomyces<\/em> species I can recover.<\/p>\n\n\n\n<p>I also prepared sub-cultures of both the aerobic and anaerobic cultures at the same time that I was inspecting the cultures on the microscope. To prepare these cultures, I transfered 0.5 mL of the initial culture to a fresh tube containing 5 mL of medium + antibiotics + cycloheximide. This was then incubated at room temperature until visible growth had occurred.<\/p>\n\n\n\n<h5 class=\"wp-block-heading\">Sub-cultures<\/h5>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"alignleft size-full is-resized\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"356\" height=\"448\" data-attachment-id=\"2943\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/pellet\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/pellet.png?fit=356%2C448&amp;ssl=1\" data-orig-size=\"356,448\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"pellet\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/pellet.png?fit=356%2C448&amp;ssl=1\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/pellet.png?resize=356%2C448&#038;ssl=1\" alt=\"Liquid cultures showing a yeast pellet\" class=\"wp-image-2943\" style=\"width:178px;height:224px\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/pellet.png?w=356&amp;ssl=1 356w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/pellet.png?resize=238%2C300&amp;ssl=1 238w\" sizes=\"auto, (max-width: 356px) 100vw, 356px\" \/><figcaption class=\"wp-element-caption\">Aerobic (left) &amp; anaerobic (right) sub-cultures of the original anaerobic culture.<\/figcaption><\/figure>\n<\/div>\n\n\n<p>The subcultures grew much more quickly than I had expected, with all four sub-cultures showing a notable pellet of yeast on the bottom of the tube after just three days (image to the left). So contrary to my enrichment plan, I ended up imaging and plating out these cultures after 3 days, instead of the planned seven.<\/p>\n\n\n\n<p>As with the initial cultures, I first inspected the sub-cultures on my microscope (slideshow, below). The aerobic sub-cultures were not usable &#8211; as expected, the aerobic-aerobic sub-culture had a lot of filamentous fungi plus some bacteria, while the aerobic-anaerobic had more bacteria than yeast. The fungi and bacteria far out umbered the yeast in both cultures, making recovery of pure yeast cultures difficult. This may also indicate an issue with the plate I prepared from the original anaerobic culture &#8211; clearly, the bacteria had survived the anaerobic culture (how else would they have emerged in the aerobic sub-culture?). This may mean that many of the colonies on the agar plate prepared from the original anaerobic culture may be bacteria rather than yeast. As such, I decided to not sample from that plate.<\/p>\n\n\n\n<div class=\"wp-block-columns is-layout-flex wp-container-core-columns-is-layout-9d6595d7 wp-block-columns-is-layout-flex\">\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:100%\">\n<div class=\"wp-block-jetpack-slideshow aligncenter\" data-effect=\"slide\"><div class=\"wp-block-jetpack-slideshow_container swiper-container\"><ul class=\"wp-block-jetpack-slideshow_swiper-wrapper swiper-wrapper\"><li class=\"wp-block-jetpack-slideshow_slide swiper-slide\"><figure><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"800\" alt=\"\" class=\"wp-block-jetpack-slideshow_image wp-image-2937\" data-id=\"2937\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_ox2.jpg?resize=800%2C800&#038;ssl=1\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_ox2.jpg?resize=1024%2C1024&amp;ssl=1 1024w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_ox2.jpg?resize=300%2C300&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_ox2.jpg?resize=150%2C150&amp;ssl=1 150w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_ox2.jpg?resize=768%2C768&amp;ssl=1 768w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_ox2.jpg?w=1218&amp;ssl=1 1218w\" sizes=\"(max-width: 800px) 100vw, 800px\" \/><figcaption class=\"wp-block-jetpack-slideshow_caption gallery-caption\">Aerobic sub-culture of the aerobic culture (aerobic-aerobic)<\/figcaption><\/figure><\/li><li class=\"wp-block-jetpack-slideshow_slide swiper-slide\"><figure><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"800\" alt=\"\" class=\"wp-block-jetpack-slideshow_image wp-image-2936\" data-id=\"2936\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox3.jpg?resize=800%2C800&#038;ssl=1\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox3.jpg?resize=1024%2C1024&amp;ssl=1 1024w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox3.jpg?resize=300%2C300&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox3.jpg?resize=150%2C150&amp;ssl=1 150w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox3.jpg?resize=768%2C768&amp;ssl=1 768w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox3.jpg?w=1218&amp;ssl=1 1218w\" sizes=\"(max-width: 800px) 100vw, 800px\" \/><figcaption class=\"wp-block-jetpack-slideshow_caption gallery-caption\">Anaerobic sub-culture of the aerobic culture (aerobic-anaerobic)<\/figcaption><\/figure><\/li><li class=\"wp-block-jetpack-slideshow_slide swiper-slide\"><figure><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"800\" alt=\"\" class=\"wp-block-jetpack-slideshow_image wp-image-2938\" data-id=\"2938\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_ox2.jpg?resize=800%2C800&#038;ssl=1\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_ox2.jpg?resize=1024%2C1024&amp;ssl=1 1024w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_ox2.jpg?resize=300%2C300&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_ox2.jpg?resize=150%2C150&amp;ssl=1 150w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_ox2.jpg?resize=768%2C768&amp;ssl=1 768w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_ox2.jpg?w=1218&amp;ssl=1 1218w\" sizes=\"(max-width: 800px) 100vw, 800px\" \/><figcaption class=\"wp-block-jetpack-slideshow_caption gallery-caption\">Aerobic sub-culture of the anaerobic culture (anaerobic-aerobic)<\/figcaption><\/figure><\/li><li class=\"wp-block-jetpack-slideshow_slide swiper-slide\"><figure><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"800\" alt=\"\" class=\"wp-block-jetpack-slideshow_image wp-image-2935\" data-id=\"2935\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_anox2.jpg?resize=800%2C800&#038;ssl=1\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_anox2.jpg?resize=1024%2C1024&amp;ssl=1 1024w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_anox2.jpg?resize=300%2C300&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_anox2.jpg?resize=150%2C150&amp;ssl=1 150w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_anox2.jpg?resize=768%2C768&amp;ssl=1 768w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/anox_anox2.jpg?w=1218&amp;ssl=1 1218w\" sizes=\"(max-width: 800px) 100vw, 800px\" \/><figcaption class=\"wp-block-jetpack-slideshow_caption gallery-caption\">Anaerobic sub-culture of the anaerobic culture (anaerobic-anaerobic)<\/figcaption><\/figure><\/li><\/ul><a class=\"wp-block-jetpack-slideshow_button-prev swiper-button-prev swiper-button-white\" role=\"button\"><\/a><a class=\"wp-block-jetpack-slideshow_button-next swiper-button-next swiper-button-white\" role=\"button\"><\/a><a aria-label=\"Pause Slideshow\" class=\"wp-block-jetpack-slideshow_button-pause\" role=\"button\"><\/a><div class=\"wp-block-jetpack-slideshow_pagination swiper-pagination swiper-pagination-white\"><\/div><\/div><\/div>\n<\/div>\n<\/div>\n\n\n\n<p>The anaerobic sub-cultures were much more promising (images in the slide show, above) &#8211; both showed a range of yeast-like cells of varying morphologies, with no obvious bacterial contamination. The yeast cells also had a range of morphologies. This may represent multiple strains\/species of wild <em>Brettanomyces<\/em>, or may indicate one (or a few) strains of <em>Brettanomyces<\/em> at different stages of growth. There is also the possibility that these may be non-<em>Brettanomyces<\/em> yeast. The dose of cycloheximide I used (50 mg\/L) is on the edge of what both <em><a href=\"https:\/\/en.wikipedia.org\/wiki\/Debaryomyces_hansenii\">Debaryomyces housenii<\/a><\/em> (also known as <em>Candida famata<\/em>) and <em><a href=\"https:\/\/en.wikipedia.org\/wiki\/Kluyveromyces_marxianus\">Kluyveromyces marxianus<\/a><\/em> can survive. While the morphology of the cells in the anaerobic cultures are not consistent with <em>K. marxianus<\/em>, <em>D. housenii<\/em> is morphologically similar to <em>Brettanomyces<\/em>. I had based my 50 mg\/L on what is standard for <em>Brettanomyces<\/em> isolation from wine and fruits. <em>Debaryomyces<\/em> do grow poorly at this concentration &#8211; one paper (which I cannot find again), claimed that only a few <em>Debaromyces <\/em>strains can grow over 30 mg\/L, and none at 100 mg\/L cycloheximide.<\/p>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"alignleft size-medium is-resized\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"300\" height=\"224\" data-attachment-id=\"2940\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/ox_anox_plate\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?fit=1205%2C898&amp;ssl=1\" data-orig-size=\"1205,898\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"ox_anox_plate\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?fit=800%2C596&amp;ssl=1\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?resize=300%2C224&#038;ssl=1\" alt=\"Fungal and yeast colonies\" class=\"wp-image-2940\" style=\"width:225px;height:168px\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?resize=300%2C224&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?resize=1024%2C763&amp;ssl=1 1024w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?resize=768%2C572&amp;ssl=1 768w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?w=1205&amp;ssl=1 1205w\" sizes=\"auto, (max-width: 300px) 100vw, 300px\" \/><figcaption class=\"wp-element-caption\">Fungal (spiky\/blue) and yeast (smooth\/white) colonies.<\/figcaption><\/figure>\n<\/div>\n\n\n<p>Given the high density of yeast in these cultures, I first used a serial dilution to prepare 1:10,000, 1:100,000 and 1:1,000,000 dilutions of the original cultures. If you don&#8217;t know how to do a serial dilution, <a href=\"https:\/\/youtu.be\/z_veqoM1nc0?t=202\">this is covered in my video on preparing wet mounts for microscopy<\/a>. I then plated 2 drops (~0.1 mL) of these highly diluted cultures onto YPD + antibiotics + cycloheximide plates. The plates were incubated at room temperature until colonies had formed &#8211; which only took 2 days!<\/p>\n\n\n\n<p>Much to my surprise, all of the aerobic plates had fungal colonies &#8211; even the anaerobic-aerobic plates. These appeared as star-like or &#8220;spikey&#8221; colonies (see image above). I confirmed these were filamentous fungi by microscopy (not shown). The fungal colonies also have a slight blue-grey hue, suggesting that they are taking up the bromocresol green. <\/p>\n\n\n\n<p>Scattered among the fungal colonies were beautiful yeast colonies. Even better, only yeast colonies were present on the anaerobic-anaerobic plates! Even more exciting, with only one exception (out of over 500 colonies), these yeast colonies were stark-white. While references and information is thin,<a href=\"https:\/\/academic.oup.com\/mmy\/article\/45\/2\/97\/1216685\"> at least one paper suggests<\/a> that <em>D. housenii<\/em> (called <em>C. famata<\/em> in the paper) will have blue\/green pigmented colonies on bromocresol green agar, and as mentioned above, I have yet to see a cell with the appearance of <em>K. marxianus<\/em>. This means that these white colonies have a good chance of being wild <em>Brettanomyces<\/em>!<\/p>\n\n\n\n<figure class=\"wp-block-image size-full\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"379\" data-attachment-id=\"2948\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/colonies\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/colonies.png?fit=958%2C454&amp;ssl=1\" data-orig-size=\"958,454\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"colonies\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/colonies.png?fit=800%2C379&amp;ssl=1\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/colonies.png?resize=800%2C379&#038;ssl=1\" alt=\"Image of the 1:100,000 diluted anaerobic-anaerobic plate, with the insert showing a close-up of the colonies.\" class=\"wp-image-2948\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/colonies.png?w=958&amp;ssl=1 958w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/colonies.png?resize=300%2C142&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/colonies.png?resize=768%2C364&amp;ssl=1 768w\" sizes=\"auto, (max-width: 800px) 100vw, 800px\" \/><figcaption class=\"wp-element-caption\">Image of the 1:100,000 diluted anaerobic-anaerobic plate, with the insert showing a close-up of the colonies. This includes the only blue yeast colony found across all plated cultures.<\/figcaption><\/figure>\n\n\n\n<p>I picked 4 colonies from each of the 3 dilution plates from the anaerobic-subculture of the initial anaerobic culture, placing each colony on a grid plate, <a href=\"https:\/\/youtu.be\/sDvGvBVw3gg?t=298\">as shown in this old video of mine<\/a>. I also cultured the only blue yeast colony I found, as I was curious what it was. These were allowed to grow for 2.5 days, producing large colonies. Once larger colonies has formed, cell morphology was assessed by microscopy. These findings are summarized below.<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"cultures\">Overview of Isolated Cultures<\/h3>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"alignleft size-full is-resized\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"695\" data-attachment-id=\"2963\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/gridplate_mirror\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/GridPlate_mirror.jpg?fit=903%2C785&amp;ssl=1\" data-orig-size=\"903,785\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"GridPlate_mirror\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/GridPlate_mirror.jpg?fit=800%2C695&amp;ssl=1\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/GridPlate_mirror.jpg?resize=800%2C695&#038;ssl=1\" alt=\"grid plate of (hopefully) wild brettanomyces\" class=\"wp-image-2963\" style=\"width:320px;height:278px\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/GridPlate_mirror.jpg?w=903&amp;ssl=1 903w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/GridPlate_mirror.jpg?resize=300%2C261&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/GridPlate_mirror.jpg?resize=768%2C668&amp;ssl=1 768w\" sizes=\"auto, (max-width: 800px) 100vw, 800px\" \/><\/figure>\n<\/div>\n\n\n<p>I isolated a total of 12 pure colonies for further analysis. All but one colony grew to the desired large size in just 2.5 days. While I was focused on putative <em>Brettanomyces <\/em>isolates, I also included the only blue yeast colony that I identified. This blue colony can be seen in position A4 in the image to the left.<\/p>\n\n\n\n<p>Much to my surprize, colony C4 also turned blue despite coming from a white &#8220;mother colony&#8221;. This colony also struggled to grow, relative to the others, suggesting that it was more sensitive to cycloheximide and therefore not likely a species of <em>Brettanomyces<\/em>. To further the identification process, I imaged all 12 colonies as <a href=\"https:\/\/www.youtube.com\/watch?v=z_veqoM1nc0\">wet-mounts<\/a>, imaging all colonies at the maximum possible magnification to resolve the cell structure as much as possible. I focused this imaging on finding dividing cells, as dividing cells are the most useful for species identification.<\/p>\n\n\n\n<div class=\"wp-block-columns is-layout-flex wp-container-core-columns-is-layout-9d6595d7 wp-block-columns-is-layout-flex\">\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:100%\">\n<figure class=\"wp-block-table is-style-stripes\"><table><thead><tr><th>Colony<\/th><th>Image<\/th><th>Notes &amp; Putative ID<\/th><\/tr><\/thead><tbody><tr><td>A-1<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2964\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/a1\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A1.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"A1\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A1.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2964\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A1.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A1.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A1.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><strong>Putative ID:<\/strong> <em>Brettanomyces<\/em>     <strong>Confidence:<\/strong> Medium<br><strong>Notes:<\/strong> Cells show classical <em>Brettanomyces<\/em> budding pattern <br>and elongated cell morphology.<\/td><\/tr><tr><td>A-2<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2965\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/a2\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A2.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"A2\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A2.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2965\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A2.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A2.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A2.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><strong>Putative ID:<\/strong> Unknown     <strong>Confidence:<\/strong> Low<br><strong>Notes:<\/strong> Cells are apiculate, which is unusual for <br><em>Brettanomyces<\/em>.<\/td><\/tr><tr><td>A-3<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2967\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/a3-2\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A3-1.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"A3\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A3-1.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2967\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A3-1.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A3-1.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A3-1.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><strong>Putative ID:<\/strong> <em>Brettanomyces<\/em>     <strong>Confidence:<\/strong> Low<br><strong>Notes:<\/strong> Occasional cells show classical <em>Brettanomyces<\/em> budding <br>pattern and elongated cell morphology.<\/td><\/tr><tr><td>A-4<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2968\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/a4\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A4.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"A4\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A4.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2968\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A4.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A4.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/A4.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><strong>Putative ID:<\/strong> <em>Schizosaccharomyces<\/em>     <strong>Confidence:<\/strong> Low<br><strong>Notes:<\/strong> This is one of the blue colonies, meaning it is highly <br>unlikely to be <em>Brettanomyces. <\/em>In addition, the cells appear to <br>divide by binary fission, which is not something done by <br><em>Brettanomyces<\/em>.<\/td><\/tr><tr><td>B-1<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2969\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/b1\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B1.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"B1\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B1.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2969\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B1.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B1.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B1.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><strong>Putative ID:<\/strong> <em>Brettanomyces<\/em>     <strong>Confidence:<\/strong> Medium<br><strong>Notes:<\/strong> Cells show classical <em>Brettanomyces<\/em> budding <br>pattern.<\/td><\/tr><tr><td>B-2<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2970\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/b2\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B2.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"B2\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B2.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2970\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B2.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B2.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B2.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><strong>Putative ID:<\/strong> <em>Brettanomyces<\/em>     <strong>Confidence:<\/strong> Low<br><strong>Notes:<\/strong> Cell morphology is consistent with <em>Brettanomyces<\/em> but <br>dividing cells were not observed and some cells appear apiculate.<\/td><\/tr><tr><td>B-3<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2971\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/b3\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B3.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"B3\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B3.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2971\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B3.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B3.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B3.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><strong>Putative ID:<\/strong> Unknown     <strong>Confidence:<\/strong> Low<br><strong>Notes:<\/strong> Cells are apiculate, which is unusual for <em>Brettanomyces<\/em>.<\/td><\/tr><tr><td>B-4<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2972\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/b4\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B4.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"B4\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B4.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2972\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B4.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B4.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/B4.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><strong>Putative ID:<\/strong> <em>Brettanomyces<\/em>     <strong>Confidence:<\/strong> Medium<br><strong>Notes:<\/strong> Cells show classical <em>Brettanomyces<\/em> budding pattern <br>and elongated cell morphology.<\/td><\/tr><tr><td>C-1<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2973\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/c1\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C1.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"C1\" data-image-description=\"&lt;p&gt;Brettanomyes nanus&lt;\/p&gt;\n\" data-image-caption=\"&lt;p&gt;Yeast clone C1&lt;\/p&gt;\n\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C1.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2973\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C1.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C1.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C1.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><strong>Putative ID:<\/strong> <em>Brettanomyces<\/em>     <strong>Confidence:<\/strong> Medium<br><strong>Notes:<\/strong> Cells show classical <em>Brettanomyces<\/em> budding pattern <br>and elongated cell morphology.<\/td><\/tr><tr><td>C-2<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2974\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/c2\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C2.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"C2\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C2.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2974\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C2.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C2.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C2.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><em><strong>Putative ID:<\/strong> <em>Brettanomyces<\/em>     <strong>Confidence:<\/strong> Low<br><\/em><strong>Notes:<\/strong> Cells show classical <em>Brettanomyces<\/em> budding pattern but <br>lack an elongated cell morphology.<\/td><\/tr><tr><td>C-3<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2975\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/c3\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C3.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"C3\" data-image-description=\"\" data-image-caption=\"&lt;p&gt;Yeasts Only&lt;\/p&gt;\n\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C3.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2975\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C3.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C3.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C3.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><strong>Putative ID:<\/strong> Unknown     <strong>Confidence:<\/strong> Low<br><strong>Notes:<\/strong> Cells are apiculate, which is unusual for <em>Brettanomyces<\/em>.<\/td><\/tr><tr><td>C-4<\/td><td><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"150\" height=\"150\" data-attachment-id=\"2976\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/c4\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C4.jpg?fit=250%2C250&amp;ssl=1\" data-orig-size=\"250,250\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"C4\" data-image-description=\"&lt;p&gt;Brettanomyces custersianus&lt;\/p&gt;\n\" data-image-caption=\"&lt;p&gt;Yeast clone C4&lt;\/p&gt;\n\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C4.jpg?fit=250%2C250&amp;ssl=1\" class=\"wp-image-2976\" style=\"width: 150px;\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C4.jpg?resize=150%2C150&#038;ssl=1\" alt=\"\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C4.jpg?w=250&amp;ssl=1 250w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/11\/C4.jpg?resize=150%2C150&amp;ssl=1 150w\" sizes=\"auto, (max-width: 150px) 100vw, 150px\" \/><\/td><td><strong>Putative ID:<\/strong> <em>Brettanomyces<\/em>     <strong>Confidence:<\/strong> Low<br><strong>Notes:<\/strong> Cells show classical <em>Brettanomyces<\/em> budding pattern <br>and elongated cell morphology. However, the colony also <br>accumulated some blue dye, and grew poorly on <br>cycloheximide, both of which are not typical of <em>Brettanomyces.<\/em><\/td><\/tr><\/tbody><\/table><\/figure>\n<\/div>\n<\/div>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"conclusions\">Conclusions &#8211; Did I find wild <em>Brettanomyces?<\/em><\/h3>\n\n\n\n<p>Clearly I have isolated several strains that I have putatively identified as wild <em>Brettanomyces<\/em>. This identification is based on five lines of evidence:<\/p>\n\n\n\n<ol class=\"wp-block-list\">\n<li><em>Rapid growth under anaerobic conditions, producing sufficient yeast to form a visible pellet.<\/em> This indicates that the yeast are highly fermentative and capable of tolerating the presence of alcohol.<\/li>\n\n\n\n<li><em>Growth in the presence of a high concentration of cycloheximide.<\/em> This is a capability of only a few species of yeast.<\/li>\n\n\n\n<li><em>Absence of blue colouration when grown on bromocresol green-containing medium<\/em>. <em>Brettanomyces<\/em> is one of the only yeast species capable of degrading this dye.<\/li>\n\n\n\n<li><em>Colony morphology<\/em>. The cell shape (ovoid-to-irregular) and pattern of cell division (cells bud from the termini of the mother cell, not the sides) is consistent with <em>Brettanomyces<\/em>.<\/li>\n\n\n\n<li><em>Vacuole<\/em>. The cells have a large central vacuole that is easily resolved by microscopy. Again, this is consistent with <em>Brettanomyces<\/em>.<\/li>\n<\/ol>\n\n\n\n<p>None of the above characteristics provide, on their own, a definitive identification. However, this particular combination of characteristics is unique to <em>Brettanomyces<\/em>. Even so, I only rated my confidence as a maximum of &#8220;medium&#8221;, as I cannot be certain of my identifications without additional tests. So while I am confident that several colonies are wild <em>Brettanomyces<\/em>, I do not yet have all the evidence needed for a firm identification nor for identification of specific species.<\/p>\n\n\n\n<p>None-the-less, b<strong>ased on these findings I am quite confident that I have isolated wild <em>Brettanomyces<\/em> from the plant rhizosphere!<\/strong> There are some interesting implications for this conclusion&#8230;but you&#8217;ll have to wait for part III of this experiment before I get into those.<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"limitations\">Limitations<\/h3>\n\n\n\n<p>There are a few limitations to how I conducted this experiment that are worth discussing: <\/p>\n\n\n\n<p><strong>Issue 1: Bias Introduced by Enrichment: <\/strong>The first of these is the consequence of using liquid cultures to enrich for <em>Brettanomyces<\/em>. My enrichment process selects for <em>Brettanomyces<\/em> by taking advantage of its ability to grow relatively quickly under fermentative (low\/no-oxygen) conditions in a liquid medium. The use of anaerobic conditions also served to eliminate non-yeast fungi and non-fermentative yeast, while cycloheximide suppressed the growth of all but a small handful of yeast species. While this approach appears to have worked very well &#8211; in that it did enrich for <strong><em>some<\/em> <\/strong><em>Brettanomyces<\/em> &#8211; it would not have enriched for <em><strong>all<\/strong> Brettanomyces.<\/em> <\/p>\n\n\n\n<p>In particular, this enrichment approach was based on the biology of <em>Brettanomyces bruxellensis<\/em>, and is unlikely to work for other <em>Brettanomyces<\/em> species. For example, <em>B. claussenii<\/em> and <em>B. anomalus<\/em> are not as quick to grow under fermentative conditions as is <em>B. bruxellensis<\/em>, and therefore may have been out-competed during sub-culturing. Likewise, <em>B. custersianus<\/em>, <em>B. naardenensis<\/em>, and <em>B. nanus<\/em> &#8211; at least by some reports &#8211; grow poorly in liquid culture and under low-oxygen conditions. Thus, my enrichment process may have eliminated &#8211; rather than enriched &#8211; these species. In other words, while this enrichment approach can be used to selectively enrich for strongly fermentative species of <em>Brettanomyces<\/em>, it would not be suitable for identifying the full range of <em>Brettanomyces <\/em>species present in a sample.<\/p>\n\n\n\n<p>In fact, it is very well possible that I started off with dozens of unique strains of <em>Brettanomyces<\/em> spread across three or four species, and ended up selecting for a single strain from one species, merely because that strain was a stronger fermenter and outgrew everything else.<\/p>\n\n\n\n<p><strong>Issue 2: Incomplete Selection: <\/strong>A second limitation is the dose of cycloheximide I used. While this dose is higher than what is used in most <em>Brettanomyces<\/em> studies, most of those studies are isolating <em>Brettanomyces<\/em> from environments such as fermented wine &#8211; e.g. environments that already enrich for <em>Brettanomyces<\/em> and which lack significant microbial diversity. After I had started this project I found papers which indicated that to truly select for <em>Brettanomyces<\/em> I needed to double the concentration of cycloheximide. As such, there is a theoretical possibility that two other species may have been isolated instead of, or alongside, <em>Brettanomyces.<\/em> The first of these &#8211;  <em>Kluyveromyces marxianus<\/em> &#8211; is not thought to be a soil organism, and rather is a lactose-fermenting yeast found on the udders of animals and in milk-derived foods (e.g. cheese &amp; kefir). It is also morphologically distinct from <em>Brettanomyces<\/em>, with none of my isolates having a morphology similar to <em>K. marxianus.<\/em> <\/p>\n\n\n\n<p>On the other hand, <em>Debaryomyces housenii <\/em>is a very common yeast that is known to live in soil, is cycloheximide-tolerant, is fermentative, has a similar cellular morphology to <em>Brettanomyces<\/em>, and divides in a similar fashion to <em>Brettanomyces<\/em>. The dose of cycloheximide I used (50 ug\/mL) is above that recommended for isolating <em>D. housenii<\/em> (10 ug\/mL), but may not be lethal to <em>D. housenii<\/em>. Information on whether <em>D. housenii<\/em> can decolourize bromocresol green was hard to find, <a href=\"https:\/\/academic.oup.com\/mmy\/article\/45\/2\/97\/1216685\">with only one paper commenting on it indirectly<\/a> &#8211; and seemingly indicating that <em>D. housenii<\/em> is incapable of decolourizing bromocresol green. But given the sparsity of information on <em>D. housenii<\/em>&#8216;s ability to declourize  bromocresol green, it remains a potential (if unlikely) organism isolated in this experiment. I also had one yeast species which appeared to divide by binary fission &#8211; if true, this would be a yeast which is only distantly related to either <em>Brettanomyces<\/em>, <em>Debaryomyces<\/em>, or <em>Kluyveromyces<\/em> (all of which are in the same <a href=\"https:\/\/en.wikipedia.org\/wiki\/Saccharomycetaceae\">Saccharomycetaceae <\/a>family of yeasts). If this truly is a fission yeast, than it is likely a species of <em><a href=\"https:\/\/en.wikipedia.org\/wiki\/Schizosaccharomyces\">Schizosaccharomyces<\/a><\/em>, from the Schizosaccharomycetaceae family of yeasts.<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"next\">What&#8217;s Next for the Wild <em>Brettanomyces<\/em> project?<\/h3>\n\n\n\n<p>That is it for this post, but it&#8217;s not the end of this project. I appear to have 7 potential isolates of wild <em>Brettanomyces<\/em>, plus 5 interesting non-<em>Brettanomyces <\/em>isolates that are worth deeper investigation (especially the putative fission yeast). <\/p>\n\n\n\n<p>I am in the process of <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2013\/04\/16\/identifying-yeasts-using-ribosomal-sequencing\/\">performing a genetic identification of all 12 strains<\/a>, which will give a firm identification of the species that I have purified. With luck, Part III of this series describing those results will be posted in one to two weeks. Once I have some firm identifications, I plan on performing some pure and mixed fermentations with any strains confirmed to be wild <em>Brettanomyces <\/em>(or otherwise of interest to brewers), to see what their flavour profile is like. Look for that post in December or early 2023.<\/p>\n\n\n\n<h5 class=\"wp-block-heading\"><strong>Other Parts to this Series:<\/strong><\/h5>\n\n\n\n<ul class=\"wp-block-list\">\n<li>Part I: <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/10\/06\/this-is-not-the-yeast-your-are-looking-for\/\">These Are Not The Yeast You Are Looking For<\/a><\/li>\n\n\n\n<li>Part II: <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/\">These ARE The Yeast Your Are Looking For<\/a> (this post)<\/li>\n\n\n\n<li>Part III: <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/06\/wild-brettanomyces-needle\/\">Wild Brettanomyces &#8211; a Needle in a Haystack<\/a><\/li>\n\n\n\n<li>Part IV: <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2023\/03\/24\/wild-brettanomyces-fermentation-test\/\">Fermentation characteristics<\/a><\/li>\n<\/ul>\n","protected":false},"excerpt":{"rendered":"<p>Not all experiments work&#8230;but some do! And that includes my second attempt to capture some wild Brettanomyces.<\/p>\n","protected":false},"author":1,"featured_media":2917,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"jetpack_post_was_ever_published":false,"_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":"","jetpack_publicize_message":"","jetpack_publicize_feature_enabled":true,"jetpack_social_post_already_shared":true,"jetpack_social_options":{"image_generator_settings":{"template":"highway","default_image_id":0,"font":"","enabled":false},"version":2}},"categories":[16,10,21,17,14],"tags":[160,205,178,177],"class_list":["post-2910","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-adventures","category-brewing-science","category-collecting-wild-yeasts","category-sour-n-wild","category-yeast-wrangling","tag-brettanomyces","tag-brewing-science","tag-hunting-wild-yeasts","tag-wild-yeast"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.3 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>These ARE The Yeast You Are Looking For - Sui Generis Brewing<\/title>\n<meta name=\"description\" content=\"Second time is the charm? 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