{"id":634,"date":"2013-08-14T00:58:00","date_gmt":"2013-08-14T00:58:00","guid":{"rendered":"http:\/\/suigenerisbrewing.com\/index.php\/2013\/08\/14\/another-wild-yeast-identification-test\/"},"modified":"2017-09-22T20:39:39","modified_gmt":"2017-09-22T20:39:39","slug":"another-wild-yeast-identification-test","status":"publish","type":"post","link":"https:\/\/suigenerisbrewing.com\/index.php\/2013\/08\/14\/another-wild-yeast-identification-test\/","title":{"rendered":"Another Wild Yeast Identification Test"},"content":{"rendered":"\n\n\n\n
<\/a><\/td>\n<\/tr>\n
Micrograph of the Wild Yeast (left) versus
\nconventional ale yeast (right)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Over a series of posts, listed below, I’ve covered my work designing a method to identify what species I’m purifying in my attempts<\/a> to isolate wild yeasts<\/a>. \u00a0This technique has concentrated on the sequencing ribosomal DNA<\/a>; stretches of DNA which are frequently sequenced for the purpose of species identification. \u00a0My first attempt<\/a> at this did not work well, but I had a higher degree of success upon re-targeting my sequencing attempts<\/a> to a different region of the ribosome.<\/p>\n

In parallel with these experiments I have been purifying a range of wild strains<\/a>\u00a0(edit: completed here<\/a>) which will soon be tested for their fermentation characteristics. \u00a0Once good brewing strains are identified, I will further characterize them – including identifying the species via ribosomal sequencing.<\/p>\n

Before I go down that road I want to test the sequencing method on a bona fide wild strain. \u00a0For this test I am using a strain kindly provided by fellow wild brewer Doc_Drive. \u00a0This yeast was featured in an earlier<\/a> post of mine as an example of suing old yeast-identification methods. \u00a0In this post I tentatively identified this yeast as\u00a0Kloeckera apiculata<\/i>\u00a0based on<\/span> its morphology and described fermentation characteristics.<\/p>\n

I have now positively identified this yeast as Kloeckera<\/i>, based on ribosomal sequencing; the full story can be found below the fold…yes, I was right!
\n<\/a><\/p>\n

\n

Approach:<\/b><\/p>\n\n\n\n\n
<\/a><\/td>\n<\/tr>\n
Something isn’t right…<\/p>\n
Left: Ladder<\/div>\n
Middle: ITS PCR<\/div>\n
Right: 26s PCR<\/div>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

As per usual, this trial starts with a DNA isolation – in this case I tried the simplest method available; a small amount of yeast was suspended in 100ul of water, and boiled for 15 minutes. \u00a0The solution was then centrifuged to pellet any cell debris. \u00a0I then PCR amplified<\/a>\u00a0both sections of the ribosome I have primers for (e.g. the ‘old’ (ITS) and ‘new’ (26s) primers), using a 49C annealing temperature and 45 amplification cycles. \u00a0When run on a gel, I got the picture on the left.<\/p>\n

The resulting bands were then purified, and sent for sequencing. \u00a0Due to the small amounts of DNA present, I didn’t get a good sequence off of the NS1 primers. \u00a0The ITS primers gave the following sequences on NCBI Blast<\/a>:<\/p>\n\n\n\n\n
PCR<\/b><\/td>\nSequence<\/b><\/td>\nIdentification<\/b><\/td>\n<\/tr>\n
ITS<\/td>\nATGTTGCTGCGTTGGGAAGCGAAGCTGGCGGCACTTTGTACGGTTTGCAGGTACTGgac<\/span>NgNATtGaAgCAAATC<\/span>
\nAGCGACAAAACTTCACCCGATTTGTGGTGTTGGCGCGTAAAGCCATTAACGTGTCTGATCAGGT<\/span>TCCGGCGAAAA<\/span>
\nCCACGTTGTTAATGGCGACCGGGCAACAAGCCGGTGCGCTGGTTGAAGCGTTGCTGGTACTGCGCAACC<\/span>ACAATC<\/span>
\nTGATTATGACCCGTCTTGAAACACGGACCANNGGGCACAGGCTGGAGGACAGACGGGGGTTGCCAGGTGGCTCT<\/span>G<\/span>
\nGGACAAGCCCAAGCTGCTCCCTGAAGGTTTCCCTCTTT<\/span><\/td>\n		Escherichia coli\u00a0<\/i>P12b<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Well shit, not what I was hoping for. \u00a0Looks like somewhere in the mailing\/reculturing chain some nasty bacteria got in – guess that’s what I get for not using an antibiotic containing plate.<\/p>\n

\n

So I streaked out my stored ‘yeast’ on an antibiotic plate, repeated the sequencing and got the following:<\/p>\n\n\n\n\n
PCR<\/b><\/td>\nSequence<\/b><\/td>\nIdentification<\/b><\/td>\n<\/tr>\n
ITS<\/td>\naaaccaactg ggattacctt agtaacggcg agtgaagcgg taaaagctca aatttgaaat ctggtacttt cagtgcccga gttgtaattt gtagaatttg tctttgatta ggtccttgtc tatgttcctt ggaacaggac gtcatagagg gtgagaatcc cgtttggcga ggataccttt tctctgtaag actttttcga agagtcgagt tgtttgggaa tgcagctcaa agtgggtggt aaattccatc taaagctaaa tattggcgag agaccgatag cgaacaagta cagtgatgga aagatgaaaa gaactttgaa aagagagtga aaaagtacgt gaaattgttg aaagggaagg gcatttgatc agacatggtg ttttttgcat gcactcgcct ctcgtgggct tgggcctctc aaaaatttca ctgggccaac atcaattctg gcagcaggat aaatcatt<\/span><\/td>\nHanseniaspora uvarum<\/i><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

It worked! \u00a0Not only did it work, but my first tentative identification of the yeast – based on\u00a0morphology & a bit of a description of where the yeast was found<\/a>\u00a0– ended up being right!\u00a0 It is hanseniasporia<\/i>\u00a0– AKA the Kloeckera<\/i> I had tentatively identified it as before.<\/div>\n","protected":false},"excerpt":{"rendered":"

Micrograph of the Wild Yeast (left) versus conventional ale yeast (right) Over a series of posts, listed below, I’ve covered<\/p>\n","protected":false},"author":1,"featured_media":0,"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":false,"jetpack_social_options":{"image_generator_settings":{"template":"highway","default_image_id":0,"font":"","enabled":false},"version":2}},"categories":[16,21,17,14],"tags":[178,177],"class_list":["post-634","post","type-post","status-publish","format-standard","hentry","category-adventures","category-collecting-wild-yeasts","category-sour-n-wild","category-yeast-wrangling","tag-hunting-wild-yeasts","tag-wild-yeast"],"yoast_head":"\nAnother Wild Yeast Identification Test - 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