{"id":690,"date":"2012-11-13T16:37:00","date_gmt":"2012-11-13T16:37:00","guid":{"rendered":"http:\/\/suigenerisbrewing.com\/index.php\/2012\/11\/13\/yeast-banking-ii-methods-to-manage-the-bank\/"},"modified":"2017-09-15T19:08:53","modified_gmt":"2017-09-15T19:08:53","slug":"yeast-banking-ii-methods-to-manage-the-bank","status":"publish","type":"post","link":"https:\/\/suigenerisbrewing.com\/index.php\/2012\/11\/13\/yeast-banking-ii-methods-to-manage-the-bank\/","title":{"rendered":"Yeast Banking II: Methods to Manage the Bank"},"content":{"rendered":"<p>As discussed in my <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2012\/11\/12\/yeast-banking-i-managing-a-large-bank\/\">last article<\/a>, I am now managing a yeast bank on behalf of the<a href=\"http:\/\/www.londonbrewers.ca\/\"> London Home Brewers Guild<\/a>. In the previous article I outlined the methods we use, and the rational behind using those methods over other conventional methods. In this post, I will outline how the bank itself is run, with detailed protocols for the clean-up, freezing and \u201cwithdrawal\u201d of the yeasts.<\/p>\n<div style=\"margin-bottom: 0cm;\"><\/div>\n<div style=\"margin-bottom: 0cm;\">As mentioned before, these methods have been implemented assuming you have access to the facilities available in a biology lab \u2013 i.e. biosafety cabinet, micropipettes, autoclave, etc. In the future I hope to post an article on how this method can be implemented using the kinds of equipment the average home brewer can access.<\/div>\n<div style=\"margin-bottom: 0cm;\"><\/div>\n<div style=\"margin-bottom: 0cm;\"><i>Methods in this article:<\/i><\/div>\n<ul>\n<li>\n<div style=\"margin-bottom: 0cm;\">Preparing &amp; freezing the yeast<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Checking for contamination<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Secondary cleanup<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">How to perform a withdrawal<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Counting yeast<\/div>\n<\/li>\n<\/ul>\n<div style=\"margin-bottom: 0cm;\"><\/div>\n<p><a name=\"more\"><\/a><\/p>\n<hr \/>\n<p><b>Preparing &amp; Freezing the Yeast:<\/b><\/p>\n<div style=\"margin-bottom: 0cm;\">The principal here is simple; fungal-derived antibiotics are toxic to bacteria, while being realtivly non-toxic to fungi such as yeast. By using a couple of these antibiotics we can clear our stocks of most contaminating bacteria (unfortunately, contamination with wild yeast is a much more difficult issue, dealt with by tossing the yeast from the bank). To achieve this, we use two antibiotics \u2013 100U\/ml penicillin and 100ug\/ml streptomycin. Penicillin kills gram positive bacteria \u2013 which includes common beer spoilers such as <i>pediococcus <\/i>&amp; <i>lactobacillus<\/i>. Streptomycin is a bacteria-derived antibiotic which kills a range of bacteria, including many gram negative (i.e. <i>Acetobacter<\/i>), gram positive, and some fungal organisms. Thankfully, <i>saccharomyces<\/i> yeast species are relatively resistant to this antibiotic, allowing us to use it to purify our cultures.<\/div>\n<div style=\"margin-bottom: 0cm;\"><\/div>\n<div style=\"margin-bottom: 0cm;\">Pure yeast cultures are then concentrated and frozen in a solution of 30% glycerol\/wort. This solution prevents ice crystals from forming during the freezing process, thereby allowing the yeast to remain viable for years.<\/div>\n<div style=\"margin-bottom: 0cm;\"><\/div>\n<div style=\"margin-bottom: 0cm;\"><i><b>Method:<\/b><\/i><\/div>\n<ol>\n<li>\n<div style=\"margin-bottom: 0cm;\">\n<div style=\"clear: both; text-align: center;\"><\/div>\n<table style=\"float: right; text-align: center;\" cellspacing=\"0\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\"><a style=\"clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;\" href=\"https:\/\/i0.wp.com\/2.bp.blogspot.com\/-OiqyBqUh8wI\/UKJyfIeYFoI\/AAAAAAAAAQo\/KV_tupKkWAc\/s1600\/innoculate.png\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" src=\"https:\/\/i0.wp.com\/2.bp.blogspot.com\/-OiqyBqUh8wI\/UKJyfIeYFoI\/AAAAAAAAAQo\/KV_tupKkWAc\/s200\/innoculate.png?resize=200%2C127&#038;ssl=1\" width=\"200\" height=\"127\" border=\"0\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">Sterile\u00a0media with antibiotics (left),<br \/>\nfresh White Labs yeast\u00a0and\u00a0washed<br \/>\nyeast (tubes in racks) as well as<br \/>\nmicropipettors, in a \u00a0biosafety cabinet<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>Prepare 50ml of 1.035 to 1.040 wort from DME, in a 250ml flask. Cap with foil and boil for 10 min or autoclave in wet cycle to sterilize. Once cooled, add antibiotics at the desired concentration.<\/p>\n<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Prepare a solution of 30% glycerol in 1.035 to 1.040 wort. Autoclave to sterilize. A large volume of this can be prepared in advance and stored in the fridge; only 2ml is required per banked yeast. For 100ml, add 30ml of glycerol to 70ml of water and 9.8g of DME.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">In a biosafety cabinet, add yeast sterilely to the 50ml flask of wort, using the following guidelines:<\/div>\n<\/li>\n<\/ol>\n<ul>\n<li>\n<div style=\"margin-bottom: 0cm;\">\n<table style=\"float: right; text-align: right;\" cellspacing=\"0\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\"><a style=\"clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;\" href=\"https:\/\/i0.wp.com\/4.bp.blogspot.com\/-eKdPc7NL5s0\/UKJygBlSXLI\/AAAAAAAAAQ4\/cjs30tKkLL0\/s1600\/shaker.png\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" src=\"https:\/\/i0.wp.com\/4.bp.blogspot.com\/-eKdPc7NL5s0\/UKJygBlSXLI\/AAAAAAAAAQ4\/cjs30tKkLL0\/s200\/shaker.png?resize=200%2C200&#038;ssl=1\" width=\"200\" height=\"200\" border=\"0\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">4 strains on the shaker<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>If starting with a yeast slurry (i.e. yeast from a White Labs tube, washed or top-cropped yeast, etc), suspend the slurry in an approximately equal volume of water or wort. For white lab yeasts, simply mix the sedimented yeast into the liquid contents of the tube. Add 500ul (0.5ml) of this slurry to the wort.<\/p>\n<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">If starting with an active liquid culture (i.e. starter, yeast cultured from a bottle, wyeast smack-pack), transfer 5ml of the active culture to the wort.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">If starting with a dried yeast, add 5-10 prills to the wort.<\/div>\n<\/li>\n<\/ul>\n<ol start=\"3\">\n<li>\n<div style=\"margin-bottom: 0cm;\">Place on a shaker, 20-24C, at 200-250RPM, for 18 to 24 hours. This will let the yeast grow to a density of 5 to 10 million\/ml. This is the point where we want to harvest the yeast, as they are minimally stressed; longer incubations will produce larger numbers of yeast, but the increasing alcohol concentration will stress the yeast, leading to poorer recovery after freezing.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">\n<table style=\"float: right; margin-left: 1em; text-align: right;\" cellspacing=\"0\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\"><a style=\"clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;\" href=\"https:\/\/i0.wp.com\/1.bp.blogspot.com\/--Vr-_k-2mPg\/UKJydY_7RXI\/AAAAAAAAAQI\/dMuV51lXiww\/s1600\/centrifuge02.png\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" src=\"https:\/\/i0.wp.com\/1.bp.blogspot.com\/--Vr-_k-2mPg\/UKJydY_7RXI\/AAAAAAAAAQI\/dMuV51lXiww\/s200\/centrifuge02.png?resize=118%2C200&#038;ssl=1\" width=\"118\" height=\"200\" border=\"0\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">Yeast pelleted<br \/>\nby\u00a0centrifugation<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>Transfer the culture to a sterile 50ml centrifuge tube. Spin for 10 min at 500g \u2013 this is sufficient to pellet all but the least flocculant of strains; if a large pellet is not obvious by the end of the centrifugation period, re-spin at 750g for 10min. If possible, avoid the higher g-forces as these can damage the yeast.<\/p>\n<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">\n<p>Remove the supernatant and suspend the yeast pellet in 2ml of 30%glycerol\/wort. Sterially transfer to two sterile cryovials, 1ml per vial.<\/p>\n<blockquote style=\"border: none; margin: 0 0 0 40px; padding: 0px;\"><p>&#8211; If desired, remove 10ul to test for contamination (see next section)<\/p><\/blockquote>\n<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Freeze at -18C to -80C. Store the duplicate vials in different freezers to insure against loss.<\/div>\n<\/li>\n<\/ol>\n<div style=\"margin-bottom: 0cm;\">\n<hr \/>\n<p><b>Checking for Contamination:<\/b><\/p>\n<\/div>\n<div style=\"margin-bottom: 0cm;\">The method I use for checking for contamination is quite simple, and can detect contaminations as low as 1:100,000 bacteria:yeast (in \u201cnormal\u201d infections, bacteria generally out-number yeast by at least 10:1). For this method you need a microscope with 40x-60x magnification, a glass slide and a cover glass.<\/div>\n<div style=\"margin-bottom: 0cm;\">\n<table style=\"margin-left: auto; margin-right: auto; text-align: center;\" cellspacing=\"0\" cellpadding=\"0\" align=\"center\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\"><a style=\"margin-left: auto; margin-right: auto;\" href=\"https:\/\/i0.wp.com\/2.bp.blogspot.com\/-YDje0Ff0fX0\/UKJye6SuHrI\/AAAAAAAAAQg\/Y5eqRLfgoSk\/s1600\/infected.png\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" src=\"https:\/\/i0.wp.com\/2.bp.blogspot.com\/-YDje0Ff0fX0\/UKJye6SuHrI\/AAAAAAAAAQg\/Y5eqRLfgoSk\/s320\/infected.png?resize=320%2C320&#038;ssl=1\" width=\"320\" height=\"320\" border=\"0\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">S-05 Yeast (large cells in middle) in a culture heavily<br \/>\ncontaminated\u00a0with\u00a0<i>acetobacter<\/i> (small, rod-shaped cells). \u00a0Yeast<br \/>\nare out-numbered 100:1 by\u00a0bacteria\u00a0in this infection<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p><i><b>Method:<\/b><\/i><\/p>\n<\/div>\n<ol>\n<li>\n<div style=\"margin-bottom: 0cm;\">Pipette 10ul of yeast-containing solution onto\u00a0centre\u00a0of the slide. For concentrated sources (i.e. pure yeast slurry), you may need to dilute 1:10 (tap water is fine) before this step.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Place cover glass over droplet, allowing yeast solution to spread between the cover glass and the slide.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Image under the microscope. Yeast will appear as spheres or buds (see above image), contaminating bacteria will be much smaller, and either spherical or rod-shaped in morphology. Debris will appear as black dots \u2013 do not confuse these with bacteria! For a comparison, an equal volume of the sterile media used to grow the yeast can be viewed.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">View at least 10 fields; if there is less than 1 bacteria per field (40x) or less than 1 bacteria per 2 fields (60x) your stock is clean enough for most uses.<\/div>\n<\/li>\n<\/ol>\n<div style=\"margin-bottom: 0cm;\">\n<hr \/>\n<p><b>Secondary Cleanup:<\/b><\/p>\n<\/div>\n<div style=\"margin-bottom: 0cm;\">Sometimes, even after culturing in antibiotic-containing wort, bacteria will still be present. In most cases this means the source of the yeast was heavily contaminated, and instead of attempting a new recovery, a new source of yeast should be found. If this is not possible, a secondary clean-up can be attempted. There are two methods that can be used:<\/div>\n<div style=\"margin-bottom: 0cm;\"><\/div>\n<div style=\"margin-bottom: 0cm;\">\n<table style=\"float: right; margin-left: 1em; text-align: right;\" cellspacing=\"0\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\"><a style=\"clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;\" href=\"https:\/\/i0.wp.com\/4.bp.blogspot.com\/-jPSKk-7l4wU\/UKFQm_204nI\/AAAAAAAAAPs\/z6JV4WIsRhc\/s1600\/yeast.jpg\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" src=\"https:\/\/i0.wp.com\/4.bp.blogspot.com\/-jPSKk-7l4wU\/UKFQm_204nI\/AAAAAAAAAPs\/z6JV4WIsRhc\/s200\/yeast.jpg?resize=200%2C200&#038;ssl=1\" width=\"200\" height=\"200\" border=\"0\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">S-05 from the infected culture (see above),<br \/>\nafter clean-up method 1. \u00a0No contaminating<br \/>\nbacteria\u00a0are present.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p><i><b>Method 1:<\/b><\/i><\/p>\n<\/div>\n<div style=\"margin-bottom: 0cm;\">This method assumes the bacteria are being inhibited by the antibiotic, and were present in the post-growth culture in an inhibited state. By growing the yeast up from a highly diluted state, these bacteria can be removed.<\/div>\n<ol>\n<li>\n<div style=\"margin-bottom: 0cm;\">Put 5ml of sterile 1.035 to 1.040 wort into a sterile culture tube.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Add antibiotics at 2X the normally used concentration.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Inoculate a small volume (10-20ul) of the contaminated yeast.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Grow for 24 hours, room temperature, with continuous shaking to oxygenate and suspend the yeast.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Transfer 10-20ul of this solution into a new tube of 5ml 1.035 to 1.040 wort + antibiotics, and culture for another 24 hours.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">Check for infection; if clean use the 1ml of the culture prepared in step 5 to inoculate a 50ml flask (with antibiotics). Culture and freeze as per usual.<\/div>\n<\/li>\n<\/ol>\n<div style=\"margin-bottom: 0cm;\"><\/div>\n<div style=\"margin-bottom: 0cm;\"><i><b>Method 2:<\/b><\/i><\/div>\n<ol>\n<li>\n<div style=\"margin-bottom: 0cm;\">Prepare a sterile agar plate of YPD yeast media, or sterile plate of 2% agar\/1.035 wort, with antibiotics at their normal concentration. <u>DO NOT<\/u> double the antibiotic concentration for this method.<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\"><a href=\"http:\/\/biology.about.com\/od\/biologylabhowtos\/ht\/streak-a-bacterial-culture.htm\">Streak\u00a0<\/a><\/span><span lang=\"en-US\">20-50ul of the contaminated yeast onto the plate.<\/span><\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">Culture at room temperature until visible colonies form (generally 24 &#8211; 48 hours)<\/span><\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">Prepare 4-5 culture tubes containing 5ml 1.035-1.040 wort + antibiotics, for each tube pick a single colony off of the plate and add to the tube.<\/span><\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">Grow 24 hours (shaking, etc) at room temperature and check for infection. Tubes showing a pure-yeast culture can be pooled, and 1ml used to inoculate a 50ml flask (with antibiotics). Culture and freeze as per usual.<\/span><\/div>\n<div style=\"margin-bottom: 0cm;\"><\/div>\n<\/li>\n<\/ol>\n<div style=\"margin-bottom: 0cm; text-indent: -0.02cm;\">\n<hr \/>\n<p><b><span lang=\"en-US\">How to Perform a &#8216;Withdrawal&#8217;:<\/span><\/b><\/p>\n<\/div>\n<div style=\"margin-bottom: 0cm; text-indent: -0.02cm;\">\n<table style=\"float: left; margin-right: 1em; text-align: left;\" cellspacing=\"0\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\"><a style=\"clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;\" href=\"https:\/\/i0.wp.com\/2.bp.blogspot.com\/-miFsvgB11Cg\/UKJydzuq29I\/AAAAAAAAAQQ\/Ylo8gHMh35E\/s1600\/flameloop.png\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" src=\"https:\/\/i0.wp.com\/2.bp.blogspot.com\/-miFsvgB11Cg\/UKJydzuq29I\/AAAAAAAAAQQ\/Ylo8gHMh35E\/s200\/flameloop.png?resize=86%2C200&#038;ssl=1\" width=\"86\" height=\"200\" border=\"0\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">Flaming a<br \/>\nculture\u00a0loop<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<table style=\"float: right; text-align: right;\" cellspacing=\"0\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\"><a style=\"clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;\" href=\"https:\/\/i0.wp.com\/2.bp.blogspot.com\/-_VUY5fhGvT4\/UKJ2DAMFx0I\/AAAAAAAAARU\/rRIFkqE53LE\/s1600\/withdrawal2.png\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" src=\"https:\/\/i0.wp.com\/2.bp.blogspot.com\/-_VUY5fhGvT4\/UKJ2DAMFx0I\/AAAAAAAAARU\/rRIFkqE53LE\/s200\/withdrawal2.png?resize=82%2C200&#038;ssl=1\" width=\"82\" height=\"200\" border=\"0\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">&#8216;Withdrawal&#8217;\u00a0ready<br \/>\nto pitch into a<br \/>\nstarter<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p><span lang=\"en-US\">Withdrawals are easy \u2013 a small amount of yeast are removed from the frozen stocks, placed into 5 to 7 ml of sterile media, and grown for 24 to 36 hours. This tube of yeast \u2013 containing anywhere from 25 million to 700 million yeast &#8211; can be pitched into a 250ml starter, then into a 1.5L starter, providing the ~100 billion cells needed for the average 5 gallon ale (an additional step-up is needed for larger volumes or lagers). Because the freezing media, growth media and starters are antibiotic free, there will be no residual antibiotics going into the beer. The withdrawal protocol will only cover the actual withdrawal process \u2013 stepping-up will be a topic of a later post.<\/span><\/p>\n<\/div>\n<div style=\"margin-bottom: 0cm; text-indent: -0.02cm;\"><\/div>\n<div style=\"margin-bottom: 0cm; text-indent: -0.02cm;\"><i style=\"text-indent: -0.02cm;\"><b><\/b><\/i><br \/>\n<i style=\"text-indent: -0.02cm;\"><b><\/b><\/i><i style=\"text-indent: -0.02cm;\"><b><span lang=\"en-US\">Method:<\/span><\/b><\/i><\/div>\n<ol>\n<li>\n<div style=\"margin-bottom: 0cm;\">\n<table style=\"float: right; margin-left: 1em; text-align: right;\" cellspacing=\"0\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\"><a style=\"clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;\" href=\"https:\/\/i0.wp.com\/2.bp.blogspot.com\/-S67T-NnklHo\/UKJyfSZMavI\/AAAAAAAAAQw\/9kHWVgbGNSc\/s1600\/loop.png\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" src=\"https:\/\/i0.wp.com\/2.bp.blogspot.com\/-S67T-NnklHo\/UKJyfSZMavI\/AAAAAAAAAQw\/9kHWVgbGNSc\/s200\/loop.png?resize=200%2C200&#038;ssl=1\" width=\"200\" height=\"200\" border=\"0\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">Culture Loop<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p><span lang=\"en-US\">All of these steps must be done under sterile conditions, using either a flame or a biocontainment hood.<\/span><\/p>\n<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">Using sterile methods, transfer 5 to 7ml of 1.035 to 1.040 wort into a sterile culture tube<\/span><\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">Using a flame, flame and cool a transfer loop. Use this to collect 10-20ul (one loops worth) of yeast from the frozen vial. Be sure to cool the loop as to not kill the yeast \u2013 to be certain, touch the loop to the inside of the cryovial before contacting the yeast. Never touch the outside of the cryovial (or any other potentially contaminated surface) before putting the loop into yeast.<\/span><\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">Swirl the yeast-filled loop in the culture tube of media<\/span><\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\">\n<table style=\"margin-left: auto; margin-right: auto; text-align: center;\" cellspacing=\"0\" cellpadding=\"0\" align=\"center\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\"><a style=\"clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;\" href=\"https:\/\/i0.wp.com\/1.bp.blogspot.com\/-Gbw6S06uLG4\/UKJ2Ck7-UhI\/AAAAAAAAARM\/gkjok2c1j-4\/s1600\/withdrawal1.png\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" src=\"https:\/\/i0.wp.com\/1.bp.blogspot.com\/-Gbw6S06uLG4\/UKJ2Ck7-UhI\/AAAAAAAAARM\/gkjok2c1j-4\/s200\/withdrawal1.png?resize=147%2C200&#038;ssl=1\" width=\"147\" height=\"200\" border=\"0\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">Withdrawals on the shaker<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p><span lang=\"en-US\">Loosely cap the culture tube to allow air transfer, and place on a shaker at 20 to 28C. Shake sufficiently to aerate, 24 to 36 hours.<\/span><\/p>\n<\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">If desired, check for contamination, then pitch into a 250ml starter.<\/span><\/div>\n<\/li>\n<\/ol>\n<hr \/>\n<div style=\"margin-bottom: 0cm; text-indent: -0.02cm;\">\n<table style=\"float: right; margin-left: 1em; text-align: right;\" cellspacing=\"0\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td style=\"text-align: center;\"><a style=\"clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;\" href=\"https:\/\/i0.wp.com\/3.bp.blogspot.com\/-BBkuq4aLya8\/UKJyeRybB7I\/AAAAAAAAAQY\/6Xtx9VJe7yw\/s1600\/hemocytometer.png\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" src=\"https:\/\/i0.wp.com\/3.bp.blogspot.com\/-BBkuq4aLya8\/UKJyeRybB7I\/AAAAAAAAAQY\/6Xtx9VJe7yw\/s320\/hemocytometer.png?resize=320%2C320&#038;ssl=1\" width=\"320\" height=\"320\" border=\"0\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">Yeast in a hemocytomere, ready to be counted!<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p><b><span lang=\"en-US\">How to Count Yeast:<\/span><\/b><\/p>\n<\/div>\n<div style=\"margin-bottom: 0cm; text-indent: -0.02cm;\"><span lang=\"en-US\">At any point in any of these processes, you may wish to perform a yeast count, in order to determine the concentration of yeast. This is simple, assuming you have a microscope with 10x to 20x magnification and a <a href=\"http:\/\/en.wikipedia.org\/wiki\/Hemocytometer\">hemocytometer<\/a>.<\/span><\/div>\n<div style=\"margin-bottom: 0cm; text-indent: -0.02cm;\"><\/div>\n<div style=\"margin-bottom: 0cm; text-indent: -0.02cm;\"><i><b><span lang=\"en-US\">Method:<\/span><\/b><\/i><\/div>\n<ol>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">Assemble the hemocytometer by placing a cleaned cover glass onto the cleaned hemocytometer.<\/span><\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">Inject 10ul of yeast between cover glass and the hemocytometer<\/span><\/div>\n<\/li>\n<\/ol>\n<ul>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">It may be necessary to dilute the yeast sample 1:10 or 1:100 to get countable numbers<\/span><\/div>\n<\/li>\n<\/ul>\n<ol start=\"3\">\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">Count the number of yeast in the four corners of the hemocytometer grid (i.e. the four areas containing the largest-sized grid).<\/span><\/div>\n<\/li>\n<li>\n<div style=\"margin-bottom: 0cm;\"><span lang=\"en-US\">The number of yeast per ml is = (number of yeast cells counted\/4) x 10<sup>4<\/sup> x dilution<\/span><\/div>\n<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"<p>As discussed in my last article, I am now managing a yeast bank on behalf of the London Home Brewers<\/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":[14,15],"tags":[166,65],"class_list":["post-690","post","type-post","status-publish","format-standard","hentry","category-yeast-wrangling","category-home-yeast-lab","tag-starters","tag-yeast-bank"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.3 - 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