Such a GREAT presentation! There are yeasts that we use all the time and those we don;t need to store or culture, but there are others that we use infrequently and those that are in danger of being taken off the market. Those are exactly what you want to do this for. Subbed...fantastic, approachable method. Cheers!
Thank you good sir. I keep rereading "Yeast" and "Brewing Yeast and Fermentation" but have no visual examples of this. I'm moving away from my homebrew shop, four hours away from the closest one. So this is vital for me.
I just wanted to say that these videos are really fantastic and exactly what I was looking for as I start my yeast bank. Thank you so much for making these!
Got some slants done on my second try. On the first attempt, I didn't completely dissolve the agar and it settled on me and I thought it might dissolve in the pressure cooker but the rack I had warped, and everything was ruined. I got a jar of starter wort to seal though so it wasn't a total loss. I do have hard water stains all over the glass so the suggestion to put some vinegar into the water might be a good one. I'm just going to use RO water next time. Thanks for the video. I tried making PDA slants but all my water evaporated when I boiled the potato. I decided to just go DME for now.
Sorry Steve, missed your post. PDA drying it is not an uncommon issue if you try to make too small a batch. Its easier to make ~1L and toss what you don't need (it only costs you half a potato), or if you want to make smaller amounts, add water throughout the boil to keep the boil volume right.
Hi Sui, I wanna thank u for these videos as they are very helpful in yeast culturing, actually I came here cause theres a mention of ur videos in the Yeast book....I am planning a mini brewery and can I still go ahead with the same procedures you've explained for commercial purpose............thank you......cheers
Hello Bryan, does storing yeast on slants cause endospores due to lack of nutrition, as you say yeast is good for storage for upto 2 years, and the other concern is due to the number of passages, does restreaking cause genetic drift in the cells.........thanks and cheers.
Great videos. I notice you don't flame the handle part of the inoculation loop, yet when sticking the loop all the way to the bottom the handle enters the slant tube. Couldn't that cause contamination?
It can if you're not careful. My handel isn't actually for holding the loups and has internal plastic parts, so I cannot flame it properly as it may melt.
Great, thorough video! Thank you very much for this! I have a quick question: could mineral oil be replaced with glycerol? Is there any particular reason mineral oil is used? Thanks again!
+Brian McConeghy Mineral oil is used to both prevent dehydration and block oxygen. I do not think glycerol would work as it is hydroscopic and also membrane-disruptive to the yeast. Used at 15-50% (in water) glycerol can be used for freezing yeast, but placing a pure solution over a slant would likely be lethal.
Thanks for the video! Very informative and to the point. Sorry ahead of time for continuing the questions over two years after you published, but........ Can you speak to any cons using water or oil in the slants that you have seen? On the surface it seems silly not to add that one step to significantly increase the shelf life. One step that, for what it's worth, that is void from other videos on the subject......
adam morrison No worries commenting on an old video. Mineral oil is the better option; water sometimes detaches the yeast from the slant, but with oil they seem to stay put. Air is the worst option, as dehydration greatly shortens the life of the culture.
Hey. Love the videos very helpful. I made my first agar slants but I used my instant pot. I later read the pressure setting is only 11.6 at around 242. Are my slants safe if nothing grows on them for a week and should I pour out my wort starter tubes I pressure cooked due to the temp difference?
@@mattblackburn1450 yep. There is perhaps a half-dozen organisms that would survive in the "window" between what your canner provides and what you get with a full 2ATA/121C canner/autoclave. None of those organisms grow in wort.
Mineral oil is a highly purified/distilled petrolium oil. If its not sold as "mineral oil" where you live, it may be sold under one of several other names: white oil, paraffin oil, or liquid paraffin, are common names used outside of north america. You'll usually find it at a pharmacy - its also the primary ingredient in baby oil (which is scented mineral oil).
Hi, impressive tutorial series !!! Thanks one more time! In the mean time, I will appreciate if you will supply some details regarding the "mineral oil" liquid. What kind of? Where to look for?
Hi Bryan. I note that you use a single colony of yeast to inoculate your starters etc. I have read elsewhere on the web (so may of course be incorrect) that using a colony formed from a single yeast as opposed to several colonies might lose some of the variation within the yeast such as flocculation behaviour, flavour and aroma profile and will not have all the characteristics of the complete 'strain'. I am sure you would not be using a single colony for inoculation if that was the case, so I am wondering what is the alternative explanation as from my limited microbiological knowledge losing some of the variation within a 'strain' by only using a single colony would seem to be a possible outcome of this method?
How many yeast I pick depends on my goals: -Cleaning an infected culture: 1 colony/tube, 5-6 colonies (e.g. tubes) total. Clean tubes get mixed into one culture. -Purifying wild strains: 1 colony -Culture maintenance: 10 or more colonies
@@SuiGenerisBrewing Thanks, so at least to some extent it is true and colonies have to be handled according to your purpose. It would be useful to do some more reading up on this, do you know any good sources of information on this subject (online advice, journal articles or books) please, but no worries if not? I can digest quite technical material if needed.
First of all thanks for your videos, I have a problem, using 2% of agar the agar doesn't solidified well I have increased to 4% of agar do you think that this will affect the slant ?
Here is a question for you. Since the yeast can only live up to a certain alcohol content, Would it not be possible to run a small line off a vat, run it through distillation the remainder being cooled and reintroduced into the VAT fermenting process, in this way keeping the yeast in their comfort zone until all sugars have been expended? making the sugar the limiting factor not the alcohol content. And with longer lives who knows what the yeast can accomplish. #Yeast lives matter# Another question, Has anyone ever reduced particle size of the ingredients into the nano realm with sonication/ultrasound which would increase their surface area while also increasing bioavailability to the yeast aiding in consumption and conversion of the ingredients. Would that speed up and increase yield in the fermentation process? I have used the ultrasound with oak and cinnamon to help age the first batch I ever made at the end of the process. The cinnamon came thru super strong so I distilled again with a little charcoal and toasted oak hoping to mellow it. It helped a little. I then used the resulting alcohol in my liposomal vitamin production which helped quite a bit in palatability and decreased viscosity. I actually drink it now instead of dreading it like the batch before it. If you should wish to try an ultrasonic unit, do not buy the harbor freight version as it is pretty weak. I bought two of the same transducers and mounted them under a heavy round stainless 9in. by 9in container. I tried the tinfoil test and the H.F. unit Put maybe 2 dozen holes in the tinfoil in 3 minute. My home made unit made confetti out of it in ten seconds. Both units cost me around eighty dollars. Just waiting to brew up some more alcohol and try it. Interested to hear your results should you think any of this interesting. I would love to hear if my theories have any merit or even turn out to be game changers.
Great video! Something's odd to me tho, that you unscrew the tubes and just leave the lids on the table and then don't flame them when screwing them back on. Is this not an issue? Also, when you put the bacteriological ring in the tube to inoculate it, you flame the ring itself, but not the whole body that also goes into the tube. Is that okay? Do you sterilize the whole body before somehow? Sorry for asking about minute details, but I want to bank my yeast for the first time and trying to minimize my chances of messing up!! Regards!
The loop is an issues that come with trying to film and talk while doing microbiology. Ideally, any part of the loop that goes into the tube should be flamed to reduce the risk of contamination - or you avoid touching the tube with the unflammed part. I didn't do a good job of either in the video. Most loops have a longer wire than mine, so usually flaming the wire is enough. As for the cap, it was placed on a sanitized bench, opening-up, close to the flame. That should keep the interior of the cap sterile due to the airflow generated by the flame (that is the purpose of the flame, after all). It is how we usually teach new microbiologists to handle caps, as it is relatively fool proof (so long as you're careful to not pass your hand or items overtop of the cap too much). Experienced microbiologists will often hold the cap in the same hand as holds the tube, using their pinky. This keeps the cap near the flame, but facing downwards. While this may be slightly less likely to become contaminated from the air, there is a higher risk of contamination from dropping the cap or accidentally sticking your finger inside of the cap. Both are fine, but the former is easier to learn.
@@SuiGenerisBrewing Just tried this and I think it went smoothly!! Slanted 5 cultures. Let's wait a couple days to see if there's no infections. I plan to use them to brew 1 bbl batches, do you have any videos or know of any resources that detail how to grow such a large pitch from a slant? I've been doing some research and I think the most efficient way would be to use sterile air in my starter steps to keep the yeast from fermenting so they reproduce manyfolds more cells per step... But I can't seem to find the specifics about how much/when to aereate, what volume steps to use, etc. Do you know of any resources that detail this process and equipment needed, that is understandable for a layman?
@@johandingler704 This is not a simple question. Simply pumping in air won’t give you growth much above what you would see with a conventional starter. The reason for this is something called the “Crabtree effect”, AKA “Glucose suppression”. When yeast are exposed to sugar concentrations above150 mg/L (specific gravity of ~1.00011), yeast will ferment the sugars, even if oxygen is present in large amounts. So continual aeration of a normal starter will not have much of an additive effect compared to conventional stirring. There is a way to force yeast to grow aerobically, called fed-batch culture, where you slowly provide the sugar to the starter, at a rate which keeps the sugar level low enough to avoid the crabtree effect. As you can imagine, such a system is not really possible outside of a lab or commercial production facility. Moreover, yeast made in this manner tend to be poor fermenters, so its not really a route you’ll want to pursue. I have grown yeast from my bank for use in a commercial brewery a few times. I generally do it by preparing the normal set of starters (a 5-10 mL culture using a loop of the frozen yeast, that goes into an ~250 mL starter, that into an ~2L starter), and then pitch the yeast into a 20L (5.5 US gal) well oxygenated, lower-gravity/IBU/colour wort beer (e.g. ~1.040 SG blond ale). The yeast cake is then made of pretty healthy yeast, which can be pitched into as much as 2 BBLs of beer upto ~1.060 S.G. If you wanted, you could make a conventional 20L culture instead of a beer, and aerate that continually, to make a large batch of extremely healthy yeast that would then be ready to pitch into a similar (or slightly larger) sized batch. One of the best things you can do to increase yeast yield is to add more available protein (or nitrogen) to the starters, as nitrogen - not oxygen or sugar - tends to be what limits yeast growth. Adding a yeast nutrient, even just DAP, at 5X (or more) the manufactures recommended dose can greatly increase yeast yields. If you do this (especially in the final stage before pitching the yeast), be sure to remove as much of the wort as possible, as yeast can make some pretty unpleasant compounds under high nitrogen concentration. A few other things that can help improve yields: -Warm the cultures to between 30 and 32C; this is the optimum temperature range for yeast growth. DO NOT go over 36C, as this will start to kill the yeast. -Aerate (stir, or use an aerator near the bottom of the fermenter) continually to keep yeast suspended. -Do not exceed starting gravities of 1.040; optimal growth occurs between 1.036 and 1.040 - above that, yields/gram sugar drop; below that and you’re wasting volume. I like this question, and its a good one to address - look to my blog in a day or two for a more fleshed-out article with citations.
@@SuiGenerisBrewing Thanks, Sui! Great info. I was actually planning of doing something similar to what you suggested in case doing pure propagation outside of a lab was unfeasible. But I didn't know you could go as far as to 2 bbl from a 5 gal cake of a normal fermentation, that's insane! Plan is to grow a normal 2L starter and pitch that into 5 gal, then wait 24 ish hour for the log phase and pitch that whole beer into 10 gals of wort, then wait another 24 hours and pitch that whole 15 gal beer into 15 gal more of wort. For context, I brew mostly lagers and have found that a very high pitch rate of 2.5x10^6 cells/ml/P works best for me, so that's a lot of yeast for 1 bbl! This sounds like too much work however 😅and I was reading about single tank propagation where they go from slant up to carlsberg flask, then 1:200 to propagator then 1:20 to final pitch, which seemed to me to be just normal starters with an intermitent areation routine, but there's probably a lot of subtlety i'm missing. So I was planning to scale that down to going from slants to 25 ml ish (sterile) on a stirr plate that sitrrs intermittently, to 6.5L (sanitized) in a modified corny keg that could aereate intermitently through a sterile filter and autoclaved aereation stone, and then pitch that into my final beer. Really looking forward to your blog post!!
@@johandingler704 Blog post is up: suigenerisbrewing.com/index.php/2022/09/20/optimizing-yeast-starters/ In terms of "steps", as a general rule, increasing your step-size 10-fold each step yields the best yeast growth. That includes going from the final "starter" (e.g. 20L of beer) to a full-sized brew (1 BBL is ~150L). Technically, you will be underpitching, but the yeast is of high viabilty and should do fine. Keep in mind, most of the yeast pitch calculators were made using yeast being repitched from batch-to-batch, not fresh from a starter/production run.
+Fredrik S You mean storing yeast slurry under sterile water? I've not tried it, but it should work for short-term storage. Without a source of food, it use for long-term storage would be limited.
+Sui Generis Brewing some more info can be found here: hbd.org/brewery/library/SterileDW1096.html I have read reports that storing in sterile distilled water in room temperature for years and then "waking them up" and to make perfectly good beer. www.ncbi.nlm.nih.gov/pmc/articles/PMC186689/, www.alsand.com/beer/yeast/store_E.html, www.mousetrap.net/mouse/brewing/dwyeast.html
+Fredrik S While there is no question that old yeast has been recovered by this method, that does not mean that most strains will last that long. Yeast have been recovered from mineral-oil slants over 30 years old, but most yeasts would not last that long, which is why I recommend 18-24 month reculturing if using that method (that method has a 50+ year history of success with all types of brewers yeast). Yeast have been stored and propagated by slanting for over 100 years - its a tried & true method with well understood dynamics in terms of viability over prolonged storage. Storage under water hasn't been tested enough with brewers yeast for me to be confident recommending it; indeed, I found a number of threads and posts of people who tried it and found it did not work. The fact that it has never been a widely adopted method in microbiology would indicate that it is inferior in some way to alternative methods, especially given its low cost. I'm not saying not to use it - I'm simply saying that I would not be willing to trust my own yeast bank to the vagrancies of a rarely used and poorly documented method of yeast storage - especially given the well established alternative methods available to us.
+Fredrik S Den här snubben har verkligen bra och sakliga klipp på sin kanal. Jämför man med memet om jästtvättning som var igång för några år sedan så är det milsvid skillnad.
Hi love the vids a question and a suggestion, question? can slants/plates be frozen? And a suggestion, making pitching yeast from frozen glycol stock. Thanks Si.
You cannot freeze slants or plates, as the ice crystals which form kill the yeast. I have a new video series in the planning stages, and one video will be on stepping up frozen yeast to pitchable amounts. Look for that in early 2023.
The maximum surface area would be achieved by rolling the tube while it cools, in order to distribute a layer of agar all over the inside surface. Granted, you would not be able to see what was inside, although you could leave a gap at the top. Tubes could also be partially rotated, leaving a viewing slot along the length. Has anyone tried this? Would it be worth doing?
The goal isn't maximum surface area, its maximum useable work area. There is a reason slants havent changed much in the ~120 years they've been in use.
It varies. Some are "extra" from a starter, other bottle dregs, and other are slurry from a brew. I don't pre-wash the yeast for slants or plates, although sometimes I do add sterile water to loosen a thick yeast cake.
@@SuiGenerisBrewing I have "propagate" bottle dregsup to 35Oml and now will be able to brew a 3liters batch. Do you think that it would be a good start to follow your yeast banking SOP with the slurry ? thanks
SO... I can start a yeast collection from a single yeast vial purchased anywhere? I just have to make a small starter , then a bigger one and then inoculate into my ( lets say) 20 l Batch? . Which yeast is the best to start a yeast bank . liquid or dehidrated? Im totally new in this field thanks for your your help!
I noticed in the video where you were transferring the old yeast to the new slant, my question is, can you do the same thing with the yeast the had the mineral oil in the slant to the new slant Agar? or do you pour out the mineral oil then scrap the yeast out of the slant and then transfer it to the new slant? thanks very helpful videos.
You can directly transfer, and you don't even need to pour off the oil. Grab a colony with your loope, allow the excess oil to drip off, then streak on the new slant. Any oil you transfer will flow to the base of the slant, leaving the yeast free to grow
Thank you for sharing your knowledge. If you had to choose, which method is the best: Petri dish, Slants or “freezing in tubes”? Or do you think we need to use all of these methods in our “home lab”? (I’m deciding on buying equipment) Thanks!
It depends on what you are trying to do. If you're only storing yeast, freezing is best and fairly easy. Slants are great if you only want to keep only a few strains. Plates are more for purifying yeast than storing them.
Hello and thank you for this awesome video! I already have all I need to make my own slants, but the agar. Here in Mexico, I have found "agar potato dextrose" (lab grade), and regular agar (the one you use in the kitchen). The first, costs $15usd for 3.3Oz, while the kitchen version is $10usd for 2.2 pounds. Which one should I buy?
Great video, but one critique: When you insert the loop into the tube, you must only insert the flamed wire. Putting the actual tool into the tube virtually guarantees that you'll touch the contaminated surface of the tool itself to the sides of the tube and/or agar surface and contaminate them. The solution to inoculating the entire surface is a longer wire.
I've never "Sanitized a handle", nor seen anyone else do so, because 1) "sanitizing" is a brewing term which only extends to killing sufficient numbers of competing microorganisms versus the intended culture (and is thus wholly insufficient for producing axenic monocultures) and 2) the wire technically should glow, albeit briefly, which is not possible for the handle using an alcohol lamp. It's a break in technique to touch anything but the flamed wire to something intended to remain sterile. I don't mean this as a personal critique- I'm watching the video because the stuff that you're doing with home untensils is really cool and I think it can save me and a lot of other folks a lot of money on yeast.
@@amschind @Adam Schindler Imagine the gall it takes to lecture an actual microbiologist on a term, and to open your argument by saying ""sanitizing" is a brewing term". If you wanted to prove you know nothing of this topic, congratulations, you succeeded. "Sanitation" has a very specific scientific definition which, in most countries (including the USA, Canada, and EU) is also codified legally. There are even an set of international standards for determining if a product or method gets certified as a sanitizer, disinfectant or steralant: for example, fungicidal activity is determined by AOAC 955.17, with fungal sanitation requiring a 3 -4 log10 decrease in fungal cell numbers after treatment. To qualify as a disinfectant it much reach a 4 -5 log10 decrease, and to sterilize a 6 log10 or better decrease (all starting with a control sample containing 5x10^6 cells). Products like starsan, 70% ethanol and iodophore typically fall into the sanitizer or disinfectant range, depending on the organism tested. I store my handle when its not in use in 70% isopropyl; hence, it is sanitized (likely disinfected) by the very scientific/legal definition of the term you claim is a brewing term. For food-based microbiology, which is what we are doing here, that is more than sufficient to limit the risk of contamination to an acceptable level. Achieving true sterility in the home is nearly impossible - with only a few exceptions, most home pressure canners don't even meet the legal/scientific requirements for that (due to insufficient pressure and temperature). Even the aseptic techniques I demonstrate in my videos, when performed in a laboratory with proper equipment, are not considered sterile techniques - which are a whole other range of methods, requiring vastly more expensive tools and facilities (hence why they are "aseptic" - " aiming at the complete exclusion of harmful microorganisms"). Over the past 23 years I've manage a collection that has grown to over 400 brewing organisms, operating largely out of my basement, using these approaches without loss of a single culture. If it was good enough for Pasteur, Koch and Reess, it certainty is good enough for us.
I'm glad that you have so much experience. I also have quite a bit, but that's not the issue. The technique in question was developed by the gentlemen you mentioned for good reason, and isn't debated. When you discuss log kill, you're describing a process which is sufficient for reduction of competing microorganisms but insufficient for indefinite maintenance of an axenic culture. Since we're discussing that sort of monoculture, log kill becomes insufficient for these purposes. It really seems like you're interpreting these comments as personal attacks and they're not: I just wanted to point out the need to get a slightly longer wire.
@@amschind lets just say that being lectured about techniques by someone who doesn't even understand common, internationally accepted, legally defined microbiological terms that are taught to first year undergrads is irritating. It also makes your claim of expertise hard to believe. This is also a video oriented at people new to micro, and I don't want viewers to think they need to buy unnecessarily expensive kit because some rando commenter made a factually incorrect statement regarding the appropriateness of a commonly used tool. Sanitized tools are more than sufficient under conventional conditions to maintain pure cultures. Its the standard used for field micro (where flames are unavailable or unsafe), and is increasingly used in lieu of burners in many facilities due to the dangers of open flames.
thanks for your videos- i was wondering if you could harvest some yeast just before it dies and then repeat until you get a super yeast. if this is possible a video would be great if not a no will do - thanks
You can continually culture yeast in that fashion, and it will lead to evolution of the yeast into something different. Whether that would be a super-yeast, or something else, is less clear.
could you use a yeast nutrient instead of wort to a) create a slant and b) re activate the yeast for pitching? We make a wheat beer and sometimes there are long time stretches between brews of this, so i don't trust the sterilised wort 100%. would be easier to make fresh before either tranfering or starting?
@@attunedosteopathy5074 There isn't really an answer to this as different brands of yeast nutrient are formulated in different ways. Most yeast nutrients will not work as they don't have everything the yeast need. An easy way to maintain stocks is dry malt extract. You can measure out enough for a slant or two at a time (0.5 to 1g for every 10 ml), and it's stable for years if kept dry. Another option is to move to lab-grade media. YPD is a ready-to-use yeast medium that can be purchased for not too much. Again, measure out the amount you need (~0.5g/10 mL), dilute in water, and pressure cook to sterilize.
Very nice video! A question I'm not finding a good answer to online. Could this technique be used for storing mixed cultures, or just single strains? :) TheYeastBay only rarely delivers to their supplier in Sweden, so having a bank of their Saison blends would be nice.
@@SuiGenerisBrewing Another question, how important is it that it's glass vials? I imagine that plastic vials "leak" more oxygen which could harm the yeast?
@@leocarlsson3753 plastic is fine - its what we use in research labs. The problem with them is that they are single use, so you need to sterilize the media, then pour it aseptically. Once the slant is used, you toss the tube. I prefer glass to avoid that waste
could you use a pressure cooker to IE autoclave lab glassware? (edit) I guess I should have finished watching the video. I thought I'd allready seen the video.
+rimmersbryggeri You found the answer, but for anyone else who sees your question...the answer is 'Yes'. A pressure cooker can be used both to sterilize media/wort, and to sterilize glassware. Same rules for each - set the pressure cooker to its highest setting, and let it run 15-20 minutes once the pressure relief valve start going. Always let it cool to below boiling (ideally to below 80C/180F) before opening.
Do the vial caps need to be on tight while in the pressure cooker? I did mine just slightly loosened, and about half the contents of every tube boiled out. Made a mess and I had to dump the slants because they were all under filled. Thanks!
I put sterile water on my slants and now most of the yeast have fallen off the slant and settled at the bottom of the tube. Is this a known issues with this technique?
Microwave them for 20 to 30 seconds to loosen the gel, dump the gel in the trash, and wash them in the sink. Once cleaned, the tubes are ready for re-use.
When steralizing it, have the ring on a little loose so that steam can escape from the jar. Once its canned the sealer part of the lid should be held in--place by a vacuum. At this point you can remove the ring completely to let the threads dry. Once the threads are dry you can store the jar without the ring, or you can put the ring on the jar finger-tight. Don't crank the ring on too tightly, as that can break the seal.
Hi, could you specify which mineral oil is most suitable for slants? Sorry to ask that, but I am from Brazil and for us there are several types of mineral oils, with different purposes.
@@mauriciocarmona3437 you can steralize with a pressure cooker although that isn't really necessary. It may become hazy if you do - it is still fine, the haze is just water suspended in the oil.
I love your videos but I have a problem making the slants using mineral oil. I followed exactly the instructions. left the slants to grow for 48 hours after inoculation and then when i came to add the mineral oil the yeast just separated away from the agar and entered into suspension in the mineral oil? Can you help me resolve this problem and suggest possible causes? Thanks.
I have not heard of this issue with mineral oil, although people have had this issue with water. Did you refrigerate the slant first? That may help. The yeast should still be fine if they come off in the oil, so long as they settle back down on the slant.
Done twice now. 1st slant was not pre-refrigerated but done straight after making the slant (after cooling). It went fine and was left for 48 hours to grow yeast. Oil did not seem to lift them once added and they are now in storage. Second slant was pre-refridgerated as I did them a week later on the first batch of slants so same agar etc. I did the same procedure and after inoculation I left them to grow for 48 hours. Once growth had begun and I could see the cultures growing I then added the mineral oil as before. The culture almost immediately freed from the agar and went into suspension. They were different strains. The first was "american ale yeast' and the second was 'british ale yeast'. Looks like the brits are being a bit awkward :D Would it be best to try to re-culture these next time I use them? Thanks
Looks like it may be a strain-specific thing. You should be able to culture from the loose yeast at the base of the slant. I'm not sure what you mean by "reculture".
There should be nothing; the first time I shot the "scene" the camera was out-of-focus, so I had to pour out the tubes and repeat the shot. I was rushing, so I didn't get the tubes clean.
Quick question.. How would I go about calculating/estimating a target amount of yeast cells that I want to get from a starter when using a banked culture?
I assume 50 million/ml for the initial growth, which seems to be close enough most of the time. I then use that number in a yeast starter calculator for subsequent steps. E.g. my initial 5 ml tubes of yeast is assumed to have 250 million yeast.
+Steve Sowinski (Airlock Sniffs) Absolutely! In fact, that is the best way to do it, as the yeast you store have a minimal chance to mutate/change before storage, ensuring that your stored yeast perfectly match the yeast in the package. Usually what I do is dump the yeast into my starter, and then use the loop to grab some of the the yeast "residue" in the tube/sachet, and use that "residual" for my stored culture. That way there is no risk of contaminating my starter, and I still get "first generation" yeast for my bank. Keep in mind that you only need a small drop of yeast to inoculate a slant - the residue in a tube/sachet after you dump the yeast out is likely enough to inoculate a dozen slants.
+Sui Generis Brewing What about "old yeast" on sale, 4-12 months old. Would you make a starter and collect yeast from it or straight from the old tube/satchet? Thanks for great video btw.
+rimmersbryggeri Yes, but they are not required. If using yeast nutrient make sure it is one of the 'complete' nutrients (often made of lysed yeast). The cheaper nutrients are usually diammonium phosphate (DAP), which is not nearly as good. As a 'rule' the good nutrient is tan coloured, while the DAP-types are white crystalline powders.
Yeah that's great. To me servomyces seems like a good reference name like aspirin is for salicylic acid and then there are lots of generic brands. But I definitely noticed a difference between wyeast nutrient and the nutient salts I have had available before.
No. I have a separate video on freezing yeast though in the same video series. Freezing yeast is quite simple, and is the method I recommend for long term storage.
Yep, although I don't know what the storage time would be. Adding a small amount of chalk to the media helps LABs grow a bit better - a fee g/L is enough.
G'day first off thanks for your video! In the very last part of your video you move yeast off a slant and onto a nice fresh one...let it ferment a bit and place mineral oil in it and fridge. Just wondering what this achieves as it's not clear (ie do you now get another 2 years or so etc). Also maybe a stupid question...but each time you move the yeast and let it start getting to work a bit...is this technically adding a generation to the yeast? (ie how often is this recommended)? Thanks
A slant, stored in the fridge with a tightly sealed cap will reliably store yeast for 6 months or so. With a layer of mineral oil, that is extended up to ~2 years. So its a good choice for storing a yeast you do not anticipate using very often but are unwilling to lose. Retrieving yeast from mineral oil slants is more difficult, so I do not recommend it for frequently used yeast. Any time you transfer from tube-to-tube the yeast will undergo some cell divisions; in terms of the number of cell divisions (and therefore potential for genetic drift), its pretty close to the number of cell divisions you would get using a starter and then fermenting a beer, so its pretty much equivalent to a 'generation' passed through a fermenter.
If you want to keep the cultures pure, pasteurization is unlikely to be sufficient. With this small of a culture, even a minute contamination would ruin it.
@@humanonearth1 these are microbiological tubes, so their lids are made of a plastic which is stable at autoclave/pressure cooker temperatures (121C). Most common plastics wouldn't survive though.
No. Glycerine will dehydrate & kill the yeast if used in its pure form. You can freeze yeast using a glycerine solution - see my video on yeast freezing for more: th-cam.com/video/o0zluMPcwrY/w-d-xo.html
I don't use slants anymore, and store everything frozen. I have two vials of each yeast, one at home and one elsewhere. The one at home is my day-to-day stock, while the off-site tube is my backup in case of a freezer failure, or if I need to replenish my home stock. Edit: I also kept duplicate tubes when I used slants.
@@SuiGenerisBrewing ok, thanks a lot. The frozen ones, do you use the full vial to make a starter? (and make new frozen vials out of that starter?) Edit: after seeing your "Freezing & Refrigerating Yeast" its clear your make a vial starter of a thawed vial. Guess you re-freeze them. Pressure cooker ordered on your advise, tomorrow I can start banking. Siked!
@@martijnblokzijl1350 I scrape a small amount of frozen yeast out of the vial, then return the vial to the freezer. One vial will make dozens of starters.
i gotcha Sui....it just seems like it would still be hot . I don't know ..maybe I am just parranoid...I cut steel a lot ...it always seems to take a while to cool ...i guess the loop is a lot quicker cooling than 1/2 inch steel ! would you be focusing this video more on small nanos or would u would suggest this for home brewers !?
Just the fridge. To maximize lifespan, fill the tube completely with oil, cap, and then seal the top with vinyl tape (e.g. electrical tape). Stored like this, most yeast will last 2+ years in a fridge. I have a video on using glycerol to freeze yeast if you want to go that route.
+Ionut-Alexandru Batrinache Depends. In some countries highly purified (i.e. medical grade/cosmetic grade) mineral oil is sold under the name of paraffin oil. That sort of oil would be ideal for freezing yeast. Unfortunately, in other countries paraffin oil refers to a clean-burning lamp oil, derived from kerosene. Using that would be acutely toxic to your yeast. So if the recommended use of the paraffin oil you have is as a laxative, skin care, or preparation of cosmetics, it will be fine. If its meant to be burned in a lamp it won't work.
Sui Generis Brewing Yes, the paraffin oil i have is bought from a pharmacy. Good to know, i will try this method. A video about freezing yeast it glycerine would be nice too :)
Fantastic video! In just 18 minutes you talk all of the mystery out of the process, nice work!
Glad you enjoyed!
Such a GREAT presentation! There are yeasts that we use all the time and those we don;t need to store or culture, but there are others that we use infrequently and those that are in danger of being taken off the market. Those are exactly what you want to do this for. Subbed...fantastic, approachable method. Cheers!
Thanks for the kind words, and I'm glad you enjoyed!
Thank you good sir.
I keep rereading "Yeast" and "Brewing Yeast and Fermentation" but have no visual examples of this.
I'm moving away from my homebrew shop, four hours away from the closest one.
So this is vital for me.
I’ll prob never do this, but it was extremely interesting and very well presented. I appreciate the time you took to share.
Liked a lot these video, its easy to follow, even with no former microbiological techniques training.
Just stumbled on your videos today and I've gotta say I'm addicted!! Very nice job explaining things.
I just wanted to say that these videos are really fantastic and exactly what I was looking for as I start my yeast bank. Thank you so much for making these!
+Todd West Thanks!
Got some slants done on my second try. On the first attempt, I didn't completely dissolve the agar and it settled on me and I thought it might dissolve in the pressure cooker but the rack I had warped, and everything was ruined. I got a jar of starter wort to seal though so it wasn't a total loss.
I do have hard water stains all over the glass so the suggestion to put some vinegar into the water might be a good one. I'm just going to use RO water next time.
Thanks for the video.
I tried making PDA slants but all my water evaporated when I boiled the potato. I decided to just go DME for now.
Sorry Steve, missed your post. PDA drying it is not an uncommon issue if you try to make too small a batch. Its easier to make ~1L and toss what you don't need (it only costs you half a potato), or if you want to make smaller amounts, add water throughout the boil to keep the boil volume right.
Really super! Bought me an whole set of equipment. Cheers from the Netherlands
Hi Sui, I wanna thank u for these videos as they are very helpful in yeast culturing, actually I came here cause theres a mention of ur videos in the Yeast book....I am planning a mini brewery and can I still go ahead with the same procedures you've explained for commercial purpose............thank you......cheers
Hello Bryan, does storing yeast on slants cause endospores due to lack of nutrition, as you say yeast is good for storage for upto 2 years, and the other concern is due to the number of passages, does restreaking cause genetic drift in the cells.........thanks and cheers.
Nice to see you doing videos again BTW non nonsense videos are needed on topics like this.
Great work on these instructional videos. Thanks for taking the time to put them up.
Great video! They are all quite helpful as I am planning on building up some yeast for storage in the next couple of weeks. Cheers!
It was pleasure to watch. Amazing!!
sorry the other 35000 viewers couldnt be arsed to click like on this excelent content.
Just what I needed... Thanks for making my efforts a lot cheaper!!
Great videos. I notice you don't flame the handle part of the inoculation loop, yet when sticking the loop all the way to the bottom the handle enters the slant tube. Couldn't that cause contamination?
It can if you're not careful. My handel isn't actually for holding the loups and has internal plastic parts, so I cannot flame it properly as it may melt.
So when you pressure cook the tubes, do you leave the caps loose or tighten? Love your vids, they help me a lot! Thanks
Sorry, just read on your blog, cap loose. 😁
thanks for your videos man
Awesome video. Thank you!
Great, thorough video! Thank you very much for this! I have a quick question: could mineral oil be replaced with glycerol? Is there any particular reason mineral oil is used? Thanks again!
+Brian McConeghy Mineral oil is used to both prevent dehydration and block oxygen. I do not think glycerol would work as it is hydroscopic and also membrane-disruptive to the yeast. Used at 15-50% (in water) glycerol can be used for freezing yeast, but placing a pure solution over a slant would likely be lethal.
An amazing video, many thanks.
great video... I'll pass the link to my homebrew club
If you did subtitles to your videos, you would would have increased the audience of viewers. Thank you for your work.
Thanks for the video! Very informative and to the point.
Sorry ahead of time for continuing the questions over two years after you published, but........ Can you speak to any cons using water or oil in the slants that you have seen? On the surface it seems silly not to add that one step to significantly increase the shelf life. One step that, for what it's worth, that is void from other videos on the subject......
adam morrison No worries commenting on an old video. Mineral oil is the better option; water sometimes detaches the yeast from the slant, but with oil they seem to stay put. Air is the worst option, as dehydration greatly shortens the life of the culture.
Hey. Love the videos very helpful. I made my first agar slants but I used my instant pot. I later read the pressure setting is only 11.6 at around 242. Are my slants safe if nothing grows on them for a week and should I pour out my wort starter tubes I pressure cooked due to the temp difference?
Your slants are fine. Nothing you'll encounter in the brewing world will survive those conditions.
Sui Generis Brewing can I still use my canned wort as well?
@@mattblackburn1450 yep. There is perhaps a half-dozen organisms that would survive in the "window" between what your canner provides and what you get with a full 2ATA/121C canner/autoclave. None of those organisms grow in wort.
Sui Generis Brewing thanks
I love your videos, they are amazing. I have a doubt with mineral oil, can you share me a brand or photo of which one should i get?
Mineral oil is a highly purified/distilled petrolium oil. If its not sold as "mineral oil" where you live, it may be sold under one of several other names: white oil, paraffin oil, or liquid paraffin, are common names used outside of north america. You'll usually find it at a pharmacy - its also the primary ingredient in baby oil (which is scented mineral oil).
Hi, impressive tutorial series !!! Thanks one more time!
In the mean time, I will appreciate if you will supply some details regarding the "mineral oil" liquid. What kind of? Where to look for?
It's also sold as paraffin oil.
Great video, Just one question. When placing the tubes into the pressure cooker do you seal them or just place the lids loosely?
Loose
Great, this is exactly the question I wanted to ask! Going to make some slants today. Do you pressure cook the mineral oil too?
Hi Bryan. I note that you use a single colony of yeast to inoculate your starters etc. I have read elsewhere on the web (so may of course be incorrect) that using a colony formed from a single yeast as opposed to several colonies might lose some of the variation within the yeast such as flocculation behaviour, flavour and aroma profile and will not have all the characteristics of the complete 'strain'. I am sure you would not be using a single colony for inoculation if that was the case, so I am wondering what is the alternative explanation as from my limited microbiological knowledge losing some of the variation within a 'strain' by only using a single colony would seem to be a possible outcome of this method?
How many yeast I pick depends on my goals:
-Cleaning an infected culture: 1 colony/tube, 5-6 colonies (e.g. tubes) total. Clean tubes get mixed into one culture.
-Purifying wild strains: 1 colony
-Culture maintenance: 10 or more colonies
@@SuiGenerisBrewing Thanks, so at least to some extent it is true and colonies have to be handled according to your purpose. It would be useful to do some more reading up on this, do you know any good sources of information on this subject (online advice, journal articles or books) please, but no worries if not? I can digest quite technical material if needed.
First of all thanks for your videos, I have a problem, using 2% of agar the agar doesn't solidified well I have increased to 4% of agar do you think that this will affect the slant ?
Should be fine, but it sounds like your agar may be of low purity.
Here is a question for you. Since the yeast can only live up to a certain alcohol content, Would it not be possible to run a small line off a vat, run it through distillation the remainder being cooled and reintroduced into the VAT fermenting process, in this way keeping the yeast in their comfort zone until all sugars have been expended? making the sugar the limiting factor not the alcohol content. And with longer lives who knows what the yeast can accomplish.
#Yeast lives matter#
Another question, Has anyone ever reduced particle size of the ingredients into the nano realm with sonication/ultrasound which would increase their surface area while also increasing bioavailability to the yeast aiding in consumption and conversion of the ingredients. Would that speed up and increase yield in the fermentation process? I have used the ultrasound with oak and cinnamon to help age the first batch I ever made at the end of the process. The cinnamon came thru super strong so I distilled again with a little charcoal and toasted oak hoping to mellow it. It helped a little. I then used the resulting alcohol in my liposomal vitamin production which helped quite a bit in palatability and decreased viscosity. I actually drink it now instead of dreading it like the batch before it. If you should wish to try an ultrasonic unit, do not buy the harbor freight version as it is pretty weak. I bought two of the same transducers and mounted them under a heavy round stainless 9in. by 9in container. I tried the tinfoil test and the H.F. unit Put maybe 2 dozen holes in the tinfoil in 3 minute. My home made unit made confetti out of it in ten seconds. Both units cost me around eighty dollars. Just waiting to brew up some more alcohol and try it. Interested to hear your results should you think any of this interesting. I would love to hear if my theories have any merit or even turn out to be game changers.
Great video!
Something's odd to me tho, that you unscrew the tubes and just leave the lids on the table and then don't flame them when screwing them back on. Is this not an issue? Also, when you put the bacteriological ring in the tube to inoculate it, you flame the ring itself, but not the whole body that also goes into the tube. Is that okay? Do you sterilize the whole body before somehow? Sorry for asking about minute details, but I want to bank my yeast for the first time and trying to minimize my chances of messing up!!
Regards!
The loop is an issues that come with trying to film and talk while doing microbiology. Ideally, any part of the loop that goes into the tube should be flamed to reduce the risk of contamination - or you avoid touching the tube with the unflammed part. I didn't do a good job of either in the video. Most loops have a longer wire than mine, so usually flaming the wire is enough.
As for the cap, it was placed on a sanitized bench, opening-up, close to the flame. That should keep the interior of the cap sterile due to the airflow generated by the flame (that is the purpose of the flame, after all). It is how we usually teach new microbiologists to handle caps, as it is relatively fool proof (so long as you're careful to not pass your hand or items overtop of the cap too much). Experienced microbiologists will often hold the cap in the same hand as holds the tube, using their pinky. This keeps the cap near the flame, but facing downwards. While this may be slightly less likely to become contaminated from the air, there is a higher risk of contamination from dropping the cap or accidentally sticking your finger inside of the cap. Both are fine, but the former is easier to learn.
@@SuiGenerisBrewing Just tried this and I think it went smoothly!! Slanted 5 cultures. Let's wait a couple days to see if there's no infections. I plan to use them to brew 1 bbl batches, do you have any videos or know of any resources that detail how to grow such a large pitch from a slant? I've been doing some research and I think the most efficient way would be to use sterile air in my starter steps to keep the yeast from fermenting so they reproduce manyfolds more cells per step... But I can't seem to find the specifics about how much/when to aereate, what volume steps to use, etc. Do you know of any resources that detail this process and equipment needed, that is understandable for a layman?
@@johandingler704 This is not a simple question. Simply pumping in air won’t give you growth much above what you would see with a conventional starter. The reason for this is something called the “Crabtree effect”, AKA “Glucose suppression”. When yeast are exposed to sugar concentrations above150 mg/L (specific gravity of ~1.00011), yeast will ferment the sugars, even if oxygen is present in large amounts. So continual aeration of a normal starter will not have much of an additive effect compared to conventional stirring.
There is a way to force yeast to grow aerobically, called fed-batch culture, where you slowly provide the sugar to the starter, at a rate which keeps the sugar level low enough to avoid the crabtree effect. As you can imagine, such a system is not really possible outside of a lab or commercial production facility. Moreover, yeast made in this manner tend to be poor fermenters, so its not really a route you’ll want to pursue.
I have grown yeast from my bank for use in a commercial brewery a few times. I generally do it by preparing the normal set of starters (a 5-10 mL culture using a loop of the frozen yeast, that goes into an ~250 mL starter, that into an ~2L starter), and then pitch the yeast into a 20L (5.5 US gal) well oxygenated, lower-gravity/IBU/colour wort beer (e.g. ~1.040 SG blond ale). The yeast cake is then made of pretty healthy yeast, which can be pitched into as much as 2 BBLs of beer upto ~1.060 S.G. If you wanted, you could make a conventional 20L culture instead of a beer, and aerate that continually, to make a large batch of extremely healthy yeast that would then be ready to pitch into a similar (or slightly larger) sized batch.
One of the best things you can do to increase yeast yield is to add more available protein (or nitrogen) to the starters, as nitrogen - not oxygen or sugar - tends to be what limits yeast growth. Adding a yeast nutrient, even just DAP, at 5X (or more) the manufactures recommended dose can greatly increase yeast yields. If you do this (especially in the final stage before pitching the yeast), be sure to remove as much of the wort as possible, as yeast can make some pretty unpleasant compounds under high nitrogen concentration.
A few other things that can help improve yields:
-Warm the cultures to between 30 and 32C; this is the optimum temperature range for yeast growth. DO NOT go over 36C, as this will start to kill the yeast.
-Aerate (stir, or use an aerator near the bottom of the fermenter) continually to keep yeast suspended.
-Do not exceed starting gravities of 1.040; optimal growth occurs between 1.036 and 1.040 - above that, yields/gram sugar drop; below that and you’re wasting volume.
I like this question, and its a good one to address - look to my blog in a day or two for a more fleshed-out article with citations.
@@SuiGenerisBrewing Thanks, Sui! Great info. I was actually planning of doing something similar to what you suggested in case doing pure propagation outside of a lab was unfeasible. But I didn't know you could go as far as to 2 bbl from a 5 gal cake of a normal fermentation, that's insane! Plan is to grow a normal 2L starter and pitch that into 5 gal, then wait 24 ish hour for the log phase and pitch that whole beer into 10 gals of wort, then wait another 24 hours and pitch that whole 15 gal beer into 15 gal more of wort. For context, I brew mostly lagers and have found that a very high pitch rate of 2.5x10^6 cells/ml/P works best for me, so that's a lot of yeast for 1 bbl!
This sounds like too much work however 😅and I was reading about single tank propagation where they go from slant up to carlsberg flask, then 1:200 to propagator then 1:20 to final pitch, which seemed to me to be just normal starters with an intermitent areation routine, but there's probably a lot of subtlety i'm missing. So I was planning to scale that down to going from slants to 25 ml ish (sterile) on a stirr plate that sitrrs intermittently, to 6.5L (sanitized) in a modified corny keg that could aereate intermitently through a sterile filter and autoclaved aereation stone, and then pitch that into my final beer.
Really looking forward to your blog post!!
@@johandingler704 Blog post is up: suigenerisbrewing.com/index.php/2022/09/20/optimizing-yeast-starters/
In terms of "steps", as a general rule, increasing your step-size 10-fold each step yields the best yeast growth. That includes going from the final "starter" (e.g. 20L of beer) to a full-sized brew (1 BBL is ~150L). Technically, you will be underpitching, but the yeast is of high viabilty and should do fine. Keep in mind, most of the yeast pitch calculators were made using yeast being repitched from batch-to-batch, not fresh from a starter/production run.
Great video, very helpful! Helped me a lot building my yeast lab at home. Have you read/tested sterile water storage?
+Fredrik S You mean storing yeast slurry under sterile water? I've not tried it, but it should work for short-term storage. Without a source of food, it use for long-term storage would be limited.
+Sui Generis Brewing some more info can be found here: hbd.org/brewery/library/SterileDW1096.html I have read reports that storing in sterile distilled water in room temperature for years and then "waking them up" and to make perfectly good beer. www.ncbi.nlm.nih.gov/pmc/articles/PMC186689/, www.alsand.com/beer/yeast/store_E.html, www.mousetrap.net/mouse/brewing/dwyeast.html
+Fredrik S While there is no question that old yeast has been recovered by this method, that does not mean that most strains will last that long. Yeast have been recovered from mineral-oil slants over 30 years old, but most yeasts would not last that long, which is why I recommend 18-24 month reculturing if using that method (that method has a 50+ year history of success with all types of brewers yeast). Yeast have been stored and propagated by slanting for over 100 years - its a tried & true method with well understood dynamics in terms of viability over prolonged storage.
Storage under water hasn't been tested enough with brewers yeast for me to be confident recommending it; indeed, I found a number of threads and posts of people who tried it and found it did not work. The fact that it has never been a widely adopted method in microbiology would indicate that it is inferior in some way to alternative methods, especially given its low cost.
I'm not saying not to use it - I'm simply saying that I would not be willing to trust my own yeast bank to the vagrancies of a rarely used and poorly documented method of yeast storage - especially given the well established alternative methods available to us.
+Sui Generis Brewing Thank you for taking time to look it up and to write a thoroughly answer to my question. Appreciated!
+Fredrik S Den här snubben har verkligen bra och sakliga klipp på sin kanal. Jämför man med memet om jästtvättning som var igång för några år sedan så är det milsvid skillnad.
Hi love the vids a question and a suggestion, question? can slants/plates be frozen? And a suggestion, making pitching yeast from frozen glycol stock. Thanks Si.
You cannot freeze slants or plates, as the ice crystals which form kill the yeast. I have a new video series in the planning stages, and one video will be on stepping up frozen yeast to pitchable amounts. Look for that in early 2023.
The maximum surface area would be achieved by rolling the tube while it cools, in order to distribute a layer of agar all over the inside surface. Granted, you would not be able to see what was inside, although you could leave a gap at the top. Tubes could also be partially rotated, leaving a viewing slot along the length. Has anyone tried this? Would it be worth doing?
The goal isn't maximum surface area, its maximum useable work area. There is a reason slants havent changed much in the ~120 years they've been in use.
really great video !! thanks a lot !!
What yeast source are you using ?? is it slurry or washed yeast or ... ?
It varies. Some are "extra" from a starter, other bottle dregs, and other are slurry from a brew. I don't pre-wash the yeast for slants or plates, although sometimes I do add sterile water to loosen a thick yeast cake.
@@SuiGenerisBrewing I have "propagate" bottle dregsup to 35Oml and now will be able to brew a 3liters batch. Do you think that it would be a good start to follow your yeast banking SOP with the slurry ? thanks
@@3mankind yep, that would be a perfect time
SO... I can start a yeast collection from a single yeast vial purchased anywhere? I just have to make a small starter , then a bigger one and then inoculate into my ( lets say) 20 l Batch? . Which yeast is the best to start a yeast bank . liquid or dehidrated? Im totally new in this field thanks for your your help!
You can start a bank from any yeast source. Dried or liquid doesn't matter - so long as they are healthy.
@@SuiGenerisBrewing thanks a Lot!!
ما هو سعر خط انتاج الخميرة الفورية
I noticed in the video where you were transferring the old yeast to the new slant, my question is, can you do the same thing with the yeast the had the mineral oil in the slant to the new slant Agar? or do you pour out the mineral oil then scrap the yeast out of the slant and then transfer it to the new slant? thanks very helpful videos.
You can directly transfer, and you don't even need to pour off the oil. Grab a colony with your loope, allow the excess oil to drip off, then streak on the new slant. Any oil you transfer will flow to the base of the slant, leaving the yeast free to grow
Thank you for sharing your knowledge.
If you had to choose, which method is the best: Petri dish, Slants or “freezing in tubes”?
Or do you think we need to use all of these methods in our “home lab”?
(I’m deciding on buying equipment) Thanks!
It depends on what you are trying to do. If you're only storing yeast, freezing is best and fairly easy. Slants are great if you only want to keep only a few strains. Plates are more for purifying yeast than storing them.
Sui Generis Brewing Wow. Thank you. You’re golden!
Hello and thank you for this awesome video! I already have all I need to make my own slants, but the agar. Here in Mexico, I have found "agar potato dextrose" (lab grade), and regular agar (the one you use in the kitchen). The first, costs $15usd for 3.3Oz, while the kitchen version is $10usd for 2.2 pounds. Which one should I buy?
Buy the cheap one; the high-grade stuff is for sensitive organismss, and is not necessary for our relatively robust brewery organisms
Thank you kindly! I will do as you suggest =)
Great video, but one critique: When you insert the loop into the tube, you must only insert the flamed wire. Putting the actual tool into the tube virtually guarantees that you'll touch the contaminated surface of the tool itself to the sides of the tube and/or agar surface and contaminate them. The solution to inoculating the entire surface is a longer wire.
Or, just perhaps, my 20+ years of working as a microbiologist means I know how to do simple tasks like sanitizing the handle first.
I've never "Sanitized a handle", nor seen anyone else do so, because 1) "sanitizing" is a brewing term which only extends to killing sufficient numbers of competing microorganisms versus the intended culture (and is thus wholly insufficient for producing axenic monocultures) and 2) the wire technically should glow, albeit briefly, which is not possible for the handle using an alcohol lamp. It's a break in technique to touch anything but the flamed wire to something intended to remain sterile.
I don't mean this as a personal critique- I'm watching the video because the stuff that you're doing with home untensils is really cool and I think it can save me and a lot of other folks a lot of money on yeast.
@@amschind @Adam Schindler Imagine the gall it takes to lecture an actual microbiologist on a term, and to open your argument by saying ""sanitizing" is a brewing term". If you wanted to prove you know nothing of this topic, congratulations, you succeeded.
"Sanitation" has a very specific scientific definition which, in most countries (including the USA, Canada, and EU) is also codified legally. There are even an set of international standards for determining if a product or method gets certified as a sanitizer, disinfectant or steralant: for example, fungicidal activity is determined by AOAC 955.17, with fungal sanitation requiring a 3 -4 log10 decrease in fungal cell numbers after treatment. To qualify as a disinfectant it much reach a 4 -5 log10 decrease, and to sterilize a 6 log10 or better decrease (all starting with a control sample containing 5x10^6 cells). Products like starsan, 70% ethanol and iodophore typically fall into the sanitizer or disinfectant range, depending on the organism tested.
I store my handle when its not in use in 70% isopropyl; hence, it is sanitized (likely disinfected) by the very scientific/legal definition of the term you claim is a brewing term.
For food-based microbiology, which is what we are doing here, that is more than sufficient to limit the risk of contamination to an acceptable level. Achieving true sterility in the home is nearly impossible - with only a few exceptions, most home pressure canners don't even meet the legal/scientific requirements for that (due to insufficient pressure and temperature). Even the aseptic techniques I demonstrate in my videos, when performed in a laboratory with proper equipment, are not considered sterile techniques - which are a whole other range of methods, requiring vastly more expensive tools and facilities (hence why they are "aseptic" - " aiming at the complete exclusion of harmful microorganisms").
Over the past 23 years I've manage a collection that has grown to over 400 brewing organisms, operating largely out of my basement, using these approaches without loss of a single culture. If it was good enough for Pasteur, Koch and Reess, it certainty is good enough for us.
I'm glad that you have so much experience. I also have quite a bit, but that's not the issue. The technique in question was developed by the gentlemen you mentioned for good reason, and isn't debated. When you discuss log kill, you're describing a process which is sufficient for reduction of competing microorganisms but insufficient for indefinite maintenance of an axenic culture. Since we're discussing that sort of monoculture, log kill becomes insufficient for these purposes.
It really seems like you're interpreting these comments as personal attacks and they're not: I just wanted to point out the need to get a slightly longer wire.
@@amschind lets just say that being lectured about techniques by someone who doesn't even understand common, internationally accepted, legally defined microbiological terms that are taught to first year undergrads is irritating. It also makes your claim of expertise hard to believe.
This is also a video oriented at people new to micro, and I don't want viewers to think they need to buy unnecessarily expensive kit because some rando commenter made a factually incorrect statement regarding the appropriateness of a commonly used tool.
Sanitized tools are more than sufficient under conventional conditions to maintain pure cultures. Its the standard used for field micro (where flames are unavailable or unsafe), and is increasingly used in lieu of burners in many facilities due to the dangers of open flames.
thanks for your videos- i was wondering if you could harvest some yeast just before it dies and then repeat until you get a super yeast. if this is possible a video would be great if not a no will do - thanks
You can continually culture yeast in that fashion, and it will lead to evolution of the yeast into something different. Whether that would be a super-yeast, or something else, is less clear.
could you use a yeast nutrient instead of wort to a) create a slant and b) re activate the yeast for pitching? We make a wheat beer and sometimes there are long time stretches between brews of this, so i don't trust the sterilised wort 100%. would be easier to make fresh before either tranfering or starting?
@@attunedosteopathy5074 There isn't really an answer to this as different brands of yeast nutrient are formulated in different ways. Most yeast nutrients will not work as they don't have everything the yeast need.
An easy way to maintain stocks is dry malt extract. You can measure out enough for a slant or two at a time (0.5 to 1g for every 10 ml), and it's stable for years if kept dry.
Another option is to move to lab-grade media. YPD is a ready-to-use yeast medium that can be purchased for not too much. Again, measure out the amount you need (~0.5g/10 mL), dilute in water, and pressure cook to sterilize.
priceless for me....NIGHTSKY BREWERY Future Ales.
Very nice video! A question I'm not finding a good answer to online. Could this technique be used for storing mixed cultures, or just single strains? :) TheYeastBay only rarely delivers to their supplier in Sweden, so having a bank of their Saison blends would be nice.
You're better off freezing mixed cultures. I have a video on that as well - its even easier than slants: th-cam.com/video/o0zluMPcwrY/w-d-xo.html
@@SuiGenerisBrewing Oh, thanks! Will look that up!
@@SuiGenerisBrewing Another question, how important is it that it's glass vials? I imagine that plastic vials "leak" more oxygen which could harm the yeast?
@@leocarlsson3753 plastic is fine - its what we use in research labs. The problem with them is that they are single use, so you need to sterilize the media, then pour it aseptically. Once the slant is used, you toss the tube. I prefer glass to avoid that waste
@@SuiGenerisBrewing Oh! I see. The vials I bought are supposed to be "autoclavable", so I'm going to try it anyway :)
Can you freeze slants by adding glycerin / water into the slant then replace with mineral oil?
Unfortunately, no.
@@SuiGenerisBrewing Thanks
I have a question please. How does yeast become airborne? Can yeast be carried out of my air lock or open fermentation vessel by the CO2?
could you use a pressure cooker to IE autoclave lab glassware? (edit) I guess I should have finished watching the video. I thought I'd allready seen the video.
+rimmersbryggeri You found the answer, but for anyone else who sees your question...the answer is 'Yes'. A pressure cooker can be used both to sterilize media/wort, and to sterilize glassware. Same rules for each - set the pressure cooker to its highest setting, and let it run 15-20 minutes once the pressure relief valve start going. Always let it cool to below boiling (ideally to below 80C/180F) before opening.
Do the vial caps need to be on tight while in the pressure cooker? I did mine just slightly loosened, and about half the contents of every tube boiled out. Made a mess and I had to dump the slants because they were all under filled. Thanks!
You want them loose. If they boiled over, that means you released the pressure too quickly.
Great videos!! So you never freeze the yeast, right?
Not with slants, but I do have another video in this series on freezing yeast, if that is something that interests you.
@@SuiGenerisBrewing thank you!
Can we use this method in mushroom culture?
You can can grow mycelium this way, but you can't store under oil like we do with yeast.
I put sterile water on my slants and now most of the yeast have fallen off the slant and settled at the bottom of the tube. Is this a known issues with this technique?
A few people have ,mentioned this issue to me, so it does happen. The yeast should still be fine.
9:45 Lid hits a non sanitary surface, maybe a chopping block. 9:53 Non sterile part of the loop holder touches the slant media. Is this good practice?
Ideally no, but if you've prepared your work area properly it shouldn't be an issue. See the earlier videos in the series for details.
What do you do with the old slants? I’m assuming clean them out somehow and put fresh agar so that the tubes can be used again
Microwave them for 20 to 30 seconds to loosen the gel, dump the gel in the trash, and wash them in the sink. Once cleaned, the tubes are ready for re-use.
If I use a mason jar with wort that I want to sterilize in a pressure cooker can I keep the 2 part lids on? And how tight?
When steralizing it, have the ring on a little loose so that steam can escape from the jar. Once its canned the sealer part of the lid should be held in--place by a vacuum. At this point you can remove the ring completely to let the threads dry. Once the threads are dry you can store the jar without the ring, or you can put the ring on the jar finger-tight. Don't crank the ring on too tightly, as that can break the seal.
Could you give an advice where to buy 16 mm x 100 mm screw-cap glass culture tubes?
Lab supply stores, ebay, or aliexpress.
Hi, could you specify which mineral oil is most suitable for slants?
Sorry to ask that, but I am from Brazil and for us there are several types of mineral oils, with different purposes.
Not sure I can help - there is only one here. It is a pharmaceutical grade oil used as a laxative and as a skin lotion. Hope that helps.
@@SuiGenerisBrewing It helps a lot. And to sterilize that can I also autoclave it at pressure cooker? Thank you.
@@mauriciocarmona3437 you can steralize with a pressure cooker although that isn't really necessary. It may become hazy if you do - it is still fine, the haze is just water suspended in the oil.
I love your videos but I have a problem making the slants using mineral oil. I followed exactly the instructions. left the slants to grow for 48 hours after inoculation and then when i came to add the mineral oil the yeast just separated away from the agar and entered into suspension in the mineral oil? Can you help me resolve this problem and suggest possible causes? Thanks.
I have not heard of this issue with mineral oil, although people have had this issue with water. Did you refrigerate the slant first? That may help. The yeast should still be fine if they come off in the oil, so long as they settle back down on the slant.
Also, do you see this with more than one strain?
Done twice now. 1st slant was not pre-refrigerated but done straight after making the slant (after cooling). It went fine and was left for 48 hours to grow yeast. Oil did not seem to lift them once added and they are now in storage.
Second slant was pre-refridgerated as I did them a week later on the first batch of slants so same agar etc. I did the same procedure and after inoculation I left them to grow for 48 hours. Once growth had begun and I could see the cultures growing I then added the mineral oil as before. The culture almost immediately freed from the agar and went into suspension.
They were different strains. The first was "american ale yeast' and the second was 'british ale yeast'. Looks like the brits are being a bit awkward :D
Would it be best to try to re-culture these next time I use them?
Thanks
Looks like it may be a strain-specific thing. You should be able to culture from the loose yeast at the base of the slant.
I'm not sure what you mean by "reculture".
Thanks
Maybe I missed this, but what is in the bottom of the tubes before you poured the agar solution?
There should be nothing; the first time I shot the "scene" the camera was out-of-focus, so I had to pour out the tubes and repeat the shot. I was rushing, so I didn't get the tubes clean.
Quick question.. How would I go about calculating/estimating a target amount of yeast cells that I want to get from a starter when using a banked culture?
I assume 50 million/ml for the initial growth, which seems to be close enough most of the time. I then use that number in a yeast starter calculator for subsequent steps. E.g. my initial 5 ml tubes of yeast is assumed to have 250 million yeast.
@@SuiGenerisBrewing Appreciate the response, thanks!
If I grab some yeast with the loop from a freshly opened package of yeast, can I go straight to a slant to store it?
+Steve Sowinski (Airlock Sniffs) Absolutely! In fact, that is the best way to do it, as the yeast you store have a minimal chance to mutate/change before storage, ensuring that your stored yeast perfectly match the yeast in the package.
Usually what I do is dump the yeast into my starter, and then use the loop to grab some of the the yeast "residue" in the tube/sachet, and use that "residual" for my stored culture. That way there is no risk of contaminating my starter, and I still get "first generation" yeast for my bank. Keep in mind that you only need a small drop of yeast to inoculate a slant - the residue in a tube/sachet after you dump the yeast out is likely enough to inoculate a dozen slants.
+Sui Generis Brewing thank you, my tubes arrived yesterday. Once the rest arrives, I will begin.
+Sui Generis Brewing What about "old yeast" on sale, 4-12 months old.
Would you make a starter and collect yeast from it or straight from the old tube/satchet?
Thanks for great video btw.
I would make a small starter from the residual in the tube, assuming it was still clean, and bank that.
Had another thought. Do you put nutrients in the agar sollution?
+rimmersbryggeri Yes, but they are not required. If using yeast nutrient make sure it is one of the 'complete' nutrients (often made of lysed yeast). The cheaper nutrients are usually diammonium phosphate (DAP), which is not nearly as good. As a 'rule' the good nutrient is tan coloured, while the DAP-types are white crystalline powders.
Sui Generis Brewing
I have the wyeast one now but you mean stuff liek servomyces?
The wyeast one is great; it is essentially the same as servomyces. Servomyces is simply a brand-name.
Yeah that's great. To me servomyces seems like a good reference name like aspirin is for salicylic acid and then there are lots of generic brands. But I definitely noticed a difference between wyeast nutrient and the nutient salts I have had available before.
CAn you freeze slants underMineral oil or glycerol?
No. I have a separate video on freezing yeast though in the same video series. Freezing yeast is quite simple, and is the method I recommend for long term storage.
@@SuiGenerisBrewing YeahI have yeas frozen allready using that method.
@@rimmersbryggeri it doesn't work with slants as the agar layer freezes, killing the yeast.
Hi, can i use molasses for agar ? Im using for starter.
It should work, but I've never tried it myself.
Would you use this technique for LAB also?
Yep, although I don't know what the storage time would be. Adding a small amount of chalk to the media helps LABs grow a bit better - a fee g/L is enough.
G'day first off thanks for your video! In the very last part of your video you move yeast off a slant and onto a nice fresh one...let it ferment a bit and place mineral oil in it and fridge. Just wondering what this achieves as it's not clear (ie do you now get another 2 years or so etc). Also maybe a stupid question...but each time you move the yeast and let it start getting to work a bit...is this technically adding a generation to the yeast? (ie how often is this recommended)? Thanks
A slant, stored in the fridge with a tightly sealed cap will reliably store yeast for 6 months or so. With a layer of mineral oil, that is extended up to ~2 years. So its a good choice for storing a yeast you do not anticipate using very often but are unwilling to lose. Retrieving yeast from mineral oil slants is more difficult, so I do not recommend it for frequently used yeast.
Any time you transfer from tube-to-tube the yeast will undergo some cell divisions; in terms of the number of cell divisions (and therefore potential for genetic drift), its pretty close to the number of cell divisions you would get using a starter and then fermenting a beer, so its pretty much equivalent to a 'generation' passed through a fermenter.
Why do you need a pressure cooker? Can't you just pasteurize it essentially be keeping a temp over 150 for a long time?
If you want to keep the cultures pure, pasteurization is unlikely to be sufficient. With this small of a culture, even a minute contamination would ruin it.
@@SuiGenerisBrewing Thx for the reply! So those plastic caps of the tubes don't melt and can take that heat in the pressure cooker?
@@humanonearth1 these are microbiological tubes, so their lids are made of a plastic which is stable at autoclave/pressure cooker temperatures (121C). Most common plastics wouldn't survive though.
@@SuiGenerisBrewing Noted. I'll look for that. Cheers!
Can I fill the tube with glycerin to put on the air??
Not glycerine, but mineral oil (paraffin oil) works well and extends the life of the slant.
In confused what does everything mean and why is he doing it?
Why not parafilm tape instead of electrical tape.. it is cheaper and easier to use.?
The only parafilm I known of is the stuff we have in the lab. It's 5 or 6 times the price of electrical tape.
can you use vegetable glycerin rather than mineral oil?
No. Glycerine will dehydrate & kill the yeast if used in its pure form. You can freeze yeast using a glycerine solution - see my video on yeast freezing for more: th-cam.com/video/o0zluMPcwrY/w-d-xo.html
How many vials for 1 yeaststrain do you use and why?
I don't use slants anymore, and store everything frozen. I have two vials of each yeast, one at home and one elsewhere. The one at home is my day-to-day stock, while the off-site tube is my backup in case of a freezer failure, or if I need to replenish my home stock.
Edit: I also kept duplicate tubes when I used slants.
@@SuiGenerisBrewing ok, thanks a lot.
The frozen ones, do you use the full vial to make a starter? (and make new frozen vials out of that starter?)
Edit: after seeing your "Freezing & Refrigerating Yeast" its clear your make a vial starter of a thawed vial. Guess you re-freeze them.
Pressure cooker ordered on your advise, tomorrow I can start banking. Siked!
@@martijnblokzijl1350 I scrape a small amount of frozen yeast out of the vial, then return the vial to the freezer. One vial will make dozens of starters.
does the heat from the loop not kill the yeast ?
scott garvey at 18:20 I talk about this. You always cool the loop on the glass of the vial, or on an empty bit of gel, before grabbing the yeast
i gotcha Sui....it just seems like it would still be hot . I don't know ..maybe I am just parranoid...I cut steel a lot ...it always seems to take a while to cool ...i guess the loop is a lot quicker cooling than 1/2 inch steel ! would you be focusing this video more on small nanos or would u would suggest this for home brewers !?
scott garvey the videos are orientated at homebrewers...hence the homemade kit and lack of lab grade materials.
yup...I gotcha . Tell ya what though...u probably have better lab skills and equipment than most nano breweries !;)
Slants with oil.can go to the freezer? or just fridge? I need yeast to be preserved for at least 2 years
Just the fridge. To maximize lifespan, fill the tube completely with oil, cap, and then seal the top with vinyl tape (e.g. electrical tape). Stored like this, most yeast will last 2+ years in a fridge.
I have a video on using glycerol to freeze yeast if you want to go that route.
Many thanks!!
Is mineral oil the same as paraffin oil?
+Ionut-Alexandru Batrinache Depends. In some countries highly purified (i.e. medical grade/cosmetic grade) mineral oil is sold under the name of paraffin oil. That sort of oil would be ideal for freezing yeast.
Unfortunately, in other countries paraffin oil refers to a clean-burning lamp oil, derived from kerosene. Using that would be acutely toxic to your yeast.
So if the recommended use of the paraffin oil you have is as a laxative, skin care, or preparation of cosmetics, it will be fine. If its meant to be burned in a lamp it won't work.
Sui Generis Brewing Yes, the paraffin oil i have is bought from a pharmacy. Good to know, i will try this method. A video about freezing yeast it glycerine would be nice too :)
A freezing video is in progress, but no ETA is set