It would surely provide extra security. Air is your enemy when storing on slants or plates. And, I once lost a culture because I did not tighten the cap. That said, no I don't parafilm or tape my slants. For short term storage on plates, I use good quality electrical tape.
Slanting yeast
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It would surely provide extra security. Air is your enemy when storing on slants or plates. And, I once lost a culture because I did not tighten the cap. That said, no I don't parafilm or tape my slants. For short term storage on plates, I use good quality electrical tape.
I am plannining on using color coded elect. tape on the plates... but I do fear sticky gooey residue... what is your concern over good quality? I was planning on the cheap Chinese stuff, unless you know of issues....
Three conditions you may want to consider when time to re-store your yeast; 1)Yeast in storage can mutate over time., 2) Possible contamination, and 3) poor yeast health. All these can be managed to some extent by dilution streaking to form single colonies before re-storing onto a slant.
One of the most common mutations is called "Petite mutants" or "respiratory mutants". As the name implies this type of mutant will form small colonies on a plate. If you have a large percentage of small colonies, your isolate may have problems. Be aware that large colonies often form as a result of growth from 2 or more cells. Typically in a healthy dilution streak plate you will see a few large colonies and numerous smaller (average size) colonies. It's the ones that are smaller than the average colonies that could be of concern.
Your slant could be contaminated and you can't see it. By streaking from your slant for single colonies, many possible contaminates would become visible. If contamination is spotted, all is not lost. You may be able to select a clean colony for re-storage. In this case, it may take least one or more rounds of re-isolation to be sure you have purified your isolate and a fermentation test may be advised.
For sure your year-old yeast are tired, hungry and in poor health. Think of a hibernating bear. Upon awakening, the first thing she wants to do is build back her reserves. By spreading out your yeast on a agar plate, they have plenty of room to get all the nutrients and sugars they need to from healthy new cells that are ready to go back to sleep and get ready for their next job.
For me it's easier to use (more flexible) and sticks better. Even with the good stuff, it often comes loose at the end in cold conditions. Like earlier advised with saran wrap or parafilm, it seems to work and seal better if stretched as applied. I don't remember gooey residue with the cheaper stuff but IMO the good stuff is worth the extra $ and does a lot of plates.
brewski09
Well-Known Member
That's some great information. Thanks. To re-ask a question just up thread do you consider it a new generation of yeast if you take it from slant to plate?
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Well, I didn't answer earlier because I really don't know. But I'll give you my thoughts. As Tally350z says, "I only think of different generations when the yeast go through the streesses of a fermentation." I would call that Fermentation Generations. If you are re-pitching yeast, it is very important to keep up with how many generations the yeast have been used. That would allow you to better manage re-pitched yeast. Likewise, if one is re-storing yeast it would be considered good management to keep track of how many time the yeast has been re-stored. So I guess you could call each re-storing a "generation". However, if you are also re-pitching yeast, I would keep those "generations" separate. I would call the first pitch to a fermentation the "first generation" regardless how many times that yeast has been re-stored.
![Craft A Brew - Safale S-04 Dry Yeast - Fermentis - English Ale Dry Yeast - For English and American Ales and Hard Apple Ciders - Ingredients for Home Brewing - Beer Making Supplies - [1 Pack]](https://m.media-amazon.com/images/I/41fVGNh6JfL._SL500_.jpg)
ColoHox
Compulsive Hand Washer
As long as you are consistent with your batch/generation/pass recording, you can use any approach that you like. Either just record fermentations, or record every time that the culture is stored or grown, etc. Here is a handly flowchart I designed to demonstrate the different recording methods:
ColoHox
Compulsive Hand Washer
That's fair. I am showing that the terms can be used in different ways. A stock would be storage (slant/frozen/washed), a "batch" of beer (round of full fermentation), then passage and generation are nearly interchangeable.
The labels (stock A&B) are just ways to keep track of the stored cultures, it is good practice to make more than one stock. So in the right side of the scheme, one of the stocks originally generated from the first starter (stock B, out of many stored cultures, ie more than just A and B) is used to ferment a batch. From that batch, yeast is washed (and/or slanted, frozen, etc) and split into two new stocks (B1, etc), then a new stock is used to ferment a new batch.
In my yeast farming practice, I follow the left side. From the original source, I make 10-15 working stocks. Each stock then ferments 2-5 batches, depending on the beer. On my last stock, I generate 10-15 more working stocks, all of which are a new generation than the previous batch of stocks, which is taken into account when fermenting new batches.
Is that more clear?
HollisBT
Well-Known Member
Has anyone tried to, or been successful at, slanting lager yeast?
And if so, how do you adjust your propagation steps? Simply allow longer (two to three times as long?) intervals for growth?
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And if so, how do you adjust your propagation steps? Simply allow longer (two to three times as long?) intervals for growth?
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Unless slanting at near freezing temperatures, perhaps such an increase is not needed. Grown at the same temps, growth rates of different yeasts seem roughly the same to me.... (limited observation).
Just did this, thanks for all the help. Used telephone brand agar, available on ebay for cheap. 7.5g in 500ml of wort.
http://imgur.com/a/a3FTo
http://imgur.com/a/a3FTo
cyberbackpacker
Well-Known Member
Thanks lol, nothing that isnt already on this great thread, but why not add more pics.
I pushed it to our club site with a bit better write up, http://laramiebrew.club/2014/10/yeast-slanting/
I pushed it to our club site with a bit better write up, http://laramiebrew.club/2014/10/yeast-slanting/
Do you think I could reanimate some slanted yeast in a tube of 40ml wort? I usually use this as my second step, with the first being 10 - 15ml. Each step last for about 24 hours so I was thinking of leaving the 40ml tube for 48 hours maybe with a few extra drags from the slant. Would this work?
HollisBT
Well-Known Member
Hey guys, I recently slanted a culture of 1217, and had after inoculating my media and letting the culture grow, I got some pretty heavy condensation on the inside of my tubes. The yeast looks very bright white and healthy on the media, and in sure that the condensation is nothing to worry about, but any input here? I just sealed the tubes and put them in my fridge, should I take them back out and loosen the caps until it dries completely?
FredTheNuke
Well-Known Member
I would leave them be. Yeast get thirsty too!
I'm pretty sure I'm most impressed with the "balled up foil" improv. Not sure why I didn't think of that. Thanks.
deeringbrewing
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Hey all. What are your procedures for plate streaking? Are you using a pure yeast colony on the loup to streak or are you diluting a pure colony in sterile water first? I'm just getting into streaking to get a pure strain and rid bacteria. I tested using a pure colony on the loup yesterday, I have been diluting first but I was getting varied results.
Some of my buddy's budding.
Some of my buddy's budding.
Nice picture. So the idea behind plate streaking (aka dilution streaking) is to isolate pure colonies. So if you have an isolate from a slant, a culture from bottle dregs, captured wild yeast, or want to isolate from a nice fermentation, etc., you can use this technique to get individual colonies that have formed from a single cell. The idea is to spread the organisms so thin that these individual colonies form. Below is an illustration of the way I do it (there are other methods out there) and below that is an image of the results.
First get a very small amount of yeast (let's say a slant) on your loop. If you can see it, it's enough. Then spread onto the first section of the plate by moving the loop back and forth without picking it up until the section is spread thin. Then flame the loop and spread from one end of the first section into the second section in the same manner, flame and move to the third section overlapping the second section. After you have spread the third section and without picking up the loop continue in a zigzag pattern as illustrated. Flame the loop and in a single swipe move through section 3 including the zigzag and again without picking up the loop continue in a zigzag pattern into the final (#4) clean area of the plate. The single colonies will most likely form in the zigzag pattern of section 3, however if things are too thick your single colonies may form in section 4.
If you were isolating from a culture (dregs, wild ferment, etc) more volume is needed, so use 3 to 6 loopfulls of the culture for the first step. Hope this helps.
deeringbrewing
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Cool pick. That's pretty much what I tried yesterday.
I'm hoping to get some isolation. Maybe I'm using too much yeast. I'm pretty much grabbing a full colony off a previous plate.
Oh and thanks for the help, much appreciated.
I'm hoping to get some isolation. Maybe I'm using too much yeast. I'm pretty much grabbing a full colony off a previous plate.
Oh and thanks for the help, much appreciated.
bionut
Well-Known Member
Hello,
I plan to slant some yeast soon, i have all the equipment needed besides the pressure cooker. This evening i found in a store a 9 litre (2 gallons?) one, with this inscription: working pressure: 0.50 bar, max pressure: 1.1 bar. Those numbers translate to working:7 psi, max: 15 psi, so, for a good sterilization i need to use it at full capacity, or near that. I will probably buy it anyway as is on offer and i could use it for cooking, but do you think that it could be used for yeast slanting also?
I plan to slant some yeast soon, i have all the equipment needed besides the pressure cooker. This evening i found in a store a 9 litre (2 gallons?) one, with this inscription: working pressure: 0.50 bar, max pressure: 1.1 bar. Those numbers translate to working:7 psi, max: 15 psi, so, for a good sterilization i need to use it at full capacity, or near that. I will probably buy it anyway as is on offer and i could use it for cooking, but do you think that it could be used for yeast slanting also?
bionut
Well-Known Member
Anybody? :fro:
FredTheNuke
Well-Known Member
Yes 15 psi is the standard for pressure cookers and provides 250°F water for sterilization. Although true laboratory sterilization requires 270+ that type of equipment is very expensive. An autoclave for example uses up to 30 PSI steam which gives almost 280°.
I'm leery of a couple of thing that may make this unit less than optimum. First the "working pressure" of 7 psi. But you realize that 15 is what's necessary so if you feel comfortable operating it at max then it should work. Caution: steam under pressure is very dangerous. This recently came to my full awareness when the overpressure plug blew out on my pressure cooker. While I was operating the unit at 15 psi, I guess it failed do to age. Anyway, it shot out a column of steam to rival old faithful. Any human flesh exposed to that eruption would have been instantly destroyed.
Secondly the size. At minimum, it will need enough capacity to hold your slants, which it should at 2 gallons. However, for propagation it is really nice to be able to sterilize your flasks (or other propagation vessels). This comes in handy because you can mix up your extract in various sized flasks (as per your step up needs) and stick them in the pressure cooker for 15 -20 minutes and it come out ready. No worries about boil over. Same with preparing solid media for plates. In addition, you may want to can wort for you propagations. I am not sure if the unit you described would hold quart jars but the standard is a 12 quart or larger.
While the pressure cooker described above will get you started, if you feel like going all way, I would recommend at least a 16 quart like this: http://www.ebay.com/itm/MIRRO-MATIC...all_Kitchen_Appliances_US&hash=item259c867663. I think (but not sure) it would hold a 2 liter flask. Or even better and what I have is this: http://www.ebay.com/itm/Beautiful-U...all_Kitchen_Appliances_US&hash=item339c57474b. This will hold a 5 liter flask.
bionut
Well-Known Member
In my country big pressure cookers are very expensive, if i could find one like that from your first link i would be fine. In 2 gallons i believe that i could sterileze some mason jars, or a 1000 ml flask... I don't know what would take to operate it at the max pressure, is there a way to minimize the pressures escape? Wouldn't the valve go off where the max pressure is reached?
darylwoodman
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First off, let me qualify my statement about the necessity of a pressure cooker by saying that I am a sterility expert. I've spend the last 17 years validating sterilization processes for the medical device industry. While most of my work is with gas, I have validated many steam sterilizers all over the globe and have peer-reviewed published literature regarding microbial lethality. The standard autoclave runs at 121°C for 15 minutes at X psi depending on the sterilizer. The pressure allows the superheating of water to temperatures above boiling. Water boils at 100°C--give or take, and still sterilizersjust slightly slower.
Now, let's talk about microbial lethality. Heat is a very good vehicle for rendering bugs dead. At 121°C, you can kill one (1) log of the absolute most resistant spore formers in about 2.5 minutes (it's decimal reduction value or D-value). Let's say this decimal reduction value is 2.5 mins. You will reduce 1,000,000 bugs (6 logs) in 2.5 mins x 6 = 15 mins.
Now, at 100°C, you are still going to get kill, it's just the D-value is going to be longer. Most everyday bugs are so much easier to kill than the resistant spores used to validate steam sterilizers. In fact, the typical D-value will probably be less than 1 minute at 100°C. That means that if you had 1,000,000 bugs in your vial, all it would take is boiling it for 6 mins at 100°C and everything would be dead. Now, run it for 15 minutes. Nothing is going to survive.
Keep in mind that with yeast slanting, you have already boiled your wort and added gelatin. Even with a non-sterile vial, you are probably starting with a handful of bugs at the most--maybe 100-1000 if your vials were dirty. Three (3) mins at 100°C will kill them. (Keep in mind that the temperature of the vial will be slower to heat than the water in the pot; the glass vial will act as a thermal barrier and slow the heat up time. You could add additional heat time, but frankly I wouldn't bother.)
You can verify sterility success 100% through inspection, that is leave your vials out for 14-days at room temperature (capped of course), and if they are free of bugs, you have a sterile product. I only use a pan, a veggie steamer to keep it off the bottom and s few inches of water.
Check out my webpage. I have a 9-step process with imagesthe whole process is less than an hour.
http://www.acapellabrewing.com/project/diy-yeast-slants/
Cheers.
Now, let's talk about microbial lethality. Heat is a very good vehicle for rendering bugs dead. At 121°C, you can kill one (1) log of the absolute most resistant spore formers in about 2.5 minutes (it's decimal reduction value or D-value). Let's say this decimal reduction value is 2.5 mins. You will reduce 1,000,000 bugs (6 logs) in 2.5 mins x 6 = 15 mins.
Now, at 100°C, you are still going to get kill, it's just the D-value is going to be longer. Most everyday bugs are so much easier to kill than the resistant spores used to validate steam sterilizers. In fact, the typical D-value will probably be less than 1 minute at 100°C. That means that if you had 1,000,000 bugs in your vial, all it would take is boiling it for 6 mins at 100°C and everything would be dead. Now, run it for 15 minutes. Nothing is going to survive.
Keep in mind that with yeast slanting, you have already boiled your wort and added gelatin. Even with a non-sterile vial, you are probably starting with a handful of bugs at the most--maybe 100-1000 if your vials were dirty. Three (3) mins at 100°C will kill them. (Keep in mind that the temperature of the vial will be slower to heat than the water in the pot; the glass vial will act as a thermal barrier and slow the heat up time. You could add additional heat time, but frankly I wouldn't bother.)
You can verify sterility success 100% through inspection, that is leave your vials out for 14-days at room temperature (capped of course), and if they are free of bugs, you have a sterile product. I only use a pan, a veggie steamer to keep it off the bottom and s few inches of water.
Check out my webpage. I have a 9-step process with imagesthe whole process is less than an hour.
http://www.acapellabrewing.com/project/diy-yeast-slants/
Cheers.
FredTheNuke
Well-Known Member
Much like alcohol flaming the sealing surface of every container in this process is overkill.
First let me say I agree with everything you're saying here. I do however want to post a word of caution as we as homebrewers delve into the world of microbiology. Boiled media will certainly be sanitized to the point where we can successfully propagate yeast with very little concern of contamination. In fact we would have a greater chance of contamination while inoculating the slant than from anything that might survive the boiling process.
However, as we get more involved in the process it becomes inviting to save wort for future use as media. Although unlikely, it is perhaps possible for Clostridium botulinum spores to survive the boiling process followed by germination and sporulation in stored wort producing the neurotoxin botulinum. Since this is the most potent toxin known to humankind, it is not something we want to take chances with. So if you want to get started slanting yeast without a pressure cooker, you can achieve success. But please don't store wort without first subjecting it to at least 15 minutes under pressure at 250 degrees F.
A couple of question on your slant preparation method:
1) How much gelatin are you using?
2) Have you tried plates with gelatin?
3) Is the consistency similar to media prepared with agar?
4) Do you have issues with condensation in your slants after subjecting them to boiling or pressure cooking. (For various reasons, including condensation, I have been preparing the media in a flask and after sterilization, adding it to sterile tubes after it has cooled to ~120 degrees F. However, like you I have a laminar flow hood.)
5) If condensation is an issue, do have suggestion to remediate the problem.
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