Slanting yeast

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I have heard of people boiling w/ some luck, but i think you're going to get more contam's and headaches then is worth it. Even working with fully sterilized agar can be quite discouraging if your handling procedure is not fully sterile, aka good glove box (or better yet flow hood).

The real big issue will be if there is a small contam you cannot see, and it makes it into your batch of beer. No good.

Really there is no getting around buying a pressure cooker if you want to work with agar/slants. But a small pressure cooker, enough for a beaker of vials, is not a large investment.
 
Would I be correct in assuming that the pressure cooker provides a means of sterilizing both the media and vials?

I am thinking about boiling the media and funneling it into the vials. Then I'll put the filled and partially capped vials into a beaker that will be placed into a boiling water bath. I will cover the bath (pot) so as to allow the steam to make contact with the vials. This should be sufficient for sterilizing the vials, right?

-or-

Could I simply boil the media and funnel it into the vials and then place the partially capped vials into a stove for a period of time? Like a ghetto autoclave?

Bottom line is that I do not own a pressure cooker, but there should be alternative means of successfully sterilizing the media and equipment.

Boiling will not sterilize. It will kill most things, so you may get lucky, but you never know.

Truethfully, though, most pressure cookers only get to 10 PSI (240F) which will kill just about everything, but not quite. To really sterilize, you need a canner that gets to 15 PSI (250F). I wonder how many people are using 10PSI with success. You may be ok at 212F, but I wouldn't count on it.
 
It isn't the steam making contact, it's the temperature of the steam. At normal atmospheric pressure, water boils at around 212F. The steam is no hotter than that. It takes about 15 PSI above atmospheric pressure to raise the boiling point of water to 250F.

This is why it is necessary to have a pressure cooker to sterilize...
 
I have heard of people boiling w/ some luck, but i think you're going to get more contam's and headaches then is worth it. Even working with fully sterilized agar can be quite discouraging if your handling procedure is not fully sterile, aka good glove box (or better yet flow hood).

I don't believe you need a glove box or flow hood for any of this kind of stuff unless you have a really unclean environment.

I do all of my yeast work on my washing machine which I clean the top of and sanitize. I make sure there are no drafts (windows or heat/air running). I use a simple alcohol burner right there and make sure not to move quickly or breathe in the direction of an open container.

Unless I was testing procedures, I've never had a contaminated plate/slant. Thinking through your process and planning out what you are going to do before you open anything is a big help.

I also will wipe everything I'm going to use with rubbing alcohol while setting it into my clean area.

If you have long hair, tie it back. Scrub up your hands and arms before starting.
 
Edit: doh! didn't realize you were saying you were having to by a PC. You can buy per-sterilized agar, but its expensive for the shipping costs if you purchase it in liquid form.

Don't be discouraged! I don't believe you need a flow hood at all. That's over kill. What you need is a nice small room (ie. bathroom) and the air is not actively circulating via air conditioning. Airborne spores of molds and bacteria are carried through the vent ducts which can cause problems contaminating your slants. That being said there are good precautions you can use to try to prevent contamination.

Practice Sterile Technique

-Pick up an alcohol wick candle and some 90% ethanol(or better yet Grain Alcohol!). High % Ethanol will help sterilize the equipment by killing most microscoping living organisms. The flame helps insure that no contaminants transfer between different samples you may work with during that one sit down time. NOTEeep each item ~1 foot from one another at all times when using them together.

- Every time you change samples (ie. Slanting Pacman and US-05 in one sitdown) you should flame your dip your tool in Ethanol for a few seconds. Let the ethanol evaporate - don't shake near the flames. When dry flame your tool for a quick second - if its metal get it red for a second then let cool.

- You can use sterilized tooth picks to do this too, but they're a bit harder to find sterilized than to buy a single metal inoculation loop. Its far easier to sterilize and can be reused. Also if you use wood, don't do the ethanol step. Just do the flame step.

- Open container 1 obtain your yeast from container 1, flame the glass mouth in the flame for 1-2 seconds while rotating the bottle to get all edges. Then close. Open slant 1, flame top if its glass,streak it on the agar slant, and close slant tube quickly after inoculation. Either toss your wooden spread stick for another or dip your metal loop in the Ethanol to start back over.


- Also be sure not to breath on the agar surface if you can help it rather breathe away from it. Its really not that bad when you get used to it. You just need some patience and a pair of latex gloves.

- Dont' allow any tool that will touch the yeast un-clean surfaces without being sterilized again. Bacteria are ubiquitous in the environment regardless of how clean you think it may be. Those same bacteria can easily utilize the same agar for quicker growth than the yeast can handle. Thats why I suggest the sterile technique.

Trust me, it's do-able. I work in a microbiology lab where we constantly culture bacteria on all types of agar. We deal with the same issues, but we find ways where we don't have to use the 1 hood available. The name of the game is being sterile and careful. Best of luck!

You guys should take a note from people who grow mushrooms. those people really know how to culture and keep around their fungus. you guys have it easy compared.
 
Any tips on handling a pure brett strain on agar? I have just acquired lambicus and it grows insanely on the plates I have just innoculated. I can tell already its a totally different behavior. Should it get different treatment/media/agar type for long term storage? Maybe something to retard its growth... Also I'm hoping water storage also works well for lambicus.
It is truly frightening.
brett_plate_1.JPG
 
i have an opportunity to pick up a nice 4.2 qt pressure cooker for $10 that is in good shape. It is kind of small but would it still work? I dont brew too much so i will only have a few slants of each strain so i wont be making more than 1o ready to go slants at a time? Just not sure it its a waste of money and i should wait for a deal on a bigger pressure cooker
 
Eric, just wanted to give a big thanks to you for this tutorial. As my brewing's come along, I've started buying bulk grain and hops, but it's still a chore to head to the LHBS every time I want to brew to snag the yeast - and not cheap at ~$7 a pop. (And with the nearest LHBS a 2 hour round trip from me, it's hugely inconvenient, I'm usually unable to get out there until brew day, which means I always have to get at least two packs, since I don't have time for a starter). Having slants in my arsenal would be outstanding.

I will say, I'm still terrified by the thought, though. Just a couple of little stray cells in one of these can happily kill a batch of beer - but only way to try is to... try!
 
so if i made 12 ml slants and was preparing with a stir plate could i follow these steps?

1 - add canned wort to slant and start a 100ml starter for 1 day
2 - day 2 pitch the 100 ml into boiled 1L or 2L started
3 - pitch into fermenter

How does one know how big of a final starter to use without counting? Is there a general rule of thumb that the yeast should be 100% viable so a 100ml into a 1L starter would be fine for a beer with OG under 1.060. My biggest concern is i use to just pitch the white labs vials and i got alot of bad off flavors from under pitching
 
ek, that's one of the issues (in my mind) that's stopping me from using slants. From what I've read, the time from slant to pitching is about a week - I believe what I read was along the lines of "if you get your starter going Monday, it should be ready for Saturday - start a couple days earlier if your OG is over 1.060."

You'd probably want to start with a 50ml starter (36 hours?), stepping to 250ml (2 days), then 1000ml (2 days) for a beer, under 1.060. If it's higher than that, add another 2 days of a 2000ml starter.
 
That is definitely more time than required. I brew ten gallon batches and I need 3-4 days.

Wednesday nightslant->10ml wort ->Thursday nightadd to 30ml (12 hours) -> Friday morningadd to 100ml -> Friday nightadd to 300ml -> Saturday morningadd to final starter volume

Every step after 100ml I do on a stirplate. I usually pitch the entire starter. For lagers I add my starter to a gallon or so of wort and let it go another 12 hours at pitch temp before adding back to the chilled full volume.
 
wow this kind of a lot of work. If i go straight from vial to canned (pressure cooked) wort could i just go to a 100ml into a 1L or do i have to worry about the little bit of bacteria that would be in the 1L flask.

Budzu What do you put your 10 ml in? also are you using canned or boiled wort?
 
I can wort from post-boil dregs. Filter through cloth then add water until 1.040.

BTW I am not using a whole slant to innoculate the starter, I'm using only a tiny sample scraped from the slant.

I have several sizes of flasks, and I'll measure out each of those additions into separate flasks before starting from a slant. Then aluminum foil on each flask and run them through the pressure cooker again. I use a 24ml vial (like you might have a slant in) for the 10ml first innoculation. then a 50 ml, 250ml, 1L, and finally a 2L (or gallon jug). I bought the set of flasks from cynmar, 5 or 6 flasks for pretty cheap. You could certainly skip some steps, but I would really suggest keeping the first addition very small and as sterile as possible.

You might go sterile 10ml -> sanitary 100ml -> full volume. Those steps just may need more than 12 hours. The main reason my steps are small is because I like to pitch the whole starter rather than decant. This ideally minimizes the off-flavors from excessive lag time in the starter.

This is just the way it works for me. I took alot of techniques from Pierre Rajotte's book.
 
I have never had in infection from building up from a slant. Ever. I have had all of: infections, off flavors, and long lag times from re-pitching saved yeast (eg. washing). My experience has been that building up fresh yeast from a slant before every brew is the way to go.

If I'm brewing 10 gallons on Saturday, I will start a slant vial on Thursday with 250mL of sterile wort. On Friday I will split half of that across two 1-2L starters, and pitch both of those on Saturday at high krausen.

Yeast grow to insanely high numbers really fast when you grow them under ideal conditions (lots of nutrients, stir plate, oxygen in the brew).
 
Thanks for that, Eric, feeling a lot less nervous about it now. My erlenmeyer flasks and glass pipettes came today, although my vials and beakers were on back order. Need to score a pressure canner as well, although trying to find as big a one as possible so I can can starter wort and take that step out of the way.

So all you're doing then is pouring a little of the sterile wort into the slant, shakey shakey, then pitch that into a 250ml, along with some yeast nutrient, on a stir plate? Or are you using pure O2 to oxygenate beforehand?

EDIT: Sorry. I just lurneded 2 reed and use da serch funkshun!

This is the best online resource IMO regarding yeast cell concentration and growth rates in wort:

http://www.maltosefalcons.com/tech/yeast-propagation-and-maintenance-principles-and-practices

If you read, you will see why I use a stir plate AND sterile wort for the first step; the method he gives you can use non-sterile wort safely but it requires adding 10mL of wort (which you could add directly to the slant vial) and wait 2-3 days before pitching the 250mL starter. Since I use sterile wort and am very very careful with my sanitation in the first step, I go straight from the slant into 250mL without any problems.
 
There has been plenty of discussion of yeast slanting in various threads, and I know there are other slanters here on the forums, but I have not seen a definitive thread which walks through the process step by step. So, last time I prepared some slants I took some photos and decided to post a tutorial here.

Although it is an advanced technique, yeast slanting has some advantages. You can share yeast strains easily by exchanging slants. Each yeast culture you buy can be used to make 25 batches or so without re-using yeast. You can save platinum/seasonal yeast strains for use year-round. You can harvest yeast from a brew buddy's starter to add to your library. Or if you go to the trouble of bottle harvesting yeast you can save it for future use. As long as you are very careful with sanitation while handling yeast slants, the risk of a contaminated batch is very low -- especially if you compare this technique to re-using yeast from prior fermentations.

I didn't make up all this stuff myself. There are some good resources out there on the web, so be sure to check them out as well before you get started. To name a few good ones:

Making Plates Slants - German Brewing Techniques
Yeast/Culturing - Brewiki
Culturing Yeast and Using Slants
Yeast Propagation and Maintenance - Principles and Practices

Is it possible to keep the DME agar agar mix after the boil? I have about 140ml of solution left and was wondering if i I can put in a plastic container and keep it in the fridge.
 
Is it possible to keep the DME agar agar mix after the boil? I have about 140ml of solution left and was wondering if i I can put in a plastic container and keep it in the fridge.

I wouldn't recommend it, it's likely to ferment. I usually mix up 250ml, which is plenty for about 20-25 slants (with the 24ml vials I'm using).
 
Is it possible to keep the DME agar agar mix after the boil? I have about 140ml of solution left and was wondering if i I can put in a plastic container and keep it in the fridge.

http://www.alsand.com/beer/yeast/index_E.html
If you check this out and click at the top "Pouring plates", it explains how you can save solid sterile agar/wort solution. I however have not had success with getting it to melt and re-liquify. It came out kind of lumpy when I tried it. Its just hard to tell when its all melted. But as far as i know, yes you can. Just be sure not to autoclave your agar solution more than once.

"Instead of pouring many plates at once, I store the solid sterile media in small 100 ml bottles, sealed with a rubber closure.
When the need arises, I simply heat a bottle until media liquifies, then pour it in 5-6 plates."
 
Did my first slants up last week and tried capturing something from the dregs of a bottle of Lost Abbey Brett Devotion and it looks like it worked!
147651197-31e0ab77508e9a1bbcf3976b0dfc0fe4.4c6c0a88-scaled.jpg
 
4997365416_40d117cfde_z.jpg


Thanks for the thread. It gave me the motivation to move ahead with this.

The red color is some food coloring I added for fun. Maybe I can change up the color each batch so I have an idea of how old they are without reading the label.

I inoculated my first two strains today.

My slants had been prepared 2 weeks ago and sat out at room temp. No signs of contamination.

Lets see how they do once they've been loaded up with yeast.

I'm glad I did this. Not as hard as it seemed. I've been thinking about doing this for a couple of years now. I've been holding back because I didn't know if my pressure cooker was able to get the pressure and temperature that I needed. But I decided that I would give it a shot and upgrade the cooker if it didn't work out.
 
4997365416_40d117cfde_z.jpg


Thanks for the thread. It gave me the motivation to move ahead with this.

The red color is some food coloring I added for fun. Maybe I can change up the color each batch so I have an idea of how old they are without reading the label.

I inoculated my first two strains today.

My slants had been prepared 2 weeks ago and sat out at room temp. No signs of contamination.

Lets see how they do once they've been loaded up with yeast.

I'm glad I did this. Not as hard as it seemed. I've been thinking about doing this for a couple of years now. I've been holding back because I didn't know if my pressure cooker was able to get the pressure and temperature that I needed. But I decided that I would give it a shot and upgrade the cooker if it didn't work out.

I know this is a little asinine, but those are some sharp looking vials :D
 
4997365416_40d117cfde_z.jpg


Thanks for the thread. It gave me the motivation to move ahead with this.

The red color is some food coloring I added for fun. Maybe I can change up the color each batch so I have an idea of how old they are without reading the label.

I inoculated my first two strains today.

My slants had been prepared 2 weeks ago and sat out at room temp. No signs of contamination.

Lets see how they do once they've been loaded up with yeast.

I'm glad I did this. Not as hard as it seemed. I've been thinking about doing this for a couple of years now. I've been holding back because I didn't know if my pressure cooker was able to get the pressure and temperature that I needed. But I decided that I would give it a shot and upgrade the cooker if it didn't work out.

Jason,

Where'd you get those vials? The ones that I've got don't have autoclavable tops and now I'm jealous of yours.
 
Jason,

Where'd you get those vials? The ones that I've got don't have autoclavable tops and now I'm jealous of yours.

I got a part number for Cynmar in the second post of this thread. See the quote below. I used the part number to do a search. The website is kind of clunky, but a direct search of the part number should find it. Once you stuff arrives, you'll get a catalog. The catalog will keep you busy for hours.

You will need some equipment:

  • A pressure cooker, 8 quarts or larger
  • A small pan (unless you are a cave man, I'm sure you already have one)
  • Filtered or bottled water
  • Dry malt extract
  • Vials. I use the 24mL vials from Cynmar, stock no. 115-27910.

View attachment 12594

Here is an update. I have growth on the slants! :mug:

5006602252_dd4e74b8bd_z.jpg
 
I got a part number for Cynmar in the second post of this thread. See the quote below. I used the part number to do a search. The website is kind of clunky, but a direct search of the part number should find it. Once you stuff arrives, you'll get a catalog. The catalog will keep you busy for hours.



Here is an update. I have growth on the slants! :mug:

5006602252_dd4e74b8bd_z.jpg

NICE! Good looking cultures too. My stuff's on the way, can't wait to get started. I think the food coloring is actually very useful, the contrast helps. I'll be using it.
 
Glad you had success. Yea your slants look great. The food coloring is a good idea I hadn't thought about that.

Everybody I know who has started doing this was surprised at how easy it is, including myself, which is what motivated the thread... I wanted to show you don't need to spend a lot of money, or time, or effort to get into yeast banking.

:mug:
 
I got a part number for Cynmar in the second post of this thread. See the quote below. I used the part number to do a search. The website is kind of clunky, but a direct search of the part number should find it. Once you stuff arrives, you'll get a catalog. The catalog will keep you busy for hours.



Here is an update. I have growth on the slants! :mug:

5006602252_dd4e74b8bd_z.jpg

About that second slant from the left, I have had similar problems. You have to control that because it will eventually push itself out of the vial through whatever crack it may find. That one will need reslanting or use it very soon.

The problem is that moisture (condensation) has found its way between the agar and the glass, and the yeast starts to grow there, pushing the agar up and away.
The solution I have found is to make sure that your slant slants enough to leave part of the bottom of the vial uncovered. This ensures, even if you have some residual moisture, that co2 can escape without disturbing the agar.
Directly addressing the problem (eliminating all condensate) is something I cannot yet do... (any suggestions anyone?)
 
I'm not sure what strategy to use against the rising agar. I did see liquid in my tubes. I believe the agar set very firm. My inoculation loop wasn't able to penetrate the surface.

One possibility would be to leave the caps loosened during the first week following the agar setting. This might allow the condensate to evaporate.

Another idea is to use less agar per tube. If the entire bottom is not covered, then you wouldn't have a plug that can be pushed out of the tube.
 
I'll try your idea of using less agar with a more aggressive tilt on the next batch of slants that I create. I've still got about 10 blanks left. I'm trying to do 5 tubes per strain. So I've got room for two more strains. I'm guessing that will last me through the year and leave some room for waste due to contamination.
 
Since others are posting pics of their bank I thought I would post mine. Here are the 12 strains I've got right now on slants. They've finally grown up enough that they'll be sealed and moved to the fridge later today.

2010-09-22_10-47-22_774.jpg
 
my slants are from last december, and still look good. but a re-slant is in order for the heck of it.

since i can't find anywhere what happens during a double autoclave, since i have some in round bottom test tubes, i will re-clave a few and see, since they don't stand up alone and are nearly full.
 
No that DOES NOT sterilize it will sanitize pretty well I'm assuming. But for something to be sterile you need heat and pressure. Well there are other ways but this is the most reasonable way for us. Don't get discouraged brother. It's like adding home brewing to home brewing. It's great. I also like when people look at me like I'm whacked when I bust out the microscope and hemocytometer.
 
i had a quick scan through the thread but didn't find if anyone had asked this- is a microwave steriliser that is intended for baby bottle suitable instead of a pressure cooker?

I've never done this. I would be interested to see the results if you try it.

Read this article that talks about using the microwave to sanitize in the home medical arena:

http://findarticles.com/p/articles/mi_qa3890/is_199905/ai_n8840314/pg_1?tag=artBody;col1

The article makes it sound like the microwave knock the bad guys out of a dirty sponge after three minutes or so on high.

I'm not going to say that it wouldn't work. But like I said, I've never tested it.

Jason
 
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