Slanting yeast

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jason.mundy said:
I haven't. I'm interested in doing it. Was a centrifuge needed to do this?
Click to expand...



No centrifuge. The way I understand it is, after growing some yeast on a plate, pick a colony and transfer it to a slant filled with 2-3 ml of distilled sterile water and that's it. Without the agar. Close the lid and even leave it outside the fridge, will last for months, maybe years in the fridge. The reason I asked is, it kinda sounds too easy. Maybe I'm missing out on something....
 
So it's my first time.. ot a couple quick questions. I didn't end up slanting until this Sunday.. and I made a petri as well just to try different things. Is this what the petri dish is supposed to look like?

Also, I just realized today that I forgot to unscrew the caps a little.. they look pretty much the same as the dish with just a little liquidy streaking. I went ahead and cracked the caps a little with a puff of air and recovered them with tin foil in the jar.. will they still be ok or should I scrap them and try again next time or with scrapings from the dish?

2013-01-23_15-37-20_334.jpg


2013-01-23_15-44-10_580.jpg
 
Roadliner said:
So it's my first time.. ot a couple quick questions. I didn't end up slanting until this Sunday.. and I made a petri as well just to try different things. Is this what the petri dish is supposed to look like?

Also, I just realized today that I forgot to unscrew the caps a little.. they look pretty much the same as the dish with just a little liquidy streaking. I went ahead and cracked the caps a little with a puff of air and recovered them with tin foil in the jar.. will they still be ok or should I scrap them and try again next time or with scrapings from the dish?
Click to expand...

Roadliner,
from what I can see in the photo, that is a lovely plate. But, I only see two, maybe three single cell colonies. That's what you are after. Collect about 10 of those and transfer by loupe to 2 ml steril wort and you will have purified (decontamiated) any yeast you may be using.
Concerning the cracking of the caps, I would say you are good, what with the egress of gas and covering with foil. If all was done with sanitary protocol, you should have no problems. Good luck in your yeast farming endeavers..

PS. Us Arkies gotta look after one another...
 
sudbuster said:
Roadliner,
from what I can see in the photo, that is a lovely plate. But, I only see two, maybe three single cell colonies. That's what you are after. Collect about 10 of those and transfer by loupe to 2 ml steril wort and you will have purified (decontamiated) any yeast you may be using.
Concerning the cracking of the caps, I would say you are good, what with the egress of gas and covering with foil. If all was done with sanitary protocol, you should have no problems. Good luck in your yeast farming endeavers..

PS. Us Arkies gotta look after one another...
Click to expand...

so all but the dots by themselves should not be used? lol guess I should have streaked a few more plates.. Next time I will :)
 
peter78 said:
Has anyone tried the "water immersion" technique from the "yeast" book? Page 198.
Click to expand...

I have. That's all I keep now. My oldest resurrection to date is a sample that was stored something like 2 years and 4 months before I re-streaked it. The yeast woke right up, as if I had stored them just a week ago.

I do keep them in a fridge, just because the unused little shelf on the door is a convenient place for me to keep them. Every bit I've read, though, says I should expect the same result if I'd tossed them in a cigar box on desk.

https://www.homebrewtalk.com/f163/2-1-2-year-old-yeast-351613/
 
there was one, but not much activity I guess.. I'd love to do some slant trading once I get my technique down.. but right now, I'm not even sure how to tell if my slants are ok to use or not...

Speaking of which.. how do I know if my slants are ok to use? i would hate to pour some nasty yeast into a batch and ruin it.
 
Saccharomyces said:
My recipe for slant media is:

1.5% agar powder
7% DME
1% Wyeast yeast nutrient

That is by weight so if you have, say, 500mL of water you will add 7.5 grams of agar and 35 grams of DME along with 5 grams of the nutrient.

The agar powder def. works better than the stuff I got at the grocery store, which after a few months in the fridge started falling apart.
Click to expand...

I'm not sure if this has been discussed in the pages after this post but I haven't gotten through all the posts yet. I mixed per the proportions above but my slants haven't set enough after 36 hours. My agar is from Now Nutrition and says 100% agar.


I boiled my DME and yeast nutrient for around 1 minute. Let cool for a few minutes then dumped in the agar powder and stirred. Then used a syringe to measure out for my vials. I pressure cooked as instructed, let cool 1 hour, tightened caps and taped (I am not looking forward to ripping tape off and retaping 36 vials! ).

The only thing I am suspicious of is my yeast nutrient. I used Fermax yeast nutrient. After boiling my wort, I got a strong ammonia smell. Perhaps Fermax is not the same concentration as the wyeast nutrient?

Edit: finally finished reading through all the pages. There appears to be a difference in the agar. Obviously the block stuff mentioned in the OP but also some flaked agar and even 2 different powder versions (a medical and the nutritional kind like I used). I also read something about getting the agar mix up to boiling temps to avoid separation while filling your vials. I did only check one vial so maybe others are more solid than the one I checked. So any input would be appreciated.

Regardless, I am curious about the Fermax issue I mentioned though.
 
chuckjaxfl said:
I have. That's all I keep now. My oldest resurrection to date is a sample that was stored something like 2 years and 4 months before I re-streaked it. The yeast woke right up, as if I had stored them just a week ago.

I do keep them in a fridge, just because the unused little shelf on the door is a convenient place for me to keep them. Every bit I've read, though, says I should expect the same result if I'd tossed them in a cigar box on desk.

https://www.homebrewtalk.com/f163/2-1-2-year-old-yeast-351613/
Click to expand...

I want to toss out there I do the oil immersion technique in YEAST now (started a couple weeks ago). I emailed white labs about it. I asked if the mineral oil used for butcher blocks was fine. And for the heck of it I inquired if 100% olive oil was ok. They never heard of using olive oil so did not recommend it. As far as the mineral oil, they just said mineral oil without being specific about 99.9% or 100%. I couldn't find any 100% mineral oil, but did find some 99.9% mineral oil for consumption (for constipation or something or other) at a rite aid or walgreens. The other .1% is vitamin E. They recommend it be sterilized before use, of course.

Anyhow, I poured some into a small mason jar and put it in the pressure cooker when sterilizing the vials and slanting media. It went in clear and came out with a white tint to it. I figure it was ok. After the slants grew appropriately, i put a few ml of oil in to cover the yeast and put it in the fridge. The only quirky thing is now I have to store the slants on an angle instead of upright so the oil stays on top of the growth area.
 
I'm sure that this has probably been answered somewhere in the 40+ pages of this thread, but as my time on hand isn't infinite, I didn't read through the whole thing, and for that, I apologize.

What gravities are used for the starting worts in scaling up these slants?

Typically, I make starters based upon the gravity and temperature of the wort that I'll be pitching to, but it seems counter intuitive to put this small amount of yeast into a starter at 1.080 or something.

Thanks!
 
Building up these slants, which may have sat for months and months, and as well for reviving/harvesting from commercial bottles, you'd ideally go lower to around 1.020 for initial buildup.

The idea is lighter wort is easier on the yeast, but you get less growth. High gravity wort will produce more yeast, but is more stressful on the yeast. In the end, it is better to have less healthy yeast then more unhealthy yeast.

So in an ideal world you'd start with 1.020 for the first buildup, then move to a regular 1.030-1.040 wort until you have enough pitchable yeast. Practically though, I don't want to make a bunch of different starter wort of different gravities so I use 1.030 for everything. Never had a problem propegation from a slant, or a bottle for that matter.
 
BullGator said:
I'm not sure if this has been discussed in the pages after this post but I haven't gotten through all the posts yet. I mixed per the proportions above but my slants haven't set enough after 36 hours. My agar is from Now Nutrition and says 100% agar.

I boiled my DME and yeast nutrient for around 1 minute. Let cool for a few minutes then dumped in the agar powder and stirred. Then used a syringe to measure out for my vials. I pressure cooked as instructed, let cool 1 hour, tightened caps and taped (I am not looking forward to ripping tape off and retaping 36 vials! ).

The only thing I am suspicious of is my yeast nutrient. I used Fermax yeast nutrient. After boiling my wort, I got a strong ammonia smell. Perhaps Fermax is not the same concentration as the wyeast nutrient?

Edit: finally finished reading through all the pages. There appears to be a difference in the agar. Obviously the block stuff mentioned in the OP but also some flaked agar and even 2 different powder versions (a medical and the nutritional kind like I used). I also read something about getting the agar mix up to boiling temps to avoid separation while filling your vials. I did only check one vial so maybe others are more solid than the one I checked. So any input would be appreciated.

Regardless, I am curious about the Fermax issue I mentioned though.
Click to expand...

Anybody?
 
BullGator said:
I'm not sure if this has been discussed in the pages after this post but I haven't gotten through all the posts yet. I mixed per the proportions above but my slants haven't set enough after 36 hours. My agar is from Now Nutrition and says 100% agar.


I boiled my DME and yeast nutrient for around 1 minute. Let cool for a few minutes then dumped in the agar powder and stirred. Then used a syringe to measure out for my vials. I pressure cooked as instructed, let cool 1 hour, tightened caps and taped (I am not looking forward to ripping tape off and retaping 36 vials! ).

The only thing I am suspicious of is my yeast nutrient. I used Fermax yeast nutrient. After boiling my wort, I got a strong ammonia smell. Perhaps Fermax is not the same concentration as the wyeast nutrient?

Edit: finally finished reading through all the pages. There appears to be a difference in the agar. Obviously the block stuff mentioned in the OP but also some flaked agar and even 2 different powder versions (a medical and the nutritional kind like I used). I also read something about getting the agar mix up to boiling temps to avoid separation while filling your vials. I did only check one vial so maybe others are more solid than the one I checked. So any input would be appreciated.

Regardless, I am curious about the Fermax issue I mentioned though.
Click to expand...

I've had problems with agar before when I didn't heat it enough to fully mix it in. I was doing a batch of 24 vials at the time and what happened is most of the agar ended up almost as a jelly near the bottom. The first 16 or so slants never set and the remaining ones were rock solid. I ended up scraping the entire batch and starting over.

This is the Agar I use, http://www.amazon.com/dp/B000MGSJ5A/?tag=skimlinks_replacement-20 It sets for me within a couple hours at most.

Now what I do is bring the water to lukewarm, add in the DME & agar and bring it up to about 190F and make sure it gets completely mixed. Sometimes I'll bring it to a boil, but most times I won't as it'll boil in the pressure cooker for 30min anyways. The only problem I run into is I end up with break material in the slants. I also do not use any yeast nutrient.
 
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Jukas said:
I've had problems with agar before when I didn't heat it enough to fully mix it in. I was doing a batch of 24 vials at the time and what happened is most of the agar ended up almost as a jelly near the bottom. The first 16 or so slants never set and the remaining ones were rock solid. I ended up scraping the entire batch and starting over.

This is the Agar I use, http://www.amazon.com/Now-Foods-6410-Agar-Powder/dp/B000MGSJ5A/ref=sr_1_2?ie=UTF8&qid=1360173191&sr=8-2&keywords=agar+agar It sets for me within a couple hours at most.

Now what I do is bring the water to lukewarm, add in the DME & agar and bring it up to about 190F and make sure it gets completely mixed. Sometimes I'll bring it to a boil, but most times I won't as it'll boil in the pressure cooker for 30min anyways. The only problem I run into is I end up with break material in the slants. I also do not use any yeast nutrient.
Click to expand...

Thanks a lot for responding! That's the exact same agar I bought. Can you tell me your ratio of water, agar, and dme?

I actually brought the dme to a boil only for a couple of seconds (to avoid break material) and then let it sit for only a few minutes while I got the agar weighed out. It couldn't have been much below 190 or 180. I think I might be doing too many slants at once as it might be cooling too much. Or maybe I need to stir it every couple slants?
 
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BullGator said:
Thanks a lot for responding! That's the exact same agar I bought. Can you tell me your ratio of water, agar, and dme?

I actually brought the dme to a boil only for a couple of seconds (to avoid break material) and then let it sit for only a few minutes while I got the agar weighed out. It couldn't have been much below 190 or 180. I think I might be doing too many slants at once as it might be cooling too much. Or maybe I need to stir it every couple slants?
Click to expand...

I've always followed the ratio in the OP. 400ml water, 35g DME, 2.5g Agar. Today I went to make up some more plates, as I plan on trying to harvest from the can's of Heady Topper I just got my hands on. I've always been annoyed by the break material that ends up in my slants/plates so I decided to try something a bit different.

I took 400ml worth of sterile wort (extra runnings from prior batches that I collected in mason jars & then sterilized in my pressure cooker) and added 2.5g of Agar and some food coloring, let it bloom for 20 min and then brought it up to 180F. I then poured my plates and they're in the pressure cooker right now. We'll see if then end result is any better..

My only annoyance I'd love to solve, is the little bit of liquid the ends up in the bottom of the slants.
 
Jukas said:
I've always followed the ratio in the OP. 400ml water, 35g DME, 2.5g Agar. Today I went to make up some more plates, as I plan on trying to harvest from the can's of Heady Topper I just got my hands on. I've always been annoyed by the break material that ends up in my slants/plates so I decided to try something a bit different.

I took 400ml worth of sterile wort (extra runnings from prior batches that I collected in mason jars & then sterilized in my pressure cooker) and added 2.5g of Agar and some food coloring, let it bloom for 20 min and then brought it up to 180F. I then poured my plates and they're in the pressure cooker right now. We'll see if then end result is any better..

My only annoyance I'd love to solve, is the little bit of liquid the ends up in the bottom of the slants.
Click to expand...

So I made some slants at my friends house Saturday. After pressure cooking and the slants were cooled, we opened the caps and set them upside down on a paper towel while waiting to inoculate. I asked if he was worried about about infecting his pressure cooked, 100% sterile slants with the paper towel, he said he's always done it this way. I have 24 slants with fuzzy yeast growing and only one is having moisture problems (slant material is pushing up). So one in 24 isn't bad.

I think my problem was I didn't boil the dme agar solution when I made them before. When we made them on Saturday, we boiled them for around 5 min.
 
BullGator said:
After pressure cooking and the slants were cooled, we opened the caps and set them upside down on a paper towel while waiting to inoculate. I asked if he was worried about about infecting his pressure cooked, 100% sterile slants with the paper towel, he said he's always done it this way.
Click to expand...

I don't think I'd ever do that, unless I was under a laminar flow hood, and even then probably still not.

if they're within the updraft they're probably fine, but why take the chance?

I have plates of bottle harvested Pacman going, once those are done I'm going to attempt to plate out some Conan directly from a can of Heady Topper.
 
So I'm still curious how to know if my slants are good or have gone bad.. right now they kind of look like a white cream has been spread on them.. almost like icing on top of the agar. But a poster a few above here said his were fuzzy :-/
 
Roadliner said:
So I'm still curious how to know if my slants are good or have gone bad.. right now they kind of look like a white cream has been spread on them.. almost like icing on top of the agar. But a poster a few above here said his were fuzzy :-/
Click to expand...


All my slants are white/creamy looking, as are my plates after growth occurs. I'd be slightly skeptical of 'fuzzy' yeast..

Here's an example of one of my WLP001 slants.

RCk2odF.jpg
 
Here is one of mine.. first set I've tried.. going to do another set tomorrow from a fresh vile of 001

2013-02-19_15-52-57_965.jpg
 
Wow, what a thread!

Many thanks to the OP for starting this.

I'm slowly accumulating all my equipment to start doing this and I have a couple of questions:

1) Is 96.6% Bioethanol OK to use (diluted to 70% I assume) for sanitizing and cleaning?
2) I'm especially interested in slanting/stabbing multi-bug yeasts/bacteria such as theRoeselare, Brett. Lambicus, Lacto and Pedio. Will slants/stabs work or do I need to do anything else special for the sour bugs?
 
Andrikos said:
Wow, what a thread!

Many thanks to the OP for starting this.

I'm slowly accumulating all my equipment to start doing this and I have a couple of questions:

1) Is 96.6% Bioethanol OK to use (diluted to 70% I assume) for sanitizing and cleaning?
2) I'm especially interested in slanting/stabbing multi-bug yeasts/bacteria such as theRoeselare, Brett. Lambicus, Lacto and Pedio. Will slants/stabs work or do I need to do anything else special for the sour bugs?
Click to expand...

Andrikos,

I'm not sure what answer to give you concerning "Multi-Bug" storing. It appears that different conditions would benefit different types of bugs/yeast.

From Chad Yakobson stated this in his dissertation:
The media recommended for use and found to be the most beneficial to this study were MYPG, WLN, and CuSO4. Additionally it was found that storing cultures at room temperature in a nutrient rich liquid substrate maintained the integrity of cells longer and can be used for repeated propagations.
Click to expand...

It appears that his advise from Brett is the opposite of what I would do for brewer's yeast.

Look here for more info:
http://www.brettanomycesproject.com/dissertation/conclusion/
 
jason.mundy said:
Andrikos,

I'm not sure what answer to give you concerning "Multi-Bug" storing. It appears that different conditions would benefit different types of bugs/yeast.

From Chad Yakobson stated this in his dissertation:


It appears that his advise from Brett is the opposite of what I would do for brewer's yeast.

Look here for more info:
http://www.brettanomycesproject.com/dissertation/conclusion/
Click to expand...

Wow, thanks for that, it looks that my "Hefe Labor" is not ready for sour bugs yet... :)
 
Andrikos,

I would recommend that you listen to this podcast as well. It's long. It's not all about Chad and the Crooked Stave brewery. You may want to just fast forward to the interview. There is a lot of gold in this. He talks about brewing with Brett. Storing Brett. And recipe ideas with the bug as well. He also talks about how he culturing different strains other than just what is available from the commercial labs.

http://thebrewingnetwork.com/shows/866

Jason
 
I may have an issue. Most of my slants started looking very good with nice creamy white yeast growth. But now most of them have seemed to get a little yeast down at the bottom of the agar-dme surface mixed in with the little bit of condensation. Well, this started fermenting and forming a krauzen.

So this started right at the time they looked good and I tightened the lids to see if they were ready to put in the fridge (I wanted to let them sit over night to see if anymore co2 escaped when I cracked the lids to make sure they were done). They were made this past Saturday so they have been sitting for a total of 5 days now. This started happening on day 3.5 or so.
 
jason.mundy said:
Andrikos,

I would recommend that you listen to this podcast as well. It's long. It's not all about Chad and the Crooked Stave brewery. You may want to just fast forward to the interview. There is a lot of gold in this. He talks about brewing with Brett. Storing Brett. And recipe ideas with the bug as well. He also talks about how he culturing different strains other than just what is available from the commercial labs.

http://thebrewingnetwork.com/shows/866

Jason
Click to expand...

Thanks for that, I'm listening to the interview right now. :)
 
FredTheNuke said:
I'm close to attempting to wake a few up from October 2011 soon here...
Click to expand...

Fred,

I think an interesting experiment would be to go out and buy a fresh vial yeast. The same type as the one you are waking up. Then try to do a split batch. One with fresh yeast, the other with propagated yeast from 2011.

I'm considering doing the same experiment with some old slants of mine.

Jamil Z had said that anything past 6 months for a slant risks mutations. I wonder how pronounced the mutations would be. Or if my palate could even detect them.
 
Jukas said:
I've always followed the ratio in the OP. 400ml water, 35g DME, 2.5g Agar. Today I went to make up some more plates, as I plan on trying to harvest from the can's of Heady Topper I just got my hands on. I've always been annoyed by the break material that ends up in my slants/plates so I decided to try something a bit different.

I took 400ml worth of sterile wort (extra runnings from prior batches that I collected in mason jars & then sterilized in my pressure cooker) and added 2.5g of Agar and some food coloring, let it bloom for 20 min and then brought it up to 180F. I then poured my plates and they're in the pressure cooker right now. We'll see if then end result is any better..

My only annoyance I'd love to solve, is the little bit of liquid the ends up in the bottom of the slants.
Click to expand...

I pulled out these plates today that I had done with 400ml of sterile wort as describe above and inoculated all of them with Pacman from a starter I had going.

Here's the first plate I pulled out.. not what I wanted to see.

Iu3F7hk.jpg


The rest of the plates look like this. I was very relieved after seeing the first one.

riYucsP.jpg


I discarded the first plate and inoculated a couple slants from the remaining plates.
 
Ive been thinking of doing some yeast slants, I just have one newb question...

What does the actual 45* surface angle achieve? is it just for a larger surface or what?
 
brewski_please said:
Ive been thinking of doing some yeast slants, I just have one newb question...

What does the actual 45* surface angle achieve? is it just for a larger surface or what?
Click to expand...

It gives you a larger surface area.

I like a much sharper angle than 45. I try to fill my about 1/3 full, and tilt them far enough that part of the bottom of the vial is exposed. A fully covered bottom will allow co2 to build up under the media and push the media to the top of the vial.
 
How important is to incubate slants (or plates in my case) at 70-80F?
I can't use my ferm. chamber to control temperature since there is amber ale fermenting at the moment, and it is 60-70 in my home where I keep my plates..
I assume it is not that big deal and it will only slow things, am I right or should I organize some sort of heating?
 
diS said:
How important is to incubate slants (or plates in my case) at 70-80F?
I can't use my ferm. chamber to control temperature since there is amber ale fermenting at the moment, and it is 60-70 in my home where I keep my plates..
I assume it is not that big deal and it will only slow things, am I right or should I organize some sort of heating?
Click to expand...

You shouldn't need a heat source. If you don't see growth, then I am wrong, but my apartment is roughly 70F all the time and I don't have issues with it.
 
Thanks.
When should I expect to see some growth?
I am planning to inoculate slants next Sunday (if everything goes well).
 

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