Slanting yeast

[NaymzJaymz]

I guess I'm asking the same question that thefost is asking.

 

[tally350z]

What are your procedures? Are you using the Agar agar from the asian market or are you using the lab quality agar? I still use the agar agar from the market and after some trial and error, I have found the ratio of waterME:Agar needs to be much higher than originally posted. Do you leave the caps loose for a few days after slanting? Need to know your procedures before we can assist accurately.

 

[05m50dan]

Gave slanting a try, here's a pic of my first attempt. I dunno, it doesn't look as good as everyone elses slants, kinda watery and not a whole lot of yeast growth. Not sure if I'm doing something wrong.

1. Make sure you thoroughly dissolve the agar agar in hot wort. My early attempts left watery portions and hard portions. If you mix thoroughly, this shouldn't happen.
2. You may need to adjust the amount of agar agar. I had to tweak this for the agar agar source I have.
3. Let vials sit with caps on loosely until condensation dissipates.

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[thefost]

What are your procedures? Are you using the Agar agar from the asian market or are you using the lab quality agar? I still use the agar agar from the market and after some trial and error, I have found the ratio of waterME:Agar needs to be much higher than originally posted. Do you leave the caps loose for a few days after slanting? Need to know your procedures before we can assist accurately.

For my slants, I was using the original recipe of 35g DME, 400ml water, 2.5g Agar. I got the powdered Agar from cynmar. It was only store loose for about a day after slanting, maybe it needed more? I boiled it for a few minutes, not sure if that was long enough.

I'm guessing I should also increase the Agar amount, but I thought that the cynmar lab grade agar would be stronger than the OPs. Should I add more DME as well?

 

[05m50dan]

For my slants, I was using the original recipe of 35g DME, 400ml water, 2.5g Agar. I got the powdered Agar from cynmar. It was only store loose for about a day after slanting, maybe it needed more? I boiled it for a few minutes, not sure if that was long enough.

I'm guessing I should also increase the Agar amount, but I thought that the cynmar lab grade agar would be stronger than the OPs. Should I add more DME as well?

I wouldn't add more DME. Just add another gram of agar agar next time. It may take a couple of batches to get the recipe right. If the slants don't set up well, don't use them.

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[tally350z]

I don't know if its proper procedure or not, but my second batch had a little liquid in it, although the slants were set well. I flamed the opening of the slant and just dumped out the liquid. I set those aside to see if I possibly contaminated them, and I didn't they were just fine. Again not sure if others do that or not.

 

[NaymzJaymz]

Thank you, sir.
Yes, I did use the Asian market agar with bad results the first time(very watery slants). Yes I did loosen the caps on the vials. I just taped the vials and noticed that the yeast seemed thicker than I realized at first, so maybe I'm okay. I sensed a very noticeable yeast smell, so that is normal, right? I did indeed increase the agar so it would gel better.

 

[HollisBT]

So, immediately after you take them out of the pressure cooker, a little condensation on the inside of the tube is ok, right? Also, some of the starter wort seems to have separated, this is also normal, right? I'm attaching pics so you guys can tell me to calm down.


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[tally350z]

I get the break in the bottom as well. It just settles out and once it solidifies, its not an issue. Leave the slants slightly open for a few days to let the condensation out and then you can seal and store.

 

[HollisBT]

I get the break in the bottom as well. It just settles out and once it solidifies, its not an issue. Leave the slants slightly open for a few days to let the condensation out and then you can seal and store.

Thanks, I'll loosen the caps when I get back home. I'm excited to finally go through with this project!


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[HollisBT]

Just got back home and it looks like the malt agar has already started to set in the slant position, am I safe in assuming that the media will be alright with the condensation overnight while I let it continue to set, and then loosen the lids tomorrow afternoon and leave them slightly loose to dry out the condensation?

Or should I go ahead and loosen the caps ASAP?


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[tally350z]

Either way will be fine. I usually loosen the cap while its setting and then let them sit. I would just loosen them and let them sit while its setting.

 

[HollisBT]

Well, out of 43 slants that I put together, all of them set except for 8 tubes... What gives there? Do you think they will eventually gel and set? Or are they just failures and I shouldn't bother with them...

 

[tally350z]

Do you remember if they were the first 8 that you poured or the last 8 or mixed? I'm not sure why only those select few wouldn't set. I would think they would eventually set. Just let them sit out for a little longer, maybe throw them in the fridge to set quicker.

 

[HollisBT]

Do you remember if they were the first 8 that you poured or the last 8 or mixed? I'm not sure why only those select few wouldn't set. I would think they would eventually set. Just let them sit out for a little longer, maybe throw them in the fridge to set quicker.

I don't recall when they were filled... If they don't set up it's no biggie I guess.



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[HollisBT]

After a few days, they still have a bit of moisture, and I'm starting to get concerned I didn't use enough agar in all of them. Should I rrdwhahb or just redo them all?


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[05m50dan]

Well, they may not be pretty, but what's is the purpose of the slants... To grow and store yeast. If the year are growing in there, and nothing else, then you should be fine. Next time add more agar to your mix.

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[tally350z]

Since they didn't set you could use the media in the tubes as the first step starter. Or you could just redo them..

 

[HollisBT]

That's a good idea, hadn't thought about that.


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[HollisBT]

So what is everyone's preferred method/recipe/ratio for preparing malt agar media? I have decided to re-do all of my slants, since I am iffy on them already, and it is easy enough.

The recipe that I used was 400ml water, 35g DME, and 2-3g agar powder. Clearly that was not enough agar for that amount of media, but I can't seem to find any consistent answers anywhere as to how much should be used...

Another question I had regarding starting a slant... When you go through the first step, do you simply add 10ml of sterile wort on top of the slant and let the yeast start there, or do you shake it to try and re-liquefy the solidified agar media? Is it easier just to take an inoculation loop and transfer the stored yeast to a new vial with sterile wort for the first step?

 

[tally350z]

For me it took a few attempts to get the ratio where I liked it. I use the cheap asian market agar agar, which like stated before is mostly sugar. It works just fine for me at a ratio of 400ml, 35g DME, 35g agar. Which I guess is a lot compared to the other agar people are using, however the slants I make are solid and they work really well.
There are different techniques to your second question but for me I have 10ml sterile vials just for the first step. I take a loop from my slant and inoculate the sterile wort and leave the cap loose to release the co2. Then step up by 10.

 

[Aunt_Ester]

I've settled on 5 grams of agar per 250ml of wort.


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[Leviathan]

I'm pretty sure the answer to this is yes but... can I begin a starter in a slant? I have some slanted WLP002 that is getting old so I plan on reslanting with the starter that I make from it. I figure that if I'm going to discard it afterwards, I might as well use all the available yeast for my brew. Can I simply add some sterile wort to the slant itself? It's a rather large test tube (200 x 25mm) and I have a backup if something goes wrong.

 

[Aunt_Ester]

I don't see why not.

I add the sterile wort to the inoculated slant, cap, and shake. Then I add the mixture back to my sterilized 50ml flask. I only do this for more headspace, but your method sounds like it should work.


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[05m50dan]

I usually add 10 ml sterile wort to the slant and shake. Then let sit for 24-48 hours until I start to see signs of fermentation. Then transfer to 90 ml of sterile wort.

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[Eugenio]

just made my slants last night using agar powder. Used the proportion of 4g DME, 1G Agar to 100ml water as well as a pinch of nutrient and a small hop pellet and it solidified fast. For the future i will run it thru a coffee filter to try and have it a tad cleaner, and will not move the petri dishes around...got some spills.

One question. I have a small pressure cooker, can I stack canning jars on top of each other and the petri dishes on top of the test tubes? Will there be any movement inside?


Im so happy to move away from washing.

 

[WQDL753]

There will be movement, There is vigorous boiling after all. You can stack if your canner is large enough.

 

[tally350z]

Your really not supposed to sterlize plates in the pressure cooker, as the process creates too much condensation. You are supposed to use dry heat to sterlize. My method is to put in a 400* oven for 90 minutes. It takes some practice on when to pour the media into the plates, as getting the temperature for each takes some practice. It took me about 5 different attempts to realize when I had to pour in order to prevent/minimize the condensation.

 

[Eugenio]

Great, thanks I'll try that next time. I'm also going to start canning the wort, I think previously I had been having some sanitation issues, hopefully this will help.


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[Aunt_Ester]

It took me about 5 different attempts to realize when I had to pour in order to prevent/minimize the condensation.

Condensation is normal. Just turn your plates upside down after they have set.



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[tally350z]

Condensation is normal. Just turn your plates upside down after they have set.

Yes condensation is normal, however there are different techniques to be used to minimize the condensation. If you use a pressure cooker with the plates already made than the you end up with a lot of big water droplets. If you use the dry head method than you only get some smaller droplets.

 

[Aunt_Ester]

Yes, you are correct to use dry sterilization for the plates. But I'm not sure how it took you five attempts to know when to pour the media. As soon as the decanter is warm enough to touch you should pour the media. Any condensation from this is okay. Just wait to set and turn them over before storing.


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[HollisBT]

The culture tubes that I bought came with paper lined lids, but some of the liners have fallen out and gotten lost. Is it safe to use these tubes without the liners? Is there a website where I can buy new liners?

 

[Aunt_Ester]

Yes they're safe. Just use electrical tape around the cap and tube to prevent them from drying out. The liners are just to make a seal.


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[HollisBT]

Is it possible to add too much agar? I redid my slants, and now I'm afraid that the media is TOO firm...

I had a good bit leftover in the pot, and it set up very firm...

Sorry I'm asking a lot of questions, I am very new and inquisitive...


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[Eugenio]

I did 1.5% agar and my slants came out perfect, there is but a hint of condensation on the slants and the plate took hold of the yeast quite easily. Tonight ill try slanting the plate.

HollisBT, im not very familiar with this but my guess is too much agar wouldn't really be detrimental. If anything, just expensive as agar costs so much, but i don't think it will really affect your slant as long as there is enough food there for the yeasts. Mine set quite fast too.

 

[Leviathan]

So I recently tried stepping up from a slant to pitchable volume in 48 hours due to a few circumstances. I don't think it was successful. I stepped up from the slant itself (added 50ml to slant)

24 hours later I added to 225ml

~10 hours later I added to 500ml

~10 hours later I added to an additional 500ml

I observed gas activity when checking the seal of the original tube after the first innoculation, however after that I noticed no apparent activity. Did I step up too quickly? I was hoping to see krausen as observed in the starters I've made from White Labs vials, however I did not see any even after vigorous swirling. I ended up pitching the starter along with a packet of dry yeast for reassurance, so I will not know definitively if the starter was adequate.

The yeast was WLP002.

 

[05m50dan]

Your really not supposed to sterlize plates in the pressure cooker, as the process creates too much condensation. You are supposed to use dry heat to sterlize.

This isn't necessarily true. Actually using the pressure cooker is s better and quicker method. I use my pressure cooker to do my plates. My lids don't make a complete seal, so its just a matter of time until the condensation evaporates. Once the condensation evaporates, use film or plastic wrap to seal it.

 

[05m50dan]

My method is to put in a 400* oven for 90 minutes.

To use dry heat to sterilize you need to rest for 24 hours then repeat this process for a total of 3x. Its called Tyndallization. Spores from bacteria and mold may survive the initial dry heat phase. Waiting 24 hours allows most of those spores to become live and active. Then you heat again and allow to rest. Then the third time should kill anything that survived the second step.
Temp doesn't need to reach 400, 212 will do for 15 minutes. Then rest/repeat.

 

[tally350z]

I have had zero contamination issues with my process, and I have let the plates sit for over a week to se if there is any. Also Tyndallization is where you boil the liquid for 20 minutes each day, for four straight days, not dry heat. Thats if you do not havea pressure cooker and can only achieve boiling temps. If you have a pressure cooker for the media, than 15-20 minutes, @ 15PSI, which is around 250 is enough to kill bacterial and fungal spores. Boiling only kills most vegetative bacteria.