Slanting yeast

[darylwoodman]

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.

1) I have used one small package--not sure what's inside. I think it's 7g.
2) I have not tried plates. I need to conserve space.
3) It is similar, but has a much lower melting point. I put my slants in my work incubator to check for contamination and went back to find a liquid solution again. I just reset them at an angle and all was good.
4) Most of the condensation seems to come out if you leave the lid slightly loose. However, some still have a small amount. Just a thought, leaving them in the incubator and then resetting them might alleviate the condensation if the lid is not screwed down (airtight). I might try this sometime.

You make a good point about the spores, and I'm not so sure what the resistance of CB is at 100°C. My assumption is that leaving at room temperature for 14-days (USP sterility) time frame would show the presence of any growth if present.

Cheers-

 

[trentm]

Thanks for the info. The possibility for a C. bot infection in stored wort is low due to the acid conditions normally found in wort. That said a mistake in preparing the wort could result in a low acid product. Here is a clip from the Wikipedia article:

"Spores are not killed by boiling, but botulism is uncommon because special, rarely obtained conditions are necessary for botulinum toxin production from C. botulinum spores, including an anaerobic, low-salt, low-acid, low-sugar environment at ambient temperatures."

With that said here is another clip: "Improperly preserved food is the most common cause of food-borne botulism."

So better safe than sorry when dealing with this organism.

Thanks for your simple method to prepare slants. However, I think I'll stick with agar. It's not too expensive, has a long shelf life, and known mixing instructions. I have heard some people are using a product available at Asian food stores call Agar Agar.

 

[ColoHox]

Thanks for the info. The possibility for a C. bot infection in stored wort is low due to the acid conditions normally found in wort. That said a mistake in preparing the wort could result in a low acid product. Here is a clip from the Wikipedia article:

"Spores are not killed by boiling, but botulism is uncommon because special, rarely obtained conditions are necessary for botulinum toxin production from C. botulinum spores, including an anaerobic, low-salt, low-acid, low-sugar environment at ambient temperatures."

With that said here is another clip: "Improperly preserved food is the most common cause of food-borne botulism."

So better safe than sorry when dealing with this organism.

Thanks for your simple method to prepare slants. However, I think I'll stick with agar. It's not too expensive, has a long shelf life, and known mixing instructions. I have heard some people are using a product available at Asian food stores call Agar Agar.

You forgot to bold the most important part of your wikipedia quote. Clostridium is anaerobic. You have to actively remove the oxygen from a culture of Clostridium for it to grow. No growth, no toxin.

And the kicker is...you need oxygen to grow your yeast.

 

[bionut]

How wise is to buy autoclave indicator tape for testing the pressure cooker sterilizing ability? It isn't very expensive, but it is worth buying (For that money i can buy a 1 liter flask.)?

 

[trentm]

How wise is to buy autoclave indicator tape for testing the pressure cooker sterilizing ability? It isn't very expensive, but it is worth buying (For that money i can buy a 1 liter flask.)?

Autoclave tape is nice to have but not really a necessity. The thing is that it only test the temperature of where it is applied. We usually apply it to the foil that covers the flask or the outside of a package. The real concern is the material you are pressure cooking. As you can imagine, the temperature of, say a liquid, would be slower to reach sterilization temperature compared to the temperature of the foil cover or the outside of the flask. I think there are products that can actually be added to the contents that will indicate the temperature of the material you are sterilizing. What is nice about autoclave tape is that it can be used to identify objects that have been sterilized so they are not mistaken for un-sterilized objects.

If you know the pressure cooker is reaching 15 psi you can also know the inside of the cooker is at 250F. Small volumes (e.g slants) will near simultaneously reach that temp. Larger volumes may require more time. If I am pressure cooking >1 liter, I give it more time for insurance.

 

[trentm]

You forgot to bold the most important part of your wikipedia quote. Clostridium is anaerobic. You have to actively remove the oxygen from a culture of Clostridium for it to grow. No growth, no toxin.

And the kicker is...you need oxygen to grow your yeast.

True, good point. But as indicated, my biggest concern is in stored wort where boiling or pressure cooking renders it near anaerobic.

 

[philipCT]

Thanks for the info, links to blogs and photos in this thread. I am sorely tempted to do some of this. My main inspiration is that it seems really cool to be able to do, and I like the idea of being able to really insure a clean culture using these methods. I have successfully cultured several yeasts from dregs and it was a real blast to get good batches from those. This seems like a logical next step.

I like the convenience of having a variety of yeasts on-hand, but I usually have to go to the LHBS to pick-up grains anyway, so picking up fresh yeast isn't a big deal. Cost savings also isn't a big deal to me given it's only an additional $6-12 per batch.

My primary holdback is that, because I really only brew about once a month, it seems a collection of even 8-10 different strains would create a lot of maintenance work (if I have to refresh the slants every six months) that may not be justified by how often I brew.

Thoughts?

 

[mbbransc]

Thanks for the info, links to blogs and photos in this thread. I am sorely tempted to do some of this. My main inspiration is that it seems really cool to be able to do, and I like the idea of being able to really insure a clean culture using these methods. I have successfully cultured several yeasts from dregs and it was a real blast to get good batches from those. This seems like a logical next step.

I like the convenience of having a variety of yeasts on-hand, but I usually have to go to the LHBS to pick-up grains anyway, so picking up fresh yeast isn't a big deal. Cost savings also isn't a big deal to me given it's only an additional $6-12 per batch.

My primary holdback is that, because I really only brew about once a month, it seems a collection of even 8-10 different strains would create a lot of maintenance work (if I have to refresh the slants every six months) that may not be justified by how often I brew.

Thoughts?

I haven't started slanting, still considering it.

That said, the reason I'm considering it is I wanted to brew up a particular IPA last week and my Conan yeast didn't make it to me in time. If I maintained slants in the fridge, that'd never be a problem.

I also am intrigued with the idea of harvesting yeast from particular expensive and difficult to acquire Belgian sours.

I've only read the first couple pages of this thread so far, but does someone have recommendations to acquire vials and beakers? Some of the links on pg 1 are still active but others are not. And I don't know if there are better (read: cheaper) alternatives now.

thx

ETA: Wow, took a couple days but I finally read through it all. Seems ebay and Cynmar are still the best bets for acquiring equipment. Definitely think I'm going to give this a go.

Question 1... is anyone here using gelatin instead of agar? Seems there are lots of problems getting agar to set correctly and gelatin is much more readily available.

Question 2... no one has really addressed bugs and brett. is agar/gelatin with wort not an adequate media for storage of brett & bugs?

Question 3 (and I think I know this one)... if I want to ranch yeast/bugs from bottle dregs, is it best to plate the dregs and try to maintain separate samples of each yeast/bug? Then grow them up separately and pitch together later? Or is there a way to maintain a mixed culture in storage?

 

[bionut]

I made some wort for agar slants with biab and it turned very cloudy? How can i clarify it?

 

[bionut]

I made some wort for agar slants with biab and it turned very cloudy? How can i clarify it?

Nobody?

 

[MichaelBrock]

Nobody?

First off, I don't personally see the need to clarify it. As long as you're sterilizing it, it should make no difference whether or not it is clear.

If however you really want clear wort for your agar (which will not be clear regardless; at least mine hasn't been) you could cold-crash the wort for a while (say 32 degrees for days) to drop out whatever is causing it to be cloudy (proteins probably).

 

[bionut]

I fixed it, I poured it through a funnel with cotton pads and i got pretty clear wort It was really cloudy as i used homemalted barley for it, cold crashing didn't worked very well. Now i will freeze the wort and used it when i need it.

 

[fizzy_nether_nectar]

This is cool I've never heard of slanting before this

 

[JonM]

I just started doing this. An inexpensive pressure cooker and the vials, etc. from Cynmar, some agar from the Asian market, and I'm in business for about $50.

As a bonus, I recently grabbed an expired yeast from the LHBS for $3 and inoculate slants with it. So - a lifetime supply of 1007 for peanuts.

 

[Lgaddy44]

I've followed the process of the OP, numerous times over the past couple years, without fail. Now I've gotten my first issue, and wonder if anyone else has seen this, or knows what this may be.

Last week, I used slants of WLP002 and WLP550, to make starters for a couple of beers I made. Did my usual process, starting the slants in 300mL, then stepping up to 1.5L starters for the brew. The night I made the big starters, I made new slants of each. I did them all at the same time (one strain then the other, immediately after).

It's been 9 days now, my 550 looks fine and normal, the 002 has red in it. Obviously this is an infection, I just dont know what. I'm also concerned now that my Imperial Pumpkin Pie Ale I made with my starter, will also be infected.

View attachment 1429448900341.jpg

View attachment 1429448911431.jpg

 

[Blue-Frog]

I have no idea, but note that they all (your 002's) seem to behave the same way, (?) suggesting it wasn't caused at the reslanting stage... assuming you used normal aseptic protocols.

Was the 002 done last and with the exact same media?

Perhaps you could make 2 more slants, leave one blank and innoculate the other with some of the yeast from the Imperial Pumpkin Pie Ale thats fermenting...

its just a thought.


Don't put much weight on my comments though;
I have my own set of problems, as you will soon see!

Blue Frog

 

[Lgaddy44]

I'm almost certain I did the 002 second, but yes, all with the same practices and same media.

The parent 002 was from a slant I previously made, with no signs of infection.

I definitely think there was a contamintation somewhere, but I am pretty strict in my process, so there's no one thing that jumps out at me.

I just hope my Pumpkin is salvageable if it shows tge same infection.

 

[Blue-Frog]

Don't do today - what you can put off till tomorrow!
So now instead of being ahead of the game tomorrow, I am behind a step!
------------------------------------------------------------------------------------
In my clever haste to save time I decided to can some media (in bulk) to work with later.
Perhaps with little thought, I added agar... and yea, it set up hard as slants do.

Has anyone canned agar laced media and used it successfuly for slants or stabs later on?

I figure it will melt and reset again more or less normally, but lacking experience, I was wondering if anyone had any tips on making the experience go smoothly.



Blue Frog

 

[butterpants]

I've followed the process of the OP, numerous times over the past couple years, without fail. Now I've gotten my first issue, and wonder if anyone else has seen this, or knows what this may be.

Last week, I used slants of WLP002 and WLP550, to make starters for a couple of beers I made. Did my usual process, starting the slants in 300mL, then stepping up to 1.5L starters for the brew. The night I made the big starters, I made new slants of each. I did them all at the same time (one strain then the other, immediately after).

It's been 9 days now, my 550 looks fine and normal, the 002 has red in it. Obviously this is an infection, I just dont know what. I'm also concerned now that my Imperial Pumpkin Pie Ale I made with my starter, will also be infected.

http://en.m.wikipedia.org/wiki/Serratia_marcescens

Pretty common in the bathroom. If you have a differential media that shows ph effects, it produces lactic acid during aerobic growth.

There's another pretty common red fungus contamination but I can't think of it at the moment.

 

[Blue-Frog]

If it was second then I would try to streak or plate a sample from the fermenting "broth"... It might give you faster indication than waiting for the beer to finish. Perhaps even a forced fermentaion if time is critical.

Don't read any further.

Well, ok, but you were warned!
This is just a wicked comment -- but the color reminds me of what is called "aka-kabi" or "red mold" where I live. It is common on shower floors and bathtub areas...
I asked some people doing home remodling here about it (when we were having work done on our house) and after looking into it, they told me it was not actually mold, but a yeast! I couldn't belive my ears but well, ok.

Anyway, Good Luck!

bf

 

[Blue-Frog]

Bacteria...? I think I was told the stuff we see locally, was a yeast
but perhaps I was told it was bacteria.... now I am not so sure.

Perhaps your slants might house Fusarium... the gusher grimlin?
Fusarium is also mentioned as being common in moist areas like baths etc.

Just came across this about testing for it:
http://www.crc.dk/flab/fusarium.htm

 

[ColoHox]

That's not Fusarium. I would say Serratia, like was mentioned already. A rare bacterial contaminant, but certainly possible. It looks like multiple organisms are growing on those slants. You might try isolating on of the creamy white colonies on a new slant or plate, if you have them.

 

[Blue-Frog]

or maybe Rhodotorula?

for a visual see 2:37~
[ame]https://www.youtube.com/watch?v=abQNl6WIdc8[/ame]

 

[ColoHox]

Yes. R. glutinis is another potential contaminant.
http://wineserver.ucdavis.edu/industry/enology/winemicro/wineyeast/rhodotorula_glutinis.html

OP of this question, you might try lowering the pH of your media (if this is something you can do) to inhibit some bacteria.

 

[Lgaddy44]

To my concern of my infected slants, I took a sample of my pumpkin brew I used the starter on (that parented the infected slants). The pumpkin shows no signs of infection. That's a huge relief to me. Although I don't quite know how, it looks like my slants were infected after innoculation, and my starter was not infected prior to pitching. Here's a shot of the slant I made from the pumpkin ale in the fermenter. As you can see, no red nasties.

Be careful with your yeast, fellas. I almost feared I'd have to do a thorough sanitation of all my equipment. Although it wouldn't hurt, it did made me think of all the stuff I have, and the time it would take to properly sanitize everything, and eliminate a spreading infection. Best to be proactive and keep things clean, rather than have an unintentional cross contamination of everything. That's a lot of headache I'm sure nobody wants.

View attachment 1430100624415.jpg

 

[butterpants]

Those look fine but you have a fingernail problem bro.... cut those claws!

 

[RJS]

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.

This looks like both too much yeast and wet plates. pluck some on a loop from this plate and re-streak. If you can see yeast on the loop you have grabbed too much.

If nothing grows in a single colony you must re-streak for optimum results.

plates need to dry out for one or two days after being made. Also, they get stored upside down, you might have been doing this already though.

 

[fc36]

This looks like both too much yeast and wet plates. pluck some on a loop from this plate and re-streak. If you can see yeast on the loop you have grabbed too much.

If nothing grows in a single colony you must re-streak for optimum results.

plates need to dry out for one or two days after being made. Also, they get stored upside down, you might have been doing this already though.

That is exactly what I was thinking too. The colonies should start to appear as fluffy white marshmallow puffs in a semi-spherical shape. They should not be oozing and wet. Drying and storing your plates upside down also keeps the plates and precious agar media from drying out too fast as aspirated moisture will tend to rise. If you store them right side up, they'll develop water droplets that will "rain" down on your media and wreak havoc when you go to pull one out and either streak a new yeast or select a healthy colony from a previously streaked plate.

 

[bionut]

What's the best practice for stepping slants to starter? I am about to use one of my slants for a batch of beer, and i am concerned about contaminating the starter.

 

[RJS]

A slant needs to be streaked onto a plate. Thats not only the best practice, but is essential. Slants and plates work together, you dont want one without the other. Brew Kaiser has the whole process on his blog.

Once you streak onto a plate and have single grown colonies, you can then pick up a colony and start a 5 to 10 ml starter. The ratio for DME to ml is 1 to 10. 1 gram of DME for every 10 ml of wort. Good to make an extra plate just in case you need to re-streak out a contaminant. I have had to do this before with my pacman strain when it got contaminated. After a re-streak I successfully got back to a pure colony, contamination free. Thus plating is vital in this process.

I sterilize everything up to 100 ml's, thats all i can fit in my pressure cooker. So i can fit my vials and mini flasks totaling 210 ml, so i make a starter wort with 21 grams DME. sterilize, cool, pick colonies from plates and dump into 5ml vials. I would do 10ml but my vials tend to get messy if liquid level goes that high. The 100 ml step up flasks just sit in a closed yeast cabinet for two days, only covered with tin foil.