Slanting yeast

[butterpants]

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.

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Streaking plates for isolation from a slant is unnecessary if your slant is prepared properly not to mention a complete waste of time in my mind.

The whole reason of making slants is to have a master culture repository with some genetic diversity to it (think of innoculating your slants with only 1 isolated colony from a plate vs 5 or 10) that you can propagate directly from by snagging a small amount with a loop and transferring to a small amount of sterile wort.

 

[RJS]

You could have a perfect looking slant and still streak a plate that shows bacteria or wild yeast.

Risks of not making a plate from slanted yeast are:infected slant, having respiratory/nutrient issues with a slant that sat and grew together for long enough.

You dont only streak for contamination but health of the cells. If they cant grow together then they have been dependent on the "lawn" from inside the slant for too long. They might be nutrient deficient and unable to grow apart by themselves. Also respiratory issues from a long stored slant and having pressure on them from growth inside the slant.

A streak and isolated colony will show vitality and viability that you cant know with, say, a month old slant.

So, I wouldnt give someone advice to just grab from the slant lawn and start it up. Eventually something will catch up to your strain. Unless maybe you re-slanted regularly.

also last thing, genetic diversity will change over time within the same slant, same strain. The point of streaking will be to increase genetic diversity from cell morphing in the slant by picking out colonies that are brand new and now, unlike the lawn where it came from, diverse.

 

[butterpants]

slant correctly, slant often

save yourself some heartache

 

[Blue-Frog]

Looking for comments from professionals... any documented sources etc.

I think that slants are by design, supposed to be grown from single colonies while starters are grown up from a more diverse mixture.

You write:
"The whole reason of making slants is to have a master culture repository with some genetic diversity to it (think of innoculating your slants with only 1 isolated colony from a plate vs 5 or 10) ...."

This is not my understanding...

Let me see if I follow you correctly...
When you slant, are you making multiple innoculations from a plate,
so that each slant has more than a single colony as it's source material?

Humm if I follow correctly, you are trying to simplify things and, as you did say...
"...that you can propagate directly from by snagging a small amount with a loop and transferring to a small amount of sterile wort."

so you are preparing a pre-mixed slant to go directly to the starter from... (?)

I am not sure.

Is this wise?

What does 'best practice methods' tell us about this method?

Opinions on "purity" vs. "diversity" ?

 

[bionut]

I made plates before making the slants. Ot seems too big of a hassle to make plates again before the starter. I will need to plan my brew day 5-7 days before to have time for the starter.

 

[RJS]

I plan 10 days in advance, from plate to fermentor. Two days each step.

This method of yeast handling is not for everyone. Its time consuming and tedious. You have to love the microbiology to sustain it.

 

[Hanglow]

What about multi strain yeasts though? Plenty of the best english strains are multi strain - the proper TT yeast, ringwood, Adnams, Bathams, Harveys etc etc . When you plate them would you have to pick a number of different colonies to ensure that you got all the different strains?

 

[RJS]

Not sure. What i would guess, since one colony is approx one million cells, they are together. A microscope would be needed at that point.

I checked Chris and Jamils yeast book for starters and there is only a chapter on starting from plates. They explain slants are only for storage.

 

[butterpants]

My slants are grown from multiple single isolated colonies on UBA media (or WLN if I want to guarantee acid production) analysed for respitory deficiency and other generally strange things (from a morphology standpoint). I get my strain diversity by picking 5-10 individual colonies and mix um all up on the slant.

 

[Blue-Frog]

What about multi strain yeasts though? Plenty of the best english strains are multi strain - the proper TT yeast, ringwood, Adnams, Bathams, Harveys etc etc . When you plate them would you have to pick a number of different colonies to ensure that you got all the different strains?

I am not familiar with these yeasts... but it is my understanding that the ideal method is to grow pure cultures of each component and blend them just before brewing use... perhaps the mixture can be used (reused?) a few times by various methods if desired, but eventually one needs to go back to the pure cultures and re-mix... otherwise (among other things), evolution might start to accenuate the differences already present and you start to get differences you can actually notice... on the other hand, it is possible the mixed cultures might continue as a "stable" mixture for very long periods of time.

 

[bionut]

Hello! I made my first starter ever; from a slant. I added 3 loops of yeast in 10 ml of wort. After 24 hours i poured the content of the vial in 500 ml of wort on my stir plate, altough i didn't see any activity in the vial that would indicate me yeast growth (i had some hot break in the vial too so i couldn't see the yeast on the bottom). After 24 hours on the stir plate it have produced a star san like foam on top (the wort was cloudy from the start bercause it was some kettle wort from a prevoius beer), and i don't know if this is a sign of fermentations or just foam produced by the continous agitation.

 

[JonM]

I usually follow a rule of ten. I inoculate 15 ml of wort then step it up to 150ml. From there, I can go all the way up to 1.5 L, but I usually don't need that much.

Point is, going from 10 ml to 500 might have been too big of a jump. But give it time. Sounds like it's doing something.

 

[RJS]

Hello! I made my first starter ever; from a slant. I added 3 loops of yeast in 10 ml of wort. After 24 hours i poured the content of the vial in 500 ml of wort on my stir plate, altough i didn't see any activity in the vial that would indicate me yeast growth (i had some hot break in the vial too so i couldn't see the yeast on the bottom). After 24 hours on the stir plate it have produced a star san like foam on top (the wort was cloudy from the start bercause it was some kettle wort from a prevoius beer), and i don't know if this is a sign of fermentations or just foam produced by the continous agitation.

Steps of ten as Jon said is good.

Letting vial sit for two days is better.

Continuous agitation will make enough foam to stick to the sides, but if the foam is rising, covering the whole surface, than that would most likely be activity.

Color is a big indicator here, look for a muddy brown turning lighter and lighter, to a cream color.

btw, congrats on not only your first starter, but doing it from a slant!?! Thats ambitious and i like that. Most would have at least done a smackpack or white labs vial. nice.

an example of a two day vial showing bottom packed with yeast lawn, the cloud pic with the flame is one day, only a small amount packed on bottom and still working, meaning little to no flocculation yet...

 

[bionut]

Thank you for your appreciation. I only brewed with dry yeast until now, that's because in my country there is only one seller of liquid yeast (wyeast), that meaning that he pretty much can make his own price for it, so i can't afford to brew with liquid yeast. All of my yeast strains are obtained from friends who bought the yeast or have it from some other friends of them. After i will get some more experience with all this starter thing and get some proper glassware (i use some jars now) probably i will buy some yeast and propagate it for a long time. The foam has covered all the surface, probably someone is having sex in there
The wort that i'm using has some hop pellets bits and hot break, so i'm not sure if the muddy look is from that or from the yeast. Tomorrow i will brew some beer (i will use some homemade caramel malt , just for fun, speciality malts aren't as expensive as liquid yeasts) and i will use the yeast from the starter if it will be any, if not, i have some dry yeast in the fridge.

LE: it seems that my starter worked, this morning i stoped the stir plate and a yeast sediment formed on the bottom . Now i need to wait to see if my sanitizing was good enough Next time i will plan my brew day better and make the starter at least 5 days before it.

 

[RJS]

Well wouldn't you know i had a surprise schedule change and had to make a starter from a vile without streaking on a plate, i couldn't afford the time. So everything was fine of course and the batch fermented well. the chances of the vile being unusable would be unlikely from my stellar paranoid cleanliness, but it still didn't feel good. Without seeing the colonies on a plate, your going in blind. You can only hope there wasn't any issue in the vile. But with the colonies on a plate you can see everything before making that commitment.

 

[butterpants]

You're so crazy now you might even go outside on a cloudy day without a raincoat! Lookout world, he's reckless as hell today

 

[RJS]

Lets not get ahead of ourselves, one step at a time. [emoji57]

 

[Blue-Frog]

How long does it take for a loop heated to a red glow take to cool down enough to safely pickup & transfer yeast?

(without using agar-touch method to help w/ cooling)

I recently had a couple of transfers that didn't take...

 

[IslandLizard]

You cool it by dipping into a bottle with 90-100% alcohol, then whisk the loop through the flame to evaporate the alcohol film that clings to it.

 

[JonM]

Just don't spill the alcohol!

 

[IslandLizard]

Just don't spill the alcohol!

Speaking from experience? Use heavy wide-bottomed low-height glass bottles for those things, think old-fashioned ink bottles. Needs a good sealing lid or it will evaporate.

 

[butterpants]

You cool it by dipping into a bottle with 90-100% alcohol, then whisk the loop through the flame to evaporate the alcohol film that clings to it.

I would use 70% for that technique... just me

 

[Blue-Frog]

Well, this does seem to be one method to remove most of the heat of sterilization, but this method represents an extra step, an additional risk and still gives a loop of iffy temperature... I think this method of alcohol flaming to cool the loop (while do-able) is probablly not typical or standard in commercial or professional microbiology labs. (?)

There seems to be a lot of youtubes saying to let cool down a bit, but they are fuzzy on how many seconds... in a few I saw, it looks like 1-3 seconds, but that seems to be too short...

Anyone here just hold the loop [in air] to cool down before transferring?

 

[RJS]

people no need to overthink this step, much more important things to be concerned with. Just touch the tip to empty space of agar medium, cools instantly. Also, if your using the nichromium (sp?) loop, then your fine with just the touch onto agar. That metal is made to heat up and cool instantly.

 

[Blue-Frog]

True;
Yet in transferring two strains onto 2 new slants ea. (4 in all),
I only had a 50 % success rate, luckily one slant for ea of the two yeast strains, so;

either I "failed" to get any yeast on the loop in half of my work,
or the loop was too hot 50% of the time.

It seems I need to adjust something to get more consistent results.

I was working fairly quickly, and suspect that the loop being too hot is more likely than having failed to pick up any yeast... but I am just guessing... is it fairly common to fail to pick up yeast?

I usually have the opposite troble and get more than actually needed.

 

[Blue-Frog]

Just touch the tip to empty space of agar medium, cools instantly. Also, if your using the nichromium (sp?) loop, then your fine with just the touch onto agar. That metal is made to heat up and cool instantly.

I have noticed that touching the tip to empty agar medium space sort of "melts" the medium... and repeated touching at slightly different areas still seems to have the same effect... (the implication being the loop is still hot.)

Perhaps I should just let the loop stay longer in the first place it touches. (?)

 

[bionut]

I cool the loop on the agar media. The agar should be sterile anyway.

 

[Blue-Frog]

I never had a problem before this last time.
That started me wondering what the minimum cool time actually is.

I was thinking some people might have an "x-seconds" rule of thumb.

That doesn't seem to be the case.

 

[IslandLizard]

Why don't you simply try it. After you pull it from the flame, wait 1 minute. Touch it to the top of your hand. Not hot, do it again, but only wait 50 seconds now. Repeat with 5-10 seconds less wait each time until it feels warm to the touch.

Then that's how long it takes, with your loop.

 

[Blue-Frog]

yea, i might try that.

I did try it the other way, touching within a few seconds after firing and found it was still quite warm when I had assumed it was ready to use.

 

[Blue-Frog]

Well After my last colony isolation last night I tried the "How soon can you hold it" test.

It seems that with my materials and methods, the loop continues to be hot for 20 seconds after removal from the flame, once it has become glowingly hot over a large area of the loop.

This supports the sense I was getting that "almost instantly" was too soon for safe use.

 

[Blue-Frog]

I am thinking about adding a few wine yeasts to my collection...

Anything one should be aware of in terms of culturing differences between beer and wine yeast?

Are White labs wine yeasts grown in wort, just like beer yeast, or do they use wine/grape juice etc.?

Are the Nutrients the same for both types of yeast or are there known requirement differences?

Humm,
I notice that the typical weight of a pkg of dry wine yeast is ca. half that of beer yeast...
What is a good pitch rate for wine ?
Is it lower than for lager, (ales?) or do wine yeast... just weigh less?

 

[NaymzJaymz]

I've been into this thread for over a year, and have made several attempts with several different yeasts. The interesting results have been in the unused slants. My first few attempts yielded slants with mold in them, to which I received replies pertaining to my pressure cooker not getting hot enough to sterilize the medium. I decided to use a roaster pan and cook my slants in an oven at a much hotter temp. The result is now I have slants that have a white growth in them, as if they had been inoculated. To what should I assign this latest failure? The medium had the lids screwed down and taped as soon as they were cool enough to touch. They were broiled at 500+ degrees for the required time. The white growth suggests wild yeast contamination. How is that possible?

 

[butterpants]

I've been into this thread for over a year, and have made several attempts with several different yeasts. The interesting results have been in the unused slants. My first few attempts yielded slants with mold in them, to which I received replies pertaining to my pressure cooker not getting hot enough to sterilize the medium. I decided to use a roaster pan and cook my slants in an oven at a much hotter temp. The result is now I have slants that have a white growth in them, as if they had been inoculated. To what should I assign this latest failure? The medium had the lids screwed down and taped as soon as they were cool enough to touch. They were broiled at 500+ degrees for the required time. The white growth suggests wild yeast contamination. How is that possible?

What about the slant lids? I'm assuming they are plastic and the tubes are glass?? I doubt you're putting plastic lids in a broiler @ 500....if not, then how are you sterilizing them?

If that's not it... care to detail your step by step method so we can critique it?

 

[NaymzJaymz]

Butterpants, the easiest way of describing my technique is to say that I've followed the directions in this thread. I did indeed put the plastic lids in the covered roasting pan I used! Naïve perhaps, but they didn't melt. I would like to know how I got what appears to be wild yeast contamination when the lids were screwed down and taped as soon as they were cool enough to handle!! Thank you!!

 

[butterpants]

Sounds like you need to buy a sterilizer/autoclave. Apparently you're filthy!

 

[jason.mundy]

My gut reaction would be to say that your pressure cooker was not getting the media up to the 15 PSI. All the biological were not killed and grew once back down to room temperature.

I do not think that your media reached oven temperatures as long as they still had moisture inside of them. You can only sterilize dry items in the oven. Water will keep your temperature down to 212F where it is in contact.

 

[NaymzJaymz]

Thanks Jason, I did not know that. It is still curious to me that the contamination in this batch looks like yeast growth, not mold like the previous problems.

 

[NaymzJaymz]

My latest endeavor is with WLP001, a very popular yeast as you all know. The yeast growth began quickly, but its a bit darker than any of my other attempts. The color is not the fluffy white as seen in the instructional photos, but the same tan color as the yeast in the vial(now a plastic pouch). Any thoughts? Thanks!!!!!

 

[bionut]

It's ok. The tan colour is normal.