# What I Don't Understand About Slanting



## Bribie G (23/11/10)

I'm going to get into slanting, particularly to cover me for the long periods when the yeast companies deign not to supply me with varieties such as 1469 to they've done that to themselves.

Now slanting seems quite straightforward, but the big glaring issue seems to be: (typical guide to slanting )

The First Step is to make up some slants - which you'll use later to grow yeast. (I make between 20-25 with this mix) Sanitize your hands, work area and utensils. 

Boil 1 cup (250 ml) water (in a small saucepan). Remove from heat, add 15 grams of dried malt extract, and stir till dissolved. Put back on the heat and boil for 10 minutes. Remove from heat. 
*Add a packet of gelatin into this "wort" and stir until COMPLETELY dissolved. *
Pour this mixture into test-tubes; Fill each about 1/4 full. Keep some empty test tubes for next stage. 
Place a pyrex jug/dish inside a large pot with lid. Place the 1/4 filled test tubes and some empty ones in the pyrex container (if you have a test tube rack which fits in your pot - this can save you some headaches as I couldn't find dishes which fit inside pots). continuation..........continuation..........

   

Ok hold everything there, I assume the tubes are now going to be cooked for a while, I thought that gelatine is denatured during intense heating. I take it that this isn't so?


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## manticle (23/11/10)

The gelatin will presumably form a jelly after cooling on which the yeast will grow reproducible cells. Most slanting I've heard about uses agar. Denaturing shouldn't be a problem though as far as I know - you're forming a substrate on which yeast cells will survive until called upon again. Jelly is made with boiling water and still sets and is still sweet.

I myself just reserve some yeast under boiled, cooled water in sanitised test tubes with a stopper, in the fridge.


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## mxd (23/11/10)

wolfy had some great picies etc.. of how to do it.


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## Bribie G (23/11/10)

I'll be using agar, I take it that during steaming / cooking the tubes don't get filled up with condensed water - as they are sitting upright?


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## AndrewQLD (23/11/10)

BribieG said:


> I'll be using agar, I take it that during steaming / cooking the tubes don't get filled up with condensed water - as they are sitting upright?



Yes they do Bribie, I always had the lids on but slightly loose, still got condensation inside, real pita.

Andrew


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## Bribie G (23/11/10)

Thanks, yes that was 'intuitive' because that's what happens when you steam food - loose lids sounds the go. I can get heaps of agar in the Valley where I work and I understand it gives a firmer substrate than gelatine.


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## wakkatoo (23/11/10)

hey bribie,

I've just started and 'slanted' my first 3 tubes of Wyeast 3068 last night. Here hoping it all went well!

I followed Wolfy's method. After the 'wort' is in the tubes, I then pressure cooked them with the lids on the tubes and the tubes standing up right in a standard pot with a Mortar, or is it pestle???, sitting on top. The wort remains liquid long enough after cooking the tubes to then lay them on their side to get the nice slant happening. Some condensation did form (1-2 drops) but I just tipped this out immediately before adding my yeast last night. I made 20 slants about a month ago and nothing has formed on the slant since then. Here's hoping the yeasties do their job now and I get some nice colonies.

Not sure if its answered your qns, but I had fun typing 

edit-spellink and gramma


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## proudscum (23/11/10)

The nice post man delivered my new 21l pressure cooker today and my collection of flasks fit in quite nicely.so i can start with a practice run on making up some slants just need to nip up to Footscray to get some Agar Agar.Just wish my brewery would hurry up and arrive!!!!!!!


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## Bribie G (23/11/10)

I can't find Wolfy's contribution in the articles, anyone have a link?


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## wakkatoo (23/11/10)

Here you are: Linky

There are links to the other two he did via this thread. Great thread and a nice bloke too!


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## proudscum (23/11/10)

this is a threat i started if you look at post#16 it may be of some help.

http://www.aussiehomebrewer.com/forum/inde...showtopic=49205


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## Silo Ted (23/11/10)

Agar culture substrate medium would be preferred over gelatine for this type of thing. 

What I don't understand about slanting is why, for less of a pain in the butt, in brewing terms, why you don't instead just keep a small sample under liquid, and if you aren't using it for three months, make a rejuvinating starter and re-stock the cool pantry for a time when you need to activate/reproduce the sample. 

But if keeping a smaller cell sample, and slanting, you would need to consider contaminants, and surely need a laminar flow hood to ensure optimum viability, without 'wild' aspects of the environment to mutate the core strain. 

Science is good, but it doesn't make better beer all the time.


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## Thirsty Boy (24/11/10)

because keeping yeast under liquid is just about the worst way to maintain a pure, viable, non-mutated version of the yeast you want to use. Whereas slanting is the next best option to cryogenic storage for exactly the same reasons.


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## Bribie G (24/11/10)

Yes, currently I keep 500ml Schott bottles full of yeast cake in the lagering fridge and when I run out of Schotties I end up with coca cola bottles full of Irish Red or even S-189 and before you know it the fridge is looking like a Chinese Brothel, following which after six weeks or so it's a PITA regenerating some of the stuff. Also as I found out in previous cold break experiment, that lovely creamy yeast cake is probably only 30% yeast, and how much of it still alive is anyone's guess - the rest is break and dead crap and although it's not going to do the next brew a great deal of harm, slanting seems to me to be a more controlled and professional way of doing things. Sure if I'm going to repitch a very similar style within a week I'll still go the Schott bottle - but that isn't going to see me over the next 2 year wait for 1469, or 1768 (remember that one, which they eke out every few years). or Pacman or.............


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## Pennywise (24/11/10)

manticle said:


> I myself just reserve some yeast under boiled, cooled water in sanitised test tubes with a stopper, in the fridge.




Sorry, a bit :icon_offtopic: Manticle, how long have you been able to store the yeast for like this?


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## Phoney (24/11/10)

Pennywise said:


> Sorry, a bit :icon_offtopic: Manticle, how long have you been able to store the yeast for like this?




Im not Manticle, but i've made starters from yeast that has been sitting in sanitized urine sample jars for 18 months+.



Making Agar Slants in Picture:

http://www.aussiehomebrewer.com/forum//ind...showtopic=47107

From Slant to Starter in Picture:

http://www.aussiehomebrewer.com/forum//ind...showtopic=46262


I bookmarked em for one day when I finally bite the bullet and go down that path.


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## RdeVjun (24/11/10)

BribieG said:


> Sure if I'm going to repitch a very similar style within a week I'll still go the Schott bottle - but that isn't going to see me over the next 2 year wait for 1469, or 1768 (remember that one, which they eke out every few years). or Pacman or.............


Hear hear Bribie, IMO it just isn't worth the farting around slanting strains that are available from retailers all year round, but with seasonals on slants, that 2 year wait (indeed if ever) reduces to about a week.
Yep, I do remember 1768 indeed, it is right up there with 1469 for ESBs! :icon_drool2:


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## porky (24/11/10)

Hey BribieG,

We make slants all the time, have done for two years now.

Have a look here:

http://www.tigereye.net.au/bluedog/slants.html

We have 23 different yeasts on hand.

Cheers,
bud


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## argon (24/11/10)

phoneyhuh said:


> Im not Manticle, but i've made starters from yeast that has been sitting in sanitized urine sample jars for 18 months+.




From what i understand... yeast can be stored under water for a long period of time... but just don't expect the strain to be exactly reminiscent of the original culture. Slanting limits the mutation by only storing very small amounts, limiting potential characteristic drift.


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## [email protected] (24/11/10)

I have been in contact with a US homebrewer who also has a PhD in microbiology and when I discussed the concept of slants and doing a yeast bank he said it is easier to manage a frozen yeast bank and that is what he does. It is the process I'm currently setting up to do. It is essentially the addition of glycerin to a water yeast blend and storing the vials in a freezer. The glycerin prevents any freezing and limited or no nutrient is present so mutation is limited compared the cryogenic storage processes the prefessionals use.

Here is a link to how it is done:

-Guide to making a frozen yeast bank-
http://www.homebrewtalk.com/f13/guide-maki...ast-bank-35891/


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## MHB (24/11/10)

Freezing is definitely the way I would go, there is lots of good information out there, you can store sensible sized (i.e. add to a starter) without any intermediate stepping up. Yeast isnt going to mutate. You can store for years. Very low risk of picking up infections.

MHB


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## Nick JD (24/11/10)

abc said:


> -Guide to making a frozen yeast bank-
> http://www.homebrewtalk.com/f13/guide-maki...ast-bank-35891/



Cheers for that. :beerbang:


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## argon (24/11/10)

abc said:


> Here is a link to how it is done:
> 
> -Guide to making a frozen yeast bank-
> http://www.homebrewtalk.com/f13/guide-maki...ast-bank-35891/






MHB said:


> Freezing is definitely the way I would go, there is lots of good information out there, you can store sensible sized (i.e. add to a starter) without any intermediate stepping up. Yeast isnt going to mutate. You can store for years. Very low risk of picking up infections.
> 
> MHB




Sweet... and the added convenience for me is i have plenty of freezer space and very little fridge space... nice
:icon_cheers:


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## [email protected] (24/11/10)

No probs Nick JD.

Does anyone know who is a good supplier for autoclavable vials? 

Argon,

That is my thoughts as well. I have plenty of freezer room.


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## drtomc (24/11/10)

I got a minimum chips 250 from ProSciTech http://www.proscitech.com.au/cataloguex/home.asp

T.


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## pk.sax (24/11/10)

abc said:


> No probs Nick JD.
> 
> Does anyone know who is a good supplier for autoclavable vials?
> 
> ...


http://cgi.ebay.com.au/ws/eBayISAPI.dll?Vi...em=110548393424

I have these, quite good quality, an ebay solution anyway. Lids are plastic autoclavable screw tops as well.


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## drsmurto (24/11/10)

abc said:


> I have been in contact with a US homebrewer who also has a PhD in microbiology and when I discussed the concept of slants and doing a yeast bank he said it is easier to manage a frozen yeast bank and that is what he does. It is the process I'm currently setting up to do. It is essentially the addition of glycerin to a water yeast blend and storing the vials in a freezer. The glycerin prevents any freezing and limited or no nutrient is present so mutation is limited compared the cryogenic storage processes the prefessionals use.
> 
> Here is a link to how it is done:
> 
> ...



Cheers for the link, that's a great guide.

Any thoughts on using a no rinse sanitiser rather than a pressure cooker? I could boil the vials in my HLT for 15 mins prior to an orthophos acid rinse?


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## TidalPete (24/11/10)

DrSmurto said:


> Any thoughts on using a no rinse sanitiser rather than a pressure cooker? I could boil the vials in my HLT for 15 mins prior to an orthophos acid rinse?



DS,
Boiled the crappers out of my autoclavable tubes (filled with sterilised water & the caps loosened sitting in a test tube rack) for a long time before getting a 2nd hand pressure cooker & experience has shown me that you need AT LEAST 30 minutes of good rolling boil to minimise the risk of tube infections but still had the odd failure. No sanitiser was used after the aforementioned. Pressure cooker definitely recommended.
Hope this helps.

TP


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## outbreak (24/11/10)

I have purchased 50x 30ml sterile tubes with plastic caps, is there any point to autoclaving these?


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## Nick JD (24/11/10)

Could the vials take 200C in the oven?

EDIT: oh, lids wouldn't regardless. 

I s'pose the other thing is you wouldn't know you had a problem until you made a starter...


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## TidalPete (24/11/10)

outbreak said:


> I have purchased 50x 30ml sterile tubes with plastic caps, is there any point to autoclaving these?



Not sure outbreak?
If they weren't specifically touted as autoclavable I might be hesitant.
Having said that, I pressure cook the sterile syringes that I use to split my smack packs ($0.50 a pop = tight arse but saving the world  ) many times over with no problems until the syringe starts to get stick from over-use. Try one & see.

TP


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## drsmurto (24/11/10)

I wonder if soaking in conc orthphos acid for 24-48 hours would be considered sterile. I use the vials to measure out the conc acid when diluting to no rinse strength so they have no problems with acid.


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## np1962 (24/11/10)

It is not only the vials that need to be sterile, the medium also requires sterilization hence the pressure cooking after adding the gelatine/agar + malt solution.
It needs to be sterile not just sanitary.
Nige


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## drtomc (24/11/10)

Or the glycerine, if freezing.

T.


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## Bribie G (24/11/10)

Yes for all we know the gelatine could be rife with salmonella and anthrax whatever


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## drsmurto (24/11/10)

:icon_offtopic: 

I understand the concept of a pressure cooker/autoclave to sterilise.

Washing with a dilute acid (orthphos) is classed as sanitary, is there a pH at which it becomes sterile? Are any of the bugs we are concerned about happy in the presence of concentrated orthphos acid (80+%)?

And yes, this still doesn't solve the issue of sterilising the medium and whilst vacuum distillation would it is a tad more labour intensive than forking out a few pesos for a pressure cooker.


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## felten (24/11/10)

I'm sure if the concentration was high enough you could sterilise, but then it wouldn't be no rinse anymore


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## Thirsty Boy (25/11/10)

I didn't read that particular guide to freezing yeast, so if itncovered this, sorry..

You need to be careful of using your frost free freezer for yeast frezing. Even under glycerol, yeast will suffer a reasonable mortality hit each time they are frozen and each time they are thawed... Yur frost free freezer will deliberately warm them up on a really quite regular basis, and each time, you are losing viability out of the stored sample.

So you either need an old fashioned non-frost free freezer for yeast freezing, or you need to take some steps to stop the freeze thaw cycle from happening to your yeast.

I froze yeast for a while, i find slants less trouble.


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## [email protected] (25/11/10)

TB,

Yes the guide does. What is recommended is the vials are placed in a styrofoam box or small esky with gel ice packs inside to counter the fridge defrost cycle.


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## Silo Ted (25/11/10)

Thirsty Boy said:


> because keeping yeast under liquid is just about the worst way to maintain a pure, viable, non-mutated version of the yeast you want to use. Whereas slanting is the next best option to cryogenic storage for exactly the same reasons.



Just back to this statement for a moment. If Whitelabs store their commercially packaged yeast under liquid for the intention of shipping to the world and sitting in a shop for 6 months or more, and expect it to be viable to the end user, that gives me faith that I can do the same thing at home with a relatively clean sample taken by top cropping a fermenting wort. 

Or is it that any mutation on the strain has already occurred once it hits the beer?


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## Bribie G (25/11/10)

Can you get little freezers - say the size of a small esky ? maybe for use in labs or something? Just a thought.


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## felten (25/11/10)

Silo Ted said:


> Just back to this statement for a moment. If Whitelabs store their commercially packaged yeast under liquid for the intention of shipping to the world and sitting in a shop for 6 months or more, and expect it to be viable to the end user, that gives me faith that I can do the same thing at home with a relatively clean sample taken by top cropping a fermenting wort.
> 
> Or is it that any mutation on the strain has already occurred once it hits the beer?


Yeah but they lose ~20% viability a month, they want you to use them fresh.


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## manticle (25/11/10)

Pennywise said:


> Sorry, a bit :icon_offtopic: Manticle, how long have you been able to store the yeast for like this?



I haven't really counted. I just use the yeast I have on hand that I want when I want. Every time I get a different type of Wyeast, I split some off into clean sterile test tubes with boiled, cooled water, label and store in the fridge. Definitely several months though.

I used to use longnecks before I got my test tubes and used yeast that was several months old with no dramas. I always make active starters which should tell you if something's wrong. Obviously I step up from the test tube amounts first.

Definitely not the best way to store yeast but it's worked well for me so far (and that's a few brews). As with all things i'll probably try slanting one day but I like to move one step at a time, especially when things work for me.

And silo - yes you can top crop, store and use the yeast later. What whitelabs do in their 'yeast lab' and what you do at home are never going to be quite the same though. You definitely can store yeast under liquid - I think what thirsty was getting at is that there are better ways (in response to you asking why would you bother?).


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## manticle (25/11/10)

Pennywise said:


> Sorry, a bit :icon_offtopic: Manticle, how long have you been able to store the yeast for like this?



I haven't really counted. I just use the yeast I have on hand that I want when I want. Every time I get a different type of Wyeast, I split some off into clean sterile test tubes with boiled, cooled water, label and store in the fridge. Definitely several months though.

I used to use longnecks before I got my test tubes and used yeast that was several months old with no dramas. I always make active starters which should tell you if something's wrong. Obviously I step up from the test tube amounts first.

Definitely not the best way to store yeast but it's worked well for me so far (and that's a few brews). As with all things i'll probably try slanting one day but I like to move one step at a time, especially when things work for me.

And silo - yes you can top crop, store and use the yeast later. What whitelabs do in their 'yeast lab' and what you do at home are never going to be quite the same though. You definitely can store yeast under liquid - I think what thirsty was getting at is that there are better ways (in response to you asking why would you bother?).


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## Nevalicious (26/11/10)

Well, the frozen yeast bank idea got me all inspired. 

I made a little trip over to my local scientific supply place (which consequently, is not at all local, but it doesn't matter, it was in work time...) and stocked up. 30 x 20ml borosilicate flat bottom vials, couple of glass pipettes etc etc. Picked up a mini cooler bag and some gel ice packs from Big Dubs, Glycerin from the pharmacy... I've 3 vials of White Labs on order, fresh from the states. I've read and re-read the frozen yeast bank link. I'm ready!!!

Bring it on!


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## Thirsty Boy (26/11/10)

abc said:


> TB,
> 
> Yes the guide does. What is recommended is the vials are placed in a styrofoam box or small esky with gel ice packs inside to counter the fridge defrost cycle.



Cool - thought i'd flag it just in case it wasn't




Silo Ted said:


> Just back to this statement for a moment. If Whitelabs store their commercially packaged yeast under liquid for the intention of shipping to the world and sitting in a shop for 6 months or more, and expect it to be viable to the end user, that gives me faith that I can do the same thing at home with a relatively clean sample taken by top cropping a fermenting wort.
> 
> Or is it that any mutation on the strain has already occurred once it hits the beer?



Well, I think its commonly accepted that the viability of those liquid yeast packs starts dropping pretty dramatically as soon as they are packaged, and after 6 months for instance, G&G starts selling them at half price - I think that there really isn't anyone expecting them to be sitting in a shop for six months or more and still be maintaining acceptable viability.

You can look at the MrMalty calculator and get an idea of how fast they drop off... And remember that MrMalty just finished writing a book about yeast with the owner of white labs... So I have a feeling that what he reckons is probably not a million miles from the truth, or perhaps his co-author might have had something to say about it.

And certainly no one would expect to repeatedly re-culture from liquid yeast "packs" that had been stored for months.

People do it, people seem to make it work - but Slants/plates have been repeatedly demonstrated to be the best way (short of cryogenics) to maintain the brewing properties of a given yeast sample in medium to long term storage.

If people are looking for easy, then there are lots of options - if people are looking for the "best" way to maintain a yeast library at home, then slanting is very probably it.

Assuming of course you do it properly.


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## felten (26/11/10)

> You can look at the MrMalty calculator and get an idea of how fast they drop off...


They reckon 20% a month, if you play with the calc you get roughly the same.


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## [email protected] (26/11/10)

Nevalicious said:


> Well, the frozen yeast bank idea got me all inspired.
> 
> I made a little trip over to my local scientific supply place (which consequently, is not at all local, but it doesn't matter, it was in work time...) and stocked up. 30 x 20ml borosilicate flat bottom vials, couple of glass pipettes etc etc. Picked up a mini cooler bag and some gel ice packs from Big Dubs, Glycerin from the pharmacy... I've 3 vials of White Labs on order, fresh from the states. I've read and re-read the frozen yeast bank link. I'm ready!!!
> 
> Bring it on!



Following through with a bit more detail on the frozen yeast bank (post 110), the guy I have been emailing in the US about this subject has some additional techniques that possibly improve the storage. He doesn't store under the starter liquid as it shown in the link in the post. He advised to pour of all the starter liquid and add a sanitised yeast nutrient water (1 cup with a pinch of nutrient) to allow the yeast slurry to be fluid enough for proper mixing with the glycerin and reduces any sugars left.

I can post all his details here if people would like to see them. There is a fair bit of detail.

Additionally, if you have a chest freezer or even some of the mini freezers (look like bar fridges) these are generally manual defrost and would help in avoiding any cyclic temperatures.


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## Silo Ted (26/11/10)

According to a 2006 BYO Magazine article, the WL yeasts are now shipped out with an average cell count of 100 billion, a lot more than what I thought to be around 35 billion. With a 20% decrease per month this would still mean there's about 26 billion cells, not far off the previous count. Not bad figures to consider if buying old stock. Also not too far off the previously shipped 35 billion. 

I almost bought one of these USB microscopes so me and a mate could look at the cell count on stored yeasts, but many reviews say that they are rubbish, and not even close to the stated specs of 400x. as well as the picture quality being poor. Would a conventional microscope be adequate to view yeast cells, and at least visually estimate cell count? Im very interested to observe the decline in viability with recycled cropped samples, and compare to newly purchased vials. 

If brewers are to take samples from an active ferment for storage purposes, isn't this going to be a new generation of healthy cells with a high count per microgram (or whatever the standard measure might be), or will hop & grain debris significantly diminish the count? Mutation aside, Im still unclear as to why you cant just keep culturing crude fresh samples in a series of starters every couple of months to perpetually maintain a yeast bank. Although I guess if you do this often enough to keep your samples viable, the cost of DME and the time involved would be greater than just buying a new vial of reliable clean yeast.


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## razz (26/11/10)

abc said:


> Following through with a bit more detail on the frozen yeast bank (post 110), the guy I have been emailing in the US about this subject has some additional techniques that possibly improve the storage. He doesn't store under the starter liquid as it shown in the link in the post. He advised to pour of all the starter liquid and add a sanitised yeast nutrient water (1 cup with a pinch of nutrient) to allow the yeast slurry to be fluid enough for proper mixing with the glycerin and reduces any sugars left.
> 
> I can post all his details here if people would like to see them. There is a fair bit of detail.
> 
> Additionally, if you have a chest freezer or even some of the mini freezers (look like bar fridges) these are generally manual defrost and would help in avoiding any cyclic temperatures.


Very interested abc,please post the details or a link. Thanks.


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## drsmurto (26/11/10)

razz said:


> Very interested abc,please post the details or a link. Thanks.



I'm also keen to see the extra details. :icon_cheers:


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## Nevalicious (26/11/10)

DrSmurto said:


> I'm also keen to see the extra details. :icon_cheers:



As am I... Cheers

Tyler


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## argon (26/11/10)

Nevalicious said:


> I made a little trip over to my local scientific supply place (which consequently, is not at all local, but it doesn't matter, it was in work time...)



Guess what i'm up to today... sweeet!!

Edit: Oh yeah +1 on the extra details


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## [email protected] (26/11/10)

A bit more detail as promised. The process is similar to the one that was posted in homebrewtalk but has a little bit more detail and some variations that are worth noting. I admire everyone's enthusiasm in getting stuck into it - it has taking me more than aa month to just start to organise some of the basics! 

This is from a friend in the US who is a homebrewer and has experience with culture labs (PhD in bio chem):

_My method for creating a frozen yeast bank isn't too difficult. The beginning supplies you need are
-Styrofoam box that fits in your freezer
-Ice Pack
-Glass/Plastic Vials (look for 15-25mL glass vials OR use 50mL Flacon tubes)
-Turkey Baster (stainless steel if you can find it)(or spend the money and buy an electric pipette and 10-15mL pipettes. though that can get pricey)
-a rack to hold test tubes
-a pressure cooker
-glycerin
-something to label with
-sterilized yeast nutrient water
-latex/nitrile gloves 
my basic procedure is as follows

-When creating a yeast starter from a new tube/pack of yeast, make a small side starter (400-600mL) with some of the yeast. You can either use this yeast or increase the starter if you want more vials. Cold crash and pour off as much of the starter wort as possible. cover and put back in fridge.

-add approx 10-15% of your vial capacity with glycerin, lightly close cap and sterilize along with turkey baster/pipette tip in pressure cooker to sanitise. Aim for about 20min. 

-Let the vials etc cool. Keep the tip of the turkey baster from touching anything.

-Add just enough sterilized yeast nutrient water to the yeast cake to allow you to suck up the yeast in the baster. Trasfer to the sterilized vials, get them full (but remember to leave head room for expansion). 

-close cap firmly (if you are not using gloves, or, even if you are, avoid touching the threading on the vial or the inside of the cap)

-invert the vials 10-15 times and then shake to allow glycerin to incorporate in the mixture. appropriately label the vials (I use packaging labels I cut up into small strips)

the info you should have is Yeast Strain, Generation (1st gen- straight from yeast tube/pack, 2nd gen- from a yeast slurry/cake or from a bottle conditioned beer, 3rd gen- yeast cake slurry from a 2nd gen vial, etc.), and date created. Store in the styrofoam box with the ice pack and place in your freezer (should be around -20 C). If you are taking it from yeast cake/slurry from a batch of beer you will have to know how to wash yeast. _

Just a couple of extra notes and clarifications that have come from discussions post this email.

* The small starter (500ml-600ml) he makes first with the fresh vial. This gives a number of advantages in that you can generally fit the starter vessel and wort into a standard pressure cooker to sterilise.
* let the small starter fully ferment out to ensure the yeast have full glycogen stores and are in good health
* It is best to store the yeast in sanitised nutrient water than any beer. I originally asked him that storing at -20 degC may cause pepite mutants as it was not below-80 C (temps used by manufacturers). The answer is to store the yeasts with the nutrient rich water which doesn't provide any potential food as pepite mutants occur during cell multiplication.
* the nutrient rich water is essentially a cup of water with a pinch of yeast nutrient and sterilised. 
* The yeast left from the smaller starter is then added to a standard starter for your main pitch.

Hope that helps. Good luck.


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## WhiteWolf (26/11/10)

Bookmarked!


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## Bribie G (26/11/10)

Sounds brilliant. One thing that strikes me, with slanting - you should keep the 'blank' slants for a week or so to see if any grow mould, so you chuck them and go with the good ones. With the tubes using the glycerine and freezing method, there is no "proving" period and I wonder if this introduces a small but significant risk?


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## argon (26/11/10)

abc said:


> _My method for creating a frozen yeast bank isn't too difficult. The beginning supplies you need are
> -Styrofoam box that fits in your freezer
> -Ice Pack
> -Glass/Plastic Vials (look for 15-25mL glass vials OR use 50mL Flacon tubes)
> ...


_


Anyone know where i can get my hands on some glycerin?_


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## [email protected] (26/11/10)

argon said:


> Anyone know where i can get my hands on some glycerin?



Try a pharmacist or chemist. Glycerin also know as glycerol, glycerine.


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## [email protected] (26/11/10)

BribieG said:


> Sounds brilliant. One thing that strikes me, with slanting - you should keep the 'blank' slants for a week or so to see if any grow mould, so you chuck them and go with the good ones. With the tubes using the glycerine and freezing method, there is no "proving" period and I wonder if this introduces a small but significant risk?




You are right about the no proving period before storage. The proving comes at the time of making the starter after storage. As you will step up the vial of banked yeast this will be your trial. Taste and smell the beer made from that sample to check OK as you would any starter and decide to continue or not.

For me as a homebrewer this technique suits me as I don't want to particularly get into yeast culturing in a major way. I don't want to keep samples for years or keep rare strains. I want to simply have some yeasts on hand and to get a little better economics from liquid yeasts at this stage with this hobby. I suppose you could look at as a stepping stone.

Who know's once you start you might not be able to stop and develop a full blown culture lab complete with stereo microscope and hemocytometer alongside your brewery!


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## razz (26/11/10)

I've been into two chemist stores and no glycerine, the missus tells me today that it's in Woolies where the vitamins are.


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## [email protected] (26/11/10)

Just rang my local chemist ( they are a compounding chemist - old school style) and they have 100ml bottle of glycerol for $5.95. They get in what every quantity you want of chemicals. As a side note they will also get lactic acid in - not cheap at $34 / 100ml.

They are located at Ashgrove for the people in Brisbane. I'm sure there are others as this is first I made a call to.


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## jonocarroll (26/11/10)

Have a peek in the cake (decorating) sections at the supermarket. I reckon that's where I've seen it.


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## Nevalicious (26/11/10)

Glycerin from Priceline Pharmacy (TTP for those in SA) was only $3.99 for 100ml! Bargain


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## Thirsty Boy (27/11/10)

Silo Ted said:


> According to a 2006 BYO Magazine article, the WL yeasts are now shipped out with an average cell count of 100 billion, a lot more than what I thought to be around 35 billion. With a 20% decrease per month this would still mean there's about 26 billion cells, not far off the previous count. Not bad figures to consider if buying old stock. Also not too far off the previously shipped 35 billion.
> 
> I almost bought one of these USB microscopes so me and a mate could look at the cell count on stored yeasts, but many reviews say that they are rubbish, and not even close to the stated specs of 400x. as well as the picture quality being poor. Would a conventional microscope be adequate to view yeast cells, and at least visually estimate cell count? Im very interested to observe the decline in viability with recycled cropped samples, and compare to newly purchased vials.
> 
> If brewers are to take samples from an active ferment for storage purposes, isn't this going to be a new generation of healthy cells with a high count per microgram (or whatever the standard measure might be), or will hop & grain debris significantly diminish the count? Mutation aside, Im still unclear as to why you cant just keep culturing crude fresh samples in a series of starters every couple of months to perpetually maintain a yeast bank. Although I guess if you do this often enough to keep your samples viable, the cost of DME and the time involved would be greater than just buying a new vial of reliable clean yeast.



It's not the cell count that is the issue - there's a hell of a lot less cells than 35billion on a slant. It's the storage and dying off process that is the issue. The properties of the yeast change as cells mutate from generation to generation, and the yeast does not maintain its brewing properties. Cells going bung, happens a lot more in stressed and dying samples and for the most part, yeast stored under liquid are stressed and dying samples. - If you culture up, harvest and re-culture from samples that have been allowed to degrade, this drift becomes rapid enough so you may well notice strain drift in as little as a couple of generations.

You probably don't want to waste your money on a microscope for counting yeast.... If you're curious, for sure. But for counting - well, a decent haemocytometer is not exactly a cheap thing and at any rate, cell counting with the stain you are likely to use (meth blue) is notoriously unreliable at cell viabilities below 80-90% - so nun less you are pretty sure that you have 80-90% live cells in your sample anyway, then you are very unlikely to be able to get an accurate cell count. And if you have 80-90% viability... You're pretty good to go anyway.


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## Bribie G (27/11/10)

Glycerine should be in the vitamins section at Woolies / Coles / IGA along with the Hydrogen Peroxide, liquid paraffin etc. I've just stocked up on test tubes with lids and a rack, and I'll definitely do some frozen ones first before I get into the slants, it just seems so much less stuffing around. 

One thing that hasn't been mentioned so far, glycerine has a very sweet cloying flavour although it's quite edible in small quantities (glycerine and honey as used by grandma) - would it taint the beer? so if making a starter from a tube I suppose I should get a good bit of yeast bred up, let it sink then pour off the liquid to get rid of the glycerine?


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## Thirsty Boy (27/11/10)

i never tasted it in any of the beers I made from yeast stored under glycerol Bribie - hardly definitive I know, but you are only talking a fw ml of the stuff after all.

and if it concerned you - you could culture up in the same way you would from a slant. Shake up tube, red hot loop into the glycerol/yeast mixture, loop into 10-15ml of wort. Go. Then its lid back onto the sample and back into the freezer.


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## Nevalicious (27/11/10)

Thirsty Boy said:


> i never tasted it in any of the beers I made from yeast stored under glycerol Bribie - hardly definitive I know, but you are only talking a fw ml of the stuff after all.
> 
> and if it concerned you - you could culture up in the same way you would from a slant. Shake up tube, red hot loop into the glycerol/yeast mixture, loop into 10-15ml of wort. Go. Then its lid back onto the sample and back into the freezer.



Thats an interesting method... 

In order for this to work though Thirsty, you would have to "defrost" the yeast/glycerine mixture to take the sample yes?? Or am I wrong in thinking that this mixture wont harden in the freezer. The reason I ask is, I thought freezing then thawing and so on is no good for yeast viability?? Constant temp should be maintained??

Also, if this would work into 15 odd ml of wort, you would wait for this to ferment out, then step up as usual??

Tyler


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## Bribie G (27/11/10)

The freezing / thawing damage to cells occurs because the cell walls burst, but apparently the glycerine prevents this from happening. The guys a Alcor maybe use glycerine when they freeze your head.


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## Nevalicious (27/11/10)

BribieG said:


> The freezing / thawing damage to cells occurs because the cell walls burst, but apparently the glycerine prevents this from happening. The guys a Alcor maybe use glycerine when they freeze your head.



Planning ahead Bribie???


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## Thirsty Boy (27/11/10)

Nevalicious said:


> Thats an interesting method...
> 
> In order for this to work though Thirsty, you would have to "defrost" the yeast/glycerine mixture to take the sample yes?? Or am I wrong in thinking that this mixture wont harden in the freezer. The reason I ask is, I thought freezing then thawing and so on is no good for yeast viability?? Constant temp should be maintained??
> 
> ...



Yeah, you are right. The yeast is better maintained by keeping a constant temperature. In my samples, they stayed primarily liquid, so it was less freezing than just very cold storage, and at any rate, a freeze thaw cycle is going to be less bad for the life of the sample than using all of it no?

I think you do want them to actually freeze for best storage though (I used to much glycerol) - and even with the glycerol, they will suffer mortality on every freeze/thaw cycle. I wouldn't re-culture from one that had been frozen and thawed, but I reckon it'd be OK to use for stepping up to a starter a few times before you binned it and started from a never thawed out version.

If in doubt - I'd go with using the whole sample though - I'm speculating not speaking from experience or a decent knowledge base.

Once you had inoculated 10-15ml of wort - you give it about a day or so to grow, then step up by 10 fold for ale yeasts and by 5 fold for lager yeasts. Thats going to give you a cell density of about 100,000,000/ml for the "grown" sample and thus a pitching rate of 10,000,000 per ml (or 1,000,000 cells/ml/plato in a 10plato/1.040 starter wort) when you tip it into the next step. Going 10ml -> 100ml - > 1000ml should give you approximately 100billion cells - or the equivalent of a brand spankingly new smack pack. Obviously if you step up by half the volume for lager yeasts, you are doubling the pitching rate - but should end up with roughly the same final number of cells/ml. Or so sayeth Chris White when I cornered him at ANHC anyway 

TB


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## Nevalicious (27/11/10)

Thirsty Boy said:


> Yeah, you are right. The yeast is better maintained by keeping a constant temperature. In my samples, they stayed primarily liquid, so it was less freezing than just very cold storage, and at any rate, a freeze thaw cycle is going to be less bad for the life of the sample than using all of it no?
> 
> I think you do want them to actually freeze for best storage though (I used to much glycerol) - and even with the glycerol, they will suffer mortality on every freeze/thaw cycle. I wouldn't re-culture from one that had been frozen and thawed, but I reckon it'd be OK to use for stepping up to a starter a few times before you binned it and started from a never thawed out version.
> 
> ...



Thanks Thirsty, this helps alot. I reckon, I might even make up my frozen yeast samples and later down the track when I require a particular strain, even pouring just half out of a vial into a starter and stepping up as required is much more thrifty than just using a $10 yeast in one brew!

Awesome thread...

When my vials of WL's roll up, i'll get things underway!!!

Tyler


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