# Yeast Culturing Equipment



## proudscum (31/10/10)

Have just ordered a plate stirrer from Digital Brewing and am thinking what size flasks do i need to buy other than a 1L ?

Good place to buy screw lid test tubes(i know i should have kept all those white labs ones)Glass or plastic not to fussed to start with-cheap is good?

How many petri dishes to do slant production?

What size pressure cooker would i need to fit a 1L flask in?

Thanks for any input,i have read a little and am about to order the new Chris White book from Amazon that came out 2 weeks ago on this subject for $14 plus postage.


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## ~MikE (31/10/10)

Autoclaving isn't as necessary for making starters IMO. 

you can happily do everything without needing petri dishes, slants are made with tubes. the tubes, get polypropylene (PP) or polycarbonate (PC) - polystyrene (PS) will melt in the pressure cooker.


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## barls (31/10/10)

ive still got some extra tubes left over from a bulk buy that i organised a while ago after some peoples pulled out. pm me if interested


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## Silo Ted (31/10/10)

Adding to Barls' offer, I have about 10 glass petri's with lids that I bought for another project but never got around to using, they were pretty cheap for me to buy, so if you want to discuss buying them off me, Im sure we can sort something out for a cheap price. I am in Sydney though, so shipping is a factor - but a mate of mine knows Barls and I think we live close, so possibly a combined shipping if youre interested?

Social networking at its best. Who needs facebook when we have AHB ?


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## Silo Ted (31/10/10)

> i know i should have kept all those white labs ones



The White labs vials are apparantly soft-drink bottle blanks before they are heat blown into a mould - that's why you notice that PET screwcaps fit them perfectly. If you find out where to source them new, please let me know, because the label gum is impossible to get off the WL empties


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## felten (1/11/10)

proscitech.com.au do all sorts of test tubes and vials, unfortunately mostly in bulk though.


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## pk.sax (1/11/10)

http://cgi.ebay.com.au/28ml-Glass-Laborato...ZQQcmdZViewItem

Found these 28ml test tubes on eBay with screw caps. All autoclavable, can't vouch for them yet as haven received and tried them but they sound the goods.


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## manticle (1/11/10)

Most basic is a vessel, some sanitiser, some boiled cooled water and a funnel. 

You can do it in stubbies or longnecks (both storage and starters). Stirplate makes things quicker but isn't necessary. A fridge is helpful for storage but i'm assuming you already have one.

For test tubes try username: sera and look in the retail thread. Otherwise I'm sure barls will sort you out.


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## elduderino (2/11/10)

> Have just ordered a plate stirrer from Digital Brewing and am thinking what size flasks do i need to buy other than a 1L ?


I lucked into finding a 3 liter flask at Goodwill years ago -- it's nice because there is room for the yeast to bubble up on the stir plate without overflowing. I would not worry about autoclaving/pressure cooking. Just dump in half a liter of water and boil for 20 minutes with foil covering the opening. I'm sure a lot of people just sanitize it.



> How many petri dishes to do slant production?


I don't think you 'need' any petri dishes to do slants. I make up slants and then after emptying my wyeast pack into the fermenter, I take a sterilized metal pick and wipe it in the pouch and then streak the slant. If you want to grow colonies on a petri dish to make sure you get a single strain, it doesn't seem like you would need more than one or two. You could also just get some little canning jars meant for jelly, as they can stand the heat of pressure cooking.


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## argon (2/11/10)

In lieu of an autoclave or stove top boiling, a baby bottle sterilizer is good for sanitizing most little microwave safe objects. Not as good as an autoclave, but works never the less.

I have one that i use for my yeast sample jars and plastic bits and pieces. i just run it through 2 cycles of 4 minutes... nukes the crap out of anything that would happen to be in there.


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## MaltyHops (2/11/10)

You will need some kind of wire loop or needle to inoculate your slants
- I've ended up using one of _THESE_ - that's right, a lobster fork  It works
well for me because I bought small diameter screw top tubes (~1cm wide)
before and using a single needle (paperclip actually) to try and poke yeast
into the agar in the tube will cause the agar to rotate annoyingly with
anything off-centre.

With a stainless steel lobster fork, you can flame the hell out of it and its
double prong tip is good for digging into the agar.

T.


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## proudscum (4/11/10)

elduderino said:


> I would not worry about autoclaving/pressure cooking. Just dump in half a liter of water and boil for 20 minutes with foil covering the opening.
> 
> 
> this confuses me a little!Boil flask in a pot of water?
> ...


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## felten (4/11/10)

they can go on the stove themselves


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## proudscum (7/11/10)

Does anyone use vocola jars to make their sterile wort in?


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## fcmcg (7/11/10)

proudscum said:


> Does anyone use vocola jars to make their sterile wort in?



Do a search for wolfy's two excellent threads ( with pics) ...making slants and from slants to starters....
I got the Chris White book at ANHC...
It's heavy going lol
Proudscum....come along to westgate , where you can have alot of your questions answered , over a beer....hehe :icon_cheers: 
Cheers
Ferg


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## Thirsty Boy (7/11/10)

OK, I'll try without any of the "you don't need a .... to do yeast" adnd give you the stuff I think you should have to do it fairly properly.

I do skip the step of plating to get a pure culture and just go straight from source yeast to slant though. I can cover plating if you really want, but someone else will be able to do it better I'm sure.

Here's what I think you "shold" have. This isn't meant to be a how to thread, I'm just putting in some of the techniques to illustrate why you need the equipment I reckon you do.

*Growing your yeast:*

To go from a slant to a pitch you need your stirplate and a few flasks/tubes. Your cultures should rise in 10 fold steps for ales and 5 fold steps for lagers so...

*For an Ale* you go. Loop of yeast -> 10-15ml wort in a little tube -> 100ml wort in a conical flask -> 1L of wort in a conical flask and that will give you about 100 billion cells, or the same as a very fresh Wyeast Pack or Whitelabs tube. From there, you want flasks big enough to go to whatever starter size you want to make. Probably 2L is enough for most reasonable-fairly strong ales.

For a Lager you go. Loop of yeast -> 10-15ml of wort -> 50ml of wort -> 250ml of wort -> 1-1.25L of wort and that gives you about 100billion cells again. And to do lager starters I think you need a 4-5L flask. You can get away with a demijohn, but best for purpose would be a 5L Erlenmeyer. They are poisonously expensive though (up to $100+)

So covering all bases .. you want as an optimum - a small tube at say 80-100ml, a 250ml Erlenmeyer, a 500ml Erlenmeyer, a 1L Erlenmeyer - and then for your "starters" a 2L & 5L Erlenmeyer.

I manage to make do with 1 x 500ml, 1 x 2L and 1 x 5L plus a couple of tubes. But life would be easier and things a touch more sterile with the extras.

*To make slants and inoculate your yeast into starters:*

A pressure cooker. A small one will do. I think its important to autoclave/pressure cook your slants and direct culturing equipment, simply boiling will do for flasks. If you are willing to pay the shekels for a big arsed pressure cooker that would be better - but even though I said I wasn't going to do the "you can get away with" thing, you can get away with just boiling your flasks.

Small tubes that are autoclavable. I use 15ml screw cap tubes from the bulk buy mentioned earlier in this thread. They are perfect.

Some agar agar from the asian supermarket. Gelatin is to hard to use and melts, you can use it, but agar is better.

An inoculation loop. I use a bit of stainless welding wire bent into a little loop at one end and a handle at the other with some gaffa tape for grip.

A little alcohol burning stove or a bunsen burner .. some sort of flame to make an updraft and to heat the loop

Alcohol to burn in the stove and to make things sterile

some dme.

*Making The Slants*

Divide your tubes in half - into one half, fill to a little less than the halfway point with wort at 1.030 mixed with agar agar. The other half of your tubes fill to a say 3/4 full with just the wort. Screw the lids on and then back them off so that they would pull off if you tried to lift them. Upright into the pressure cooker inside a coffee cup or something so they dont fall over. Pressure cook them for a while.. to long is no harm, so I just cook the shit out of them.

After the cooker cools enough for you to touch, open the lid and as you take each tube out, tighten the lid. Wear gloves and try to get it done while things are still really hot. The agar ones get laid on their sides to give an angles surface, try to get the maximum surface area you can. These are your slants. The wort ones are just a source for pre-sterilised wort for your initial 10-15mls - its a PITA to try and cook that small an amount up on spec.

For both tubes and slants - keep them at warm room temp for a week before use, so that if anything is going to grow, you will see it and can toss that one away.

All that gets done in advance - tubes and slant will last indefinitely, or at last for a very long time.

*Innoculating from a yeast pack onto a slant.*

Wear a facemask - I tie a bandana over my face. Light the little alcohol stove and when you work, try to work close to and sort of over the flame so you are in the updraft it creates.

Use alcohol to wipe down the outside of your yeast pack, dip into the alcohol and set on fire the blades a pair of scissors. Cut open your yeast pack. Heat up your inoculation loop in the flame till its red hot, dip it into the yeast pack, wipe the loop on the surface of the agar in a slant. Tighten the lid onto the slant. Repeat for all the yeast packs or slants you want to make.

You should use sterile techniques as best you are able, I wont detail them here as you can google them.

Slants at room temp for a few days, to make sure the yeast starts to grow and nothing else does... then into the fridge at 4C. Re-culture every 4-6 months for best performance.

*To use a slant*

Flame on, mask on, lid of slant dipped in alcohol, lid of tube of wort dipped in alcohol. Flame loop and cool by dipping into alcohol - drag loop across surface of inoculated slant to pick up some cells, then stir it into the tube of wort. let it grow.

If your sterile technique is good, you can use the same slant again and again. I tend to discard it after one use so I make several slants of the same yeast when I am culturing. If you intend to do likewise, then you can do what I mainly do, which is ditch the loop for getting yeast into wort, and just pour some wort directly into the slant itself.

I think thats it - less than a perfect sterile technique and proper micro people out there are probably flinching/laughing - but its served me pretty well, isn't as much trouble as it sounds and on the scale of "too slack -- proper" I think its on the "proper" side of the balance point.

Warning - you have both highly flammable alcohol and an open flame... do not set yourself on fire. That would be bad.

TB


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## felten (8/11/10)

bookmarked!


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## BrenosBrews (8/11/10)

I just wanted to add that the Chris White book has more information about yeast than a homebrewer probably needs to know :lol: 

Great guy & it was awesome to talk to him one on one for a fair bit during ANHC


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## proudscum (8/11/10)

BrenosBrews said:


> I just wanted to add that the Chris White book has more information about yeast than a homebrewer probably needs to know :lol:
> 
> Great guy & it was awesome to talk to him one on one for a fair bit during ANHC




Thanks heaps for that its great.Hopefully by the end of the week i will have all my glassware and tubes.Was given a lge Meyer gift voucher sometime ago so will see what pressure cookers they have.That leaves some agar agar to get plus a top up can of gas for by small chefs blow touch and a spray bottle for sanitizer.Am keen to get some vacola jars to make sterile wort to have on hand.

When i find a new toy to play with i go nuts and all.
If the short course that i may be teaching on brewing kicks off then the ATO will get my receipts as they will all be tools of my teaching trade.


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## FreeBaseBuzz (24/11/10)

Thirsty Boy said:


> ... Heat up your inoculation loop in the flame till its red hot, dip it into the yeast pack, wipe the loop on the surface of the agar in a slant. ...



to ask a couple of stupid questions.. 

you dip the loop into the yeast pack while it is still red hot? I assume it's a liquid yeast pack?


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## Thirsty Boy (25/11/10)

FreeBaseBuzz said:


> to ask a couple of stupid questions..
> 
> you dip the loop into the yeast pack while it is still red hot? I assume it's a liquid yeast pack?



Yes, liquid culture. No need to slant a dried culture, they are already in a stable storable form.


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## Amber Fluid (11/7/12)

Thirsty Boy said:


> Yes, liquid culture. No need to slant a dried culture, they are already in a stable storable form.



Trying not to state the obvious here but I'm going to, why doesn't the hot loop kill the yeast it collects?
I am assuming that it cools on contact with the liquid, but still kills off a few cells although too little amount to worry about.


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## therook (11/7/12)

Amber Fluid said:


> Trying not to state the obvious here but I'm going to, why doesn't the hot loop kill the yeast it collects?
> I am assuming that it cools on contact with the liquid, but still kills off a few cells although too little amount to worry about.



Spot On


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## Wolfy (11/7/12)

Amber Fluid said:


> Trying not to state the obvious here but I'm going to, why doesn't the hot loop kill the yeast it collects?
> I am assuming that it cools on contact with the liquid, but still kills off a few cells although too little amount to worry about.


Yes, that's the theory - the loop is so small/thin that it cools virtually instantly.
However, I've had a few yeast-culturing attempts fail because I did not cool the loop well enough.


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## piraterum (11/7/12)

Wolfy said:


> Yes, that's the theory - the loop is so small/thin that it cools virtually instantly.
> However, I've had a few yeast-culturing attempts fail because I did not cool the loop well enough.




Simple solution get some disposable (sterile) 1uL or 10uL Inoculation Loops. They are cheap to buy and a box will last you a lifetime :beerbang: 

Available from plenty of Laboratory / Scientific suppliers like e.g. Livingstone International http://www.livingstone.com.au/

No affils etc etc plenty of suppliers to choose from


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## Wolfy (11/7/12)

piraterum said:


> Simple solution get some disposable (sterile) 1uL or 10uL Inoculation Loops. They are cheap to buy and a box will last you a lifetime :beerbang:


The problem with the disposable ones is they can't be heat sterilized and so are really single-use-only (and if I used them for each slant I make, that's about 300 a year).


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## piraterum (12/7/12)

Wolfy said:


> The problem with the disposable ones is they can't be heat sterilized and so are really single-use-only (and if I used them for each slant I make, that's about 300 a year).




They come in boxes of 1000 so that should last you a while :icon_cheers: 

Not to mention save a lot of f**king about with a wire loop, which have a much higher contam risk than using a gamma sterilised disposable one


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## Wolfy (12/7/12)

piraterum said:


> Not to mention save a lot of f**king about with a wire loop, which have a much higher contam risk than using a gamma sterilised disposable one


There is a bit of messing around with a wire loop, however I don't think much of anything can survive the loop being heated to red/white-hot ... can it?


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## benno1973 (12/7/12)

piraterum said:


> Simple solution get some disposable (sterile) 1uL or 10uL Inoculation Loops. They are cheap to buy and a box will last you a lifetime :beerbang:



Similar theory, I use sterile syringe needles. Cheap to buy a box of them and lots of brewing uses. I just suck a little into the syringe and squirt that into the slants/testubes.


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## MaltyHops (12/7/12)

Wolfy said:


> Yes, that's the theory - the loop is so small/thin that it cools virtually instantly.
> However, I've had a few yeast-culturing attempts fail because I did not cool the loop well enough.


Another simple solution is to keep a vial of vodka to dip the hot loop into to cool it (assuming there's no chance of the vodka blowing up - hmmm must test this sometime :huh: ).


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## troopa (12/7/12)

Is 40% vodka enough to sterilise or does it need to be higher in alc% ?


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## MaltyHops (12/7/12)

Troopa said:


> Is 40% vodka enough to sterilise or does it need to be higher in alc% ?


This _homebrewtalk poster_ writes "You need 80% concentration of
alcohol for 45 seconds to sanitize. Your cheap vodka is likely 40%
alcohol (80 proof) and way too low a concentration for sanitizing
unless you have very, very long contact times (but it's not going
to grow stuff on its own or harm already-sanitized stuff, so it's
okay for filling airlocks and such)."

There's also _brewingtechniques article_


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## Wolfy (13/7/12)

Troopa said:


> Is 40% vodka enough to sterilise or does it need to be higher in alc% ?


Isopropol (rubbing) alcohol (comes in a green bottle with a crocodile on the front) is what I use, it's 80% and probably cheaper than vodka (also use it for a clean heat-source for our fondue).


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## Lord Raja Goomba I (13/7/12)

Kaiser Soze said:


> Similar theory, I use sterile syringe needles. Cheap to buy a box of them and lots of brewing uses. I just suck a little into the syringe and squirt that into the slants/testubes.


Free with a diabetic in the house.

Goomba


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## Darkman (13/7/12)

Wolfy said:


> Isopropol (rubbing) alcohol (comes in a green bottle with a crocodile on the front) is what I use, it's 80% and probably cheaper than vodka (also use it for a clean heat-source for our fondue).



Wouldn't Metho be just as effective?


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## piraterum (13/7/12)

Wolfy said:


> There is a bit of messing around with a wire loop, however I don't think much of anything can survive the loop being heated to red/white-hot ... can it?




Some environmental mould spores are resistant to dry heat and alcohol. The other thing to consider is the rest of the loop is not being heated so there is potential for contamination from the handle.

Metho is fine to use for flaming e.g. dipping a loop in metho and flaming it. If you are using alcohol to sanitise a 70% ethanol solution is the most effective concentration. Using higher concentrations (80%, 100%) is less effective. There are plenty of peer reviewed papers demonstrating this.


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## kieran (15/7/12)

MaltyHops said:


> This _homebrewtalk poster_ writes "You need 80% concentration of
> alcohol for 45 seconds to sanitize. Your cheap vodka is likely 40%
> alcohol (80 proof) and way too low a concentration for sanitizing
> unless you have very, very long contact times (but it's not going
> ...



While the 40% vodka is too low, sterilising in 80% (by volume) alcohol for 45 seconds is complete overkill (like using an H-bomb to kill a mouse) and a waste of time. 
A 1 second double-dip/shake and flame is more than enough to kill even the most pervasive bacteria and yeast. As for spores, if you're worried, before the first dip flame the loop until it is glowing. Then let it cool, dip and flame. That technique has served me well for over 10 years in the lab, with controls, and perfect bug growing environments (so any contamination had a great chance of kicking-off).

Methylated spirits is denatured alcohol, and it's cheap. It's also usually available in 4L jugs at Bunnings. Dilute that to ~80% by volume (which works out to about 70% by weight), and a quick dip and flame and you're done.
Of course, pure alcohol is the best, but for a back yard homebrew environment, "metho" is just fine.


side note.. has anyone with access to it, used ampicillin/kanamycin/choramphenicol on home plates to kill off any bacteria? Not sure how easy it is to get in the home for regular brewers though. Ampicillin is pretty well controlled now, as an S2 drug so that probably won't fly, but I don't think the others are?


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## manticle (15/7/12)

kieran said:


> (like using an H-bomb to kill a mouse)




Fucker won't ever piss in my pantry again though will he?


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## MaltyHops (15/7/12)

kieran said:


> .
> Methylated spirits is denatured alcohol, and it's cheap. It's also usually
> available in 4L jugs at Bunnings. Dilute that to ~80% by volume (which
> works out to about 70% by weight), and a quick dip and flame and
> ...


I'd always been put off by the "POISON" printed on bottles of metho
and since using for yeast cuturing would end up in the beer we drink
- or would the amount ending up in the beer be so low as to be
negligible?

Pure alcohol is also not easy to get.


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## manticle (15/7/12)

Flaming will drive that tiny amount away. Many a kitchen I used to work in would rinse clean cutlery in hot water and a touch of metho.


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## Wolfy (15/7/12)

MaltyHops said:


> I'd always been put off by the "POISON" printed on bottles of metho
> and since using for yeast cuturing would end up in the beer we drink
> - or would the amount ending up in the beer be so low as to be
> negligible?


I'd be curious to know if _any _poison - that one can readily buy in a shop - is potent enough to be harmful if 1 drop is diluted into 20L of wort that is then boiled for an hour.
_(Botulin toxin is one of the most powerful known toxins: about one microgram is lethal to humans, but the botulinum toxin is destroyed by thorough cooking over the course of a few minutes)_.

Besides as *manticle* said the metho is burned away in the flaming process.


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## Golani51 (15/7/12)

Is there any reason to use an innoculation loop over a syringe for example?
I would have thought a syringe ideal. It doesn't get messy, costs pennies and can be bought at any chemist, sterile, and the yeasties are stored inside so there would be no issues with contamination from air and dust. A drip or two on each slant and all done.

R


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## Golani51 (15/7/12)

BTW Is anyone doing a yeast swap (any forum topic on it I dont know about?)?
I am happy to get a new smack pack to exchange for some slants.


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## Wolfy (15/7/12)

Golani51 said:


> Is there any reason to use an innoculation loop over a syringe for example?


A rounded-end loop is better for streaking slants and plates because it allows easy collection of the yeast sample and will not cut/scar/indent the surface of the agar when streaked (so the yeast colonies will grow on the agar surface and not in the 'holes' or 'grooves' cut by the tip of the sharp syringe-needle).
If taking samples of liquefied yeast in solution a syringe-needle might be more useful.


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