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General Category => Yeast and Fermentation => Topic started by: Thirsty_Monk on January 08, 2010, 07:35:51 PM

Title: Yeast Culturing
Post by: Thirsty_Monk on January 08, 2010, 07:35:51 PM
I just made my supply purchase at http://cynmar.com.

Code: [Select]
12030895  GLASS ALCOHOL LAMP w/CAP, 4oz      US$3.40
CHA80228  DEHYDRATED AGAR POWDER, 30g      US$8.25
11527906  CLEAR GLASS VIALS w/AUTOCLAVABLE CAP, 12ml 12/PK      US$5.00
11527908 CLEAR GLASS VIALS w/AUTOCLAVABLE CAP, 16ml 12/PK US$5.75
13225101 NICHROME INOCULATING LOOP, 25 GAUGE WIRE, 8" TOT LENGTH US$1.50
13225110 NICHROME INOCULATING NEEDLE,25 GAUGE WIRE, 8"TOT LENGTH US$1.40
12030036 TEST TUBE BRUSH, WHITE NYLON, 3"BRUSH, 8" TOTAL US$0.70
13220697 GLASS PETRI DISH, 75x15mm,10/PK US$7.25

Kai and his "Yeast culturing gear" http://braukaiser.com/wiki/index.php?title=Yeast_culturing_gear was very helpful.

I also have "First Steps In Yeast Culture"
(http://new.midwestsupplies.com/media/catalog/product/cache/1/image/265x/5e06319eda06f020e43594a9c230972d/f/i/first-steps-inyeast-culture-rajotte.jpg)
read "Yeast Propagation and Maintenance: Principles and Practices" http://maltosefalcons.com/tech/yeast-propagation-and-maintenance-principles-and-practices
and listen yeast culturing episodes on Basic Brewing.

Anything else I should be aware of?
Thank you
Title: Re: Yeast Culturing
Post by: Hokerer on January 08, 2010, 07:52:47 PM
Anything else I should be aware of?

30g of agar powder will last a good while but, once you do use that up, if you have a local Asian food store, you can usually get 25g of agar powder for about a buck twenty-five.
Title: Re: Yeast Culturing
Post by: Kaiser on January 08, 2010, 08:02:39 PM
Quote from: Geoff Henderson
Anything else I should be aware of?
 

Keep a back up (dry yeast for example) on hand until you are confident.

Yes, agar from those lab places is expensive. I have yet to go to an Asian food store for that. I couldn’t get it at a health store.

Kai
Title: Re: Yeast Culturing
Post by: a10t2 on January 08, 2010, 09:18:11 PM
30g of agar powder will last a good while but, once you do use that up, if you have a local Asian food store, you can usually get 25g of agar powder for about a buck twenty-five.

Making your own from gelatin and DME works perfectly well for me, and I get some peace of mind from culturing the yeast on a maltose substrate.
Title: Re: Yeast Culturing
Post by: mrdrysdale64 on January 08, 2010, 10:53:13 PM
30g of agar powder will last a good while but, once you do use that up, if you have a local Asian food store, you can usually get 25g of agar powder for about a buck twenty-five.

Making your own from gelatin and DME works perfectly well for me, and I get some peace of mind from culturing the yeast on a maltose substrate.

What proportions of gelatin and DME do you use? I assume you mix and then autoclave or put them in a pressure cooker? I need to try this. I have been kicking around the idea of culturing for a couple of years now. This forum is getting me motivated to do so many things!  :)
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 09, 2010, 12:16:13 AM
Greg Pietsch recipe:
    * 100 grams H2O
    * 1.5 grams agar
    * 1.5 grams Fermaid K
    * 12 grams light DME
http://saccharomycessafari.blogspot.com/2009/08/today-im-making-up-some-new-blank.html

Kai's recipe:
    *  100 ml wort
    * 3-4 g agar
http://braukaiser.com/wiki/index.php?title=Making_Plates_Slants

Maltose Falcons:
1.5-2.0 grams per 100 ml liquid
http://maltosefalcons.com/tech/yeast-propagation-and-maintenance-principles-and-practices
Title: Re: Yeast Culturing
Post by: Hokerer on January 09, 2010, 12:40:08 AM
Kai's recipe:
    *  100 ml wort
    * 3-4 g agar
http://braukaiser.com/wiki/index.php?title=Making_Plates_Slants

This is the ratio that works best for me (4% that is).  I find it easier to squirt 5ml of wort into as many of the 12ml vials as I want slants.  I then weigh out enough agar to come out to about 4g for every 100ml.  Divide it up by eye into equal portions (and hope nobody walks in while I'm pushing white powder around with a razor blade) and dump a portion into each vial.  Shake it up good and then pressure can them.

If I mix in the agar first, it starts to set before I get all the mixture measured into the vials and if I use less than the 4% ratio, my slants "slump".
Title: Re: Yeast Culturing
Post by: Kaiser on January 10, 2010, 03:01:14 PM
If I mix in the agar first, it starts to set before I get all the mixture measured into the vials and if I use less than the 4% ratio, my slants "slump".

That approach works well for me. But the solution has to be somewhat hot to remain liquid.

Kai
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 10, 2010, 03:45:43 PM
Thank you for advice about solid media.

Any advices about streaking yeast on petri dishes?
Some people suggest to:
Sterilize inoculation loop,
Get yeast sample,
Streak the whole plate,
Sterilize inoculation loop.

and some people suggest:
Sterilize inoculation loop,
Get yeast sample,
Streak 1/3 of plate,
Sterilize inoculation loop,
get another yeast sample,
Streak 1/3 of plate,
Sterilize inoculation loop,
Streak 1/3 of plate,
Sterilize inoculation loop.

Where would I use inoculation loop and where would I use inoculation needle?
Title: Re: Yeast Culturing
Post by: Kaiser on January 10, 2010, 05:58:12 PM
Any advices about streaking yeast on petri dishes?

give different approaches a try. Nowadays I simply keep streaking many lines next to each other. At some point the spead of cells gets so sparse that you get nice single colonies. Re-sterilizing the needle between streaks or repeated dillution in drops of sterile water sounds cool but is way to much work for me. In addition to that it increases the time the plate is exposed to the air which is something I try to minimize.

Quote
Where would I use inoculation loop and where would I use inoculation needle?

needles are for stab cultures and streaking plates. loops are for picking colonies, scraping lawn and inoculating slants. At least that's what I use them for.

Kai
Title: Re: Yeast Culturing
Post by: hankus on January 10, 2010, 07:57:59 PM
Here's something that is related to the culturing topic-harvesting yeast
I have begun serious yeast ranching after rinsing and after washing a yeast cake realized that when I tilted the carboy I would be pouring through the crud that had built up on the carboy walls and neck so I came up with a clean transfer system which MAY work for top cropping
I have a 8" segment of SS tube from a cut off dip tube which I immerse in a bottle of boiling water and let it sit there loosely covered with AL foil until the bottle cools.I attach one end to a hose (PURGED with hot water) which then goes to one of 2 tubes in a #12 stopper plugging a sterilized mason jar.The other tube is connected to a hose ending in a harbor freight hand pump-about 6 bux.when HF pump pulls on the system the vacuum created draws and the clean SS tube picks up the goods in a relatively clean fashion and delivers to the Mason jar(s).
If someone tries this for top cropping please report back
Title: Re: Yeast Culturing
Post by: roffenburger on January 12, 2010, 02:11:01 PM
Thank you for advice about solid media.

Any advices about streaking yeast on petri dishes?
Some people suggest to:
Sterilize inoculation loop,
Get yeast sample,
Streak the whole plate,
Sterilize inoculation loop.

and some people suggest:
Sterilize inoculation loop,
Get yeast sample,
Streak 1/3 of plate,
Sterilize inoculation loop,
get another yeast sample,
Streak 1/3 of plate,
Sterilize inoculation loop,
Streak 1/3 of plate,
Sterilize inoculation loop.

Where would I use inoculation loop and where would I use inoculation needle?

If you are looking for purity, use the second method. This is streaking for isolation. Streak your first 1/3, sterilize (and cool)  your loop. Then make a couple passes back into the first third and into the second 1/3. sterilize and repeat.
Alternatively, you can streak quadrants, sterilizing between the third and fourth quadrant.
Or...you can make one pass right down the center of the plate and then zig-zag perpendicular to your first pass all the way down the plate.
Lots of options.

If you just want lots of growth, the first method is a way to do it. You could even make a suspension and do a spread plate.

BTW, disposable plastic loops are awesome! I didn't think I would like them when I started this job (after exclusively using ni-chrome loops), but I prefer them now. Umm....I just checked the price on them, and it may not be very practical for home culturing...scratch that idea.

Title: Re: Yeast Culturing
Post by: Kaiser on January 12, 2010, 02:14:21 PM
I found that even w/o sterilizing between streaking sections I can get good colony isolation. But then again, I never had formal microbiology training.

Kai
Title: Re: Yeast Culturing
Post by: roffenburger on January 12, 2010, 02:31:39 PM
I actually don't sterilize between sections either ::)
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 14, 2010, 03:10:51 AM
I got my stuff today.
I will be making plates and slants tomorrow.
I was surprised how small were the petri plates and vials.
I guess I will have to get used to it.
Title: Re: Yeast Culturing
Post by: Hokerer on January 14, 2010, 03:21:30 AM
At least the loop is really small also.  You can get a lot of "loopfulls" of yeast off of one slant  :)
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 14, 2010, 08:27:02 PM
I made my first plates.
Everything looks O.K. so far.

I used DME in my recipe and now I have hot break in the solid media.
It just does not look very pretty :(
Should this be a concern?

Next time I can make a wort up front and remover break from it before making plates and slants.
Title: Re: Yeast Culturing
Post by: Kaiser on January 14, 2010, 08:30:31 PM
Should this be a concern?

No. But it will be easier to spot growth if the media doesn't have bits in it. You are right in that you may want to remove the break next time.

Kai
Title: Re: Yeast Culturing
Post by: roffenburger on January 15, 2010, 01:23:53 AM
I made my first plates.
Everything looks O.K. so far.

I used DME in my recipe and now I have hot break in the solid media.
It just does not look very pretty :(
Should this be a concern?

Next time I can make a wort up front and remover break from it before making plates and slants.

I don't know if you plan on it, but I would suggest leaving the plates at room temperature for about a week to serve as a sterility check. Sorry, that was kind of unrelated to you question, but its a step I like to take.
Title: Re: Yeast Culturing
Post by: Kaiser on January 15, 2010, 01:41:45 AM
I do that too and used to have a fairly high fail rate if I opened the plates before taping them shut. That act of opening them up used to be for flicking off condensation. Now I don't worry about it the moisture and rarely get spurious growth on any of them. 

Kai
Title: Re: Yeast Culturing
Post by: enso on January 15, 2010, 11:55:27 PM
Thirsty monk what is the shipping from cynmar if you don't mind me asking..

Thanks!
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 16, 2010, 02:55:41 AM
it was almost $10.
Quite high if you ask me.
Title: Re: Yeast Culturing
Post by: enso on January 16, 2010, 02:27:10 PM
it was almost $10.
Quite high if you ask me.

Thanks.  Yeah, sort of steep, though I have seen higher!  I am not surprised really though as their prices are considerably lower than many other suppliers I have looked at.  Usual internet retail tactic I guess.  Low prices offset by high shipping charges!

Still, not bad though I suppose
Title: Re: Yeast Culturing
Post by: Kaiser on January 16, 2010, 02:59:37 PM
In cynmar's defense, their primary business are customers who buy larger amounts. Just look at the 500 ml flasks. They only come as 6 pks. If you have a club, organize a club buy. Or safe up until you can get everything you want. If you want to start you may also make slants using baby food jars or small mason jars. Metal wire works as innoculation loop and needle. That's how I got started before I bought more specialized lab supply.

Kai
Title: Re: Yeast Culturing
Post by: Hokerer on January 16, 2010, 03:23:24 PM
Thirsty monk what is the shipping from cynmar if you don't mind me asking..

Just looked at my cynmar order from back in August.  Shipping was $9.95 on $59.15 worth of stuff which I don't consider overly steep.  And yeah, like Kai says, their quantities are geared toward bigger orders.  That's not a bad thing, though, as I've found tons of uses for the 12-pack of 150ml beakers :)
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 17, 2010, 12:06:25 AM
I am not complaining.
I wanted stuff from there so I bought it there.
My order was only $33 + $9.95 shipping.
I got everything what I ordered in timely matter.

I fired my Alcohol lamp first time and flamed the inoculation loop first time.
I do not see anything unusual on plates or slants so I am exited to streak some yeast on it.
I brew tomorrow so it might be Monday  ;D
Title: Re: Yeast Culturing
Post by: tom on January 17, 2010, 01:24:03 AM
Here's a little light reading on brewery yeast handling and bacteria identification:   http://brewingscience.com/PDF/BSI_brewers_lab_handbook.pdf
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 19, 2010, 07:24:48 PM
Here's a little light reading on brewery yeast handling and bacteria identification:   http://brewingscience.com/PDF/BSI_brewers_lab_handbook.pdf

Thank you for the link. I will check it out :)

I just streak my first two plates with WLP838 Southern German Lager.
We shall see in couple of days if I grow something.
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 22, 2010, 08:31:44 PM
Here is a picture of one petri dish:
You can see two streaks and some individual colonies.
(http://farm3.static.flickr.com/2691/4295472471_1b5b91411e.jpg)

and then I made more plates and slates:
Plates are in sandwich ziplock bags (they are supposed to be sterile).
Standing tubes are 10ml wort for first step starter.
(http://farm3.static.flickr.com/2794/4295472479_23bbbd5950.jpg)
Title: Re: Yeast Culturing
Post by: dean on January 24, 2010, 09:25:56 PM
How long can you keep yeast in slants and how many steps are needed for a typical ale (1.050ish)?  I read where Kai said using wire is okay to start with so I may give it a try if I can find some damn agar within 50 miles of me.   >:( :D
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 24, 2010, 09:43:52 PM
How long can you keep yeast in slants and how many steps are needed for a typical ale (1.050ish)?  I read where Kai said using wire is okay to start with so I may give it a try if I can find some damn agar within 50 miles of me.   >:( :D

I think they said that you can keep yeast on slant for 1/2 year and then re-culture.
When building it up you could do something like this: 10 ml, 80ml, 400ml, 2000ml
Each step is about 2 days.
Title: Re: Yeast Culturing
Post by: Hokerer on January 24, 2010, 11:14:37 PM
if I can find some damn agar within 50 miles of me.   >:( :D

Check out your nearest Asian Foods type of grocery store.  A package (25g) should only set you back a buck or two.

(http://farm4.static.flickr.com/3067/2311167536_fa7eccdd7a.jpg?v=0)
Title: Re: Yeast Culturing
Post by: a10t2 on January 25, 2010, 04:41:59 AM
I may give it a try if I can find some damn agar within 50 miles of me.   >:( :D

it's pretty easy to make your own growth medium from DME and gelatin.
Title: Re: Yeast Culturing
Post by: dean on January 25, 2010, 04:16:52 PM
I may have to use gelatin, so far I haven't been able to find agar but I've got one more place to check.  Do gelatin cultures keep as long as agar?  It seems like it would be hard to spread, tearing the gelatin surface rather than mixing in... but then I don't really know if agar is more maleable for lack of a better word than gelatin either?
Title: Re: Yeast Culturing
Post by: denny on January 25, 2010, 04:18:44 PM
When I was doing it, I found I needed to reculture slants at least every 6 months.
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 25, 2010, 05:42:25 PM
Just to report progress.
This is after one week from streaking:

(http://farm5.static.flickr.com/4008/4304343578_3af79f9f2e.jpg)
We have some individual colonies.
Now I have to inoculate some slants and try to make some 10ml starter.
Title: Re: Yeast Culturing
Post by: dean on January 26, 2010, 03:08:20 PM
Wow!   :o  I'm definitely no expert but it looks to me like you have a Lot of really fine looking colonies there!!   8)
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 27, 2010, 05:12:49 PM
I streak out WY2308 Munich Lager on Monday:
I have a lot of yeast in there but there are still some individual colonies.

(http://farm3.static.flickr.com/2686/4309589638_9ca97b1f81.jpg)
Title: Re: Yeast Culturing
Post by: yeastmaster on January 28, 2010, 04:39:49 PM
Just to report progress.
This is after one week from streaking:

(http://farm5.static.flickr.com/4008/4304343578_3af79f9f2e.jpg)
We have some individual colonies.
Now I have to inoculate some slants and try to make some 10ml starter.

That is a pretty nice looking plate!  Looks like things are going pretty well for you.  How much agar did you use?  I noticed that you had several recipes including mine up above and that I use a lower concentration of agar than Kai.  That concentration works well for me, it is easy to pour.  Next time I make up media I might try a higher concentration to see how it works for me.
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on January 28, 2010, 05:06:09 PM
I made plates twice so far.

First time I started with your recipe but the amount of DME gave  me too high wort.
So I deluded it to 1040.
After sterilizing I got hot break in there and it did not look too pretty.
I used plates and yeast likes them.

Second time I use BSI (Brewing Science Institute) recommendation:
Wort agar (instead of commercial nutrient agar) = 30ml house wort, 70ml water, 2g agar; sterilize for 15 minutes at 15psi.
I saved wort after boil (on regular brew day) that did not fit into fermenter.
Plates and slants are nice and clear but I got much more condensation this time.
I did not use these plates yet.
May be next week I could play with some WLP001 :)
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on February 03, 2010, 08:27:53 PM
Today I played with yeast.
I had WLP838 on plate for about 3 weeks and
I had WY2308 on plate for about  10 days.

I streak new plates, made two slants for each yeast and made one 10 ml starter from each yeast.

The WLP838 had pretty large colonies.
When I picked one singular colony for streaking or slanting it did not want to smear nicely.
Is it because my plate is drying out?
Any recommendation how to do this?

WY2308 had smaller colonies and it did not want to smear nicely but it was not as pronounced as WLP838.

It should be the same yeast so they should have similar properties.
Title: Re: Yeast Culturing
Post by: Kaiser on February 03, 2010, 09:59:34 PM
It should be the same yeast so they should have similar properties.

Maybe they are not. One way of identifying yeasts for example is the shape of their giant colonies. Though the differences may be more pronounced between ale and lager there may even be differences between closely related strains.

In the end it may not matter much how well they smear as long as they leave a trail of yeast cells.

Kai
Title: Re: Yeast Culturing
Post by: dean on February 03, 2010, 10:15:09 PM
I received my innoculation needle today... that sucker is HUGE!   :o  I was expecting something with a fine wire and point.  The whole thing must be 10 inches long and most of it is needle (closer in diameter to a coathanger really though!)  Well, I haven't been able to find any agar locally so I'm wondering if anybody has tried cornstarch... like making gravy but thicker so it stiffens more when its cooled.  Its generally clear and it seems corn is related closer to barley than grass maybe?  Anybody tried it?
Title: Re: Yeast Culturing
Post by: enso on February 03, 2010, 10:35:00 PM
I received my innoculation needle today... that sucker is HUGE!   :o  I was expecting something with a fine wire and point.  The whole thing must be 10 inches long and most of it is needle (closer in diameter to a coathanger really though!)  Well, I haven't been able to find any agar locally so I'm wondering if anybody has tried cornstarch... like making gravy but thicker so it stiffens more when its cooled.  Its generally clear and it seems corn is related closer to barley than grass maybe?  Anybody tried it?

I would try unflavored gelatin.  Corn starch will not solidify.
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on February 04, 2010, 02:48:44 AM
I received my innoculation needle today... that sucker is HUGE!   :o  I was expecting something with a fine wire and point.  The whole thing must be 10 inches long and most of it is needle (closer in diameter to a coathanger really though!)  Well, I haven't been able to find any agar locally so I'm wondering if anybody has tried cornstarch... like making gravy but thicker so it stiffens more when its cooled.  Its generally clear and it seems corn is related closer to barley than grass maybe?  Anybody tried it?

I would try unflavored gelatin.  Corn starch will not solidify.
How about 4oz from Amazon? It is $8.30.
Agar Agar Powder, 4 oz.
http://www.amazon.com/Agar-Powder-4-oz/dp/B00015UC48/ref=pd_sbs_indust_3
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on February 04, 2010, 02:51:17 AM
It should be the same yeast so they should have similar properties.

Maybe they are not. One way of identifying yeasts for example is the shape of their giant colonies. Though the differences may be more pronounced between ale and lager there may even be differences between closely related strains.
Another reason to get microscop.

In the end it may not matter much how well they smear as long as they leave a trail of yeast cells.

I will be watching  ;D
Title: Re: Yeast Culturing
Post by: Kaiser on February 04, 2010, 03:08:11 AM
Another reason to get microscop.


no microscope needed for that. I'm talking about the colony shape they form on a plate when they grow really large.

Kai
Title: Re: Yeast Culturing
Post by: dean on February 04, 2010, 03:10:01 AM
I received my innoculation needle today... that sucker is HUGE!   :o  I was expecting something with a fine wire and point.  The whole thing must be 10 inches long and most of it is needle (closer in diameter to a coathanger really though!)  Well, I haven't been able to find any agar locally so I'm wondering if anybody has tried cornstarch... like making gravy but thicker so it stiffens more when its cooled.  Its generally clear and it seems corn is related closer to barley than grass maybe?  Anybody tried it?

I would try unflavored gelatin.  Corn starch will not solidify.

Oh yes it will... I've seen it gel up more than a few times.   :D
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on February 04, 2010, 03:13:10 AM
Another reason to get microscop.


no microscope needed for that. I'm talking about the colony shape they form on a plate when they grow really large.

Kai

I see.
Plates are week or so apart and I chilled later plate sooner so that might be a reason why colonies are smaller.
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on February 04, 2010, 03:54:39 PM
Both of yeasts are pretty happy in 10 ml wort.
Granted I shaked it about 2 hours ago.

(http://farm5.static.flickr.com/4072/4330527958_52e8a5f45c.jpg)
Title: Re: Yeast Culturing
Post by: Kaiser on February 04, 2010, 04:00:35 PM
I see.
Plates are week or so apart and I chilled later plate sooner so that might be a reason why colonies are smaller.

I innoculated a plate with the itent to grow a giant colony. Let's see how large it will get or if I get mold first.

Kai
Title: Re: Yeast Culturing
Post by: roffenburger on February 04, 2010, 05:11:28 PM
I see.
Plates are week or so apart and I chilled later plate sooner so that might be a reason why colonies are smaller.

I innoculated a plate with the itent to grow a giant colony. Let's see how large it will get or if I get mold first.

Kai

What is the purpose of the giant colony? Just for fun?
Title: Re: Yeast Culturing
Post by: Kaiser on February 04, 2010, 05:18:07 PM
What is the purpose of the giant colony? Just for fun?

Yes, pretty much just for fun.

Kai
Title: Re: Yeast Culturing
Post by: Kaiser on February 04, 2010, 05:41:24 PM
here are some pictures of giant yeast colonies: http://prfdec.natur.cuni.cz/~zdenap/gallery.html

But they are not brewing yeasts and are also gown on stained agar.

Kai
Title: Re: Yeast Culturing
Post by: Kaiser on February 04, 2010, 06:51:44 PM
Here is some more info: http://brewingtechniques.com/library/backissues/issue2.3/morris.html

Maybe I should start an endavour to create a library of giant colony pics for a number of the yeast strains I culture.

Kai
Title: Re: Yeast Culturing
Post by: Thirsty_Monk on February 04, 2010, 07:09:12 PM
here are some pictures of giant yeast colonies: http://prfdec.natur.cuni.cz/~zdenap/gallery.html
Those Czechs.
Who would think of growing giant yeast colonies :)
And by the way I can read the original version!!!

I will also read article from brewing techniques.
Thank you
Title: Re: Yeast Culturing
Post by: Kaiser on February 06, 2010, 11:05:35 PM
If you are looking for purity, use the second method. This is streaking for isolation. Streak your first 1/3, sterilize (and cool)  your loop. Then make a couple passes back into the first third and into the second 1/3. sterilize and repeat.

I gave this a try the other day and besides making my plates look more prefessional it does seem to give me better colony isiolation.

Thanks,
Kai

Title: Re: Yeast Culturing
Post by: roffenburger on February 07, 2010, 12:50:20 AM
If you are looking for purity, use the second method. This is streaking for isolation. Streak your first 1/3, sterilize (and cool)  your loop. Then make a couple passes back into the first third and into the second 1/3. sterilize and repeat.

I gave this a try the other day and besides making my plates look more prefessional it does seem to give me better colony isiolation.

Thanks,
Kai


hehe. Glad it worked out for you.
Title: Re: Yeast Culturing
Post by: Kaiser on February 09, 2010, 05:17:32 AM
hehe. Glad it worked out for you.

I sure am a convert. Look how nice it came out:

(http://braukaiser.com/wiki/images/5/57/Streaking_grown_plate_2.jpg)

I'll have to take some time during the next days and update my Wiki with that technique.
Title: Re: Yeast Culturing
Post by: roffenburger on February 09, 2010, 04:35:35 PM
I know I mentioned sterilizing the loop between quadrants, but its really not necessary. I mentioned it because most people not familiar with the techniques tend to take up an entire colony and streak it, which isn't needed. If you only take a very small amount, you don't have to flame your loop between quadrants to get isolated colonies.

Here are the two main streaks I do. I like to make a big third "quadrant" (I guess its not really a quadrant if I only do three sections...).
(http://i46.tinypic.com/k19fd1.jpg)

Title: Re: Yeast Culturing
Post by: Thirsty_Monk on February 09, 2010, 05:25:02 PM
Thank you guys.

My last streaks are just a mess so I need to practice just a little more.

I have grown nice yeast up to 100 ml.
So the growing side seams to be working.

I used 16ml vials to do first step (10ml).
I think bigger vial would be better.

For 100 ml I used Mason Jars.
I was growing two strains.
For one strain I had 250 ml jar and for the other I had 500 ml jar.
I think I like 500 ml better for this step.
For next step I would probably use 2000ml flask with 800 ml wort.
Title: Re: Yeast Culturing
Post by: enso on February 10, 2010, 05:54:46 PM
On streaking technique.  I did my first plates last week.  I used a technique involving 2 plates.  Streak one in thirds turning the plate approx. 60° each time.  Then without flaming the loop restreak a second plate the same way.  I got excellent results for my first time.  I realize it is a bit of a "waste" of a plate but it gave me many good single colonies on the second plates despite the fact that I believe I had too much yeast on the loop to begin with.

Only thing I now wonder about is this.  Do you need to harvest the colonies before they grow too large?  Meaning, should you use the plate as soon as colonies are evident?  Mine went about 5 days at room temp before I streaked them on slants and into distilled water, and the colonies were quite thick and wide.  Maybe ~1 mm?
Title: Re: Yeast Culturing
Post by: Kaiser on February 10, 2010, 06:12:39 PM
Only thing I now wonder about is this.  Do you need to harvest the colonies before they grow too large?  Meaning, should you use the plate as soon as colonies are evident?  Mine went about 5 days at room temp before I streaked them on slants and into distilled water, and the colonies were quite thick and wide.  Maybe ~1 mm?

Depending on the depth of the agar, the colonies don’t grow much larger than what you see in the pick I posted. At that point you can pick them.

What I like about the technique where you flame the loop between streaks is that it can handle a very large range for the number of viable colonies on the loop. If you happen to have a lot it may take until the 4th section that you get good isolation. Wile you may have good isolation already in the 1st section if there only a few live ones on your loop.

Kai
Title: Re: Yeast Culturing
Post by: roffenburger on February 10, 2010, 06:21:17 PM
Its not really necessary to step up from a single colony. Streaking for isolation is helpful for making sure the culture is pure. If you can see that your culture is pure, take a couple loops full and put it in your starter wort. I don't see any benefit is growing from a single cfu.

Edit: I see that you're culturing on slants---:) In this case just barely touch the colony. You really don't need much. The colonies don't have to be large to use them, but give them a reasonable amount of time to grow (2 days or so at room temp)
Title: Re: Yeast Culturing
Post by: enso on February 10, 2010, 11:24:38 PM
Its not really necessary to step up from a single colony. Streaking for isolation is helpful for making sure the culture is pure. If you can see that your culture is pure, take a couple loops full and put it in your starter wort. I don't see any benefit is growing from a single cfu.

Edit: I see that you're culturing on slants---:) In this case just barely touch the colony. You really don't need much. The colonies don't have to be large to use them, but give them a reasonable amount of time to grow (2 days or so at room temp)

Oops.   :-[

I picked up the whole colony (maybe 2?) for the slants.  Overkill I guess.  I suppose they will get quite populated eh?

Should I make new slants once these grow?

In this case I did want to isolate as the two yeasts I was culturing had been obtained from bottled beer.  A bottle of Ommegang Abbey and a bottle of Victory Helios.  They appear (without a microscope) to be nice and healthy.
Title: Re: Yeast Culturing
Post by: roffenburger on February 11, 2010, 12:50:19 AM
you'll be fine.