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Old 08-04-2008, 05:15 PM   #1
ThaDutchMasta
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Default Culturing Yeast On Agar Plates

Ive been learning how to culture yeast on plates for preservation and isolation and was wondering if theres anyone of the boards with any more information about the subject.
thanks,
dutchy

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Old 08-04-2008, 05:23 PM   #2
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I believe there was a thread about this somewhere, I will search for it.

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Old 08-05-2008, 12:20 AM   #3
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There are a few other science guys on here. Although I've never cultured yeast onto Petri dishes with agar, I've done it with bacteria and have some general microbiology lab knowledge. I've thought about doing it with yeast and have an idea of how I would do it. Is there something specific you want to know?

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Old 08-05-2008, 12:33 AM   #4
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Agar is such a unique term that a forum search will likely return only useful results.

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Old 08-05-2008, 01:08 AM   #5
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I have some here:

Making Plates and Slants and Growing Yeast from Plates

I have to get on the ball and add an article about inoculating plates and slants though, but you can also find that on the web.

Kai

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Old 08-05-2008, 02:23 AM   #6
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You can make your own malt agar using DME and gelatin. I've had good success with it.

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Old 08-05-2008, 01:26 PM   #7
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Agar is likely available (cheap, like 10x as cheap) at the local Asian market than from LHBS.

I bought both dishes and tubes, and I culture in tubes so far because I understand they are less vulnerable to dehydration and contamination. My understand is that plates are perfect for doing a 4-quadrants innoculation in order to get a nice single cell colony, which you then culture in a tube.

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Old 08-05-2008, 08:24 PM   #8
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Hi!

I use plates to isolate colonies (4 quadrant streaking as mentioned earlier) and to make sure that the source of the culture is not contaminated.

My method is to start by innoculating 2 to 4 plates from the same source, based on how "rare" the source is. Then, after incubating for several days, I select the strongest/cleanest colonies in each plate to innoculate 2 or 3 slants per plate.

Assuming that the source was not contaminated and my sterile technique was, well, sterile, I could have up to 12 possible slants. In reality, I usually only bother making 4-6 slants.

When I am down to two fresh slants, I innoculate another couple of slants when I open the 2nd to last slant. This way, I always have a known clean culture in my "yeast bank"

I use medical-grade agar (expensive, but I can't find Asian market agar locally) and DME:
- 40g extra light, hopped DME to 450ml water (around 1040 SG after boiling)
- boil for 5-10 min
- 1 tbsp agar
- boil for 5 min
- put into a glass, auto-clave safe medium bottle
- place into pressure cooker for 30min @ 15psi
- pour into plates and test tubes
- microwave extra agar/dme medium to boiling for 1 min and refrigerate for later use

This last step is just to raise temp to > 200degF before cooling.
I microwave the agar/dme medium or place back into the pressure cooker to sterilize before pouring more slants/plates.

I use hopped DME for the anti-bacterial properties of hops. If I'm out of hopped DME, I just place a medium sized hop pellet into the 400ml of water and make sure to boil for at least 10 min.

I'm not sure if my technique has improved or if it's the hops, but since I started hopping my agar media, the number of bacterial contaminations has dropped significantly!

My next improvement will be to place the hopped DME into the pressure cooker for 30min @ 15psi (without the agar) and then chill overnight before boiling and adding agar. This should precipitate the hot/cold breaks and result in much clearer agar medium.

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Old 08-08-2008, 02:20 AM   #9
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Quote:
Originally Posted by N3WWN View Post

My next improvement will be to place the hopped DME into the pressure cooker for 30min @ 15psi (without the agar) and then chill overnight before boiling and adding agar. This should precipitate the hot/cold breaks and result in much clearer agar medium.
wouldn't that result in an unsterilized medium?
do you inoculate your plates/slants in front of a flowhood or in open air?
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Old 08-08-2008, 04:26 AM   #10
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Quote:
Originally Posted by ThaDutchMasta View Post
wouldn't that result in an unsterilized medium?
do you inoculate your plates/slants in front of a flowhood or in open air?
I'm just talking about chilling the DME and water. I'd decant the wort off the break, bring to a boil and then add the agar and proceed with the pressure-cooker "auto-claving" and pouring.

Even without the auto-claving, there should only be a slight chance of contamination if my technique is good. But I'd rather play it safe.

I inoculate in open air, but close all windows and doors. I also turn off fans and make sure the dog is in another room.

My sterile technique also involves keeping the propane torch burner on at all times to flaming the mouths of test tubes, sterilize the loop, working in the flame area, etc.

It may be overkill, but I also wear a cheap painters mask... the kind you can get 5 for $5 at most hardware stores. This keeps my direct breath off the equipment and baffles my breath air currents.

SWMBO sure thinks the mask increases the mad scientist factor (MSF) even though all of the tubes and plates, etc generate a pretty high MSF already!

Is it possible that I am being way too careful? I'm sure of it!

Here's a quick rundown of sterile technique, if you're not familiar with it:
http://structbio.vanderbilt.edu/chaz...o/sterile.html
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Planning - Cranberry wine, Tea Crabapple Lambic, Pinot Noir
Primary - 20 oz Rum in the Raw (experiment) - 1.150 OG
Secondary - 1 gal Chokecherry wine
Secondary - 5 gal California Uncommon (dry hopping)
Bottled - Cherry Apfelwein
Aging - 1 gal Still of the Night Mead - 1.132 OG
Drinking - ESB, Root Beer, Tank It Down Ordinary Bitter
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