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

I believe there was a thread about this somewhere, I will search for it.

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?

Agar is such a unique term that a forum search will likely return only useful results.

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

You can make your own malt agar using DME and gelatin. I've had good success with it.

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.

My yeast ranching adventures are starting to be documented on this crummy draft webpage:
http://www.mousetrap.net/~mouse/brewing/yeast.html

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.

N3WWN said:

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.

Click to expand...

wouldn't that result in an unsterilized medium?
do you inoculate your plates/slants in front of a flowhood or in open air?

ThaDutchMasta said:

wouldn't that result in an unsterilized medium?
do you inoculate your plates/slants in front of a flowhood or in open air?

Click to expand...

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. :cross:

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/chazin/wisdom/labpro/sterile.html

N3WWN said:

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/chazin/wisdom/labpro/sterile.html

Click to expand...

its definitely not enough to be considered sterile in my opinion, but still im sure you have no problem propagating the yeast through the use of careful transfers to isolate and remove them from contaminants. I would consider using a flowhood as the only way to go about creating a sterile environment, on top of autoclaving and most importantly - an extremely clean mad scientist. Additionally, spraying a 10% bleach solution high into the air throughout the room, three times 15 minutes apart, before you inoculate new plates or transfer to slants would be another helpful way to minimize contaminated plates and cultures.

I didn't mean to claim that my technique was actually sterile, just that it is sterile enough! This is why I make multiple cultures from the same source.

If the source is contaminated, all (or at least most) of my cultures will be, too. If a contaminate sneaks into the process, hopefully it will not infect at least one of the cultures.

You hit upon one thing that I did forget to mention!

I spray all surfaces with 90% alcohol several times before starting work. All items used in the process, such as funnels, test tube clamps, tubes, stoppers and caps, petri dishes, etc that can be autoclaved are autoclaved. I also spray my freshly washed hands with the alcohol before touching anything.

While I don't have the money or room for a flowhood right now, but it's certainly something to consider for the future.

Though, I would have to think that a flowhood would be best used in a less sterilized than how I operate. A HEPA filter is not 100% and neither is any sterile technique that I can think of... outside of a clean room.

Again, I'm not saying my technique is completely sterile or that the way I do things is the best or only way to do them. I'm just passing along what works well for me.



Speaking of what works well for me, I am also using some 3 section petri dishes for my initial streaks from a yeast source. Even if the source is slightly contaminated, I find the contamination usually appears in one section first which gives me time to transplant some promising colonies from the other sections to another three section dish to increase the chance of finding a clean culture.

Still not 100% sterile, but it works for me and haven't had any contanimations migrate across sections so far! :cross: