Cleaning up an Agar Culture

Nan's Nook : Archives : Cloning : Agar                                                                   Also See: Isolating on Agar
Posted by: brainbreath Aug 17 03, 06:05 PM GMT

Isolating a Pure Agar Culture

This isn't my idea, it's taken from GGMM by paul Stamets, I thought a visual tek would be helpful smile.gif . The following is a fairly simple, yet effective, alternative to multiple transfers on agar used to isolate a pure culture from a contaminated plate.

What you will need:
Glass tubing (I bought mine from
1 Small Jar/Lid
Aluminum Foil
Parafilm( or cling wrap)
a Skewer

To begin you'll need to soak the millet overnight, the same as making grain spawn.

Posted by: brainbreath Aug 17 03, 06:10 PM GMT
When the millet has been soaked and rinsed, it's ready to be loaded in the tubes.
I start by plugging on end of the tube with a small piece of aluminum foil, inserted about 1/4" into the tube, in order to leave space for the agar being transfered.1
I then wrap the end with a piece of parafilm (cling wrap should work-I haven't tried it though) and mark it with an L so there's no confusion about which end to load later on.2 wink.gif

Load the tubes with millet so that it's loosely packed, you want the mycelium to be able to run through the tube as fast as possible. I load it by hand, a pinch at a time, and tap the end on the table till the seed falls to the bottom, add another pinch, tap, etc. This is where the skewer becomes necessary, sometimes the seed will stick part of the way down the tube. If it does use the skewer to loosen it up enough to slide down the tube(using the point along side it works best, Don't pack it down!)3

When the tube is filled, cover the end with a small piece of aluminum foil. 4
Then seal with film.5
Repeat this process till you have all the tubes filled.

Posted by: brainbreath Aug 17 03, 06:12 PM GMT
I wrap them in aluminum foil both individually, then the whole lot of them, as an added precaution to keep moisture and possible contams (post sterilizing) out. Sterilizing the tubes @15 psi for 1 hour is sufficient, unless you're sterilizing them in a packed PC.

Posted by: brainbreath Aug 17 03, 06:14 PM GMT


Posted by: brainbreath Aug 17 03, 06:16 PM GMT
After the tubes have been sterilized and cooled you're ready to transfer. Cut a piece of the culture that isn't in direct contact with the contam, and transfer that to the "L" end of the tube. The mycelium should be facing the millet in the tube.
There most likely will be spores present from the contam, even though there isn't any live tissue being transfered-It isn't a major concern though. Because live mycelium is being transferred, and the spores need time to germinate, in most cases the myc has run 1/3 or more of the tube before germination-leaving no food for the contam. biggrin.gif

Reseal the end of the tube.

The process should be complete now (sometimes it is necessary to do multiple transfers.) the tube will need to be incubated till the mycelium has run the length of the tube, emerging from the end a pure culture, which is then transfered to agar.

Posted by: Nanook Aug 18 03, 12:08 AM GMT
Great Tek, great post wink.gif biggrin.gif

Posted by: No Logos Aug 18 03, 09:31 AM GMT
This tek is better than filling the little tubes with agar media! Good one.
Stamets doesn't use antibiotics much, but I like kanamycin in my plates. It doesn't kill anything fungal, but it does get rid of most bacterial contams. Kanamycin can't be autoclaved, tho. Stamets does mention gentimycin, which can be autoclaved, but I don't have much experience with it. Anyone else?

Posted by: Nanook Aug 18 03, 09:48 AM GMT

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