Stipe-n Cap said:

Fungi are totipotent. Totipotency is the ability of a cell to regenerate or develop into any other cell or into a complete organism when grown from a single cell or tissue culture. For those of you who propagate cacti pups, you'll recognize the term.

Even in the mature fruitbody, every hypha retains its totipotency which in turn can produce the entire organism from any part of the whole. This is why culture expansion and cloning work, totipotency is the underlying mechanism.

The leading edge is simply a grouping of quickly expanding hyphae. The leading edge is in a state of rapid expansion but is not necessary to select for due to the totipotent nature of the organism, any part will suffice; however, allowing the culture to fill out a bit will allow sufficient time for any lagging contaminants to incubate/grow, while simultaneously allowing the culture time to reveal which growth is most robust/healthy.

Leading growth/edge transfers are best reserved for known pure cultures that have been sufficiently worked to produce the desired outcome.

Here's an excellent example from a germ plate streaked with MS solution:



Leading edge is not necessary, selecting away from contamination is the primary objective at this stage.

The same applies here:



When clean growth is achieved, select for rapid/healthy growth from the leading edge once the morphology has been given time to develop:

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MycCycle said:

I know some people wrap cling wrap around their SABs to seal the holes. I’d prefer having a permanent non-waste solution to simply attach some kind of lid that doesn’t disturb the still air in the box.

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Ahh gotcha. I thought you were wrapping the SAB before doing any work

Stipe-n Cap said:

Fungi are totipotent. Totipotency is the ability of a cell to regenerate or develop into any other cell or into a complete organism when grown from a single cell or tissue culture. For those of you who propagate cacti pups, you'll recognize the term.

Even in the mature fruitbody, every hypha retains its totipotency which in turn can produce the entire organism from any part of the whole. This is why culture expansion and cloning work, totipotency is the underlying mechanism.

The leading edge is simply a grouping of quickly expanding hyphae. The leading edge is in a state of rapid expansion but is not necessary to select for due to the totipotent nature of the organism, any part will suffice; however, allowing the culture to fill out a bit will allow sufficient time for any lagging contaminants to incubate/grow, while simultaneously allowing the culture time to reveal which growth is most robust/healthy.

Leading growth/edge transfers are best reserved for known pure cultures that have been sufficiently worked to produce the desired outcome.

Here's an excellent example from a germ plate streaked with MS solution:



Leading edge is not necessary, selecting away from contamination is the primary objective at this stage.

The same applies here:



When clean growth is achieved, select for rapid/healthy growth from the leading edge once the morphology has been given time to develop:

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MycCycle said:

I know some people wrap cling wrap around their SABs to seal the holes. I’d prefer having a permanent non-waste solution to simply attach some kind of lid that doesn’t disturb the still air in the box.

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Dkeppel said:

Now I'm curious if you have a link to your thoughts on the subject CB?

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CocaineBuffet said:

For me: I noticed that when I checked back in some early edge growth transfers that the donor plate had growth that looked more enticing than the first and decided to transfer from that and I would usually see the kind of growth I would prefer grow out from that "newer" transfer

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Phaethon said:

The satellite colonies starting from the edge are mold, which is probably a sterile technique issue (hovering hand over a plate, e.g.). You can try transferring some of the mycelium from the central colony that's far away from the mold, but I'd start over from the master plate.

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Thanks, figured as much. I tried to be as careful as I could in my SAB, but 3 out of 3 contamed plates is a huge fail

Is the master plate even safe to use anymore? Or is it likely that I contaminated it too in the process?

Also, I noticed that the mold is starting from the outside edge of my plates and working inwards. Does the fact that I used cling wrap to wrap my plates instead of parafilm have anything to do with that?

stuffedmushrooms said:

Phaethon said:

The satellite colonies starting from the edge are mold, which is probably a sterile technique issue (hovering hand over a plate, e.g.). You can try transferring some of the mycelium from the central colony that's far away from the mold, but I'd start over from the master plate.

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Thanks, figured as much. I tried to be as careful as I could in my SAB, but 3 out of 3 contamed plates is a huge fail

Is the master plate even safe to use anymore? Or is it likely that I contaminated it too in the process?

Also, I noticed that the mold is starting from the outside edge of my plates and working inwards. Does the fact that I used cling wrap to wrap my plates instead of parafilm have anything to do with that?

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I've had my share of bad sessions. It gets better with practice.

There's a chance the master plate has mold spores on it, but I think you'll probably be able to get a clean culture from it unless you left it out in open air or something equally drastic.

The fact that the mold is all from the side could mean it was introduced when you wrapped it. Cling wrap is fine though, lots of people use it. Wrapped in SAB, right? I think I'd suspect more something with putting the lid on after the transfer - did it maybe scrape the rim a bit? Or did your scalpel handle tap the rim?

Phaethon said:

stuffedmushrooms said:

Phaethon said:

The satellite colonies starting from the edge are mold, which is probably a sterile technique issue (hovering hand over a plate, e.g.). You can try transferring some of the mycelium from the central colony that's far away from the mold, but I'd start over from the master plate.

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Thanks, figured as much. I tried to be as careful as I could in my SAB, but 3 out of 3 contamed plates is a huge fail

Is the master plate even safe to use anymore? Or is it likely that I contaminated it too in the process?

Also, I noticed that the mold is starting from the outside edge of my plates and working inwards. Does the fact that I used cling wrap to wrap my plates instead of parafilm have anything to do with that?

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I've had my share of bad sessions. It gets better with practice.

There's a chance the master plate has mold spores on it, but I think you'll probably be able to get a clean culture from it unless you left it out in open air or something equally drastic.

The fact that the mold is all from the side could mean it was introduced when you wrapped it. Cling wrap is fine though, lots of people use it. Wrapped in SAB, right? I think I'd suspect more something with putting the lid on after the transfer - did it maybe scrape the rim a bit? Or did your scalpel handle tap the rim?

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Appreciate the tips to improve my sterile technique. And here I thought I had it all figured out! Still much to learn

I reapplied the lid in the SAB, but wrapped it outside - that could have been the issue. I flamed the blade, but didn't do the same to the handle of course. Do you sterilize the scalpel handle as well (alcohol perhaps)? Is there a way to sterilize the rim of the dish itself?

Phaethon said:

Wrapping in the SAB will probably help. Plates are often vented (small gap between rim and lid), so if you move them around too much in open air you can get spore laden air in there.

(Flaming still needs to happen outside SAB in case that's not obvious. Besides the risk of igniting alcohol fumes if you're overzealous, fire results in a lot of moving air, which defeats the purpose of the still air box. Just move the scalpel back into the box before it cools.)

I don't sterilize the scalpel handle. I just wipe it down with an iso soaked paper towel before flaming the blade.

No need to do anything with the rim. It's sterile out of the bag (or PC if it's a reusable dish) and will stay clean if you don't touch it with anything but the lid and don't let it contact open air.

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Great advice, thanks!

salubrious said:

In terms of clean growth how am I looking here?

Nat T4




Thanks for the help 🙏

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