c10h12n2o said:

Happy to help

What was your agar recipe?

I really love the streaking technique posted here, REALLY speeds up the process of isolating cultures and cleaning contams. I've gotten an isolate in 3 transfers like this (!)

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c10h12n2o said:

Happy to help
...cleaning contams. I've gotten an isolate in 3 transfers like this (!)

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Does this mean, T1 thru T3? And if so does that mean you are using the same streaking technique with myc?

And maybe I can sneak this question in, no one seems to want to touch it.
My GT isolates keep looking "just fuzzy", am I just isolating contam?
           

nosf3r4tu said:

c10h12n2o said:

Happy to help

What was your agar recipe?

I really love the streaking technique posted here, REALLY speeds up the process of isolating cultures and cleaning contams. I've gotten an isolate in 3 transfers like this (!)

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PDA, just potato flakes, honey and agar.

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Inthepit said:

Glad you're back too!

c10h12n2o said:

Happy to help
...cleaning contams. I've gotten an isolate in 3 transfers like this (!)

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Does this mean, T1 thru T3? And if so does that mean you are using the same streaking technique with myc?

And maybe I can sneak this question in, no one seems to want to touch it.
My GT isolates keep looking "just fuzzy", am I just isolating contam?
           

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c10h12n2o said:

I dont like using swabs directly to agar, I know it works, I just cant consider them sterile. I use individually wrapped swabs from medical supply places so I guess they are sterile, I just love using a loop

When I'm using a swab I normally scratch it with my loop above zone 1, then streak that area to spread them out, then flame and streak the next zones (using bacticinerator or flame between zones)

That way I'm not introducing food for contams onto my swab, and less chance of introducing contams from my swab onto the agar

Also I usually do 4 or 5 zones for the streak. You could probably do the same with a new sterile swab, but unlike a loop, most spores would probably end up on the swab rather than the agar, so idk how well it would work. I'd just make a loop and either scrape it like I do or swab section 1 then use the loop to spread it out and create the other zones

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c10h12n2o said:

I dont like using swabs directly to agar, I know it works, I just cant consider them sterile. I use individually wrapped swabs from medical supply places so I guess they are sterile, I just love using a loop

When I'm using a swab I normally scratch it with my loop above zone 1, then streak that area to spread them out, then flame and streak the next zones (using bacticinerator or flame between zones)

That way I'm not introducing food for contams onto my swab, and less chance of introducing contams from my swab onto the agar

Also I usually do 4 or 5 zones for the streak. You could probably do the same with a new sterile swab, but unlike a loop, most spores would probably end up on the swab rather than the agar, so idk how well it would work. I'd just make a loop and either scrape it like I do or swab section 1 then use the loop to spread it out and create the other zones

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Inthepit said:

Some myc just refuses to go rhizo on agar, but I'd be surprised to see that across that many cultures . How many transfers in are you from the original print/clone?
It looks like I'm at T5 and needing to go T6. My labeling system matured as the plates evolved.
   
Funny thing, this pin is my first mushroom in 45 years! OMG!
   

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c10h12n2o said:

I would use fine cheesecloth rather than a coffee filter. There are pics of the way I do it in the guide.  Never tried a coffee filter , cheesecloth does the job just fine

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c10h12n2o said:

I would use fine cheesecloth rather than a coffee filter. There are pics of the way I do it in the guide.  Never tried a coffee filter , cheesecloth does the job just fine

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c10h12n2o said:

WARNING: this is a little bit less than kosher, but i will provide the reasoning every guide says to let the PC zero out on its own. naturally the pressure falls once you kill the heat, and once it hits zero it goes below zero, then eventually back up. in my early work with agar, i had some contamination due to opening the PC when the pressure was negative, which led to sucking contams into my agar. what i like to do now is a little impatient, but i have not not seen any problems from it in the 15k to 20k plates i have done with this method. so here is what i like to do: i carefully watch the pressure as it falls, then right as it gets to zero, right before it crosses the line, i pop the vent. this means there is a small hiss of positive pressure being released from the valve (which is good, because it means nothing was sucked in), with less and less pressure as you get closer to zero, with negative pressure building if you wait too long (which sucks in contams). so right before zero, i put on protective gloves/mits, pop the valve, open the PC, and immediately tighten the lids on the media bottles and remove them

lids tightened and bottles removed from PC

c10

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