I'm looking for any input anyone is willing to give. This is my first go at this and I'm learning a lot. If you look at my current results you can probably immediately tell how much of a new guy I am. Bear with me.
Agar is a standard MEA recipe with 5g potato flakes added, no dye, PC'd for 40 min. I used a SAB for all of these. I wiped everything down with 91% ISO but am told 70% is better. I will use that moving forward. I purchased a syringe from a
sponsor. First run was 5/29 and second run was 6/9. Initially my temp control was terrible - ranging from 64-86F. I've fixed that problem; pretty steady at 80F now. I did not use the syringe needle on the 5/29 run and had control issues, getting way more liquid out than I wanted. I used the needle for the 6/9 run and had much better control. Flame sterilized between plates - loop and needle. From what I see, the bacterial contamination follows the liquid. They are just absolutely UGLY. I had no mycelial growth until the temperature issue was fixed then a couple of plates from both the 5/29 and 6/9 germinated at the same time - almost appearing to grow from under the bacterial cultures. Here are pictures for evidence of what I'm seeing here.
This one has me baffled (well, they all do). You can see it follows where my loop drew the liquid. The pink hue has me thinking some sort of fungal contamination. The rest just appears bacterial. No signs of anything mycelial - mold or target fungi.
These two are the same plate, obiously. The first just shows a bright white patch of mycelium germinated from the end of where the smear started. In the SAB (closer look) it appeared like the target species versus white mold. It could also be a pin mold. I transfered it today to a fresh plate to see what happens. The second picture is with the lid off to show the bacteria on the smear pattern. I was hopeful the fanned out area by each end of the Z would develop mycelium. It hasn't so far.
I just can't figure out where the hole in my technique is or why it would appear it stuck to just where the liquid touched. They syringe was kept in the refrigerator, wrapped. I wiped it all down with 91% ISO before using it on both occasions. I flame sterilized the needle and the loop between each plate as well - again, I didn't use the needle on the 5/29 run and ended up with way to much liquid. Any input would be greatly appreciated. While I expect to carry the contamination over, I did my best to isolate some of the mycelium in a couple of the plates and transferred each to a fresh plate. We will see what happens next. An upside, I'm enjoying the learning process and trying to sort out where things are derailing.
Edited by coorsmikey, 21 June 2020 - 04:50 PM.
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