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OfflineInsultingLizard
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Couple questions about cultures
    #26591806 - 04/10/20 06:41 PM (3 years, 9 months ago)

I have a couple questions about my cultures.

First, here's a small test I made. I just cut a small piece of a veil and placed it on the plate. Didn't even do it in the SAB or sterilized the scalpel. I see two good sectors but my question is, should I bother transferring the one at 12 o'clock, being so close to the mold, or should I just not risk it?


Second, a couple days later I made four plates with spores. I put a cap in a sterilized jar for 24 hours, then removed it and rubbed the print with the inoculation loop (after flaming it and cooling it in the receiving plate), then dragged the loop through the agar in a zigzag motion. Now, five or so days later, all four plates look like this:

Is this normal, or did I fuck up somewhere?


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Offlinerumfor69
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26591875 - 04/10/20 07:08 PM (3 years, 9 months ago)

The second plate looks like a bunch of bacteria I'm thinking.

The first plate I'd transfer from the 7-8 o'clock growth looks
the best. I dunno about the 12 o'clock region.


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OfflineInsultingLizard
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26592596 - 04/11/20 02:33 AM (3 years, 9 months ago)

I mean, it's fairly obviously bacteria. My question is whether that's normal. I'm really not sure where I might have screwed up to contaminate 4 out of 4.


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Offlinerumfor69
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26592640 - 04/11/20 03:22 AM (3 years, 9 months ago)

Are you using an SAB or a flowhood?

Spores aren't sterile themselves, when applying them
directly to a plate it's kinda better to just place
them in a spot rather than dragging like that. Each
spore placement is a dice roll on contamination and
germination. A plate like yours you could do 3-4
individual placements per plate. Instead of the whole
plate being once dice roll from dragging.


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OfflineInsultingLizard
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Re: Couple questions about cultures [Re: rumfor69]
    #26592678 - 04/11/20 04:02 AM (3 years, 9 months ago)

I'm using an SAB. I always wash it in the shower then spray it with disinfectant (2-phenyphenol and ethanol) before using. I also wear gloves and tyvek sleeves to go in.

Hmm... I had read streaking was the right method to transfer spores, but I guess I used a print rather than solution. I'll try just touching the agar rather than streaking next time.


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Offlinepoisoned
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26592691 - 04/11/20 04:12 AM (3 years, 9 months ago)

Streaking is definitely a better method to inoculate agar. You're spreading out the endospores too and since the count should be way lower than the shroom spore count, you usually end up with some areas with clean growth.

It's not entirely clear where did you obtain the spores and how were they hydrated. I think that's where the issue is coming from.


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OfflineInsultingLizard
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26592710 - 04/11/20 04:31 AM (3 years, 9 months ago)

Quote:

where did you obtain the spores and how were they hydrated


I made the print from one of my mushrooms in a sterilized jar. Actually, you give me an idea. I did notice some water in the jar after making the print that I figured was from the PCing, but maybe it was from the mushroom. Should I let all the water evaporate before using the spores next time?


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Offlinerumfor69
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26592728 - 04/11/20 04:50 AM (3 years, 9 months ago)

Also SABs aren't perfect, they help a lot and can be very successful
but it's not like a flowhood where only a VERY small amount of micron
particles are around. SABs have a somewhat limited success rate sometimes.

The water in the jar with the print probably wasn't the best but it
should've been sterile, until it came into contact with a not sterile
cap and spores.

Just try again it's really all you can do. I've never used a streak
technique and have lots of success with spores.


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OfflineInsultingLizard
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26592778 - 04/11/20 05:36 AM (3 years, 9 months ago)

I know an SAB is no sure thing, but I'm just shocked I didn't get a single clean culture.
Plus it's strange that the veil should have been even dirtier than the spores, yet that culture was clean, other than the mold, even though I took practically no precautions there. A spot where I wiped the scalpel has even germinated now! Bizarre.


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Offlinepoisoned
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26592793 - 04/11/20 05:57 AM (3 years, 9 months ago)

How did you get from that print to the plate?

I doubt it was your print that was dirty. Something else was dirty somewhere during that transfer from print to agar.


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Offlinerumfor69
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Re: Couple questions about cultures [Re: poisoned]
    #26592806 - 04/11/20 06:17 AM (3 years, 9 months ago)

If you placed actual live veil tissue on the agar it probably just grew
mycelium like you made a clone. The spores might not have even germinated.


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OfflineInsultingLizard
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26592826 - 04/11/20 06:42 AM (3 years, 9 months ago)

Quote:

How did you get from that print to the plate?

I doubt it was your print that was dirty. Something else was dirty somewhere during that transfer from print to agar.



For every dish:
1. Flamed the inoculation loop.
2. Moved it into the SAB.
3. Cooled it with the receiving dish.
4. Dragged it through the print.
5. Streaked the dish.

Quote:

If you placed actual live veil tissue on the agar it probably just grew
mycelium like you made a clone.



No doubt. Making a clone was my intention. I wanted to see what would happen if I attempted to clone a mushroom with basically no prophylaxis.


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Invisiblerickyswamps
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26592836 - 04/11/20 06:50 AM (3 years, 9 months ago)

You don't want to get the spores like that that.  First you make a clean print.  Check out bod's tek on spore prints.  Then, you get the spores from the dry print.  The bacteria comes from touching the cap directly.  To clone a mushroom you'll have to split it open to get tissue that is clean.  I've never had bacteria from a spore print.


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Offlinepoisoned
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Re: Couple questions about cultures [Re: rickyswamps]
    #26593043 - 04/11/20 09:21 AM (3 years, 9 months ago)

Maybe the print wasn't dried correctly and bacteria grew on it?


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OfflineInsultingLizard
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26593065 - 04/11/20 09:33 AM (3 years, 9 months ago)

Quote:

Check out bod's tek on spore prints.  Then, you get the spores from the dry print.  The bacteria comes from touching the cap directly.



Do you mean this one? I don't see anything that prevents the caps from touching the foil directly. eatyualive's tek does use a grid to hold the caps above the foil. I won't be able to try anything like that for several weeks, unfortunately; lots of businesses are closed because of the quarantine. For now I'll try transferring the cap after a few hours, as well as letting the print dry.

Quote:

Maybe the print wasn't dried correctly and bacteria grew on it?



Well, it wasn't dry, as I said, but it was new. It was barely more than 24 hours since I had placed the cap in the jar to print. It's not like it was left for several days to culture.


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Invisiblemushhead
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26593105 - 04/11/20 09:51 AM (3 years, 9 months ago)

To start I am no expert on Agar however here is how I think this went down.
You got yourself a clone from the growth of myc off a live veil (Awesome btw I intend on attempting that). The plate with all the bacteria was caused by the spore print itself containing live bacteria, you gave yourself the same reaction as from the veil plate, only this time it was bacteria. I would dry your print next time. I do this by just allowing it to sit in my fridge for a few days after I take the print.


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OfflineInsultingLizard
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26593116 - 04/11/20 09:59 AM (3 years, 9 months ago)

Yeah... It's a shame I made a bunch of syringes off that print that are now all suspect. It was a nice and dense print, too!


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Invisiblemushhead
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26593159 - 04/11/20 10:23 AM (3 years, 9 months ago)

Quote:

InsultingLizard said:
Yeah... It's a shame I made a bunch of syringes off that print that are now all suspect. It was a nice and dense print, too!




Obtain a microscope, if you know what you're looking for you can determine if you have contams or not. In a sterile SAB I would make five plates, on plate empty and sterile as control and the other four I would squirt spores from your syringes onto them. Provided given time and you'll have your answer and the possibility of getting clean cultures.


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OfflineInsultingLizard
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26593704 - 04/11/20 03:03 PM (3 years, 9 months ago)

I do have a microscope, but for the life of me I can never see bacteria at all, even at a 1000x magnification. Of course, I don't have oil for the 100x objective, but I've still managed to get some pretty clear images even through air:

(Disregard the speckles. Those are scratches on the eyepiece lenses.)

EDIT: Actually, I have been able to see something I believe was bacteria on a dish that was just teeming with them, but focusing properly on a culture is extremely difficult, so couldn't even take a picture. But my point was about spotting them on slides.


Edited by InsultingLizard (04/11/20 03:08 PM)


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Invisiblemushhead
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Re: Couple questions about cultures [Re: InsultingLizard]
    #26595869 - 04/12/20 02:04 PM (3 years, 9 months ago)

Quote:

InsultingLizard said:
I do have a microscope, but for the life of me I can never see bacteria at all, even at a 1000x magnification. Of course, I don't have oil for the 100x objective, but I've still managed to get some pretty clear images even through air:

(Disregard the speckles. Those are scratches on the eyepiece lenses.)

EDIT: Actually, I have been able to see something I believe was bacteria on a dish that was just teeming with them, but focusing properly on a culture is extremely difficult, so couldn't even take a picture. But my point was about spotting them on slides.




I have the AmScope B120C-E1 which is perfect for spotting those devils.


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Meditation Principles

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