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InsultingLizard
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Couple questions about cultures
#26591806 - 04/10/20 06:41 PM (3 years, 9 months ago) |
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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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rumfor69
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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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InsultingLizard
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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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rumfor69
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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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InsultingLizard
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Re: Couple questions about cultures [Re: rumfor69]
#26592678 - 04/11/20 04:02 AM (3 years, 9 months ago) |
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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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poisoned
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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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InsultingLizard
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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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rumfor69
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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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InsultingLizard
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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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poisoned
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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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rumfor69
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Re: Couple questions about cultures [Re: poisoned]
#26592806 - 04/11/20 06:17 AM (3 years, 9 months ago) |
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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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InsultingLizard
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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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rickyswamps
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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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poisoned
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Re: Couple questions about cultures [Re: rickyswamps]
#26593043 - 04/11/20 09:21 AM (3 years, 9 months ago) |
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Maybe the print wasn't dried correctly and bacteria grew on it?
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InsultingLizard
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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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mushhead
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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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InsultingLizard
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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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mushhead
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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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InsultingLizard
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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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mushhead
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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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InsultingLizard
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Well, the syringes are definitely contaminated. I made (A) four dishes with one of those (two by directly squirting on the dish and two by streaking with a loop), and (B) two dishes with one of the vendor syringes I still had. There's nothing visible on the surface of the B dishes, but the A dishes are already showing a sort of "raised" texture everywhere the water touched. I'm sure in a few days they'll look like the dish from the OP.
I also transferred from the clone dish and it's looking nice and rhizomorphic:
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mushhead
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Quote:
InsultingLizard said: Well, the syringes are definitely contaminated. I made (A) four dishes with one of those (two by directly squirting on the dish and two by streaking with a loop), and (B) two dishes with one of the vendor syringes I still had. There's nothing visible on the surface of the B dishes, but the A dishes are already showing a sort of "raised" texture everywhere the water touched. I'm sure in a few days they'll look like the dish from the OP.
I also transferred from the clone dish and it's looking nice and rhizomorphic:

An now you know for sure, I love agar for this reason. When I make new syringes I always scope them out and test on agar before knocking up anything significant.
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InsultingLizard
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Re: Couple questions about cultures [Re: mushhead]
#26598428 - 04/13/20 04:38 PM (3 years, 9 months ago) |
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Oh, for sure. This is why I started doing agar in the first place. It's a bit of an up-front cost, but worth it to eliminate some of the guesswork.
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mushhead
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Premix MEA is only about $15 tops, petridishes you can get in bulk for an amazing price, or you could invest in Jelly jars which eliminates the need to replace plastic petri dishes. Build a flow hood or a simple SAB and away you go. Agar is simple and cheap ^_^
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InsultingLizard
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Well, a lot of that stuff I can't find here and I can't import it, so... For now I'm making do with cat food agar and the dishes I have left, which I don't even know when I'll be able to re-stock, since all the lab supplies stores are closed (unless I buy something ridiculous like 500 or 1000 units).
EDIT: On second thought, I might just do that.
Edited by InsultingLizard (04/13/20 05:35 PM)
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mushhead
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Quote:
InsultingLizard said: Well, a lot of that stuff I can't find here and I can't import it, so... For now I'm making do with cat food agar and the dishes I have left, which I don't even know when I'll be able to re-stock, since all the lab supplies stores are closed (unless I buy something ridiculous like 500 or 1000 units).
EDIT: On second thought, I might just do that.
You should always get bulk, it'll save you time and cash in the long run. Also any kind of small mason jar, jelly jar, half pint jelly jars even work well with agar and can be sterilized like any substrate jar.
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InsultingLizard
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Re: Couple questions about cultures [Re: mushhead]
#26623031 - 04/23/20 05:53 PM (3 years, 9 months ago) |
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A little update: I managed to get clean spores by modifying my jars I use for printing. Here's what they look like now:
 The wire in the center is a paperclip to hold the cap over the glass without touching it; I stab it so it will come out through the stump of the stipe, and then I bend the wire so the cap won't slide off. The holes allow evaporation, and on top there's some micropore tape to keep things clean and to hold the paperclip in place.
All the spores I got from a jar like this germinated cleanly.
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