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redhandmat
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What kind of contam is this guys? 1
#26531111 - 03/12/20 02:05 PM (3 years, 11 months ago) |
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Hello guys! (cross post from contam forum) First thank you all for the help you are giving this newbie. I have tried 3-4 times to grow mushrooms, and always failed, this is my second agar attempt. So never seen a contam like this (thinking it must be contam, because looks nothing like mycelium). Could you guys help me understand. Here was my procedure:
tldr: Inoculation loop, spore print to PDA, Nocced , showed sign of "something" (see picture). Today it looks like the pictures attached.
1:I followed recipe from the forums: prepared agar with some dextrose, drop of honey, potato starch and ofc agar powder, and green colorant. 2: poured into 2 small glass food containers that were washed with soap, chlorine, and scrubbed with 75% ethanol/alcohol (gonna buy petri for next time).
2: I put paper towels on the containers, wrapped them in aluminum foil together with utensils also wrapped in aluminum foil. Sat in PC 15 psi for 60 minutes. Put the PC away for four days as I couldn't nocc the containers.
3: I prepared a kind of SAB (the front side for the hands was too open) in the bathroom as we have a surface there were I can work. I cleaned the entire bathroom. then sprayed the 75% disinfectant everywhere, took a shower.
4:(Right after the shower, I disinfected everything again. Took out the containers from the PC, they looked clean and no water inside. Put the containers upside-down on top of each other, disinfected the outside with 75% and loosened the lids. Torched the loop red hot, touched the agar surface (with container upside down), scraped the print then scraped the agar surface. Repeat for second container. Wrapped them in saran even though the lid has good seal, put them above fridge as it has good temp.
5: it showed a little bit of growth of something that does not look like myc (I hadnt checked for a few days before this date). Growth wasn't green or anything so I wanted to see how fast it would grow.


6: (Today) SI continued checking but it didnt grow as fast as I would expect contam to grow. Today its been 12 days since the first growth and it looks like the pictures:





You may now wonder what happened to the second container? Well it had no growth at all for 20 days, and today it showing the begining of something. No idea what that something is. Only time will tell.
So Im starting to think that my spore print is contaminated, because everything has failed, no matter how extreme I have been with cleanliness.
Anyways! what do you guys think it may be? And please give me your feedback on what you think I need to do/dont do. I will definitely get petri!
Edited by redhandmat (05/16/20 05:40 AM)
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A.k.a
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Re: What kind of contam is this guys? [Re: redhandmat]
#26531189 - 03/12/20 02:52 PM (3 years, 11 months ago) |
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Looks like myc with bacteria. You might be able to transfer and then get some clean growth.
Maybe hot pour or sandwich it.
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LAGM2020     
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redhandmat
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Re: What kind of contam is this guys? [Re: A.k.a]
#26532051 - 03/13/20 03:31 AM (3 years, 10 months ago) |
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Quote:
A.k.a said: Looks like myc with bacteria. You might be able to transfer and then get some clean growth.
Maybe hot pour or sandwich it.
Thank you for your answer! I just ordered some glass petri so I can make transfers when its time. But I will probably do a hot pour in the mean time. I searched the forums and found RR's post about hot pours. If Im understanding it right I need agar with low amount of nutrients and pour at around 60 C.
IF anyone else has some ideas please share. I cant believe there actually is myc in there.
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LadysKnight
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Re: What kind of contam is this guys? [Re: redhandmat] 2
#26532120 - 03/13/20 05:01 AM (3 years, 10 months ago) |
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They don't look too dirty, I would just take several transfers from clean looking edges.
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LogicaL Chaos
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Re: What kind of contam is this guys? [Re: A.k.a]
#26532258 - 03/13/20 08:16 AM (3 years, 10 months ago) |
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Quote:
A.k.a said: Looks like myc with bacteria. You might be able to transfer and then get some clean growth.
Maybe hot pour or sandwich it.
Definitely agree. Looks bacterial but does have mycelium (the white fuzzy growth). Shouls clean up nicely with some transfers, maybe make antibacterial agar
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redhandmat
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Re: What kind of contam is this guys? [Re: LogicaL Chaos]
#26532558 - 03/13/20 11:30 AM (3 years, 10 months ago) |
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Quote:
LadysKnight said: They don't look too dirty, I would just take several transfers from clean looking edges.
Quote:
LogicaL Chaos said:
Quote:
A.k.a said: Looks like myc with bacteria. You might be able to transfer and then get some clean growth.
Maybe hot pour or sandwich it.
Definitely agree. Looks bacterial but does have mycelium (the white fuzzy growth). Shouls clean up nicely with some transfers, maybe make antibacterial agar 
These are amazing news guys. Y'all probably havent had anyone be this happy to have mycelium with contams. I bought a scalpel today, just waiting for the petries to arrive after the weekend.
I found this antibiotic for fishes called "Nifurpirinol" (http://www.theaquariumwiki.com/wiki/Nifurpirinol) Im gonna search for information if its viable for agar and mycelium growth. Other types of antibiotics will be very hard to find because my country has strict laws. Thank you all!
Edited by redhandmat (03/13/20 11:39 AM)
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LadysKnight
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Re: What kind of contam is this guys? [Re: redhandmat]
#26532632 - 03/13/20 12:28 PM (3 years, 10 months ago) |
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You don't need antibiotics. Black tea agar works great to freeze bacteria in place. But I think you can clean these up with just a couple transfers.
https://www.shroomery.org/forums/showflat.php/Number/23860431/fpart/1/vc/1
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redhandmat
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Re: What kind of contam is this guys? [Re: LadysKnight]
#26534077 - 03/14/20 07:25 AM (3 years, 10 months ago) |
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Quote:
LadysKnight said: You don't need antibiotics. Black tea agar works great to freeze bacteria in place. But I think you can clean these up with just a couple transfers.
https://www.shroomery.org/forums/showflat.php/Number/23860431/fpart/1/vc/1
Thank you for the suggestion. One last question, should I take the transfer from the edge of the mycelium? or should I instead try to get a part of it without the agar, meaning just scrape off a bit that looks good?
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Neowynd8

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Re: What kind of contam is this guys? [Re: redhandmat]
#26534129 - 03/14/20 08:00 AM (3 years, 10 months ago) |
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Take a wedge of agar and myc from the edge of growth.
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redhandmat
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Re: What kind of contam is this guys? [Re: Neowynd8]
#26542372 - 03/18/20 09:55 AM (3 years, 10 months ago) |
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Update.
I followed more or less the advice of everyone in this thread.
1: Made a weak nutrient agar solution as poposed by RR in a thread where the grower had bacterial issues. As advised in this thread I infused the water with black tea, but didn't actually add black tea powder to the solution.
2: I wrapped the agar bottle, utensils, and the new petri dishes in aluminum foil and put them inside the PC.
3: I thoroughly PC'ed everything for 45 minute, then for 30 minute, and then again for 45 minute. I just wanted to be certain, and I was cleaning the workplace in the mean time so didn't want the agar to cool on me.
4: I set up the workplace. I know I should get an appropriate glovebox, but I have a set up that more or less mimics the purpose of a glovebox so not much air current is about and minimizing the contams. I have a workbench and a shelf above it. So I use plastic to create a kind of box between them. I later put on even more tape than you see. And I layer the plastic with some of it working as a curtains so that even when I put in my arms later it keeps everything covered. I have washed everything with soap and chlorine, including the walls, bench, floor, below the shelf, etc. The towel I have in there is sprayed down with chlorine and disinfectant. I spray a layer of iso all over, both inside and outside and let it sit:

5: I bring in the pc inside the bathroom and wipe it down with iso. Take a shower, brush teeth, change clothes. Wipe everything down with iso again. And start working. I never hover my hand over the dishes, and thoroughly clean the scalpel in between by wiping the blade with iso and then burning it thoroughly.
6: I have made a transfer from the leading edge as suggested to two petri dishes. I blank nocced the third dish to check if the blank dish shows contams later. I lastly hot poured over the original container. once all of this was done I put saran plastic over the petri, and then put them inside zipbags. They are now stored up and down and waiting for the mycelium to grow.

Hotpour:

Now lets wait and see what happens. Thank you all, and Ill keep you updated.
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redhandmat
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Re: What kind of contam is this guys? [Re: redhandmat]
#26550170 - 03/22/20 04:30 AM (3 years, 10 months ago) |
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Update on the transfers. So 4 days later this is how the transfers are looking. Both are showing some growth, but one of them is showing much more. I'm hopping its the right kind of growth I really hope you guys will share your opinions and advice. Thank you again!

Left petri high res (the black is just from burning the scalpel, and probably a light trick, because it doesnt look really that way irl) (as you can see in the above picture):

Right petri high res:

Would really appreciate all feedback on the progress.
Edited by redhandmat (03/22/20 08:14 AM)
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redhandmat
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Re: What kind of contam is this guys? [Re: redhandmat]
#26550824 - 03/22/20 11:41 AM (3 years, 10 months ago) |
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Bumping in the hopes that someone will give some help if they see the thread.
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Zifozonke
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Re: What kind of contam is this guys? [Re: redhandmat]
#26550851 - 03/22/20 11:55 AM (3 years, 10 months ago) |
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Hard to tell from your pics but personally Id work with the dish on the right- look at area between 1&2 oclock & let it grow out abit and maybe take some transfers from there ...that first one looks abit dodgy to my eye.
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Sunny Skies
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Re: What kind of contam is this guys? [Re: Zifozonke]
#26550908 - 03/22/20 12:39 PM (3 years, 10 months ago) |
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Wait a day or 2 with the plate on the right..1 o'clock looks promising as buddy said above.
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redhandmat
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Re: What kind of contam is this guys? [Re: Sunny Skies]
#26550925 - 03/22/20 12:55 PM (3 years, 10 months ago) |
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Thanks for the feedback guys!
Quote:
Zifozonke said: Hard to tell from your pics but personally Id work with the dish on the right- look at area between 1&2 oclock & let it grow out abit and maybe take some transfers from there ...that first one looks abit dodgy to my eye.
I too felt it was a bit dodgy, first time I tried agar I got a mold that looked a bit like it. Ill keep an eye out and transfer 2 o'clock on the right one if it continues growing out well.
Quote:
Sunny Skies said: Wait a day or 2 with the plate on the right..1 o'clock looks promising as buddy said above.
Will do mate, thank you for the feedback.
I think Ill even do a no-pour since I have glass petri dish. at least it will lead to one less vector for contamination.
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redhandmat
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Re: What kind of contam is this guys? [Re: redhandmat]
#26556313 - 03/25/20 03:25 AM (3 years, 10 months ago) |
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Im in need of big help guys. Super confused by the results of the transfer and the hotpour. I will present the pictures of the results so far and ask if my reasoning for what to do next sounds ok or not. Really relying on your expertise here  Results of the hotpour (if pictures are not good just let me know and I try opening it in a glovebox to take a better pic):


As you can see what I'm guessing is the mycelium (hopping really), has broken the surface of the hotpoured agar. If you guys agree that this looks like mycelium I will then transfer it asap. However, I may not have enough petri dishes so if you deem that one of the bellow cultures is just contamination I will clean it & PC for the purpose of transferring the hotpour mycelium.
The results of the two transfers from previously are looking like this now:

Both of these growth dont look good to me at all, I have taken a small piece of the dish to the right that was white and looked like mycel and transferred to see in a few days if I get anything good from them. But guys overall these dont look like other pictures of mycelium I have seen on this site. But my experience is almost 0 here, so Im thinking you guys are the only hope for ever getting to grain and then to a shoebox. What is your opinion? Please dont hesitate to leave a comment. Thank you again and again for your time and help.
PS! Im wondering if the weak nutrient of the agar could explain this: I have no sugars (honey or dextrose) and no potato starch, only agar powder with a small amount of yeast.
Edited by redhandmat (03/25/20 03:36 AM)
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Sunny Skies
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Re: What kind of contam is this guys? [Re: redhandmat]
#26556320 - 03/25/20 03:51 AM (3 years, 10 months ago) |
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Look up Agar is easy as 1-2-3 and try that recipe, seems to work for me with zero issues apart from being clear..bacteria will out run mycelium on the plate..try another transfer or 2,it will clean up. good luck and soo happy to see you going the agar route..it makes life so much easier in the end.
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redhandmat
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Re: What kind of contam is this guys? [Re: Sunny Skies]
#26556372 - 03/25/20 05:25 AM (3 years, 10 months ago) |
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Quote:
Sunny Skies said: Look up Agar is easy as 1-2-3 and try that recipe, seems to work for me with zero issues apart from being clear..bacteria will out run mycelium on the plate..try another transfer or 2,it will clean up. good luck and soo happy to see you going the agar route..it makes life so much easier in the end.
Thank you for taking the time and letting me know about that recipe. But any feedback on the growth? Am I even growing mycelium on the dishes? and the container hotpour growth? does that look like a candidate for transfer? Is that better than the petries? Should I toss the petries?
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InfiniteDreams


Registered: 10/25/19
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Re: What kind of contam is this guys? [Re: redhandmat]
#26556378 - 03/25/20 05:37 AM (3 years, 10 months ago) |
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Those pours look way too thick to me. You don't need that much agar.
The first picture look like fuzzy mycelium. You may want to try lowering the nutrient content and going with a thinner poor.
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redhandmat
Dude

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Quote:
InfiniteDreams said: Those pours look way too thick to me. You don't need that much agar.
The first picture look like fuzzy mycelium. You may want to try lowering the nutrient content and going with a thinner poor.
Thanks for your reply mate! the hotpour had to be thick because the mycelium had some height, so needed to submerge it. As for the original pour, I totally agree. It was my second attempt at agar so just poured But going forward, as I did with the petri dishes I will keep it thin. As for the nutrient, these agars barely have any nutrient, its just water agar and a trace amount of yeast. But Ill be ordering some LME to do proper agar pours for future transfers.
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