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JSE
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Spores on Agar Hand Holding Please!
#28086933 - 12/07/22 04:03 AM (1 year, 3 months ago) |
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Hi All
I am hoping for a little hand holding if anyone would be so kind. This is my 3rd attempt at going from spores to agar. I think my sterile tek is getting a little better each time. Hopefully. So I am hoping to make better progress this time than on previous attempts.
I have dropped spores into 6 agar jars. A couple of weeks ago. 3 are doing nothing. 3 are doing something. These are the 3 that are doing something:
Looking at these plates, I don't see what I understand as being Rhizomorphic mycelium. It could be Tomentose mycelium, but equally it could just be general shit. It isn't clear to me how to identify Tomentose mycelium growth from contam - I have searched online. Any tips would be greatfully received. I see that there is SOME contam - obviously the orange splodge. So I would really appreciate help in determining if any of these is viable, so that I can transplant to a clean agar jar sooner rather than later - before any contam gets out of hand.
Thanks so much.
Edited by JSE (12/07/22 04:05 AM)
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ruawakeyet
Registered: 05/03/21
Posts: 1,862
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Re: Spores on Agar Hand Holding Please! [Re: JSE]
#28087146 - 12/07/22 09:42 AM (1 year, 3 months ago) |
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Are those 8 oz. regular mouth jars?! Aren't those difficult to work in? I use 8 oz. wide mouth jars, those aren't easy to use and they are even shorter. I would first recommend switching to shorter jars, or real petris if that is possible. (I haven't made the switch yet, but I really want to, just have to wait for the budget.)
Anyway, mycelium will usually be disorganized on germination plates, so distinguishing rhizomorphic vs. tomentose growth will be difficult at this stage.
All you really need to be worried about is isolating mycelium away from contaminates, and the mycelium will become more organized with future transfers.
I would take as many transfers as you can manage from that second plate shown. You want to grab as small a piece of mycelium each time that you can.
The first plate has a lot of contamination other than that orange dot. There is bacteria or yeast that has been spread by condensation, you can see it on the top part of the plate.
The third plate looks pretty suspect to me also, I would try not to use anything from that one either.
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ruawakeyet
Registered: 05/03/21
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Re: Spores on Agar Hand Holding Please! [Re: ruawakeyet]
#28087151 - 12/07/22 09:46 AM (1 year, 3 months ago) |
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Also, are you starting off with a spore syringe, or a spore print?
Syringes are difficult unless you can get only one or two drops on the agar, but they usually squirt all over instead.
If you are starting with a print, use a sterile knife blade or even a sterile bamboo skewer, (pointy end), and collect a small amount of spores. Run the tool on top of or through the agar in a straight line, and stay away from the edge.
This will give you the best odds of having many leading edge sectors available to transfer, and will help organize the mycelium even on the germ plates.
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JHOVA
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Re: Spores on Agar Hand Holding Please! [Re: ruawakeyet] 1
#28087296 - 12/07/22 12:03 PM (1 year, 3 months ago) |
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Spores to agar is tough especially with an average syringe thats dirty. You can try Brf pucks prepped a little less wet. You wanna use a shallow container not a tall 1/2 pint. It's very hard to angle your scalpel with tall glasses.
Keep in mind your fingers and scalpel are dirty. If it touches the inside most likely you just contammed your culture.
The josex brf puck method works because verm stops bacteria from spreading fast like on agar.
Get some pasty plates from the grocery store or Holy grail plates from bezos.
-------------------- π
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JSE
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Re: Spores on Agar Hand Holding Please! [Re: JHOVA] 1
#28088201 - 12/08/22 05:56 AM (1 year, 3 months ago) |
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Thank you guys for your help.
So the jars I am using are small I think 4oz - these: https://www.ballmasonjars.co.uk/collections/all-products/products/4-pack-ball-mason-quilted-design-preserving-jars-135ml-regular-mouth-with-recipe-insert-8003
I bought them because I figured they would last well, and be practical for sterilising for no-pour tek. But I do see that people use the little condiment pots for no-pour tek too so I will investigate that as well as the links you have sent. I am in the UK so will need an equivalent. I try not to buy from Amazon.
These plates were made direct from spore prints - I used a bent piece of wire that I heated will BUT I scraped off of the al foil into the jar and after reading your reply I see I should have gathered the spores into the wire and then wipe into the agar rather than scraping from above. Thank you.
So on your advice, I will take transfers from various places on the second plate. Wish me luck!
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DERRAYLD
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Re: Spores on Agar Hand Holding Please! [Re: JSE]
#28088213 - 12/08/22 06:13 AM (1 year, 3 months ago) |
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You will have to sterilize your entire piece of wire if you're using those jars or you will be using a dirty tool to inoculate.
That's why we use thin dishes to limit the exposure of the tool to the agar.
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JSE
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Re: Spores on Agar Hand Holding Please! [Re: DERRAYLD]
#28088215 - 12/08/22 06:17 AM (1 year, 3 months ago) |
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Thank you dude. I am in the UK so I will look for some containers I can use in the pressure cooker for no-pour tek. Maybe the little condiment pots!
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ruawakeyet
Registered: 05/03/21
Posts: 1,862
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Re: Spores on Agar Hand Holding Please! [Re: DERRAYLD]
#28088220 - 12/08/22 06:21 AM (1 year, 3 months ago) |
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Yeah, that's my bad on guessing the jar size being used. They looked like the tall ones from the picture.
Both those, and the 8 oz. wide mouth jars are not ideal for agar. I use the 8 oz. wide mouth jars, they at least provide a larger diameter to work with. I make them work, but they are just not ideal. They force me to hold the jar at an angle to avoid hovering my hand over the jar, and placing transfers cleanly is very challenging. They often times get stuck to my blade, so I have to tap the blade, then the segment of agar lands where it lands, usually bounces around a couple of times.
I'm really wanting to switch to glass petris. Pre-pour (no-pour) is totally doable in them, and of course they are reusable like jars.
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DERRAYLD
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Re: Spores on Agar Hand Holding Please! [Re: JSE]
#28088231 - 12/08/22 06:30 AM (1 year, 3 months ago) |
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Give that a try, it's important you find a dish that limits you hovering of the dish when innoculating.
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JSE
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Re: Spores on Agar Hand Holding Please! [Re: DERRAYLD] 2
#28088244 - 12/08/22 06:51 AM (1 year, 3 months ago) |
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OK, I just splashed some cash on 10 glass petri dishes I am a bit fed up of waiting weeks and weeks only to find I don't have something useable!
I'm in a real rush to grow right now as my previous grow is inexplicably weak. a good 2.5g to feel anything at all, weak.
Edited by JSE (12/08/22 06:52 AM)
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Shrimps
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Re: Spores on Agar Hand Holding Please! [Re: JSE] 1
#28088247 - 12/08/22 06:54 AM (1 year, 3 months ago) |
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glass petri is a great choice
-------------------- I am not crazy, I prefer the term mentally hillarious. Agar - The way to go! Clean spawn checklist Proper Surface Moisture Recognizing and dealing with contamination π
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π
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JSE
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Re: Spores on Agar Hand Holding Please! [Re: DERRAYLD]
#28165659 - 01/31/23 08:53 AM (1 year, 1 month ago) |
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Thanks so much guys.
I started again with a few different strains, from spore to agar about 3/4 weeks ago.
I wonder if you could please help me identify - and also understand better how to identify - which mycelium to transfer for best results?
5 plates did absolutely nothing. There were spores in there so I don't know why they did nothing but I'm assuming I can wash them out. Is it normal for a proportion of plates to do nothing?
These ones did something:
This one clearly has significant contam in the bottom but the mycelium has really gone crazy. Is this ok to transfer from?
This one obviously is contaminated too but the mycelium might be ok?
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ruawakeyet
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Re: Spores on Agar Hand Holding Please! [Re: JSE] 1
#28165702 - 01/31/23 09:25 AM (1 year, 1 month ago) |
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Looks like you have a lot to work with. Take as many transfers as you can from the leading edge from various colonies. With that many germ plates, you could potentially take well over a dozen transfers from different locations.
That first APE plate looks a little over grown, so it might be too late to get a transfer from a leading edge. In that case, just find some spots that aren't right near the edge of the plate.
That contaminated plate could give you some transfers from the healthy colonies, but I wouldn't open that in front of a hood. If you only have a SAB, I would do that one last, and clean your SAB well afterward just for precaution. If you have enough healthy plates, I wouldn't even use that contaminated one.
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JSE
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Re: Spores on Agar Hand Holding Please! [Re: ruawakeyet]
#28165704 - 01/31/23 09:30 AM (1 year, 1 month ago) |
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Thanks so much. Your positive prognosis of the plates is a lot better than I had imagined it might be! Can I please clarify - when you say leading edge you mean cut a section that includes some of the outer boundary of a patch?
I have 10 new plates I will put in the PS tonight so should be able to make transfers tomorrow.
That first ape one has lots of contam at 6 o'clock. I don't know why the mycelium grew so quickly compared to the others though, it was done at the same time. Could this indicate that this is good genetics?
Edited by JSE (01/31/23 09:31 AM)
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ruawakeyet
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Re: Spores on Agar Hand Holding Please! [Re: JSE] 1
#28165725 - 01/31/23 09:51 AM (1 year, 1 month ago) |
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Looking at it again, it's hard to tell what exactly is going on with that APE plate. If you think there is some mycelium there, I would try transferring some spots.
By leading edge, I would take transfers from the areas marked in red. I like to take squares, but most people use little triangles since it requires one less cut. Either way works.
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BiggDaddyCane
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Re: Spores on Agar Hand Holding Please! [Re: JSE] 1
#28165762 - 01/31/23 10:11 AM (1 year, 1 month ago) |
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Make 20+ Agar plates. An keep making transfers.
You'll never truly know if that sector your looking at is clean until you transfer it to a clean agar an see how it grows.
Never trust the first plate your Myc is germinating off. No matter how clean it looks. An if your carefully an crafty enough you could take a sample of clean myc from that contam plate but dont risk it.
Always have a few extra plates for transfers. Just keep making transfers.
Here's a bunch of my plates from transferring 4 times from agar.
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JSE
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Re: Spores on Agar Hand Holding Please! [Re: ruawakeyet]
#28166095 - 01/31/23 02:53 PM (1 year, 1 month ago) |
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Thank you - I would never have thought to take from the almost invisible leading dge - I would have took from the thicker more visible growth!
How would I identify whether whatever is growing in the APE one is mycelium? I have no idea, it isn't green but it is thick white fluffy growth
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JSE
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Re: Spores on Agar Hand Holding Please! [Re: BiggDaddyCane]
#28167628 - 02/01/23 01:48 PM (1 year, 1 month ago) |
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Those plates are beautiful! How many transfers have you made to get there? And how long does the mycelium take to populate the whole plate? These little splodges have taken weeks to establish!
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