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Rosen_Rot
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20days slow growth on Agar
#23948852 - 12/22/16 11:26 AM (7 years, 1 month ago) |
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These are the 3rd transfers from around 20 days ago. I am not exactly sure what is up with the slow growth, some of them even appear to be somewhat fluffy even though I've been focusing transfers on rhizomorphs. The plates below are transfers and a continuation from this thread it's with the same recipe and incubating temps of 22C
I have some back up agar plates so I can always restart. I am not sure on how to proceed from here on out or whether if they are good to use considering their slow growth. Perhaps maybe transfer them to the a higher concentration of nutrients?
Here are some pictures
Transfers A






Transfers B






Back up Plates
I started transfering from the top and bottom right plates. The ones on the left are my back up.
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"The internet has one rule; use or be used" - Bjeldiablo ''there is no loneliness, only moments where contentment is fleeting'' SBJs "The Basics" 3iRiS9 "Cirque du freak" B+ BONANZA
Edited by Rosen_Rot (12/22/16 12:13 PM)
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enlightenment
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Re: 20days slow growth on Agar [Re: Rosen_Rot]
#23948861 - 12/22/16 11:30 AM (7 years, 1 month ago) |
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What recipe did you use? How long did you pc the agar?
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Rosen_Rot
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Its just a 3% concentration of crushed wheetabix with agar, I think around 10g agar and approx 2g crushed wheetabix. PC'ed for 40mins, poured infront of laminar, bottom up....etc
I see I have not hyperlinked my other thread to my OP
I'll fix this
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"The internet has one rule; use or be used" - Bjeldiablo ''there is no loneliness, only moments where contentment is fleeting'' SBJs "The Basics" 3iRiS9 "Cirque du freak" B+ BONANZA
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enlightenment
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Re: 20days slow growth on Agar [Re: Rosen_Rot]
#23949057 - 12/22/16 12:28 PM (7 years, 1 month ago) |
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I have no experience with wheetabix agar. I have a lot of experience with MEA. It's my first choice nutrition for agar.
I PC agar bottles for 20 - 30 minutes.
The dishes look much darker than the first dishes. It would be interesting if you make your agar mix again and PC for ~ 25 minutes. It might be slow growth caused by the Maillard reaction. It would be a nice move if you test it and update this thread.
Edited by enlightenment (12/22/16 12:37 PM)
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Rosen_Rot
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Which dishes are you referring to as ''well'' ?
Also the change in color is not in the agar but rather the different ways I took the photos. The back up plates were taking with flash while the TA and TB plates were taking on aperture priority setting using window light. Some of the plates have the reflection of the camera lens, giving them that half moon shadow
The back up plates are made with the same recipe, same concentration and same pc time.
I was thinking of doing another batch of agar and start with the back up while these finish out but it sure as hell they are taking their sweet time.
You think I can transfer the above plates [ TA AND TB ] onto new agar pced at a lesser time will give them better growth?? How much of the sample would you suggest I take? Cut all of it or just a small segment?
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"The internet has one rule; use or be used" - Bjeldiablo ''there is no loneliness, only moments where contentment is fleeting'' SBJs "The Basics" 3iRiS9 "Cirque du freak" B+ BONANZA
Edited by Rosen_Rot (12/22/16 12:45 PM)
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enlightenment
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Re: 20days slow growth on Agar [Re: Rosen_Rot]
#23949095 - 12/22/16 12:50 PM (7 years, 1 month ago) |
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Quote:
Which dishes are you referring to ''well'' ?
the backup plates.
Quote:
The back up plates are made with the same recipe, same concentration and same pc time.
I never had a comparable decrease in growth after one transfer.
Quote:
You think I can transfer the above plates [ TA AND TB ] onto new agar pced at a lesser time will give them better growth?? How much of the sample would you suggest I take? Cut all of it or just a small segment?
You can give it a try. It would be better to transfer from the backup IMO. I am still not completely sure about the reason for the slow growth.. I would transfer pieces of this size:

Can you make a picture from a backup dish next to a TA/TB dish?
Edited by enlightenment (12/22/16 12:56 PM)
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Rosen_Rot
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These are 3rd transfers, the back up are 2nd transfers.
I will try tomorrow, grab the best looking plates from each batch and inoculate it onto the new agar. I'll start with my back up as well. Thank you for your suggestions
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"The internet has one rule; use or be used" - Bjeldiablo ''there is no loneliness, only moments where contentment is fleeting'' SBJs "The Basics" 3iRiS9 "Cirque du freak" B+ BONANZA
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enlightenment
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Re: 20days slow growth on Agar [Re: Rosen_Rot]
#23949117 - 12/22/16 01:00 PM (7 years, 1 month ago) |
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Quote:
These are 3rd transfers, the back up are 2nd transfers.
I know. I noticed slower/weaker growth after 7+ transfers but never that rapid from one transfer to another. OK. Keep us posted.
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Rosen_Rot
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I have just finished making the transfers and I believe I know what the problem was. The agar medium that I had before was too watery, coupled that with the low concentration of nutrients than it's a wonder why the growth was so slow. It was very soft, slippery and hard to cut which made the new transfers rather tedious.
The agar I have now transferred them to has a more solid consistency and added 5.2g wheetabix instead of 2. The puck is more jelly like and more solid than the previous plates.
Hopefully this should yield some good results
I will post here again on their progress in a week
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"The internet has one rule; use or be used" - Bjeldiablo ''there is no loneliness, only moments where contentment is fleeting'' SBJs "The Basics" 3iRiS9 "Cirque du freak" B+ BONANZA
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Mad Season
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Re: 20days slow growth on Agar [Re: Rosen_Rot]
#23951070 - 12/23/16 08:30 AM (7 years, 1 month ago) |
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Agar recipes should always be 2% agar and 2% nutrients (same weight between the 2)
10g agar and 5g weetabix? I'm assuming with 500ml of water. Well it might work I guess. I'd still probably do 10g if it was me.
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Munchauzen


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Re: 20days slow growth on Agar [Re: Mad Season]
#23951173 - 12/23/16 09:23 AM (7 years, 1 month ago) |
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My agar recipe has different values for agar and nutes. Works fine. Its even based off a labratory recipe from a scientific wholesale distributor that sells their own premixed pda
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Rosen_Rot
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Re: 20days slow growth on Agar [Re: Mad Season]
#23951280 - 12/23/16 10:20 AM (7 years, 1 month ago) |
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My recipes were 20g agar 20g nutes with 500ml and I cut that by half cause I had a lot left over after I was done pouring the plates, so it's with 250ml of water PC'ed for 20mins
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"The internet has one rule; use or be used" - Bjeldiablo ''there is no loneliness, only moments where contentment is fleeting'' SBJs "The Basics" 3iRiS9 "Cirque du freak" B+ BONANZA
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Rosen_Rot
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Re: 20days slow growth on Agar [Re: Rosen_Rot]
#23998165 - 01/10/17 12:57 PM (7 years, 20 days ago) |
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*UPDATE*
So it's been around 18days since doing the transfer A and B
No activity what so ever so I am basically abandoning this set



I've did some transfers from the back up as Enlightenment suggested
There seems to be better activity and the mycelium seems to be growing healthily however I am finding the growth rate to be really slow
These are pictures from 4th January



My apologies for the late update, been busy with uni assignments
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"The internet has one rule; use or be used" - Bjeldiablo ''there is no loneliness, only moments where contentment is fleeting'' SBJs "The Basics" 3iRiS9 "Cirque du freak" B+ BONANZA
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leschampignons
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Re: 20days slow growth on Agar [Re: Rosen_Rot]
#23998306 - 01/10/17 01:51 PM (7 years, 20 days ago) |
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I think there are likely preservatives in wheetabix. Like maybe BHT. Look on the back of the carton and see if it says treated with BHT to maintain freshness or something like that.
EDit: just looked up BHT and apparently it's not a preservative but an antioxidant used to prevent oxidation of compounds in the food by atmospheric oxygen, so that's prob not the problem. It could be some other preservative though
I also agree with enlightenment that the agar looks different (darker) in the failed/slow plates.
Edited by leschampignons (01/10/17 01:59 PM)
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Mycolorado
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Re: 20days slow growth on Agar [Re: Mad Season]
#23998510 - 01/10/17 03:19 PM (7 years, 20 days ago) |
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Quote:
Rosen_Rot said: My recipes were 20g agar 20g nutes with 500ml and I cut that by half cause I had a lot left over after I was done pouring the plates, so it's with 250ml of water PC'ed for 20mins
Did you accidentally add 20g nutes to 250ml? If so, that's the issue...8% is way too high.
Quote:
Mad Season said: Agar recipes should always be 2% agar and 2% nutrients (same weight between the 2)
Not sure why you would suggest this...different nute concentrations serve their purposes as well as different agar concentrations, and there is absolutely no reason the agar and nute must be equal.
Edited by Mycolorado (01/10/17 03:20 PM)
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Mad Season
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Re: 20days slow growth on Agar [Re: Mycolorado]
#23998558 - 01/10/17 03:48 PM (7 years, 20 days ago) |
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You are correct, but it's my go-to for any new recipes, and has always given me results. If it fails on 2%/2% something is wrong. Also I've had shit sideways, upwards, or myc lying down on agar surface, it makes literally no difference. I really have no idea what's going on because I've never used any of these recipes, but at least start off with something proven.
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Mycolorado
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Re: 20days slow growth on Agar [Re: Mad Season]
#23998786 - 01/10/17 05:08 PM (7 years, 20 days ago) |
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Fair enough.
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PinPornProducer
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Re: 20days slow growth on Agar [Re: Rosen_Rot]
#23998835 - 01/10/17 05:22 PM (7 years, 20 days ago) |
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Quote:
Rosen_Rot said: These are the 3rd transfers from around 20 days ago. I am not exactly sure what is up with the slow growth, some of them even appear to be somewhat fluffy even though I've been focusing transfers on rhizomorphs. The plates below are transfers and a continuation from this thread it's with the same recipe and incubating temps of 22C
I have some back up agar plates so I can always restart. I am not sure on how to proceed from here on out or whether if they are good to use considering their slow growth. Perhaps maybe transfer them to the a higher concentration of nutrients?
Here are some pictures
Transfers A






Transfers B






Back up Plates
I started transfering from the top and bottom right plates. The ones on the left are my back up.

Those plates are acting bacterial to me, the plate labeled transfer A, right above the A leading edge of myc, is that yellow or just pic quality? I'm thinking the edges of the plates you took transfers from a harboring bacteria. Even the bottom left plate, leading edge 8oclock, looks yellow
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Rosen_Rot
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I believe that is the pic quality. I took these pictures on the window ledge of my lab with Aperture priority setting on my DSLR camera. The dotting in the agar is the ground wheetabix flakes while the black edging is run off from my sharpie liquifying in response to alcohol in the air of the tote that I store them in. Meaning I wipe the inside of the box with ISO everytime I take the plates out and since there was no way where the ISO could evaporate it liquified my labelling, which is why I switched to pencil 
And it could be that I added 20g of nuts to 250ml of water I slashed everything in half from the 20/10/500 recipe so everything came to 10/5/250. Since there is 10g of agar and 5g of wheetabix would this constitute as 15g of nutrients?
If so that could be the issue
Is it possible to use 10g of agar with potato extract WITHOUT the dextrose?? I wanna try this next to see if I have any success
Also my temperatures have been fluctuation from 22C during the day to 5-8C during the night cause I stupidly forgot that I had plugged in the fan heater to the same timer as the lights 
This is why you don't get stoned before doing culture work kids
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"The internet has one rule; use or be used" - Bjeldiablo ''there is no loneliness, only moments where contentment is fleeting'' SBJs "The Basics" 3iRiS9 "Cirque du freak" B+ BONANZA
Edited by Rosen_Rot (01/10/17 11:06 PM)
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Orbit
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Re: 20days slow growth on Agar [Re: Rosen_Rot]
#23999874 - 01/10/17 11:21 PM (7 years, 20 days ago) |
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Ya low temp doesn't help.
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