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Agar Work


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#1 Microbe

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Posted 22 November 2014 - 03:36 PM

I want to start a thread where i can document my agar work from germinating to isolating. Maybe TR can combine my agar experiment thread with this one or maybe it should stay seperate i dont know. Anyway it was really hard to take pics and conduct the process and it also felt weird and almost as if my technique was non existent. I have seen some very nice threads documenting agar work, maybe they had a second person taking pictures.

Any questions or suggestions on what anyone would like to see just let me know. I will be posting inoculation of plates taking myc from a master slant and transferring from some EC plates momentarily.


Edited by Zen_, 26 May 2016 - 04:46 PM.
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#2 Microbe

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Posted 22 November 2014 - 04:53 PM

INOCULATING 4 PLATES FROM MASTER SLANT FOR MASTER SPAWN JARS.

I execute all standard aseptic technique in front of a flowhood.

I lay out my utensils on a bleach alcohol soaked papertowel and fold it over to cover them up.

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I grab my PDA plates. I like to use ziploc bags instead of micropore tape, parafilm, and etc. It is easy for me to use and increases efficiency. I bought a 1000 for 7 bucks i think from Amazon.

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I spray the bag down with alcohol then remove it and place in a container. Most of the time i toss them and use new bags but this time i did not. If im reusing the bags i make sure to open them with ooening facing away from the air flow. If im using new bags i load my container with the quantity i need and spray with alcohol before putting the lid on.

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I wipe every plate down with alcohol.

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I grab my container i use to store my master slants, a deli container filled with pollyfill.

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I select the slant i want to use and wipe it down with alcohol and remove the micropore tape while leaving the pollyfill in. I bore out my caps and pack with pollyfill before covering with micropore tape.
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I place it in the holder that has been cleaned and steralized.
Its cramped but we are ready to go. Everything you see has been cleaned. Once i get everything laid out i spray everything with alcohol again and let it sit on there.

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This was hard to do and take pics but i pulled it off. My only concern is it required me to leave the lids off longer then usual.

I lay out 4 plates that i want to inoculate. I work front to back so that if anything where to drop off of me and make it down to the working surface the plate has already been inoculated.

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When ready to go i grab my loop and flame steralize it. For photography purposes i set it so i didnt have to hold it. I would never recommend doing this as it is a blow torch and it gets knocked over it could be really bad.

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I then touch the agar in the plate that is going to receive the transfer. This cools it down and helps the myc to stick to the loop.

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I then go to the slant and smear the surface.

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Take the loop with the myc now on it and go back to the plate and smear the center.

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I do this for all 4 plates making sure i flame steralize the inoculation loop between plates.


I put a fresh piece of micropore tape on my slant and it will go into incubation to recover and colonize the agar surface again.

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I lable my plates with any information i need. If isolating or making alot of transfers i use a spread sheet i made that tracks plates and transfers utilizing diagrams and space to input information such as temp, nutrient agar ingredients, colonization times, and etc. But for this instance just strain and date is fine. I wrote iso on the tag but it is not a true monoculture but rather a very agressive, nice fruiting specimen.
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I would say after a few more transfers it would be a true mono.

I will incubate these at room temp under my flowhood. Its a mess right now and requires some organization.

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At the end of my session i wipe down my flowhood and replace the dust cover.

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Edited by Microbe77, 22 November 2014 - 04:57 PM.

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#3 Microbe

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Posted 22 November 2014 - 05:14 PM

TRANSFERRING EC SPECIMEN TO OBTAIN A NICE RHIZO CULTURE.

I go against the grain and absolutley will not just noc up grain jars with just any mycelium. I dont isolate either before fruiting but just simply want to have a culture that IMO will give me good results and a base culture as i like to call it in which i can isolate from there pending fruiting results.

I wont be talking about sectors or anything that technical. I am making my selection based solely on gut feeling here or firing from the hip.

IMO these specimens are not worthy of going into any of grain jars.

Plate 1
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Plate 2
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Plate 1 this is where i elected to take a specimen for transfer.

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I transfer the specimen and try to place directly in the center of the plate.

Plate 2 this is where i elected to take a specimen for transfer.

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Again i try to place directly in the center of the plate.

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I place in bags and label each plate and then will also be incubating these at room temp under my flowhood.

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#4 Microbe

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Posted 22 November 2014 - 05:40 PM

SOME INFO ABOUT MY NUTRIENT AGAR.

I use PDA mainly and use a standard tek in preparing it. I add agar at a 2% ratio to the infusion made from boiling sliced potatoes and dextrose is added at a 1.5% ratio. I add black food coloring as it makes it very easy to see growth in the beginning stages rather it be a contamination or myc and that otherwise you would not be able to see on regular or clear agar without holding at a weird angle while shining a beam of light. If you have not tried this yet then give it a try and you will see exactly what im talking about.

I steralize at 15 PSI for 20 minutes for 500 ml or less of nutrient agar.

I use a no pour method using a ghetto version of a no tilt jar designed for LC. It can be found here https://mycotopia.ne...ilt-agar-jars/.

This was the first area of study for me when getting into this hobby. I have spent countless hours conducting agar work. The benefits are many and allow you to be very selective on what goes into your spawn jars. Agar work is hands down my favorite process to conduct and i absolutely love experimenting with it.
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#5 AGAMA

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Posted 23 November 2014 - 09:28 AM

Agar work is hands down my favorite process to conduct and i absolutely love experimenting with it.

And it shows in your extreme attention to detail.
As I hope to get off my ass this year and give agar a go,
this thread is very inspirational to me.
Thanx for sharing, Microbe.
(Cool gloves. I've only seen black nitrile gloves
associated with the mortuary sciences, but do
have a pair stashed for their novelty value myself.)


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#6 invisibilitysyndrome

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Posted 23 November 2014 - 10:56 AM

Hey microbe 77
I started some plates fer the firsr time on the 21st
Pe6 tly sg30 falbino.
Whats your experience with time frame for signs of first growth..number of Days?.
Love this tthread since im pretty much started same as you on here...good timing....thnx
Im in a gb no flow hood. Any tips?
I already made the mistake of pouring too hot and closing lids too soon however turning plates upside down cured most of the condensation. .......

#7 invisibilitysyndrome

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Posted 23 November 2014 - 10:58 AM

Agar work is hands down my favorite process to conduct and i absolutely love experimenting with it.

And it shows in your extreme attention to detail.
As I hope to get off my ass this year and give agar a go,
this thread is very inspirational to me.
Thanx for sharing, Microbe.
(Cool gloves. I've only seen black nitrile gloves
associated with the mortuary sciences, but do
have a pair stashed for their novelty value myself.)

Most tattooers have blk gloves and now most auto parts stores have em too....seems majority of non "medical" gloves are black these days. Pink too fer breast cancer. Anyways just an aside.....cheers
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#8 CatsAndBats

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Posted 23 November 2014 - 11:07 AM

I'm with Agama, truly inspirational work.


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#9 hyphaenation

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Posted 23 November 2014 - 11:55 AM

To say you've come a long way would be an understatement... Very inspiring, excellent work and presentation.
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#10 hyphaenation

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Posted 23 November 2014 - 12:03 PM

*Archive Material*
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#11 Microbe

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Posted 23 November 2014 - 12:21 PM

Hey microbe 77
I started some plates fer the firsr time on the 21st
Pe6 tly sg30 falbino.
Whats your experience with time frame for signs of first growth..number of Days?.
Love this tthread since im pretty much started same as you on here...good timing....thnx
Im in a gb no flow hood. Any tips?
I already made the mistake of pouring too hot and closing lids too soon however turning plates upside down cured most of the condensation. .......

Have not worked with the sub-strains you mentioned. As far as growth and time frames there are many variables such as temp you incubate at, inoculation method, aggressiveness of the strain, vigor, and so on.

IME these are the average time frames that i have observed.

Spore to agar 10-14 days for germination.

Spore Solution to agar 5-7 days for germination.

Transfer colonized agar plug, 1-2 days for recovery then 5-7 to fully colonize. I use 60 mm plates also. Again these are averages and have had plates colonize in 3 days and have had it take 3 weeks!

I dont worry to much about condensation and have a few issues from time to time when im in a hurry but it almost always dissipates and gets absorbed back into the agar. The key is timing, you should be able to hold the container containing the nutrient agar comfortably to your cheek. Stacking your plates also helps as it keeps the lids warm so condensation doesnt develop except on the very top plate. I hope that makes sense and i explained it right.

Hope this helps!

Edited by Microbe77, 23 November 2014 - 12:33 PM.

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#12 invisibilitysyndrome

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Posted 23 November 2014 - 01:14 PM

Right on. Dint realize that spore to agar was longer than ms solution. Good to know. One plate from 20 has some type of infection. All others show nothing and since ive used spore im not concerned now. So i wait and watch this thread. Cheers and thnx
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#13 Microbe

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Posted 23 November 2014 - 01:37 PM

Right on. Dint realize that spore to agar was longer than ms solution. Good to know. One plate from 20 has some type of infection. All others show nothing and since ive used spore im not concerned now. So i wait and watch this thread. Cheers and thnx

Typically, unless the spore print is very fresh, it needs to hydrate before it can germinate. So it usually takes longer then if it were already hydrated.

Can you post some pics of your plates?

Edited by Microbe77, 23 November 2014 - 01:38 PM.


#14 Microbe

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Posted 24 November 2014 - 09:43 AM

My contaminated agar experiment thread has been updated.

https://mycotopia.ne...r-experimenting
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#15 hyphaenation

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Posted 24 November 2014 - 11:10 PM

Must be your settings, looks good from here. Hope you get it sorted, its cool shit!



#16 wharfrat

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Posted 24 November 2014 - 11:54 PM

I'm not getting any of the pictures you guys are talking about ... Grrrr

 

attachicon.gifAAA Agar Work - Fungi- Magic Mushrooms - Mycotopia.png

if it's supposed to show pics, it doesn't on mine either. but it does not show a place card



#17 hyphaenation

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Posted 24 November 2014 - 11:58 PM

Tried it on two browsers and both show pictures... what browser /OS's are you guys using?



#18 wharfrat

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Posted 25 November 2014 - 12:11 AM

nice write up microbe! i must have missed this, i been really busy lately.. bookmarking!


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#19 Microbe

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Posted 25 November 2014 - 02:35 AM

*Archive Material*

What is the meaning of this? Isnt everything archived? Whatever it means, i am very humble that something i contributed is going to be available for a long, long, time..... if that is what *Archive Material* represents anyway.


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#20 invisibilitysyndrome

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Posted 25 November 2014 - 09:49 AM

20141125_094129.jpg 20141125_094152.jpg
Can take more. I use para film..
Looks like one tly shows growth and a b plus as well.
The b plus is a newer print so probly started faster due to this
When i say growth i just meen tiny whisp that u can only see at an angle so no pics

Edited by invisibilitysyndrome, 25 November 2014 - 09:51 AM.

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