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Agar questions...


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#1 Stroker

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Posted 06 January 2020 - 09:16 PM

When starting spores on agar, what's the minimum amount of transfers I should do before going to grain?
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#2 FunG

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Posted 06 January 2020 - 09:32 PM

You can get a useable culture in your first dish if the print is clean.

If you want to play it safe then one transfer to a new dish will do it.

So yea, the answer is one (unless the print is dirty and then you need to do probably around 3-4 transfers to isolate out healthy mycelium from contaminates)
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#3 onediadem

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Posted 06 January 2020 - 10:18 PM

I always transfer at least once to make sure nothing is hiding, and I also want the best strands.


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#4 Stroker

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Posted 08 January 2020 - 08:37 PM

Thanks, also curious. The stain I'm getting is called Melmak, only available in swabs. Should I flame sterilize tweezers and pull apart the swab a little at a time., placing just a small amount of cotton swab strands in each agar dish? Or what would be my best bet using a swab?

#5 onediadem

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Posted 08 January 2020 - 09:01 PM

Yes, you don't want to use the whole q tip on the agar. Too many opportunities to contam.



#6 Stroker

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Posted 08 January 2020 - 10:28 PM

Thanks, onediadem!
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#7 roscoe

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Posted 09 January 2020 - 12:46 AM

When starting a culture on agar, you want to spread (S-shaped, zig zag, etc) very few spores across a large part of the dish.

 

This allows you to see different genetics, allows you to sector out specific desirable genetics, allows you to better see contaminates, and allows you the ability to avoid contaminates when making transfers as you attempt to clean up a culture.

 

If you do not want to use the whole swab to streak the plate, then cleanly remove some of the swab and then streak the plate.

 

It is always best practice to make several plates, to increase your chances at success.  It also helps you to determine what, if any vectors of contaminates you are dealing with.

 

If your sterile technique is on par, and all of the plates contaminate straight away, then you can be relatively certain that the print is dirty.  Still workable, but a valuable data point.

 

If one or two plates contam, then it might be a problem with your sterile technique.  Again, a valuable data point.

 

It is also best practices to make at least one transfer to another plate before you go to grain, even if your first plate did not contam.

 

If you are transfer from a contamed plate, it is wise to transfer a couple more times before going to grain.

 

An exception to this might be, if your first plate did not contam, you can make an agar transfer to a very small amount of grain.  A half pint jar with enough hydrated popcorn to just cover the bottom, for example.  If that colonizes quickly before it contaminates, you can shake it up vigorously and wait a day or two for it to recover quickly and clean.  If it does, I would feel comfortable transferring that to a larger amount of grain, and expand from there if all looks well.

 

This works because the popcorn is large, and mostly one color, (unlike WBS) and there will be only one layer, so it will be easy to detect contams.


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#8 Moosecaboose

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Posted 09 January 2020 - 01:30 AM

I have started trying to test my Spore syringes with agar just recently. I also use liquid cultures but have read that they are not worth the time. Yet Ive only has 2 fail out of like 20. But everything i have ready about spores on agar is the zig zag or something like that for better isolation and visibility. I do wish there was way more info on the subject of starting spores on agar. everything i have found seems vague and dismissive of the practice as far as home cultivation goes at least.



#9 onediadem

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Posted 09 January 2020 - 01:52 AM

I actually prefer working with liquid cultures. Very easy to make them with agar wedges also.


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#10 Moosecaboose

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Posted 13 January 2020 - 05:52 PM

I actually prefer working with liquid cultures. Very easy to make them with agar wedges also.

See I do as well. But when I was looking up some more info on them and testing them on agar, The majority of what i found was people thrash talking Liquid Cultures saying things like the wort mistake a newbie could make is having a successful liquid culture, and blah blah blah. But In my experience with them (Liquid culture) Ive used honey and water i boiled for 10+ mins and the only time i got contams was when i made two cultures without boiling the water first. I don't really think Liquid cultures are that useless and don't mind them at all. But to each is own i suppose right.



#11 onediadem

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Posted 13 January 2020 - 09:10 PM

I have always used LC's. The people who bash them probably failed due to incorrect prep, or lids so now they are experts on what not to do.


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#12 clumsy

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Posted 09 April 2020 - 09:21 PM

I PC'd a 1 liter flask with 600 ml water + agar powder. After 45 minutes cooking, I was left with 500 ml in the flask. The rest was at the bottom of the pressure cooker. How can I avoid this? Should I dissolve the agar power prior to pressure cooking?



#13 Choices

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Posted 09 April 2020 - 11:09 PM

I heat my water on stove top and mix/dissolve agar mix or recipe in water.

Here is where I like no pours. You can take mixed solution and pour in to jelly jars and PC. If not no pour i used a 700ml bottle with a poly fill cap and PC then pour plates in SAB or in front of hood. Works for me (no pours). The only downfall is viewing sample even after using PP5 clear homemade lids.

#14 Fungi2b

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Posted 10 April 2020 - 02:28 AM

I mix the agar, pour the plates,stack and wrap in foil then p.c my plates. I do a bunch and keep the extras wraped in foil and in the fridge upside down.

#15 Arathu

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Posted 10 April 2020 - 08:12 AM

Pyrex_Agar_001.jpg

 

My current set-up is now all Pyrex. Brother Needles , et al. (thanks dudes) got me going down the "true" path but MANY systems will work for you. I did no pour in jelly jars for years.

 

Here I have a 1000 ml capped Erlenmeyer flask, cap is drilled and a filter disc fitted under the inside of the cap. When in the PC an aluminum foil cover is wrapped over the top.

 

Ingredients (what ever recipe your are testing at the time) are thoroughly mixed right in the flask using a magnetic stirrer and then PC'd. I also have a nice mortar and pestle for pulverizing CATatives.....

 

After the sterilization run the entire PC is taken to the flow hood and left to cool down then opened in the air stream and the flask placed on another magnetic stirrer and cooled to pouring temperature

(I've had good results at about 55C checked/monitored by non contact IR thermometer). 

 

I still have to get a nice set of stainless sterilization caddies for my glass pitri plates as aluminum foil makes things a pain in the ass and these bad boys BREAK.....

 

This year I'm actually going to set my laboratory up in earnest and get moving on making LARGE amounts of plug spawn for knocking up logs....

 

I've put first plates to grain (been burned more than once doing that) and gone as far as a dozen transfers or more depending on what I'm trying to accomplish AND also the work itself.

 

Test plates and jars are always better than trashing quarts or filter bags of grain spawn. Contamination is always an issue we have to be vigilant towards.

 

Agar opens up a vast world of mycology for us. Good growing vibes folks...

 

A


Edited by Arathu, 10 April 2020 - 08:13 AM.

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