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Agar For Beginners?


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#21 Microbe

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Posted 31 May 2017 - 10:47 AM

I notice only a couple mention ph adjustment, is it assumed the others are neutral? In practice do you check ph of each batch?

I do not check ph. Recipes are design to achieve a target ph and is related to ingredients and even using tap water, most will remain near the intended ph level. That being said not all agars are created equal and some recipes are selective or restricted meaning only a specific organism can grow on it or as far as ph, encourages or gives a specific organism an advantage to grow.

Ph should be under 6 most of the time, any higher and the media would favor bacteria.

Edited by Microbe, 31 May 2017 - 11:04 AM.


#22 Microbe

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Posted 31 May 2017 - 11:01 AM



Thanks Microbe , I'll paste them to my recipes file. I'm going to have to try one soon might as well move on from ready made PDA or MEA. Do you have a favorite/ I see one with an antibiotic, I was wondering about those , good for cleaning up a field collected print? Nice to see a culture broth in the recipes. Hoping to get more equipment soon, like a flow hood thinking of n 12" X 18" along with a mag stirrer I want another pressure canner .....

I'll study some of your threads too, re-read and take notes. just about anything a person wants to learn can be found here @ Topia.

Lots of studying to do........peace


My favorite agar is still PDA. I supplement it accordingly to make it selective for the species im working with.

Antibiotic agars such as Sabouraud agar is good for cleaning up wild cultures. Bacteria imo is easy to isolate away from. Molds, not much we can do there.

Do you want a portable flow hood design? If not and if you have the space, i woukd go with at least a 2'x2' especially if you work with bags.

#23 Heirloom

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Posted 31 May 2017 - 12:17 PM

Thanks Microbe, I'll go with the 2'x2', building my own box and ordering the filter from a mycology supply vendor. I'm looking to try one of the agar recipes today or tomorrow.

#24 Microbe

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Posted 31 May 2017 - 02:35 PM

Thanks Microbe, I'll go with the 2'x2', building my own box and ordering the filter from a mycology supply vendor. I'm looking to try one of the agar recipes today or tomorrow.

Ebay has excellent prices on lab quality filters. My first filter i purchased was a 2'x2'x1' and it was filter designed for a air scrubber unit, the kind they use for mold remediation, fire and water restoration and etc. It cost about 400.00 after shipping and weighed about 75 lbs but it will last a cultivator a life time.

Here is a excellent deal!!!!! One of the better deals i have seen in awhile.

Look at this on eBay

https://www.ebay.com...09809095&chn=ps

Ebay links i share never work. Search ULPA cleanroom filter. It should be the first to pop up.

5555c3c291ef8e75dbdbea0d4a4a21e0.jpg

Edited by Microbe, 31 May 2017 - 02:48 PM.

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#25 Heirloom

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Posted 31 May 2017 - 03:00 PM

Thanks I'll check out ebay. Glad you said 2x2 that should be all I need , I only want to build once and be happy. it's to easy to go to small less expensive then regret it later.I expect it will last for quite a while. man your's sounds heavy, I might have to put it together where it will sit.

#26 Microbe

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Posted 31 May 2017 - 03:07 PM

Thanks I'll check out ebay. Glad you said 2x2 that should be all I need , I only want to build once and be happy. it's to easy to go to small less expensive then regret it later.I expect it will last for quite a while. man your's sounds heavy, I might have to put it together where it will sit.

I dont have it anymore. Tenderfoot is the proud owner of it now and it us a beast. My current filter doesnt even weigh 15 pounds but it sits a top a cabinet with another cabinet on top to house the blower. It is heavy also but has casters mounted to it so i can move it.

#27 darkshogun

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Posted 31 May 2017 - 03:22 PM

I don't currently have an inoculation loop. Can I use a scalpel in place of an inoculation loop for now to transfer samples to agar plates and in between agar plates?


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#28 425nm

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Posted 31 May 2017 - 04:11 PM

I don't currently have an inoculation loop. Can I use a scalpel in place of an inoculation loop for now to transfer samples to agar plates and in between agar plates?

You most certainly can. You can also make an innoculation loop out of a chop stick and nickle chrome wire.
Although the they can substitute for one another they are better at slightly different tasks.
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#29 darkshogun

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Posted 31 May 2017 - 07:08 PM

I know this is probably covered in other threads, but once a mycelium culture is developed on an agar plate, and assuming it is stored in the darkness at room temperature (not in the fridge, my operations are clandestine), how long will they typically last?



#30 Microbe

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Posted 01 June 2017 - 12:59 PM

Inoculation loops are ideal for reaching into slants or precision selection of in agar plates and makes it simple to leave the agar behind. They are tool you should have and are inexpensive for generic. I prefer single strand nichrome wire instead of loops.

As far as how long cultures on agar will last is not easily answered. There are so many variables and to name a few, how aggressive the culture is, nutrient content, and depth of agar.

At room temp the mycelium metabolism is very active. It will continue to consume the nutrient agar and it will be depleted long before the culture dies.

If i were to store at room temp i would consider the nutrient and O2 free distilled water method then inoculate plates from it on a need to have basis.

If you must store on plates then i would pour as deep as possible. This would give a little more food but you will be surprised just how quickly a aggressive culture approaches the south glass. The depth in this case is more for moisture.

Another thing i would do is to go a little heavy on nutrient content @ 5%-8%. This will slow down the growth and consumption as well.

Also i would add some peptone or a cheap substitute such as soybean powder. This will spike the nitrogen content slowly growth down even more. The amounts will have to be determined by you and your culture. You will have to ease it in to higher nitrogen contents. I seen cultures take more then two weeks to colonize a 65 mm plate which otherwise would have grown in out 7 days.

The nitrogen will provide you a thicker mycelia mat. Nitrogen does encourage more biomass and this probably the most critical imo. Once a plate is colonized you will gradually start seeing the mycelia mat thinning out. You wont really notice until you see your agar surface through the mycelium.

Finally i would store in ziploc bags or another air tight container. All though i do this in addition to parafilm to stop the fridge from drying out my plates, a arid room can also dry out the plates.

Keep in mind your at room temp, the metabolism is very active so you will need to open the bag or container at least once a week depending on the size of the container/bags and quantity of cultures in there, you may need to do it more frequent.

I would recommend making slants also of master cultures also. You will have much higher maintenance then you would using cold storage but you should easily be able to pull off 4-6 months before having to transfer with a very slight slant in the tube. Also i would simmer some hobby sticks in a dextrose solution so that you have a chance to pull life from a slant that appears to had completely died off. Remember all it takes is a single cell to start a new colony or two compatible if so happened to pull a single monokaryotic cell, but if you can pull that off with scaple, heck if you can pull that in anyway, come to my house and help me breed some strains!

I dont see why you couldnt pull off at least a month after complete colonization of the plate.

It will be difficult maintaining a extensive library so work with only a few strains and print them in addition to slanting them. When your ready to work with another strain or few strains, stop expanding the current one on plates. This will leave you only transferring slants once every 3-6 months, maybe longer and your plates of just a few strains once every 45 days or so. Who knows you might get 90 days on plates after complete colonization.

Last thing to discuss is expanding. Slants are designed to give a relatively large enough area to develop a nice colony on the surface while minimizing cell division. By design they provide much more food and moisture respectively.


Distilled water storage. I would use a bat and cat jar also known as jelly jars. Put 2 ships on the lid and some shards of glass or something comparable to rip apart the mycelium.

Fill the jar with 2-3 ounces of water and pc. This will create a vaccum inside the jar assuming it is sealed properly.

Harvest mycelium without getting any agar, or as minimal amounts as possible as this only works with 0 nutes and 0 O2 present or trace amounts of each. If there is O2 then the culture will search for food and if there is nutes the culture will need O2 for cellular respiration.

So once you harvest your mycelium, place it in another jelly jar with glass shards. I use shards from a broken crown royal bottle. You only need 1 ship and a ge filter for this jar or you can go with 2 ships and use a airport syringe for this step.

Shake the hell out of the jar with the mycelium in it like it owes you a bunch of money.

Draw the myc water into a 10 cc syringe. Make sure you can see the mycelium in the syringe and hold it upright so that the mycelium settles in the bottom of the syringe.

When ready stick the syringe into the jar under vaccum and it will pull the contents of the syringe into the jar while any O2 that enters will be what was dissolved in the water which is ok.

Or you can buy glass vacationers and they will do the same thing. If they come with a chemical such as K3 EDTA a anticoagulant agent, you will need to wash and rinse it out.

I have stored cultures in slants before realizing this chemical was sprayed on the inside of the vacutainer. The mycelium was able to tolerate it but im not sure if it would be a food source or not.


I bought some tubes on ebay for super cheap to realize that they were polyethylene and the purple lid meant they had K2 EDTA. So i cant wash and steralize. I will be testing these to see if i can use these for distilled water storage. I figured i will fill several and then check viability a year later. If it works then the plastic sterile tubes would be the way to go.


If you use the jar then you can shake it up even more to dilute it. When your ready to grow it out again grab a few cc'ss and hit some plates up.

This method was designed for spores and or live cultures of NON spore producing fungi but a study that covered a span of 4 years showed that 95% of the 150+ or so cultures of various species, a dozen cultures from a dozen different species from the Basidiomycetes division were viable at 4 years.


Happy agar work!

Edited by Microbe, 01 June 2017 - 01:00 PM.

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#31 Heirloom

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Posted 01 June 2017 - 02:52 PM

Another informative post, thanks for sharing your experience Microbe
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#32 darkshogun

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Posted 01 June 2017 - 09:43 PM

Yes, thank you very much Microbe, very valuable information for my later work.


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#33 darkshogun

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Posted 01 June 2017 - 10:39 PM

x


Edited by darkshogun, 01 June 2017 - 10:40 PM.


#34 darkshogun

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Posted 12 June 2017 - 10:42 PM

Great info in this thread. I got my agar plates in the mail today (I decided to go with the pre-poured plates to ease into the process of agar work, since I'm a beginner with it). I'm going to transfer some mycelium from a cake that has colonized and not yet fruited. I didn't get inoculation loops yet so the plan is to sterilize a small sharp knife, take a small slice of mycelium off the cake and transfer it to the agar. Should I press the mycelium into the agar, or rub it into the agar, or should I just lay the piece of mycelium on the agar and leave it alone?

 

When I get my next big shroom I'm also going to attempt to clone as Microbe suggested.



#35 peacefrog

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Posted 14 June 2017 - 04:53 AM

Lots of good info here and another great agar thread.

Not much more I can add that brother microbe hasn't already touched on.

To answer your question, shogun:

You just want to place the tissue very gently on top of the agar surface. I recommend to just nudge the tissue on the edge of the blade, not stabbing it, when transferring to agar. Then with very deliberate motions, open the Petri just enough to get the tool in, make contact with the surface ever so gently and with a circular slight twisting motion of the tool, the sample will easily fall off. My lids are only off a second or less for each transfer like this.

Then just let her go and transfer away from any contamination, if they are present after colonization begins. I would also recommend transferring at least 1-3 times even if it appears clean. This will be just a precaution in case you may have some comtam hitching a ride.

Good luck and I promise once you see how easy and effective agar is, you will kick yourself for not starting earlier lol.

Good vibes.
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#36 darkshogun

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Posted 14 June 2017 - 03:51 PM

Lots of good info here and another great agar thread.

Not much more I can add that brother microbe hasn't already touched on.

To answer your question, shogun:

You just want to place the tissue very gently on top of the agar surface. I recommend to just nudge the tissue on the edge of the blade, not stabbing it, when transferring to agar. Then with very deliberate motions, open the Petri just enough to get the tool in, make contact with the surface ever so gently and with a circular slight twisting motion of the tool, the sample will easily fall off. My lids are only off a second or less for each transfer like this.

Then just let her go and transfer away from any contamination, if they are present after colonization begins. I would also recommend transferring at least 1-3 times even if it appears clean. This will be just a precaution in case you may have some comtam hitching a ride.

Good luck and I promise once you see how easy and effective agar is, you will kick yourself for not starting earlier lol.

Good vibes.

lol thanks PeaceFrog, I probably will kick myself, I don't know how I made it this long without working with agar. It's probably a good thing I am growing PE since it basically forced me to start, and I doubt I'll regret it. There happened to be a good specimen of a shroom that had grow out just as my agar dishes arrived, so I removed a small slice of tissue from inside the stem and placed it gently inside one of the petri dishes as you suggested. I will update my progress.


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#37 darkshogun

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Posted 18 June 2017 - 10:34 PM

And we have lift off. Mycelium is growing from the stipe tissue I place on my first agar plate.


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#38 Arathu

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Posted 18 June 2017 - 11:05 PM

And we have lift off. Mycelium is growing from the stipe tissue I place on my first agar plate.

Awesome..........I'll be in the air stream for some time tomorrow for sure. I have a ton of transfers to do so welcome to a new world opening up.

 

You're gonna love what you can do with this............. :biggrin:

 

A


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#39 darkshogun

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Posted 21 June 2017 - 02:05 PM

Ok this is embarrassing, but after tripping on PE every other day I got overly paranoid, became concerned, read too much into the remarks and behavior of the people around me, and destroyed everything - agar, prints, shrooms, supplies. I guess this is a testament to the power of this strain. But it's better to be safe than sorry. I will resume my studies in 3 weeks time. :tinfoil:

 

PS: Am I correct in assuming I can shoot spores from a syringe directly onto an agar plate and get mycelium?


Edited by darkshogun, 21 June 2017 - 02:07 PM.


#40 CatsAndBats

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Posted 21 June 2017 - 03:14 PM

Ok this is embarrassing, but after tripping on PE every other day I got overly paranoid, became concerned, read too much into the remarks and behavior of the people around me, and destroyed everything - agar, prints, shrooms, supplies. I guess this is a testament to the power of this strain. But it's better to be safe than sorry. I will resume my studies in 3 weeks time. :tinfoil:

 

PS: Am I correct in assuming I can shoot spores from a syringe directly onto an agar plate and get mycelium?

 

 

I don't even know how to address that first part..

 

 

If you're going from syringe to agar, just a drop, it's not the easiest thing to get a drop out. One can also cut a lil divot in the agar and try to put the drop in the divot.

 

If you do "squirt too much", try swirling it around the surface of the agar IMHO


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