Nan's Nook : Archives : Cloning : Agar

Fungusmaximus

Iggy(ignuf) and I, are working w/ malabar prints that he has supplied. Goal: try and get some good fruits from this new strain, that we have had limited success with I have had none, as of yet 0% . Although IGGY has grown some w/ success.

He will be posting a log of the isolation, w/ some backup from me.
I will be following along doing the same experiments, and cloning/printing etc...
We will see what happens

Iggy

OK here is my homemade glass petri tek Maybe someone else has tried this or done it before but this is how I go about it. First I mix my malt agar according to the directions. Then I pour it into my 4oz. jelly jars. Make sure you get the ones without the lip (they do come with a lip sometimes) I find these to be easier to work with.

I only pour enough in each jar to cover the bottom as you would any other petri dish. Like so

Place lid on jar with the seal (gasket) part up. I prep my lids by drilling a small hole in the center. Small because we don't want to have too much air but just enough to prevent the condensation that you get in a air tight enviroment. Not to mention our culture needs to breath. Then put a good tape that can withstand the heat from the pressure cooker over the whole (here I used some clear packaging tape, I have found scotch tape has a tendancy to melt down). Like so.......

Next completely cover with a large sheet of aluminum foil, so as to keep the moisture out of the jar where we put the lid on upside down. Then pressure cook for 30 min @ 15 psi. I let mine cool overnight in the p.c. I don't open it until I am ready for the innoculation process.

Now it's time for the glove box part My friend Fungusmaximus has built a few easy to make and damn nice ones. I just made this just for you guys! I know this is not the best glove box but it will accomplish what I want. Also this is something simple that any poor boy can make with supplies around the house. Rubbermaid bins work good also just keep in mind you need to be able to see what you are working with. I always was my hands all the way to my elbos with anit bac. soap. I used the 10 gal. fish tank for its clarity for the pictures Keep in mind I do have a HEPA air purifier running 24/7 in this room I'm working in Ok, now put on a pair of laytex gloves. First I sterilized the whole inside and out with iso. alcohol (91%) Wiping dry with one paper towl (thus creating the largest alcohol wipe ) Then I gathered 3 small plastic trash bags to cover the opening. I taped the trash bags to the opening at the top and bottom, pulling the middle one tight to remove all slack in the plastic.

I know I need to clean my room. I have perlite and verm and all sorts of stuff in the way Next we tape the side bags on leaving just enough slack to allow enough play in the plastic to let our hands in and out.

Then the other side. Be sure and tape on the bottom too. I only left it slack in the previous pic to show the placement. Now we have something to prevent any unwanted drafts and to keep our breath off our work Your breath (my breath too!) is one of the largest sources of bacteria in this line of work. I actually gargle with mouthwash and then wear a dust mask when working. This glovebox is only a temporary fix. If you use it again I suggest starting from the begining and using all new plastic.

OK now we get going! First wash hands and arms again. Then put laytex gloves on!!!!! Laytex gloves are cheap so please don't cut any corners and think washing your hands will do a good enough job. Now lay out all supplies inside your glove box. Be sure to sterilize ( I recomend a flame, aclohol lamp) any tools before placing them in the glove box. Pictured below I removed the foil, band, and tape inside the glove box and layed everything else to be used out. We have here some polyfil (found in craft stored/dept. and most pillows ), agar filled petri jar, steril malabar print, sterilized scalpel, and 2 coffee filters. I opened the foil print inside the glove box BTW

Now we can innoculate the agar. I do this by lightly scaping the print while holding it over the agar so the spores fall onto the agar. It normally only takes one or two light scrapes. If you use an innoculation loop you just rub it in the print and then rub it across the surface of the agar in a figure 8 pattern. You will have to reapply the spores 2-3 times (be sure to flame the loop each time before rubbing on the print. This way you burn all the agar off of it. Any small amount of agar in your print will ruin it for good ) Let the hot loop cool for a minute before touching any spores! Sorry I don't have a pic of this step for you but I just don't have enough arms to snap the shot I think you get the picture though

Now if you scraped the spores I hope you tried to spread them evenly accross the surface. This will help keep even growth and colonize the surface quicker (to avoid contams) We will get into isolating the rizomorphic myc. later on. First we are hoping to create a pure and sterile culture. In the pic below maybe you can make out the black specs (spores). It really only takes a few, just try to spread them out. I believe that the fewer spores the more likely you are to create the rizomorphic growth.

Now we want to dot the i's and cross the t's Cover the jar with the lid (still upside down) leaving the band off for now. Cover the hole and most of the lid with a thin, but not see trough layer of poyfil.

Now cover with the coffee filters (2 thick) and secure in place with the band. Make sure the band goes on evenly as the filters tend to set it off track (crossthread) easily

Now tear the excess off the edges or trim with a box cutter. However you choose. Lable the strain and date. and place in the incubator. I like to keep mine between 80-85F Just try not to exceed 90F and cooler temps=slower growth=higher risk of contams =
Here we are ready for the incubator BTW the lable is tapped to the bottom Hope you enjoy my info. It will be updated as the prodject progresses
Oh yeah, in case your wondering the agar cooled at and angle (from leaving in the pc), no it's not still a liquid I can't be perfect at everything, or anything now that I think of it

Steveoi812

Now for my first question. Iggy, when the cultures are done colonizing, what do you do with them. Dosent that agar turn kinda gel like or solid almost, or does it stay in a liquid form? Sweet tek man I would like to get into this kinda thing myself. This would be a great tek to follow step by step. Two thumbs up from steveo! Sorry if I messed up your thread.

Iggy

As soon as it cools it is a solid/gel Full of nutrients and ready to grow! You didn't mess up the thread man. This is an educational experience for all, including me. I have dabbled in the past with this but now I am ready for the next level You just treat your agar culture as you would a new cake. Keep in the dark and warm, somewhere where it can breath just a little but not in a draft

Steveoi812

ok so once the entire agar sollution has colonized how do you frut it out? Just introduce light to it or do you transfer it to some kind of substrate?

Iggy

Transfer a nice rizomorphic cutting to another petri. Then let that grow out. You should get a picture perfect rizomorphic as hell dish then. If not keep transfering unitl you get at least a nice sized section of rizo. But yeah the final idea is to transfer a wedge (or cutting) of rizomorphic myc. to your substrate (I find grain to be best for this). Birdseed, rye berries, wheat berries, or millet are some of the common grains used with good results. Then use colonized grain as spawn to compost substrate for best results. But you can just place the grain in a tray, pouring the grain until a nice 2" depth. and then allow a couple days covered in incubation to recover, then case Isolation of the culture allows more even flushes and pinset. You should have the mojority of fruits that look the same or similar in size. Hopefully XXX Large!

Steveoi812

Thanks for clearing that up for me iggy.  What are the benefits of starting a culture such as this as opposed to just picking a good looking fruit and cloning it into a liquid culture? I guess you get a wider spectrum of fruits to pick from when cloning. Great thread you guys. This has risen my interest in agar strain isolation 10 fold . Thanks guys
 

Iggy

Unlike a liquid culture you can seperate a contam from the culture If the agar starts to contam you just slide it in your glovebox and transfer a clean viable section of the culture to a whole new petri Then just throw away the contam Also I prefer to clone to agar rather than liquid, mainly for this reason. Then hopefully you can isolate the strongest rizomorphic growth from your cloned tissue.

Fungusmaximus

Isolating mycelium from contams can sometimes be difficult.
Here is a very very helpful agar recipe, Peroxidated Agar


You CAN'T use spores w/ h2o2 agar! You need live tissue cultures.

I use that recipe when making regular agar too. Just add the full 150ml h2o.


Mix your dry ingredients first, here is 3grams agar/3grams dextrose, in a cruet from wal mart 1.47$, the metal funnel was 3$.

Then add 150ml potato h2o, this one has a bit of Bee pollen mixed in, ingnore the yellow color.

stuff neck w/ polyfil and foil the top, PC for 15min @15psi and after removing from the PC HOT it goes straight to my glovebox. I swirl it to ensure its mixed well, I let mine cool to about
140-130 degrees before pouring.

HOT AGAR!!

UPDATE!

almost 100% colonized in 4 days! No shaking!!

Agar : Cloning : Archives Main : Shroom Glossary : The Nook

Posted by: BigWoolyMammoth May 01 03, 02:01 AM GMT

Works great, better than petris in some ways (IE, easier to sterilize without melting )

Posted by: repobob May 01 03, 09:33 PM GMT

Thanks DirtyWOP, Yea, the Tyvek would be pointless if the lid had no hole. Oh well, thats why I ask advise from such smart people Is air important with agar? Could I just "can" the 1/2 pint with metal lid seal down. Then when I'm in the glove box open it up, innoculate then plastic lid with hole and tyvek. I hope I,m not making this more than it is I'm just nervous and don't want to screw it up. I don't understand Molester Bob

Posted by: ninja May 01 03, 10:22 PM GMT

ASIDE FROM TAKING UP TOO MUCH ROOM, THE 1/2 PINTS WOULD DO FINE

Posted by: repobob May 03 03, 12:04 AM GMT

Thanks Highroller. I read the PM before I checked this thread. You da man. Now I understand Molester, sounds like a workable solution. Thanks. Bob

Posted by: repobob May 04 03, 12:26 AM GMT

Great idea Camelot. Would a standard hole saw cut plexiglass? I'd be a little concerned that it would crack. Perhaps on a drill press with a piece of wood for backing. I have some plexiglass and several different size hole saws, I'll try cutting some Monday. Does agar require more air than would be in the jar? If not the hole could be easily closed with silicone. I'm just thinking that there would be less chance for contams if you could seal the center hole from the drill bit. This is getting interesting Bob

Posted by: Molester May 04 03, 02:53 PM GMT

QUOTE (repobob @ May 04 03, 12:26 AM GMT) Great idea Camelot. Would a standard hole saw cut plexiglass? I'd be a little concerned that it would crack. Perhaps on a drill press with a piece of wood for backing. I have some plexiglass and several different size hole saws, I'll try cutting some Monday. Does agar require more air than would be in the jar? If not the hole could be easily closed with silicone. I'm just thinking that there would be less chance for contams if you could seal the center hole from the drill bit. This is getting interesting Bob I use a standard hole saw with no problems. I'm glad I didn't do my agar the way I said. Those small lids are the perfect size to get stuck in a widemouth. Suppose they can be fastened to the second lid, but I'm gonna buy a bigger holesaw when I can afford it and cut me some lids. Props to Camelot!

Posted by: Camelot May 08 03, 09:11 PM GMT

Thanks guys...glad the NeoMyc could contribute something useful. repo - the acrylic, although pretty tough as Mol indicated, is kind of brittle and could possibly crack when subjected to the hole saw. Get a "fine-tooth" hole saw and perhaps use the drill press (vice hand-held dirll) for stability and keeping the bit straight. I would also go a step further (bare in mind I'm pretty anal when constructing things ) and sandwich the acrylic between two thin pieces of wood (1/4" plywood, scrap, something cheap)...clamp lightly if possible, then drill press. Low speed (to reduce heat), low pressure (reduce stressing the acrylic) Should work like a peach Can't answer the agar question...not that advanced yet. (still working the cake tek ) However, you could plug the center hole. I would use epoxy instead of silicone though...tougher and should be less affected over time in the PC. I used epoxy to plug up some noc holes in some "pre-made" jars that were very asymmetrically placed...works great. You can get the "dual-syringe" epoxy kits at any store that sells h/w...lowes, home depot, wally world, etc. Makes it easy to mix and sets in 5 minutes...PC-able in about 6 hours. One additional thing - rough cut your acrylic sheets before using the hole saw. That way if it does crack you don't risk the crack running the whole length of the sheet and ruining it. If you do get a crack, you can stop-drill it to keep it from going further=drill tiny hole at the end of the crack) The sheets I got at Lowe's are 18 x 24 x 3/32 and cost about $15/per. I estimate you can make about 18 caps per sheet...24 if you cut them real close together. Good luck bro! Cam

Posted by: repobob May 08 03, 10:24 PM GMT

Camelot, some great ideas. Sandwiching the glass between two boards should help alot. That will make it much easier. Thanks Nan, That is exactly what I was looking for. Tried plexiglass without success,even with a drill press on low speed. The sandwich idea may help, been to busy this week to look for lexan, hope to have some time this weekend. I really appreciate all the help on this, you guys are great.

Posted by: Molester May 12 03, 06:45 PM GMT

Extruded acrylic will melt in the PC. I thought I'd try printing on it so I cut up a bunch of slides from some scrap pieces. PCed them in a jar for 20 mins, they were all warped and stuck together.

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