48 replies to this topic

[jrh]

Once upon a time, I grew some B+ mushrooms. Then I wanted to do it again, and that's when my foray into mycology started unraveling.

 

To be fair, I used an all in one grow bag to grow those mushrooms, but I didn't know anything about FAE (fresh air exchange) vs humidity or how using a heating mat can dry out everything while still making it look like there's plenty of moisture everywhere. I feel bad for those poor things... they were alternately dehydrated and drowned, but they got out a couple of flushes, and my husband and I got a few trips.

 

Then I simply tried to grow some more. After failure upon failure upon failure, it turns out at some point I had started cultivating trich instead of cubensis. Boy did it suck to learn that.

 

So yesterday I sterilized all my cultures (except for one LC (liquid culture) that looks so clean and pure), trashed everything that was in progress, and threw all my jars and lids into the dishwasher.

 

I'd still like to have some mushrooms, though, so now what?

 

1st Priority -- A Clean Culture 

 

Right now my highest priority is to get a clean culture of some kind of cube. I have a LC of Purple Mystic (from Hawk) in the fridge that looks so pretty and white it could almost be a snow globe. 

 

 

Is it clean though? How do I know?

 

I also have a few janky spore syringes that I'm sure are dirty, and hopefully I'll be getting a fancy spore print in the near future.

 

Agar

 

The budding mycologist can use agar to verify that cultures are clean. If they're not, clean mycelium can be transferred away from contaminates until you end up with a clean culture. Agar is great for other things, but keeping my priority in mind, I want to focus on getting a clean culture.

 

I'm only now starting with agar because I only recently got a SAB (still air box). To use agar, you really need some way of working sterile -- usually a SAB or a flow hood. My SAB is fairly small, though, so I'm going to try a version of agar that does not need to be poured inside of a SAB.

 

Pasty Plates

 

Pasty plates are basically agar which has been cooked with nutrients, spooned into containers, then sterilized in a PC (pressure cooker).

 

For my agar containers, I decided to use 1/2 pint wide mouth jars. 

 

For my agar recipe, I used 3 mg agar agar powder, 2.5 mg LME (light malt extract), a pinch of nutritional yeast, and 150 ml of sterile oat water (water I had cooked oats in then sterilized). I also added some food coloring, but instead of getting a purply color, I ended up with something more pinkish with light flecks from the yeast.

 

 

This was only the first issue I encountered when making my Pasty plates. I didn't quite use enough agar in each jar, so in some there is little to no agar right in the middle of some jars. Also, none of the jars are level. I'm assuming this is because of how the jars sit on the rack in my pressure cooker, and I'm not sure there's much I can do about that. In addition, I use a small, electric pressure canner that reaches 15 psi. It looks like an instant pot ate another instant pot. It's not very smart, so instead of turning down the heat way down once pressure is reached, it just continually vents pressure throughout the cook. This means I have to add a lot of water, relatively speaking, to keep the pot from boiling dry during longer cooking times. Since my jars were essentially empty, I'm guessing some of my jars may have been floating for at least part of the time.

 

I cooked my jars at 15 psi for 35 mins (since I added the yeast, I wanted to sterilize for a slightly longer time.) One of my jars ended up taking on water, but I ended up with 7 jars of perfectly useable (hopefully) though irregular agar.

 

 

My first priority is to test my LC. I'll do that today. I guess you're supposed to add a drop or two of liquid then use an inoculation loop to streak it across the plate? Maybe I'll do that for 2 jars and then just put a drop or two in the middle for the 3rd jar.

 

That leaves me 4 jars and several syringes. I'll just pick one of them, whichever I can see more spore clusters in, I guess. Or maybe I should do 2 syringes with 2 jars each.

 

Question

 

Should these jars be stored upside down to culture? What if there's liquid running around in there from an overenthusiastic syringe?

 

[ElPirana]

Looks like you’re well on your way to finding a clean culture!

Question
 
Should these jars be stored upside down to culture? What if there's liquid running around in there from an overenthusiastic syringe?

Store the jar right-side-up to grow out your culture. It doesn’t really hurt anything if the agar is not perfectly level in the jar. Also, unless you plan to make more agar jars soon, don’t inoculate all your jars right away...you want some clean jars readily available to make transfers.

As for working in a SAB, you might consider a larger tub...nice thing is that they’re cheap. I’ve found many benefits to a larger SAB:
The additional space allows you to fit more supplies in the SAB and keep things organized while working. If you ever want to try a grain to grain transfer, it can be nearly impossible in a small SAB if you’re using quart size jars. A large SAB allows you to have larger diameter arm holes, which has its own benefits...mainly if the diameter of arm holes are too small, then as you insert/remove your arms into the box and displace the air, then the air moves in/out of the box at higher velocity than when the holes are larger. More air movement churning around inside the SAB is bad.

[Auhron]

I love how that Jar says made in USA, and has metric (mL) increments on the side.

[jrh]

Also, unless you plan to make more agar jars soon, don’t inoculate all your jars right away...you want some clean jars readily available to make transfers.

 

So maybe 2 jars for the LC and 1 jar each for two syringes. That gives me one free jar for each variety without making more agar.

[jrh]

Poured some petri dishes today. It wasn't bad, except my agar must have been warmer than I thought because I've got plenty of condensation even though I stacked as I went.

 

 

I learned my SAB isn't level on the bottom, although there is a ridge in the middle running the length of it that is more level than the rest. For me it seemed easier to have my empty plates on the left, move a stack of 3-4 to the middle, pour each dish starting from the bottom until the mini stack is done, then move that stack over to the right.

 

When it sets, is the agar supposed to be soft-ish, as in the inoculation loop slices through it or cuts grooves really easily? Or more firm and bouncy?

 

 

[ElPirana]

When it sets, is the agar supposed to be soft-ish, as in the inoculation loop slices through it or cuts grooves really easily? Or more firm and bouncy?

I imagine if the agar is too soft it’ll be a little hard to swipe the surface.

You can make your agar more or less firm. There are some recipes that if you follow exactly, it ends up a little on the soft side IMO. Just adjust your recipe the next time you make agar plates and add one or two grams extra agar agar. It doesn’t take much to change the consistency.

[ElPirana]

Also, unless you plan to make more agar jars soon, don’t inoculate all your jars right away...you want some clean jars readily available to make transfers.

 
So maybe 2 jars for the LC and 1 jar each for two syringes. That gives me one free jar for each variety without making more agar.

That should be ok. You’ll figure out your own preferences as you do it. I tend to make at least a couple transfers out of each plate.

[jrh]

Poured some ketchup cups. There's not much to do when you're waiting for spores to germinate and start growing.

 

 

Oh, and I noticed that room was only 70 degrees f, so I ordered a little space heater.

[PJammer24]

Storing them upside down helps to slow evaporation and keeps condensation from dripping down onto your culture. I prefer to store mine upside down.

[jrh]

I'm storing my petri dishes and jars upside down, and the cups I've inoculated are also upside down, 4 to a sandwich bag. I can break up the big ziploc bag of cups into smaller bags of 4 and store them upside down as well.

 

So, it turns out my first mycelium (I'm not sure yet it's a cube, but it's definitely not a yeast or bacteria, which is what I'm getting from the el cheapo spore syringes I have) I've been able to grow is from mushroom crumbs from the empty ziploc bag I was keeping them in.

 

 

The weird lines are because I took the photo through a magnifying glass with a fluorescent light attached, I think.

 

I transferred 3 of these to petri dishes, one in the center of each dish. Assuming those grow out, I just need to figure if they're cubes or my nemesis trich.

[jrh]

And everything in that cup is trich or worse.

 

I threw away my 3 red cups like that, but still have the 3 transfers I made because all I really have to do is watch my trich grow.

 

Going to change my name to TrichFarmer.

[jrh]

I'm starting to get more interesting things in my agar cups. There are little tufts of white in cups that hadn't been doing anything or that had either a dry looking film, or slimy looking blobs or films. I guess it's too early to see what if anything will happen with the little white tufts. Maybe they'll trich out too. Maybe cube spores take longer than trich spores or bacteria and yeast, so I need to wait out the gross stuff to see if anything good shows up.

 

I would like your opinion on the following, though.

 

 

This is one of the cups where I sprinkled residue from a ziploc bag that had my B+ stash. I threw out a few cups made in a similar way a few days ago because they all had very healthy crops of trich. Then again, that was a different batch of agar. Maybe I should have let them go longer. Oh well.

 

Anyway, this cup has 2 growths. Any chance the fluffy one is something I'd want to keep?

Edited by jrh, 13 December 2020 - 07:00 AM.

[ElPirana]

 

This is one of the cups where I sprinkled residue from a ziploc bag that had my B+ stash. I threw out a few cups made in a similar way a few days ago because they all had very healthy crops of trich. Then again, that was a different batch of agar. Maybe I should have let them go longer. Oh well.

 

Anyway, this cup has 2 growths. Any chance the fluffy one is something I'd want to keep?

Sorry jrh,  but it doesn't look good.  Looks like you have two types of mold there.
 

[cujoloki]

Looks like cobwebs and trich/mold. How old Is the spiderwebby stuff? That particular cobweb may stall out but iv seen that stuff take over a Petri real fast. Transfer the cobweb s if you have a good feeling about it and have a hunch or whatever. It'll sporulate on you if it is the cobwebs. I think anyway. Maybe it won't take over the air where you open it. 90% sure it's a cobweb competing against a healthy competitor. More than likely the trich is eating whatever actual mycelium media is there and it's all gone. Could just be trich though. Love the variables in this field. Trich likes to eat whatever is clever if found. Its early for me though. Still asleep.

[jrh]

lol, at least I'm consistent.

 

I should have a spore print arriving tomorrow, so hopefully I can get something from that. :) I'm not giving up yet.

 

(^o^)♫ I'm dreaming of a white Christmas!

[cujoloki]

At least a snowy new years I hope. I see that you put your cups into a bag, that's smart! I just stack mine in towers of 10 and play a fucked up game of jenga and move slow af while I work.

[jrh]

Actually, I've moved them into sandwich bags, 4 cups per bag. That way I can store the cups upside down. It also gives me an easy way to glance at 4 cups to see if there's anything I should take a second look at.

 

 

 

Speaking of second looks, trich or not trich?

 

 

 

[cujoloki]

Usually a colony that size would show green by now and be blobbier than that.

[jrh]

I'm not sure what to make of this. It's the same stuff as in the BRF cake but on agar. It looks sort of like pillowy white mounds in the center of the agar. It's hard to get a focus through the glass.

 

 

Once part of it hit the glass, though, then you can see some structure.

 

 

Is this just another random fungus mycelium?

 

 

 

 

[jrh]

This is a streak plate that I made by

 

1. sucking some mycelium from the bottom of a potentially contaminated LC

2. shooting it into a little pinch bowl

3. using an inoculation loop to try to grab a bit of mycelium and streak it on the agar.

 

I have a JMF jar and Z-Strain jar that aren't nearly as advanced, but the growth looks the same. Pillowy white mounds. It doesn't look fuzzy or like there's white lint everywhere like the cobweb mold did.

 

These are all dated 12/8 so just over a week old.

Edited by jrh, 16 December 2020 - 12:53 PM.