How is my first Reishi agar dish looking?

[Merlyn]

Hey everyone,

So ive just begun experimenting with agar and Reishi LC.
I used a glove box (modified SAB) to inoculate the petri dishes of malt agar.

I was very thorough in my sterilization with 70% isopropyl and now i am storing the dishes in a container (with the lid slightly open) surrounded by ambient daylight.

I messed up a little when i was inoculating with LC and put a few too many drops on the agar (instead of just 1 or 2 drops).

I also noticed the condensation on the dishes is quite high (but now have some methods which i will implement in the future to reduce condensation.)

The dish looks healthy to me.
How does this dish look to you? (4 days after inoculation)

[Kiwii]

are there more pictures? e.g. from the bottom? or side? through all the drops on the lid i can only make out some hyphae (which is good i reckon)
why do you use ambient daylight?

[Merlyn]

Heres some more photos (the quality of the photos isnt great and the condensation doesnt help make any things clearer)

Using ambient light…Well I thought that the mycelium might like some indirect light to grow. Its pretty much enough light just to be able to read in. Nothing intense.

Again, excuse the bad quality of photos…

[Merlyn]

Yeah im still researching the most optimal way to store agar dishes while they colonize. Any ideas?

Like i mentioned earlier, theyre in a lunchbox container (lid slightly open), about a meter away from my window. Slight light coming through but not directly

[Kiwii]

hej so yeh the condensation makes it tricky, but i see mycelium growing we also used reishi fungi for our insulation experiment (check the C2C report uploaded under resources). we had it growing on different substrate though (spelt husks and beach wood chips) in a climate chamber with optimal growing (not fruiting) conditions.

As far as i know light is not necessary as fungi do not photosynthesize. although light does come in when you want to go over to fruiting stage. basically you would try to immitate nature: mycelium growth happens mostly in summer (warm temp) underground (dark) and when all the leafs start to fall in autumn more light gets through to the suface, it gets colder and wetter. this is when most fungi fruit. hence, i would suggest placing the agar dishes into a cupboard in a room you keep quite warm until you seek fruting bodies. then the light and lower temp comes in.

[glyph]

Hey @Merlyn, welcome to the forum and thanks for sharing your progress!

Inoculating agar from LC can definitely be very tricky! I’ve had my share of oops moments when doing that. All-in-all, it looks like you’re off to a good start.

The mycelial growth towards the center of the plate looks healthy to me, though I’m slightly suspicious of those spots of growth around the periphery of the plate. I recommend making transfers from the leading-edge of the mycelium as soon as possible (to at least three new plates - the more, the better). Expand the mycelium through 2 - 4 fresh plates until you’re certain you have clean growth (no contaminants).

As @Kiwii mentioned, your mycelium does not require light during this vegetative phase. A warm, dark location will do the trick. The ideal temp for Ganoderma lucidum during this phase is approximately 24°C (this is true for most species grown for food or medicine), though anywhere in the range of 21° - 27°C is good. You do not need to keep the lunchbox lid slightly open. There is more than enough oxygen in the sealed container to meet the requirements of the mycelium. Most saprotrophic species (decomposers) are used to growing under conditions with elevated carbon dioxide levels.

Looking forward to seeing how your experiments progress!

[Merlyn]

Thanks @glyph and @Kiwii for the awesome input and information.
I just made 3 fresh plates of agar, with regard to your advise.

I flame sterilized my scalpel before alcohol was added to the glovebox.
Then i proceeded to spray it done some more from the inside.
However to be honest i was a little worried about the alcohol fumes possibly killing the mycelium when i opened the lids - so i waited a little bit (as patiently as possible) for the alcohol to evaporate a little.

Then i unwrapped the mother agar dish to transfer from into the new fresh agar. Seems like im getting a little more used to the gloves now.
Before it was extremely tricky to take off and put on micropore tape…

I made cube incisions into the mycelium agar towards the rhizomorphic growth away from the cloudy looking “mycelium”, extracted and gently placed them into the center’s of each new petri dish - on top of the fresh agar (except for one, which fell to the side off my scalpel mistakenly)

I must say the whole experience was quite stressful, just exposing my dishes like that, even in the still air.
I was also wondering about the black soot formed on the scalpel after flame sterilizing, couldnt that potentially contaminate the agar?

I didnt want to wipe the scalpel clean before extraction just in case i contaminated it.
But anyways a little bit of black residue from the burnt scalpel was left on the agar.

I have now stored the 4 dishes (including the original mother) in my cupboard, all sealed up.

Tomorrow I will send photo’s. When the mycelium is a little more settled for its photoshoot.

Thanks guys for the assistance so far, will keep you posted as it develops

[Merlyn]

This is the 3 fresh agar plates i prepared, with a picture of the mother agar i transferred from…

!

Mother dish.
As you can see condensation is going crazy, looks like a bloody rainforest in there!
image|375x500

[glyph]

Hey @Merlyn, thanks for the updates.

@Merlyn

I must say the whole experience was quite stressful, just exposing my dishes like that, even in the still air.
I was also wondering about the black soot formed on the scalpel after flame sterilizing, couldnt that potentially contaminate the agar?

I didnt want to wipe the scalpel clean before extraction just in case i contaminated it.
But anyways a little bit of black residue from the burnt scalpel was left on the agar.

Haha yes, plate work was very stressful for me too at first! Lots to think about. I can tell you though that it becomes much more relaxing and enjoyable as you gain experience, practice your technique and develop muscle memory.

Nothing to worry about in terms of the black soot. I imagine you’re using an alcohol lamp? At some point in the future you might consider switching to a handheld butane torch. That will allow you to rapidly sterilize your blade without any residue. That being said, the alcohol lamp is perfectly fine - just takes a little longer.

You mention working in a glovebox. Is it a plastic tub with rubber gloves attached to the armholes? Or is it a still air box (SAB) with armholes but no attached gloves?

Don’t worry about the condensation. It’s a bit of a bother (not being able to see the agar and mycelium clearly) but it won’t cause any major issues, unless you’re getting so much condensation that it’s forming large water droplets. I generally store plates upside-down if they’re going to be in storage for a while (weeks or months). That ensures that no water drops or puddles end up on the surface of the agar.

One trick is to stack the plates on top of one another right after you pour them and before you wrap them in tape. You can also add a cup of hot water to the top of the stack. This seems to reduce the amount of condensation in each plate.

Keep a close eye on your mycelia as they expand across the agar. Hold each plate up to the light - you’re looking for any spots or discolouration away from the mycelium itself. If you see any spots, it’s likely that they are contaminants. If you encounter contams, transfer the healthy mycelium on to new agar as soon as possible. Keep running until you get clean cultures.

I know it’s quite a lot to process and learn but you’re doing great!

[Merlyn]

I am using a sealed DIY glove box; a plastic tote with rubber gloves attached to pvc armholes. @glyph

So I struggle a little with sterilizing the scalpel outside the glove box. I think my future technique will be flaming the scalpel inside the glove box then spraying down the glove box with alcohol from the inside. This time i moved the sterilzed scalpel from outside to inside the box, i know this wasnt ideal. But unfortunately my glove box was fuming with alcohol gases and reside; frankly didnt feel like bombing myself over inoculation.

Anyways. These 3 dishes in the pictures are 5 days old. One in particular seems to be doing better then the other 2 transfers. I have also included the original petri dish.

I am curious to find out if those white spots around the original “mother” are: bursts of mycelium or contamination? Hmm.
After all, I ended up squirting a few many drops all over the show hehe

I will be shifting over to a butane torch, to escape the residue. Thanks for that.
Another friend of mine told me the same trick with the hot water glass, ill give it a bash next time i prepare agar.

The whole process has been quite rewarding just in the experience of watching these beautiful networks grow on a 2D plane.

[glyph]

@Merlyn

I am using a sealed DIY glove box; a plastic tote with rubber gloves attached to pvc armholes

I recommend removing the rubber gloves completely and using the tote as a still air box (SAB). The attaches gloves do not offer a significant advantage and only serve to get in the way and reduce your dexterity. You’ll find it much easier to sterilise your scalpel blade once the gloves are removed; simply place your alcohol lamp outside the front-left or front-right of your tote and move your hand holding the scalpel outside when needed (moving slowly to avoid major air perturbation).

More SAB tips - not all strictly necessary but they’ll give you something to think about (roughly in order):

• Clean your work surface with warm soapy water
• Soak a freshly-laundered towel in a weak bleach solution, wring-out the excess water and place this on your work surface (if your work surface is wood, rather use the lid of the tote as a base - upside down - and place the towel on top of it)
• Place a clean baking tray or similar object on top of the towel (this gives you a raised surface to work on; any contaminants in the air will fall past the tray and land on the towel) - stainless steel is best
• Use warm soapy water to clean the inside and outside of the tote prior to each work session
• Wipe everything that’s going to be inside the SAB with 70% alcohol solution and place it on the tray
• Place the tote (upside down) onto the towel, with the tray inside; don’t worry about spraying the tote with alcohol since you just washed it with the warm soapy water and none of your items are coming in direct contact with the tote
• Using a spray bottle with water and a few drops of liquid detergent, spray 2-3 times inside the SAB - the idea here is that the water+detergent will adhere to any dust or other relatively large molecules in the air and pull them to the bottom of the SAB
• Close all doors and windows in your workspace to reduce ambient airflow as much as possible (no fans on)
• Allow a couple of minutes for the air in the SAB to settle before starting to work
• Keep a spray bottle handy with 70% alcohol solution and wear nitrile gloves while working in the SAB
• Practice your sequence of movements in your mind before you begin working
• Move slowly yet decisively - try to ensure that your hands never move over the top of open plates

You can achieve 95-100% success rates in a SAB with proper technique. It may take years of practice, but you will get there with determination and mindful movement.

Back to your plates: the spottiness in your last image looks a bit suspicious to me. The growth in your third image looks more promising, especially toward the top-right corner. I would transfer from that leading edge to new plates. Don’t worry about cutting a big cube; you only need a small piece. Aiming for efficiency in all aspects of your practice will save you a lot in the long-run. Good luck!

[Merlyn]

Those SAB tips are amazing. Thank you for sharing

Ill be doing smaller chunks of agar wedges in the future, first time doing this and didnt realize they can be much smaller and triangular

As for the glove box: Would I not be exposing my tote to more contamination if i removed the gloves?
Since airflow through the exposed holes could potentially take contaminants inside my glove box from the outside.

Another mycologist also told me I should remove the gloves so there’s obviously validity to that but just trying to grasp the concept further. I will take off the gloves regardless

[glyph]

@Merlyn

As for the glove box: Would I not be exposing my tote to more contamination if i removed the gloves?
Since airflow through the exposed holes could potentially take contaminants inside my glove box from the outside.

This concept / technology can be quite counter-intuitive at first. I highly recommend reading this page from the Shroomery: Still Air Boxes, Gloveboxes, and Flowhoods. I’ll highlight some key points here:

A still air box (SAB) works on the principle of still air. A SAB does not have to be sanitized, and cannot be sterilized. You cannot sterilize something that’s in open air, the air we breath is loaded with contamination. Air inside the still air box is well, still. Particles that encounter still air lose their velocity quickly and tend to fall straight downwards. Thus by cleverly working in a SAB and never moving any non-sterile things over the top of sterile things you can achieve great success.

A glove box is just like a SAB but it has attached gloves. Attached gloves means you create a “piston effect” when you move around creating more air currents. Since you cannot sterilize a glove box you will have little to no success trying to attempt working like that. You can sanitize the inside of a glove box but remember sanitizer does not kill everything and you will still be at risk. A still air box doesn’t have to be sanitized to work properly - it works on design and physics.

A glove box also inhibits you from being able to flame your tools outside of the box. which is a huge disadvantage as you will need multiple pre-sterilized disposable tools.

There is a section at the top of the linked page differentiating between disinfection, sanitation and sterilisation; the difference is vital for understanding the how’s and why’s of effective cultivation technique.

[Merlyn]

This is very helpful information and will be implementing these procedures in my next run

Thank you.

I actually learnt a very hard lesson with regards to agar. I used parafilm that had been soaked in bleach water to wrap my agar plates. Turns out the bleach water seeped through the lids and got into my plates. Lost quite a few dishes to such a silly mistake. But i guess its part of the learning process…Next time ill be sure to dry the parafilm before wrapping the petri dishes

Made me even more inclined to potentially invest in a laminar flow hood, to reduce the amount of disinfectants used during the inoculation process.

Anyways thanks everyone for all your advice. Please dont feel inclined to respond to this message, i just wanted to express my gratitude

[glyph]

There’s no need to bleach the parafilm. Peel-off the paper and fold the plastic lengthwise so that the outer surface is on the inside, then wrap your plate.

Some photos to illustrate (I’d usually be wearing gloves and performing this process inside a SAB).

The “inner” side of the plastic is clean.

Once you’ve peeled away the paper, fold the parafilm so that the outside, previously-exposed surface is inside (note: I usually peel the paper off completely before doing the folding; I left it on here for demonstration purposes).

You end up with a folded piece of parafilm that looks like a cigarette rolling paper. The outside surfaces are the clean ones. Now simply wrap your petri dish - no bleach required.

[notplants]

Place the tote (upside down) onto the towel, with the tray inside; don’t worry about spraying the tote with alcohol since you just washed it with the warm soapy water and none of your items are coming in direct contact with the tote

I was revisiting this amazing thread, and I’m wondering about your SAB method @glyph

do you have a picture of this setup with the towel and tray by any chance?

I’ve been using a SAB with arm holes cut into the lid, on its side, that looks like this:

I generally clean and spray down the whole thing with alcohol before using, and try to close all windows and not be too frantic while using it.

Is the point of the towel as the bottom that it traps more falling particles which have fallen to the bottom of the SAB than if it was some other type of ground (like the bottom of the plastic box)?

I’m curious to try to decrease my contam rates, and about this towel method, especially now that I’m trying to do agar in a SAB

[glyph]

@notplants

do you have a picture of this setup with the towel and tray by any chance?

I don’t have a photo but I just drew a diagram to try and explain:

The white bit is the towel. It must be larger than the opening (top) of the tub. It helps to prevent any air flowing through tiny gaps between the rim of the tub and the table. The black lines represent the tray. The blue bit is the tub. With this method, the arm holes have been made in the side of the tub and the tub is oriented upside-down (so that the bottom of the tub is the uppermost surface).

[notplants]

this drawing is helpful, thank you!

I missed before in the writing that the tray is actually something which small things and particles can fall through — just a sort of platform