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Hello every one.
Way back in 2018, I wanted a method of taking tissue samples from wild mushrooms in the field as well as at home. I love to walk through and explore nature. Sometimes finding mushrooms that I would like to sample for study but not always do I want to carry all these mushrooms back home for work in my lab. With many "Back to the Drawing Board moments and designs" the Open air agar pots came into existence 0ctober 2019.
If you have ever tried transferring tissue samples in a still air box you will start to understand the problems faced with outdoor or indoor tissue sampling or cloning. The air is full of nature, fungal spores and bacteria are everywhere and as soon as you open the petri dish to transfer the sample. All of nature wants to join the party.
Of course the great main goal of cloning a single mushroom for reproduction as liquid mono culture is that you will get an exact replica of that mushroom. Liquid Mono Cultures will yield better results than Multi spore mushroom grows, have shorter fruiting cycles and replicate the characteristics of the clone parent. Ever seen a mutant that you wanted a whole monotub of?
The only way I had of taking clean samples and transferring them with perfect results up to then was to remove the mushroom, bracket or fungus from nature and drag the poor thing into my lab for dissection. In my mind all mushrooms were fruits and so free for the picking. Then I read that some Brackets like the Fomitopsis pinicola, Red belted Bracket fungus, grow a new layer of tubes each YEAR giving the appearance of a white lip at the outer edge of the bracket........ This Bracket had 7 layers. That means that this Bracket had existed for 7 years before meeting me.
How could I remove this little life form from its home just to satisfy my Mycological interest.. The only way to sample this beautiful specimen was to take a tissue sample from the bracket without removing it from the tree stump. So with a 14g needle, alcohol swab and an Open Air Agar pot I was able to do just that. Take tissue samples in open air... ANY WHERE.
To find out how to clone your favorite mushrooms for replication, please read on......
****As a warning. If you are not 100% sure of the species you are working with, you should never consume wild mushrooms****
Open air agar pots come fully sterilised with two injection ports and a filter patch for air flow. Using our famous Blue Smurf Agar to feed any tissue OR spore you would choose to sample
Choose the Fungus that you would like to clone. For this example I had decided to take a sample from the largest of the brackets pictured.
You choice should be 100% free of visual contamination or damage. If you would like dense canopies in your mono tubs. Try and clone the biggest and proudest mushroom from a CLUSTER of mushrooms. The cluster characteristics and large size will be carried forward with your tissue choice.
As we need to work fairly quickly, it is always best to connect your 14 gauge needle to your syringe so it is ready ready for use. To do this. Simply open the needle pack and syringe packet. Now screw the needle into the end of the syringe but leave the protective needle cap in place. (Pictured right in orange). I always take with me: 8 x Open air agar pots, 8 x 14G needles and 8 x 20 ml syringes.
The Open Air Agar Pots have an 2-3 month shelf life if kept cool and secure. After this time the agar inside tends to be too dry to work with.
Take a trusty alcohol swab and clean the area that you want to take a sample from. You want to wipe the area to try and kill as much surface contamination as possible. The chemical action of the alcohol and abrasive action of the swab will do a great job.
When you have wiped the sample area and before the alcohol dries. Simply remove the orange cap from the needle and insert the needle into the Fungus. For this species I inserted the needle to a 3cm depth. Screwing the needle into the fungus will help cut a better core sample.
Leave the needle INSIDE the fungus.
Also at this point I have not sucked any air into the syringe. This will be done just before removing the needle from the target. By slowly drawing air into the syringe while the needle is still inside the tissue of the fungus will allow the fungus to act like an air filter. Alternatively, you may draw air slowly into the syringe when the needle tip is inside the Open Air Agar pot where the air is sterile. We will need air inside the syringe to help spit the core sample out and onto the agar surface.
With the needle still inside the fungus and safe away from contamination. We can turn our attention to the injection ports located on the lid of the Open Air Agar Pot.
This is where the core sample needle will be inserted to transfer the core sample to the agar surface. Give the port a good wipe. The pots come with an Aluminium foil layer to help protect the pot from possible contamination but a wipe of the port is good practice.
When you are ready, pull the needle out of the Fungus and quickly insert the needle into the cleaned injection port. When the needle is inside the pot. You need to now gently pull some air from inside the Agar pot into the syringe ready to push the core sample back out of the needle and onto the Agar surface. Draw the air slowly or you will pull the core sample into the syringe.. Of the 20ml available air space inside the syringe. Please slowly draw 3-5ml of clean air so you can push out the core sample.
With a quick push of the syringe plunger, the air inside the syringe will eject the core sample on to the agar. If the core sample does not come out the first time. Slowly draw 10ml of air from the agar pot and again with a quick press of the plunger the core sample should eject.
When the core sample is on the agar. You may remove the 14g needle and inspect your sample.
Now that you have successfully captured the sample all you need to do is clear the area of any human rubbish
Always, always, always. CLEAN UP YOUR RUBBISH.
It will not take long to make sure the area is as clean and clear as when you arrived.
Taking all your rubbish home will make sure the area is there for everyone to enjoy.
Now, its time to go home and incubate your agar pot to help the sample grow.
For my studies. I have found that incubation temps of 20-27c work great for most species and strains.
I will leave my pot to incubate for ten days before having a look at how things have grown.
So after incubating the Open Air Agar Pot at 26-27c for ten days. The mycelium has spread from the core sample from two sides. The far side is where most of the sample fell. On the side closest to the camera is where a piece of the core sample landed and this side is the least contaminated.
Of the 4 main colours inside the Agar pot we have.....
1: Blue- under white-This is the Agar colorant use in SporeBuddies Blue Smurf Agar.
2: White- two half moons-This is the colour of the target Mycelium.
3: Black- far side-This is a fungal contamination known as Aspergillus niger or black mold. We don't want this...
4: Yellow- near side-This is a bacterial infection and could be one of many unwanted bacteria.
I will now take the Open Air Agar Pot to my flow hood where I can safely open the agar pot to cut and transfer a small sample from the cleanest area of target Mycelium. In this case, I will cut a sector from the half moon closest to the camera.
2-4 Transfers of mycelium to new agar dishes should give me a clean sample.
We have a great selection of Agar and laboratory equipment for sale in our shop. If you cant see what you need please let me know..
Known as the penny bun and Porcini meaning "LITTLE PIGGY" in Italian
Found growing on the tip of a dead Ash branch. 20m away from the Lake
Captured in Exeter, United Kingdom and taken 170 miles back home for study.
Mushroom species found in the wild may possible be poisonous. It is never advised to consume Wild Mushrooms unless you are 100% positive of the species.
****The above information serves only as educational information and should not be followed. The author accepts no responsibility for your actions****
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