My intent: a liquid culture solution of cloned tissue, in sterile distilled water, for inoculation and/or storage purposes.
My initial considerations:
- I hate mixing/pouring agar and dealing with Petri dishes (pre existing neurotic condition based on past work experience – best not to ask). Also, I have an irrational fear of Parafilm.
- Every time source material touches air, even HEPA filtered air, there is a contamination vector introduced. I want access to my master culture via a flamed syringe passing through a self healing injection port. Petri dishes/slants involve multiple opening/closing rituals and the dreaded Parafilm.
- IME, throwing chunks of tissue straight into a liquid culture produces very unpredictable results and is not a satisfactory solution.
- Mycelia (in distilled water) has been shown to remain viable after extended periods of storage. Ending up with macerated mycelia in distilled water is very much desirable.
Begin by mixing up a liquid culture broth of your favorite sugars and/or additives. I like light malt extract and dextrose (1:1 by weight) at a %4 dilution in tap water with just a pinch of bee pollen. Any LC recipe should work (honey, Karo or Lazlo TEKs should be fine). All you need is sugar and water in the right percentages. I have seen pure Karo and pure dextrose both work adequately. The sugar(s) you use are less important than the dilution ratio. Not sweet enough results in slow growth; too sweet results in no growth whatsoever. Note: a proper liquid culture broth is just barely sweet to the taste by human standards. An LC will have almost no smell.
Add 9mL of your LC broth (syringes work great for measuring small volumes) to the bottom of a pint or ½ pint jar. I’m using ½ pints for these pictures. Put three glass marbles in the jar to aid in swirling (and later macerating) the mycelium. Shards of broken glass work well for shredding the tissue later, but tend to get in the way of gentle swirling. IME, shards result in slightly slower colonization but also slightly less tendency for syringes to clog later. To me, marbles are superior because I cut my clumsy ass fingertip while cleaning out a “shard jar” (clogged syringes are way better than infected index fingers, IMHO).
Don’t add more than 9mL of LC! The idea is to have a dry island in the center with nutrient pooled all around. It’s kind of a castle/moat situation. Once your jar is filled put it in the PC for no longer than 25 minutes at 15psi (if you PC for too long the sugars will caramelize and the LC will not work).
You’ll notice from the pictures that my jar lids all have tyvek vents in the center. I always use lids with tyvek vents to avoid a vacuum forming in the PC. Vacuums can suck in contaminants.
In your most sterile environment, tear a mushroom stem in half. If you let the stem air dry for 18-24 hours before tearing, the inner materials will shrink away/differentiate from the outer stem and make tissue extraction easier. You can see this shrinking/pulling away in the picture below. I should have taken a closer shot.
Using a brand new sterile blade, excise a pencil eraser sized piece of tissue from the center of the stem. Dull blades make this difficult, time consuming and more prone to contamination. To make your shroom surgery quick and efficient you should use a brand new blade. This is a total non-sequitur, but can anybody out there give me a dime store explanation as to why flaming blades makes them dull?
Spear the tissue chunk lightly with the blade and place it in the jar. Open the lid as little as possible and for as short a time as possible. A few practice runs will make this FAR easier. Ideally, you want to open the jar, dunk the tissue in the LC then tap/drop it into the center of the jar (in one smooth motion) while opening the lid only about ¾ of an inch. In less than three seconds. Easier said than done! The neophyte will probably find it easiest to spear the chunk, open the jar just a little and scrape the chunk off on the inside (sterile) edge of the jar.
Try to end up with the mushroom chunk in the center (dry) area of the jar. If the chunk starts over at the edge, just shake and/or tap it back to the center. The process will not fail if you can’t get the chunk back to the center (it just colonizes a little slower). Do NOT open the jar to reposition the chunk, as it isn’t that important! Please note, the chunk below has been stained blue to make it easier to see. That size chunk seems to work quicker than smaller pieces, by the way.
Put your sealed jar in the incubator and swirl (GENTLY) once every day for 2 weeks. This will also work (just slower) with no swirling at all. When swirling, the idea is to lap nutrient up around the edges of the growing mycelia, not to drench it. Overly wetting it by shaking violently will make it grow MUCH slower. A cool ring pattern, like rings in a tree trunk, will be visible on the bottom of the jar after a while. During the growth process, any contaminants are easily identified by carefully examining the jar from the sides and bottom (a flashlight helps). Anything that looks remotely non-mushroom like should be disposed of immediately. Note that some darkening/browning/bluing will happen to the bottom of the clone tissue chunk after a while.
Below is pictured a clone jar after 2 weeks in the incubator. You will note that almost all the food source is gone. This jar actually had three chunks of starter tissue placed in it. Three little pieces didn’t seem to expand nearly as quickly as one big piece, however. The biggest chunk of starter tissue is still recognizable as the oblong beige lump just off center. The two smaller pieces dissolved almost unrecognizably into the cloudy structures to the right and top of center.
You should NEVER open your clone jar to look at it like this! I have done so to take a picture (this culture will be disposed of). I removed the marbles because my camera’s auto focus kept centering on them instead of the mycelia.
It is redundant to repeat it over and over, so from here on assume that syringes are flame sterilized red hot before insertion into any sterile jar. Also, they syringes are NOT fully inserted. Only the ¼ inch of metal that gets red hot goes inside the jar.
If you are clumsy, like me, “blowtorch” type refillable butane lighters make a great substitute for alcohol lamps when flaming syringes. I will invariably turn over and/or burn my forearms on alcohol lamps.
Using Hippie3’s airport syringe to equalize the pressure, a 60mL syringe is filled with sterilized water (use distilled for long term storage). It takes a second or two after you finish pulling up the plunger for the pressure to equalize completely (submerge the airport needle and wait for the bubbles to subside). If you pull out the 60mL syringe too soon, some unfiltered air might get sucked in through the hole in the silicone injection port. As always in this hobby, patience is a virtue.
Again using the airport syringe to equalize pressure, the 60mL of sterile water is injected into the clone jar. Do this a second time (120mL total). If you are using a pint, inject 240mL of water. You will have more than enough mycelium for a pint of clone water after 2 weeks of healthy growth.
The jar is now shaken VIOLENTLY for 8-10 minutes (the longer the better) until it looks like the picture below. The longer/harder you shake, the less easily your syringe will clog. Don’t worry about damaging the mycelia (tearing it to tiny shreds is your objective).
A syringe (again using the airport to equalize pressure) is now filled with clone water from the jar. Once filled, the syringe will look like the picture below.
The syringe will clog with mycelia as you fill it. Just squirt a little back into the jar, shake, and begin drawing again. Sometimes you can fill the syringe in one clean pull. Sometimes it takes 19 tries. Just be patient and keep working with it until you get the results pictured below. This is a close up of a clone water syringe with great potential:
This syringe of clone water can now be used to inoculate whatever your heart desires. A small amount goes a long way, especially if you use the clone water to start a liquid culture. Don’t clone clones, though. Go back to spores at least every third or fourth generation. We aren’t growing orchids. Our mushroom of choice drops (in most cases) abundant spores that are easily collected, stored and germinated. There is no reason to toy with the possibility of senescence. Also, over the years, going back to spore will encourage a strain to adapt to your grains and fruiting environment. They will form a relationship with you…
If your objective is long term storage of the culture, leave the jar in your incubator for 3-5 weeks until all the nutrients are consumed (other than some condensation, the jar will be pretty dry inside). Please note that clone water from jars which have eaten all their food seems to colonize jars much slower than clone water drawn while mycelium is actively growing.
If fast colonization is your objective, shoot/shake before all the food is gone (2-3 weeks). If storage is the objective, wait for ALL the food to be gone before you shoot/shake (3-5 weeks). Storage should be in the fridge (not freezer) in an air tight and light proof container.
I can’t personally testify to the long term efficiency of sterile water storage but the work of other mycologists indicates that cultures in sterile distilled water should remain viable for 5+ years. If all the food is gone before you shoot/shake, your culture should go into suspended animation and stay viable for at least 2 or 3 years (in the fridge).
Your mileage may vary and reports of your experiences are infinitely appreciated!