I have a few questions and I'm interested in liquid culturing - I’m new and don’t want to clutter your thread, but you have great experience and I’m wondering about some things brought up on this thread:
Any thoughts on sterile water vs nutrient solution for storage? An analogy might be use use of refrigerated agar slants for long term storage.
The scientific article cited referred to a ‘cork punch’ used to extract mycelium from agar. I have had good luck with one of these sets - cheap, autoclavable (or I just flame and dip in sterile water to cool it in between samples). See attached image - can't get it to load inline unless it's a URL.
Blender question or warning - Blenders are indeed used to disrupt cell membranes to emulsify cells for analysis. Be careful, I guess about hitting the myc too hard with the blender. I thought about using a blender but then decided not to, but then I do think it’s a good idea to break up the myc to at least some extent for a liquid culture. What are your thoughts on this? Run blender briefly? Low speed? Modified blades?
I noted your malt conc was 10 gm per dl. I have read on this forum that perhaps 5 gm per dl was a “max” although I guess we are talking about grams % and not molarity or osmolarity. Malt, I guess, is a mostly a starch, which would have a large molecular weight and thus less osmolarity than, for ex., dextrose. The hazard with high conc might be osmotic effect on the cells (?)
Can you give a reference to the flasks with the screw tops? Have not heard of that. Erlenmeyer, I presume?
Thanks for all your work.
For long term storage in liquid media it cant have any O2 or Nutrients. If it has O2 then it will want to conduct cellular respiration which requires energy and thus will eventually starve to death. If there is nutrients then again it will want to conduct cellular respiration which requires O2, thus it will suffocate.
To get around both of these you sterilize a jar, tube , etc of distilled water with no GE hole and this will create a vaccum. Then you harvest the mycelium from a agar plate leaving behind the agar and blend it thank you needles for my blender lids and motor, then you draw into the syringe and when you inject the container with the sterile distilled water under vacuum, it pulls in the mycelium water, which also needs to be distilled as i forgot to mention, the contents will be pulled in by the vacuum.
The only O2 in the water will be disolved and minimal. Note the O2 molecule making up the H2O is not a available source of O2 for the mycelium just the free O2 molecules.
Simply stated the culture seases all metabolic functions which puts it a state that goes beyond hibernation and more like a antimated state of suspension.
Spores can be stored the same way. I posted a link somewhere around this site where a experiment was done with a dozen cultures of many species and over 90% were recorded at the 4 year mark when the experiment was concluded. Could they go longer? Absolutely. Some species may not keep as long or not at all but im certain Cubes will keep a very long time, probably a decade.
My buddy said Peter McCoy talks about it in his Rodical Mycology Book which i have yet to read after he gifted me a copy a few years ago(I have ADHD and cant focus very long) but this lead me to do some research and it is limited but there are some good studies out there if you dig deep enough.
I believe this can replace master slants because they require maintenance and although its slow, there is still metabolic activity thus the culture is aging and needs Fresh air exchange periodically.
Finally and not to get to far off topic, you can also use cryo- protectants such as a food grade propylene glycol. I dont have the chart handy but it doesn't take much to lower the freezing point of liquid to below freezing. I want to say 3% gets you down somewhere around 27° i think. Even though PG has antifungal properties, at that concentration it is not toxic and can actually be a food source. H8gher concentrations beyond 5% probably needs to have the culture rigorously exposed to it slowly upping the concentration but would be worth it if you can get the culture into sub zero temps without harming it.
The only down side to this and this speculation, im not sure this training will be passed on to subsequent generations. The PG being recognized as a food source probably would but that is irrelevant as the goal is to put it in a state where all metabolic activity halts so no eating allowed. The other and is fact, the culture needs to slowly and i mean very slowly brought back up to room temp. You can't just toss it in a incubator.
This IMO would be superior to all other long term storage methods and can easily be done at home with a deep freezer. I started a trial but abandoned it for some reason, maybe my LC contaminated i don't remember but it has not been forgotten.
Hyphae are single cell wall thick including the septa that bonds the colony or all the individual hyphae, it doesnt take much to destroy that infrastructure and happens frequently in many processes. The colony will repair itself and grow back together or begin to form individual colonies once put back into a nutrient media.
On the contrary, my favorite thing becauae I see both sides of most things, i can certainly see those individual hyphae that get obliterated not making it through the long term storage but keep in mind were not creating inoculate and you will see large strains of mycelium after the blending process. For simple conversation, even if only a few strains of mycelium survive, its successful because the goal is to bring in back to its normal metabolic functions then can be expanded back to create another healthy colony with X amount of mycelia mass.
@needles one of those BD 50' s , the flange at the top broke durung shipping. Thats the one I'm giving you when come through
thanks for letting me throw a little info out there on the subject.....
Sent from my LGLS992 using Tapatalk