I started on a new hobby several months ago. I’m trying to grow Chinese Paddy Straw mushrooms (volvariella volvacea) in semi-sterile laboratory conditions. These mushrooms are delicious fresh in the button stage tasting like olive oil and cashews to me. For various reasons they are not available fresh in the United States and only a compatively tasteless canned mushroom is for sale here.
The problems are basically harvest (the preferred “egg” stage only lasts 8 hours so harvest must happen 3 times daily), the shelf life of the egg is 3 days maximum so farm to market to consumer must happen in a big hurry like milk before refrigeration in the old days), biological efficiency is low (typically 20% of dry mass of food is turned into mushrooms), they die in low temperatures, and require 85F-95F temperature and 90%-95% relative humidity to grow well (only a few spots on the gulf coast of the US are suitable for outdoor cultivation). Anyhow, I love a sciency engineering challenge like this. Plus I like growing things.
I didn’t want to be anal about clean-room procedure so I add ampicillin to all my agar recipes (available without prescription as Fish Cillin). Antibiotic agar is commonplace but not with ampicillin as it breaks down at temperatures well under pasteurization and WAY under autoclave temps. On the other hand autoclavable wide spectrum antibiotics are hideously expensive and ampicillin is dirt cheap in comparison. So I add a step to pouring petri dishes which is to add ampicillin to fresh agar when temperature falls below 140F. (Plates not used right away must be refrigerated or the ampicillin will begin breaking down even at room temperature in a week or so.) This doesn’t give you a lot of time to pour before the agar starts to congeal. As a result I get no bacterial growth on culture plates and thus do not have to worry about sub-micron air filtration. So I do all my innoculation on a dusty desk in a dusty room in front of a HEPA breeze from a cheap Walmart room air filter and just wipe down the immediate area with alcohol and use an alcohol lamp to sterilize a scalpel. My only real problem child is penicillium spp. (blue-green bread mold) which if I’m too careless its big old 18 micrometer spores that are everywhere drop like a depleted uranium balloon right through the HEPA breeze. It germinates in about 3 days on agar and being an imperfect fungi quickly produces massive quantities of spores 24 hours after that.
Volvariella is quite aggressive and if a reculture is being performed from an active volvariella colony it will cover a petri dish in 3 days and be climbing the walls trying to escape before any penicillium can hatch. However, long term storage of commercial (read very aggressive) strains of volvariella is problematic. Unlike every other mushroom in the world where cultures can be stored at near freezing its mycelia dies at any temperature below 45F. So I developed a 48F refrigeration strategy using a modified household refrigerator to put the mycelia into stasis. Real commericial labs either reculture once a week (eventually resulting in senescence of the strain from accumulated random micromutations and a major pain in the arse) or use liquid nitrogen storage (well beyond the hobbyist) or work with a less aggressive strain with far less biological efficiency (mass ratio of food to fruit) where cultures will store at room temperature for months without dying. So far my method works fine but after 90 days in stasis (as long as I’ve had my first cultures in cold storage) it takes the mycelia 5 days to come out of stasis. That’s enough time for pencillium to blossom and spread.
So anyhow, I was just a few minutes ago looking at some recently innoculated plates from 90 day-old volvariella in stasis and I wasn’t scrupulous about wiping everything down with alcohol before innoculation. About half the plates got penicillium contamination. Volvariella is racing the penicillium to occupy the open real estate.
Looking at the contaminated plates it struck me there’s a whole new meaning for the popular phrase “culture wars”! 😉
HAHAHAHAHAHA!!!! I’m such a geek.
As long as I’m thinking about it I should probably expound on how my understanding of targeted micromutation and natural selection has led to understanding and success working with vv colonies.
Several observations to note about these rapidly reproducing colonial eukaryotes capable of both asexual and sexual reproduction.
1) Asexual reproduction over scores of generations results in senescence – a lack of vitality. The members of the colony and their cloned offspring basically get old and die. Allowing them to sporulate (sexual reproduction) results in some of the spores having renewed vigor.
2) Continued use of the same food source like PDA (potato-dextrose agar) results in a colony that can digest PDA like the dickens but is crippled on MEA (malt-extract agar). The longer the food source is unchanging the greater this effect. Periodically changing the food source keeps them able to digest a range of foods with minimal learning curve for a new source. The final food source is often added to agar (like powdered rice straw) so they don’t “forget” how to digest what the colony is eventually going to be given for a fruiting substrate.
3) The colony dies rapidly once it has engulfed a food source if conditions are not suitable for fruit formation. If the temperature is lowered just short of deadly the colony remains alive in a state of suspended animation.
Now we form an hypothesis based on the evolutionary concept of rapid directed mutations in response to stress. This is a known way that bacteria rapidly acquire antibiotic resistance. Bacteria turn up the mutation speed on selected genes in response to antibiotic poisoning. If a eurkaryote has the same ability lets hypothesize that vv colonies can rapidly mutate their repertoire of digestive enzymes in response to the environment.
Now apply it to the observations (not having a genetic lab I can’t really test these explanations but if they lead to practical results they’re useful nonetheless).
1) Presuming a rapid mutation rate which never gets completely turned off vv accumulates errors in the genes specifying its digestive enzymes in clonal reproduction. It eventually loses the ability to digest food as the enzymes deteriorate. Sexual reproduction performs a “reboot” operation where a standard set of alleles reemerges from which the process of fine tuning of the enzymes through rapid mutation can start over.
2) Presuming a rapid mutation rate which never gets completely turned off enzymes not critical for the food source in question deteriorate through genetic drift (no selection pressure) while the ones critical to the food source continue to be refined by high natural selection pressure. Changing up the food source before the drifting genes become too dysfunctional keeps the entire suite fresh and frosty but not optimal for any one food source.
3) Colony dies rapidly in culture after it has covered the entire plate. Assuming a stress response of rapidly increased mutation of digestive enzymes when it hits the glass wall of the petri dish and starts climbing up the sides (it’ll fill the entire inside of the dish hunting for food). The colony is basically trying to learn how to digest glass. Rapid mutation of digestive enzymes without success eventually leads to production of enzymes which digest the vv colony itself. It poisons itself. Lowering the metabolic rate to near zero with cold temperature halts the rapid mutation rate and keeps it in the state where it’s not stressed out from lack of fresh food.
One last note here. It has never once occured to me that in a vv colony’s rapid mutational search through digesitive enzyme space to digest an unknown food source the mutation will produce teeth and claws so it add the hunting of live rodents to its potential food sources.
HAHAHAHAAHAHA!!!! Random mutation and natural selection have obvious limits in practical application. No matter what they tell you there is absolutely no practical benefit in applying macroevolutionary fairy tales to experimental biology. None. Zip. Zilch. Nada.
Now if you’ll excuse me I’ve got to run out to the store a pick up a couple dozen petri dishes and cook up a pint each PDA and MEA. My culture stockpile is running low… and the culture wars shall continue.