@Lando and I were discussing what actually counts as a “master culture”, and where to start counting number of generations from master to bulk.
Should we only count generations since sexual reproduction? Or if we clone mycelium from a fruiting body, is that also a sort of fresh start?
What is the industry standard for this?
I’m current looking to see what Stamets has to say about this…here are some excerpts relating to grain generations (this also relates to your other thread about rhythms):
The first time mushroom mycelium is transferred onto grain, that container of spawn is called a grain master, or G1.
The next generation of grain jars is denoted as G2. Each grain master can inoculate 5 to 20 times its mass. Many start with narrow mouth quart mason jars for grain masters, and use 1/2-gallon or 2-liter jars for second-generation spawn. Third-generation spawn is typically in bag form and is sold to consumer-growers.
A standard inoculation rate would be 1 quart (liter) grain master to 5 1/2 gallons, in other words, a 1:10 expansion. […] Liquid-inoculation techniques allow a much greater exponent of expansion than the traditional method described here.
Third-generation grain spawn is inoculated in exactly the same fashion as second-generation grain spawn. However, contamination is likely to go unobserved. Some large spawn laboratories successfully generate fourth-generation spawn. However, contamination outbreaks discourage most from pushing this expansion any further.
Most laboratories do not fully realize the potential of every culture. In many cases, spawn expansion is terminated at G2. Many spawn managers choose not to “chase the optimum”. Few laboratories are large enough to accommodate the end result of the methods described here.
For those who have the appetite, I highly suggest reading this book cover-to-cover.
Stamets on sexual reproduction and senescence:
Every sexually reproducing organism on this planet is limited in the number of its cell replications. Without further recombination of genes, cell lines decline in vigor and eventually die. The same is true with mushrooms. When one condiers the exponential expansion of mycelial mass, from two microscopic spores into tons of mycelium in a matter of weeks, mushroom mycelium cell division potential far exceeds that of most organisms. Nevertheless, strains die and, unless precautions have been taken, the cultures may never be retrieved.
Culture slants are like “backups” in the computer industry. Since every mushroom strain is certain to die out, one is forced to return to the stock library for genetically younger copies. Good mushroom strains are hard to come by, compared t the number of poor performers isolated from nature. Hence, the Culture Library, aka the Strain Bank, is at the pivotal center of any mushroom cultivation enterprise.
McCoy notes that “lack of environmental stress” is also a contributing factor in senescence:
The long-term preservation of a culture is a critical skill for maintaining viable genetic stocks of the species and strains that you work with. The vigor of a mycelial network is at its peak early on in the aseptic practice before it slowly senesces due to a lack of environmental stress. The cultivator should seek to preserve backups (“snapshots”) of this early, non-senesced stage by keeping small amounts of the young mycelium in small, refrigerated containers.
So there’s a hint there that introducing stress may be a means of reintroducing vigor to a strain - perhaps through epigenetic processes - though I imagine this depends on the species in question. One of the things I do to maintain vigor is to switch-up the substrate I offer to my cultures; this was something I was taught by my mentor Taki-runa in Peru. I think of it as introducing fun and novelty. We’d all be a bit bleak and lethargic if we were eating and doing the same thing every day.
So in terms of agar, try to have 3-4 distinct media recipes in action and ensure that a given culture is cycled through these different media. The same goes for liquid cultures - switch it up from time to time.
And one more response, this one on naming of generations when it comes to cultures on agar:
The Stamets “P” Value System
The Stamets “P” value system is simply an arithmetic scale I have devised for measuring the expansion of mycelium through successive inoculations from one 100 x 15-mm petri dish to the next. […] The Stamets “P” Value (SPV) benefits cultivators by indicating how close to the origin their culture is at any point in time by simply recording the number of petri dishes the mycelium has grown through. When a culture has been isolated from contaminants, usually in one or two transfers, the first pure culture is designated as P1. When the mycelium has filled that dish, the next dish to receive the mycelium is called P2. Each culture is labeled with the date, species, collection number, strain code, “P” Value, and medium (if necessary). Thus, a typical example from one of my culture dishes reads:
Agaricus blazei
P5
2/12The date 2/12 refers to the time the medium was inoculated. Spawn created from such young cultures, in contrast to one grown out 100 times as far, produces more mushrooms. The “P” value system is essentially a metric ruler for measuring relative numbers of cell divisions from the culture’s birth. I have strains in my possession, from which I regularly regenerate cultures, that are 10 years old, and kept at a P2 or P3.
For purposes of commercial production, I try to maintain cell lines within P10, this is, within ten successive transfers to medium-filled petri dishes.
And here he mentions what my mentor taught me:
The slowing of mycelium may also be partly due to media specificity, i.e., the agar formula selectively influences the type of mycelial growth. To ameliorate degenerative effects, the addition of extracted end-substrates (sawdust, straw, etc.) favors the normal development of mycelium. The introduction of end-substrate acquaints the mushroom mycelium with its destined fruiting habitat, challenging the mycelium and selectively activating its enzymatic systems. This familiarity with the end-substrate greatly improves performance later on. Parent cells retain a “genetic memory” passed downstream through the mycelial networks. Mycelia grown in this fashion are far better prepared than mycelia not exposed to such cultural conditions. Not only is the speed of colonization accelerated, but also the time to fruiting is shortened.
@glyph that bit on pure agar’s influence on degeneration has me finally modifying my agar brew. had conceptually mostly been thinking of it in terms of training for immediate substrate spawn run but very little in terms of long-term potential. very very cool.
brb, culturing strains of green mold in various intensities for future “deathmatch” plates