'sblood! you just found it lying there? the Leitz as well? curious as to why you sold the better of the two of thats the case? I guess the 8-50x might have been dumped just because its pretty shit as far as magnification goes. Still, really nice find. I'd have sold both, as my own goes up to 200x, if I'm using the oil-immersion lens, shows up individual plant/animal/fungal cells really well, never tried viewing a bacterial colony before but you reminded me, so I may as well leave a blank plate of PDA out and see what decides to infest it, just so I can take a look, its not like I'm terribly pressed for time, not that it takes long, most of the time would be spent in making up the agar (might just use gelatine/boiled down potato broth, maybe next time my family eat mashed potatoes, I'll tell my old man not to toss the water. The reason for using gelatine would just be to avoid opening a new pack of agar so its sterile for doing actual work, rather than simply buggering about out of curiosity, for when contaminants actually matter, rather than being the entire POINT being to grow whatever the hell happens to be floating around at the time.
Need the agar for my Claviceps project, complicated, inconsistent, frustrating, finicky little bastard fungi that they all are. Getting cultures to produce is difficultish but doable....for a while at least.
Little fuckers once a sucessfully mutated into an alkaloid producer strain (working with common rye ergot currently, Claviceps purpurea, but I'd very much like to get hold of a strain, (ANY strain, even wildtype, like the C.purpurea I found growing not far from where I live, meaning I have to do all the mutagenesis and selection myself, over and over and over and.....) of Claviceps paspali, because theres a ton less work involved if one does isolate a decent strain and manage to stabilize it, mainly,much less chromatography afterwards, because C.paspali doesn't produce all the peptidic ergot alkaloids that C.purpurea does/CAN do dep. on strain. So no need to run a column after first subjecting the lot that comes out of a successful project to hydrolysis in alcoholic KOH, which given the sensitivity of ergot alkaloids to ..well...pretty much everything-high PH, low PH (IIRC theres something like a PH variation safety margin, of sorts of 0.2 either way past the ideal mark that sits between
a successful hydrolysis to lysergic acid and it completely, irreversibly tipping your whole yield down the john. They don't much like heat either although they are far more sensitive to UV light which causes lysergic acid and its derivatives to epimerize to the useless lumi- forms, via rearrangement of an internal double bond which is not accessible to chemical manipulation, making degradation via epimerization, unlike simple optical isomerism (lysergic acid and its derivatives have four, possibly 6 enantiomers, D- and L- forms of lysergic acid, isolysergic acid, each forming s pair, and D/L-lumi-lysergic acid/derivatives, not sure if the two epimers are normally counted as enantiomers or not, or indeed, if lysergic acid itself forms the lumi-derivatives, I don't think so in the former case and just don't know about the latter, since the process of formation, IIRC involves not just UV exposure, but addition of H2O as well, whixh would make them not isomers of the parent compound.)
Susceptible (very) to oxidizing conditions, even the residual chlorine in tap water is enough to destroy it/them.
Aside from analytical chromatography, I've no experience, never run a prep-scale column, hell I'd have to buy the column too.
On top of that there are three or four other issues, the two most important are that the strains (at least, C.pupurea does) become in some way senescent with multiple subcultures, another is that alkaloid producer strains with rare, rare exceptions, are also those that do not form conidia (asexual spores of ascomycete fungi) in culture making it extremely difficult to consistently maintain a monoclonal lineage, which is a bugger as then new sclerotia could simply be farmed via letting it go through its normal, parasitic lifecycle in the plant hosts as nature intended, which is meant
to revitalize tired old strains, and then one could just harvest the ascospores by germinating the sclerotia.
The other main issue is mechanical stress, everything from blending up mycelium to prepare seed cultures (ergot farming, in-vitro is done in two separate stages, the seed phase and actual culture require different media, because phosphate levels, both availability and whatever the fungus has by way of intracellular reserves are critical. High phosphate levels mean vigorous growth but sod all productivity whereas low levels, either naturally or lowered still further by poisoning phosphate metabolism using arsenic (As is abit different to most heavy metals in that it isn't sulfhydryl groups it binds to and buggers up, like lead, cadmium, mercury and thallium, not primarily anyway, but it being close to phosphorus, gets uptaken but cannot be used in place of it, at least not by most organisms, and it cannot substitute P in DNA either as the arsenodiester bond (analogous to the phosphodiester making up the backbone of DNA) isn't stable under biological conditions an.d would hydrolyze very quickly. Thats one mode of toxicity in mammals, though it also fucks over pyruvate dehydrogenase and one of its cofactors, inhibiting cellular energy production; although some bacteria
are able to use arsenates to oxidize their food, the As(IV) being reduced to As(III) in doing so.
But whilst not so close it can play the same role phosphorus does in vivo, its close enough to compete with it, thus inactivating processes that depend on phosphorus, and there is no shortage of those!
So one amongst many tricks to increase yield, is to do the seed cultures as usual in normal high-phosphate medium but to spike the production medium with arsenic compounds, preferably the more toxic arsenites as opposed to arsenates (arsenic in the +3 oxidation state vs +5 in arsenates), low phosphorus levels seem to trigger the resting/non-growth/possibly reproductive stage, which is the
only time the fungi produce goods worth having, in the natural, parasitic cycle, alkaloid production is correlated very strongly with cessation of sphacelial growth and sclerotia formation, so knocking out much of the capacity to utilize phosphate, presumably, although I don't know for certain, forcing it to depend on accumulated reserves of intracellular phoshate to fuel the very basic bare bones needed, such as DNA/RNA synthesis, triggers a switch in vitro from sphacelial/anamorphic growth phases to the productive stage, corresponding to sclerotial growth in nature.
Other such tweaks to the media include adding a surfectant like Tween (polysorbate-80), propylene glycol or 2,3-butanediol to the mix, which acts on a physical level by altering cellular membrane permeability, allowing lipophilic alkaloids to exit the ergot cells, they seem to act as a self-regulatory negative feedback mechanism, that is to say, ergot alkaloids inhibit their own biosynthesis. I don't
(yet) know the specific enzymes mediating that process, if I did, I'd be willing to invest in a custom-made retroviral vector bearing an antisense oligo or a morpholino, etc. or coding for an siRNA, etc. to
perform a targeted knockdown of whatever the regulatory pathway is, at one of its critical steps. The effect of surfectants on the other hand is simply enhancing transport of alkaloids from the cellular
space to the culture media, thereby taking the inhibitor away from the regulatory pathway they bind to before they can retard alkaloid synthesis.
Enhancing mechanical resistance to physical trauma via encapsulation in calcium alginate polymer is another one, this vastly improves the stability of the strain against becoming senescent, but at the price of reducing oxygen uptake, which is an important factor, the beads MUST be tiny, a couple of mm at most because after a certain, quite shallow depth they essentially become nearly anoxic, providing enough to survive but not to produce. I'm currently looking into an electro-spray method of particle formation, by forcing the sodium or potassium alginate solution through a fine needle under
great pressure, the needles and the solution of CaCl2 they are to be sprayed into to induce polymerization, and with diced up myc in the mixture, or better yet, if productive conidial strains can be isolated [they exist, they are just really rare, and the ones big pharma has, they aren't exactly handing out to anybody who just turns up at their door cap in hand, begging for a sample. Like hell they would.) then that could further improve yield I bet by avoiding the mechanical shock inflicted during the process of liquidizing the mycelium, then whatever is in the master solution gets encapsulated
within the beads formed when the alginate forms its calcium salt and polymerizes.
Adding up to 50% of a perfluorocarbon as an emulsion can greatly enhance oxygen uptake in encapsulated myc, both the sum total and the depth it penetrates to. I assume rigging a medical oxygen
concentrator, or building a simple electrolytic cell to bleed in a continuous supply of enriched air would improve yield too although I haven't tried it, haven't tried the encapsulation technique itself yet
either actually. I still need to find out which PFC has the highest capacity to dissolve O2, and then buy some of it. Seemingly both mechanical stability and alkaloid yield is increased up to about 8% alginate but the TYPE of alkaloids produced is altered the higher you go, the more of clavines, intermediates between the initial synthesis of dimethylallyltryptophan, used to construct the ergoline
ring structure and lysergic acid, but not much use on their own; and the less lysergic acid peptides you get. PFC emulsions are an attempt to address this.
And tryptophan itself acts as its own negative feedback mechanism, high levels in the medium seem to reduce uptake and incorporation into alkaloids, but selection of a mutant strain auxotrophic
for tryptophan and then reversion to a regular, non-auxotroph strain appears to uncouple the production and/or use of the same from its regulatory element/s allowing MUCH higher alkaloid yields than a
strain not so treated.
There are MANY other such tweaks and tricks that can be deployed to increase yields, way too many to list them all, or even a sizeable proportion of them here (I've been reading up on these fungi and
how to grow/mutate the buggers for about 4 maybe 5 years now, and that was before ever so much as preparing a plate.
and as such, have an awful lot of research journal articles printed out, many more stuck up in my head, and there is plenty for me to be chewing on, plus a fair few ideas of my own, like protoplast fusion experiments, and induction of polyploidy via the gout drug (and notorious poison) cochicine, originally derived from the underground parts of the autumn crocus, Colchicum autumnale, which causes cells to leave polyploid progeny upon division and replication (so I assume if its to do anything useful it would need adding during the actively growing phase.
novel and interesting work going on on one of the clandestine chem/biotech forums I'm a member of, perhaps the most interesting thing being attempting to stop the senescence via altering DNA histone acetylation/methylation since some of the userbase there deduced the mechanism to be not a genetic mutation, but epigenetic changes, aiming to knock this out by targeting histone acetyltransferases using such things as anacardic acid (the corrosive, irritant component of the violently caustic dark oil found between the inner and outer shell layers of unroasted cashew nuts. Although anacardic acid itself seems not to be practical due to solubility issues, DMSO seeming to be one of the few nontoxic solvents that will dissolve the stuff, but apparently the DMSO just sits on top of the plates and somewhat excludes O2 by being a mechanical barrier. Personally I am sitting on the fence w/ this one, waiting to see if it ends up being practical and worthwhile investing.
So...yeah...theres a hell of a lot to it, not quick and easy money for any aspiring acid chemist, actually the reason *I* am trying is more for the microbiology experience, learning and getting better at it in general. That and a stable, high-producing strain of either C.purpurea or C.paspali would be worth a lot of money to the right people. Hell, there are some people I'd send some to ASAP just to make
sure the cat is let most dramatically and irreversibly, out of the bag, so to speak. Once it got sent to other growers it would be impossible for LE to completely squash its use/distribution.
Getting to work on not on LSD, but on the less well known, more exotic analogs of it afterwards, or making some money off the lysergic acid itself, those would just count as a bonus, reward for hard work and a pat on the back for a job well done, although of course given the value of LA, even 1g would be the equivalent of maybe 500-600 hits of acid, depending on the skill of the chemist
making use of it, and upon the specific lysergic acid amide/s being produced, and its always nice to have food on my table and fresh equipment and consumables for the lab.
For some reason a lot of the people on that forum didn't understand that as a motive at all; that I get as much out of the actual project as I ever could from it ever ending up becoming profitable.
What sort of camera would you recommend for taking photomicrographs the way you suggested, Parts? by simply sticking a camera against the one of the eyepieces and taking a shot.
And for a digital camera what sort of settings, exposure times, film speed emulation, flash, light compensation etc?
IMO the reason those hair-thin spines penetrate so easily is BECAUSE they are so small, that often results in a finer point than a macroscale object could have easily.
Take a look at a magnified nettle stinger thats not been discharged and one that has. The silica stinging hairs are so damn fine that they break at the slightest touch, and the tips are so fine
that the points are sharp as hell. And when they break, the end is basically a tiny little fine hollow syringe, with a needle tip made from broken glass, and whilst fragile they are way beyond
razor sharp. The finer the point or edge, the sharper it usually is. IIRC, broken flint can very often produce flakes with a monomolecular edge. Flint is actually used apparently in certain surgical procedures requiring very delicate cuts, they are brittle, unlike steel, but even the sharpest surgical blade cannot even begin to compare to a truly sharp edge of broken flint, if its as
fine and sharp as it can get, so can be used for very delicate work requiring extremely fine cuts that a normal metal blade couldn't hope to perform, and they are nonmagnetic too which can be an advantage.
And IMO they don't draw blood just because of their being so fine, the wound itself likely closes up instantly, and/or is too small to permit the passage of a red blood cell. I'd bet even if you
could get some several inches long and drive them in all the way, the would still wouldn't bleed. Although don't hold me to that, its just a guess. Not that it can be tested anyhow.
