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The Corroboree

Darklight

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Posts posted by Darklight


  1. Go for it. I'd recommend bulking up your sterile spawn using the limesoak/ sugar cane mulch tek and stuffing the trunk with some of that once it colonises.

     

    I isolated a local pink oyster strain I found fruiting on a tree fern a few years back. Some strains can def handle the ambient conditions and contams, if you have one of his feral blues that can work too

     

    Mushrooms can't save the world tho, sorting our own shit out is a much higher priority. Expecting another genus to do it for us is bloody lazy

    • Like 2

  2. On 7/19/2018 at 4:46 PM, RonnySimulacrum said:

    Ok, I have been MIA, but hell I needed it. This year has been very challenging for me personally to date due to unforeseen circumstance and sadly is likely to be very challenging for me yet.

     

    Ronny my friend, I hope everything goes smooth as silk for you and all your loved ones. I so admire you, your work, and the community you have been a big part in making

     

    Not sure if I can make the event this year, could go either way. It'd be a hell of an event, the caravan park is a great venue and the owner sounds like a top bloke :) I wanna meet them and shake their hand

     

    Take it easy on yourself

    • Like 2

  3. On 3/7/2018 at 10:13 AM, Darklight said:

    The actual lab phase for this stuff is facile and fast. It's the monitoring and logging which kills it for mutation work.

     

    Rightio, the above sums it right up.

     

    Interesting conclusions have been drawn, experiments from both the Surflan and pure Oryzalin batches  above are still underway

     

    The regen plants from the pure Oryzalin batch are of a different species to the Surflan batch. So yeah, some difference in response to be expected. Also, the first species had undifferentiated ( not embryonic ) callus as it's parent material. The latter species had apical nodes as it's donor material. This has def affected the selection process, I might not explain it well enough to do it justice, pls see below.

     

    Kill curves which escalated over doseage for the first species. A good sign. Progressive failing at 30 days was observed in the second species, which was only treated at a single rate. Also a good sign. Something's whacked em, especially obvious since untreated controls from both groups showed no variance from normal growth in-vitro

     

    Both treatment batches above maintained sterility after exposure- no further worrying about dirty Surflan needed.

     

    However none of the Oryzalin/ callus batch are showing signs of morphological changes at deflask. And the Surflan/ apical nodes are definitely showing morphological stress that only a few are starting to recover from now.

     

    Since Surflan is just Oryzalin powder + PEG, there's not the scope on this work to discuss the possible contribution of the PEG to the difference in results. I'm not convinced this is even an issue. For the purposes of the screening process I'm looking at two very different sets of results ( species and donor material aside ) and it seems to come down to exactly how the survivors of the treatment are selected for monitoring.

     

    This could be because of the length of time both treated parent stock could survive exposure. The callus regens were grabbed pretty much as they showed up, over a 2-4 week period, and the remaining callus died off ( possibly exposure rate for the callus was too high )- and thus while the kill curve looks great- what's persisted is Surflan survivors rather than transformed polyploids.

     

    Callus is a bit more sensitive to external influence than organised tissue is.

     

    I can't remember the application rates or whether they were comparable for the two species/ treated tissue types. Will look it up later

     

    Deflasked plants for the Oryzalin batch ( total of 18 survivors. 2 controls, 14 low range Oryzalin and 2 high-range Oryzalin  ) for this species/ treatment will go in the ground for long term monitoring of gross morphological changes to indicate polyploidy. It's the best I can hope for in this species, I've fucked around enough with wretched bloody microscopy teks to check the leaves, but my microscopy is shit, it's fiddly, and after all it won't make any difference to plant sales unless ploidy confers distinct traits which are visible to the buyer ( the entire point of the experiment in this plant- and yes, such distinctions do matter at the start when you're setting these things up )

     

    In contrast, ALL of the 1:200 24hr Surflan/ apical node treatment species showed significant distress not long after my last post. Discolouration from the Surflan hit them to varying degrees, and this became even more pronounced after a temperature rise on the grow shelves ( hint- keep your Surflan-treated tissue at a constant temperature. Growth almost completely ceased even after 2 subcultures to try to get them away from any excreted Surflan metabolites

     

    Growth in some of the remaining Surflan treated apices is now slowly resuming. Control, untreated apices from that experiment are fine and kept growing normally during this time. All leaves from apices in the treated group dropped or severely discoloured, new growth is scarce and severely stunted. About 30% failed at +30 days and another 20% failed really quickly after a temp rise. However root development is occurring in a few surviving apices and I'm hoping leaf regrowth will follow

     

    This Surflan batch is a way off deflask yet, hopefully the tells of ploidy will be more visible in this species.

     

    It could well be that cells/ tissue which has been rendered polyploid by these treatments takes a while to recover- that by grabbing the earlier survivors of the first ( Oryzalin ) treatment I was unlikely to get any polyploid individuals. The four month recovery rate for the second apical ( different species and parent material, Surflan treated ) batch would bear this out. At this point this idea is just that, but I have read ( mostly informal )  accounts of bothvigorous early, and of late recovery from Oryzalin/ Surflan treatment being indicative of ploidy. As a result of my ( minimal, early stage ) project here I'm more inclined to suspect it's the latter. But we'll see

     

    Am confident the application rate for 1:200 Surflan/ apical node batch is sufficient- maybe a tad high if anything. I woulda/ shoulda/ coulda run a lowed dose batch alongside the controls but CBF. Maybe next time

     

    So yeah, there it is... it's not just the actual wetwork, its the monitoring and logging which is the killer here. I won't know about the Oryzalin batch for 2 years. The second species isn't even close to deflask yet, so maybe 2 years from today- if there are any survivors

     

    Apologies for the lack of organisation or succinctness in the post, I only got my head around some of it last week

    • Like 3

  4. Yup, it's a big one

     

    Best for the day, and the year- to one of the most incredible people I've ever had the pleasure of being friends with.

     

    It's been a tough haul for you the last few years, harder than most imagine, and yet here you are, kicking on, helping, giving space here, contributing to the field generally in some unique and outstanding ways

     

    Thank you, wishing whatever you can eat most that closely resembles cake. And drinks

     

    Love your work old friend <3

     

    DL

    • Like 2

  5. This might not be relevant in your case, but Catha here is horribly prone to borer.

     

    Symptoms here look almost bacterial- leaf splotch and wilt, tends to start closer to the bore hole site wherever that is, and work it's way up to the tips

     

    Cut the branch below the wound site as soon as you see it and throw the branch well away from the parent plant- it spreads fast, especially on fresh growth and can invade entire old branches

     

    Usually not fatal but can set growth back a season or two if it's bad

    • Like 1

  6. On 3/15/2018 at 11:46 AM, obtuse said:

    but i am very concerned with the idea that specimens are leaving the country and not being deposited with local herbariums.

     

    Its important that we support local opportunities for research

     

     

    Yup, this /\/\/\/\/\

     

    I'm not sure if there is some process involved in herbarium/ info submission which would give preference to the first institution to log the data- I did bump up against something of the kind a few years back but didn't pursue the specifics.

     

    But the grumpy taxonomist ( is there any other kind? ) at the other end gave me a stern reminder that we need to remember to submit first to Australian institutions if we want them to get recognition and funding.

     

    Which is totally fair enough IMO

     

    Alvalabs in Spain do a great job of the sequencing for a reasonable price, but I think there are now local services which offer identical services at similar prices.

    • Like 2

  7. I have an old Gelman Sciences Australia Laminar Flow cabinet class I. Model CF43S

     

    From the serial # it looks like it was made in 1988.

     

    Fuck it's been a corker of a unit. If you're purchasing one new, or even secondhand, I'd recommend them. Of course Gelman Sciences Australia no longer exists the way it did, the company still exists as Gelaire. I have yet to contact them to see if they have any info on the model, but from experience with other equipment the chances of any documentation from that era are fuckall.

     

    Last few months the fan has started to make bearing whine noises- especially when it's hot ( I don't have aircon in the lab right now, dammit ). Too broke to bring in a specific technician to replace the fan and test the filter.

     

    I'd like to at least find somewhere to buy a similar unit for cheap and install it myself, or to compare the cost of me replacing it with a non-genuine part against the cost of getting a tech down here ( incl. travel time ) to do the whole thing and under warranty

     

    I've looked online but can't find a manual, unsurprising since 1988 wasn't a great year for online PDFs ;)

     

    Anyone here working with these, or similar units who could advise? Would it be just a case of looking at the diameter of the fan and picking something off Alibaba? Brands to avoid? Anything else I need to know?

     

    Took the pre-filter off, it looks to be bolt-on. It could well be a standard blower fan and I might be able to buy something cheap and slot it in the old unit's place. As quickly as possible and on a cool day so as to prevent anything chunky from getting into the airstream/ the seals from warping.

     

    The only way to make sure it's still a closed system would be to do the old waft-with-a-stick-of-incense and to check it with some open petri dishes for 30s, 1 min, 3 min etc. And panic if I warped the seals during replacement and then fork out for a technician to visit

     

    There's a compliance and info plate which can't be seen unless I fully pull the top steel plate off the unit. If I can avoid that, I will. That shit never ends well when you're working alone and it's mission critical

     

    How realistic is this whole scenario? I know a few here have had problems with the airstream in smaller portable units when seals warp or shift and allow pathogens to find a place to live

     

    Have pics, put em up later

     

    Happy birthday to my beautiful Gelman Sciences Australia Horizontal Laminar Flow Cabinet. 30 years, 20 of them with me :D


  8. Update: all explants from the 1:200 Surflan soak treatment remained sterile despite the lack of filter sterilisation for the Surflan solution

     

    This is not atypical for chem stocksolutions, many of which don't support microscopic life if kept reasonably stored and unopened, but it's good to know. Also I don't need to run the experiment again- how good is that :)

     

    The thing that hasn't happened, and that I was hoping for, is a kill rate. It shows uptake of the Surflan and gives a good indication of the effectiveness of the application rate. I didn't run a gradient ( multiple concentrations simultaneously, or same concentration for different time points ) because I couldn't be arsed. A kill rate of about 30-50% at some concentration would be expected to provide some level of ploidy- tho it might only leave you with survivors and you'd still need to monitor for gross morphological changes and signs of chimerism.

     

    1:200 would have been a pretty high rate from previous experience with Oryzalin solution ( Oryzalin is the active ingredient in Surflan ) in the agar media and using callus as a starting material rather than apical tips- I've seen that in two other species. But good quality callus is difficult to optimise media for in this species and I was going for a fast and dirty protocol, so I tried the web-standard protocol. Looks like it's not as effective.

     

    All explants are fine- no Surflan damage at +9 days and under lights. There's still time for damage to show, but it's becoming less likely as time progresses. Not expecting new growth to show for another month

     

    Tl:dr: If I want to try apical node soak again I'll need to run the soak at higher concentrations- ideally with a kill curve plan

     

    The actual lab phase for this stuff is facile and fast. It's the monitoring and logging which kills it for mutation work.

    • Like 1

  9. On 2/27/2018 at 11:16 PM, obtuse said:

     

    a bit serious, na i find him too flippant.  misquoting papers and making things sound like they agree with his research.

     

    With you 100% on that Obtuse.

     

    I can't quite decide if his self-promoting is causing others to misreport his work and/ or mis-attribute the work of others to him personally. Or worse, if he does the mis-attribution himself.

     

    That issue was really bought home to me when I was doing some mycoremediation research with a bunch of other crew for a fledgling project, which frankly died in the arse after the client realised that yes, Mushrooms Can Maybe Save The World, but that it wasn't going to be as easy as it sounded and he'd actually have to pay for some proper research. Which may not give him the results as seen on Youtube.

     

    Half the stuff or more Stamets quoted was done at least a decade previously by others- this was published work at the time too. I never heard mention of it in any of his work I read or heard which led me to the papers, but a quick Google made it really obvious once I stopped shaking my head around certain incongruities of dates and facts.

     

    Personally I'm well over the cult of celebrity around some speakers. Maybe it's needed in order to raise awarenesses ( ha! ) of their special subjects- a certain amount of celebrity is more of a media honeypot. But often there's not enough at the back of it to justify the hype.

     

    This could also be as much a criticism of audiences as much as of celebrity- too many people looking for pub flair and dinner party convo and easy fixes who CBF with their fact checking or sticking with something longer than five minutes to get a deeper understanding.

     

    But a professional presenter of information has a responsibility of conveying accuracy, conferring credit where it's due, and not slipping into hype. And he's failing at that- instead of encouraging a community of colleagues he's fuelling consumption of dumbed-down sound bytes, unjustified speculation and increasing sales figures.

     

    Look, the bloke's written some great books, runs a good business as far as I can tell, and is probably kind to old ladies and small kittens. Good on 'im. More power to his writing arm. He's bought the issues of the importance of fungi to the public hive-mind in a way nobody else has. That's a breath of fresh air. But it doesn't make him Mushy Jesus or anything.

     

    It's Planet Of The Spivs out there in every field- science included- and certainly psychedelic science. But it doesn't have to stay that way :)

     

     

    • Like 7

  10. Just now, Darklight said:

     

     

    Fuggit. That wasn't the Surflan solution I got from Teon that I used for that experiment, it was some old pure Oryzalin stock for

     

    The main issue here being sterility. I've read that Oryzalin doesn't survive autoclaving and so the pure Oryzalin I used for the callus experiment was dissolved then filter sterilised and added to media post-autoclave.

     

    Given that I'm out of 0.2uM syringe filters, and I'm crossing my fingers that the Surflan is as close to sterile as practicable, being dissolved in something hostile to life like DMSO. So I just pipetted it raw into some sterile distilled water flasks using sterile tips.

     

    This'll be the first place things can fuck up if the Surflan has fugly spores of anything in it.


  11. On 1/17/2018 at 5:36 AM, Darklight said:

     

    Pending.  I did mine in-vitro and am having trouble deflasking them

     

     

    Fuggit. That wasn't the Surflan solution I got from Teon that I used for that experiment, it was some old pure Oryzalin stock for micropropagation. Threw it in some callus regen mix I had for a species I have a full callus regen protocol for, but because the species is variable in morphology while in culture I need to wait until plants are deflasked and in the ground to monitor for gross changes of horticultural interest. While they're in the jars they could be anything. The kill curve was nice over the Oryzalin gradient tho, so I'm hoping for some happy results.

     

    Since I don't have a flow cytometer handy I thought I'd save time and check the stomates under a microscope against the controls, but that wasn't possible with my very limited microscopy skills. Couldn't see a damn thing. So we're back at wait-and-see. For a bunch of reasons results should be in by Xmas

     

     

    I only just finished running the wetwork for the Surflan experiment a minute ago, and fuck it's the most virulent orange solution I've ever seen. Fair burned my eyes out with colour. I ran it using in-vitro apical tips, because this other species doesn't have a working callus culture protocol yet. And I used tiny tips because I don't want to give any cells the chance to encourage other cells to revert back to diploidy.

     

    Everything survived the 1:200 soak for 24 hrs ( I used the lower level for all explants- in-vitro plants have their stomates more open as they are adapted to a high humidity environment ). Actually I was surprised how much they were in good nick, given I didn't throw any nutes or sugar in there and if I'd spent 24 hrs in a place that orange I would have died of bleeding eyeballs :)

     

    I wasted a fuckton of time looking for an optimal protocol for this then realised I should go for the simplest one as I haven't run the experiment at all before, so I'm not expecting superlative results. First time is indeed lucky if something good happens :) There are a couple of minor things I'd do differently but overall it was just a cut and soak on the orbital shaker for 24 hrs, 3x rinse in water, recut ends and place in media

     

    Normally I eschew anything with this much liquid handling if I can avoid it, throwing round multiple liquids over multiple explant batches is a prime way to introduce contam. Fingers crossed we didn't get any.

     

    Writing up the notes now. There were 46 explants treated. Interested to see if there's a kill rate. Wish me luck :)

     

    Thanks Teonacatl for the chance to give this a whirl <3

    • Like 1

  12. Ha! Found em.

     

    They'd sent some NTS 4/20 and some NTS Bio-N. I know why they labelled it the way they did, because proprietary blends can change over time.

     

    Everything triangulates nicely via notes and cached web pages and backup drives. So yeah, lab notes still winning :D


  13. About a gazillion years ago  ( OKOK it was 2013 ) a lovely kind forum member sent me some microbial fertiliser product samples. I have the envelope but the address is faded and lost to time and fridge mould, but it was a NSW postcode.

     

    From memory there were 2 aliquot tubes in it. I still have one, labelled Azotobacter.

     

    The other tube is missing, maybe used up. I can't remember what it was labelled as.

     

    Was about to start some plant rooting experiments for some putative mutants which are proving a right bastard to deflask. Looking back at the 2013 notes they say that the control species was successfully rooted in-vitro using Nutritech NTS 4/20. The deflasks survived well and went on to propagate so well we abandoned micropropagation altogether

     

    Nutritech NTS 4/20 is a bacterial product. It's been discontinued, but it did contain Azotobacter.

     

    There's a chance that the lost test tube contained the proprietary blend NTS 4/20 and the Azotobacter is a pure product from another source

     

    The other option is that the tube labelled Azotobacter is NTS 4/20 and the lost tube was something else

     

    Fucked if I can find the original PM via search engine. And I'm about to load up the autoclave.

     

    If it was you, you lovely person, do you remember what was in the other tube?

     

    And do I owe you anything for the trade? Did I send you anything?

     

    I lost a bunch of references to the trade during an OS crash this month- my scratchpad didn't back up as well as I was hoping.

     

    This is entirely the reason I keep lab notes. But even these weren't sufficient in this instance.


  14. 3 minutes ago, MeanGreen said:

    Especially when you consider that big "false kratom" debacle back in the early 2000's where my compatriots Bruno & friends at ebotashop flooded the market with false kratom (M. parvifolia I believe). This could have caused everyone to just brush off kratom as ineffective and killed the market in the egg...

     

    There were vendor pages online back then which incorrectly ( and possibly knowingly ) pushed the psychedelic properties of M. speciosa and those most definitely contributed to the TGA scheduling. Mitragyna speciosa is not psychedelic, had never been touted as psychedelic prior to the existence of those few web pages, and the incorrect publicity was not scientifically evaluated, but was used directly as evidence nonetheless during the TGA scheduling

     

    By the time Jon Hanna's expose on false kratom came out it was too late to do anything

     

    Flawed data met lack of oversight and flawed process and produced poor outcomes

     

     

    3 minutes ago, MeanGreen said:

     

    I think it's extremely unfortunate that Australia decided to ban it, especially during an opiate epidemic. How can you consider an opioid which doesn't cause lethal respiratory depression as not having any medicinal potential is beyond me...

     

    It also has potential as an anti-inflammatory, anti-tumour etc. It was a cruel and irrational scheduling, and because of the bureaucratic specifics of the scheduling process back then we were not permitted to respond or object to the scheduling. I believe that's changed for all new introductions, T would know better than I, but it's not retrospective

     

    If we'd not been hamstrung by the scheduling we were on track to do so much more good research

     

    In practical terms a helluva lot of good science gets proven wrong over time ( which is entirely the point ) and so many claims made back then could have been fortified by extra research or dropped as spurious

     

    Our facility, our towns and region really lost out on this. Research programs and commercial opportunities all happened off shore. Quite a few overseas companies have done seriously well out of it and should continue to do so for some time. Good on 'em. We were uniquely placed to do all that here, in a region of high biodiversity, appropriate climate, high unemployment and fair access to some first rate research facilities. But hey, no.

     

    I still want that small, private research lab where we can rigorously train citizen scientists in the love and practice of quality empirical data generation and never be de-funded by any bastard government again. I want it so bad I can taste it. So much talent going to waste.

     

     

    3 minutes ago, MeanGreen said:

     

    Could you share a bit about what the selection process was for the Rifat clone? Was it simply the most vigorous sprout of the batch of seeds?

     

    Erk. It was the only one which survived. We bit our nails for a year or so until it was proven both stable in micropropagation and active. Long story snipped

     

    3 minutes ago, MeanGreen said:

     

    If you do a talk about this at EGA I might just have to fly my ass over there haha :)

     

    Seriously, you should come to an EGA anyhow. They're deadset brilliant. Best crew ever. And if we are accepted for an EGA presentation on this and T agrees to co-present with me again I may hold you to that statement :D

    • Like 6
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