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The Corroboree

Darklight

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Posts posted by Darklight

  1. Posted

    Yes by true colonisation I am referring to active production of carbohyrases and lignin peroxidases and active growth. What I suspect is happening is that the fungi are merely savaging free dissolved sugars and simple polysaccharides for exploratory growth, or still using the agar plug. My point is that is difficult to tell from a morphologically, whether the substrate is being metabolised or not... at least at this point in time.

    I've experienced this in experiments as well.

    Best thing you can do is wait, especially if it's a small plug + a large amount of substrate. The inoculated plug will lose it's resources and the mycelium won't find food elsewhere

    You can also run the experiment backwards just to be thorough- add some identical sterile substrate to a colonised agar plate and see if the growth pattern is the same

    Also run controls, just in case there is something weird with the substrate. It happens.

    • Like 1

  2. Posted

    Yup. Bulk up on household stuff if you can see a hard time coming too

    Sucks to run out of shampoo, conditioner, toothpaste, bog rolls, cleaning products and laundry detergent when you're trying to look good job hunting. Especially if a few of them run out at once

    All that suff lasts a long time. If you know the next few months will be unwaged or hard, go to a cleaning products place and stock up

    • Like 3

  3. Posted · Edited by Darklight

    I use a $20usd (shipped) blue plastic probe from Aliexpress, this is wired up to a simple pH circuit and fed into an ADC and to an Arduino monitored via the serial out

    This sits in a saltwater frag tank all day and the data gets logged to a xively feed; when the lights come on there is a nice shift from the photosynthesis kicking in, and it's all exactly the same as the colorimetric 7 to 8.9 test kit that I have which is highly regarded (sailfert)

    As for probe calibration, Im sure you're aware that 99% of calib is done by cleaning the probe/keeping it clean

    I use a 12 bit ADC and this generates a value between 0-4096 which is directly proportional to pH

    The main calibration factor used is drift, which is the measure of how different the probe's reading is to a "new" probe if that makes sense

    Nice workaround :)

    I might have to risk a probe shipped from China as long as the specs match mine. I do need it now tho and shouldn't risk the shipping lag esp if it doesn't work, but $ is the limiting factor this week

    As for the rest, to set something up similar to that would be more than the cost of a manufacturer's probe from Amazon and take me a few weeks to work out, it's not an option right now as I don't have Arduino capacity

    But yeah, I love all that DIY stuff when I have time

  4. Posted

    .... and the answer is NAAAAHHH

    Not all BNC connector pH probes will work with all meters

    It could be because the one I borrowed to use on my unit was a combines pH and temperature meter, whereas on my unit the pH and temp are separate probes off separate plugs

    I couldn't calibrate the meter while it was on there, there was too much difference between the electrode reading and the buffer pH for the calibration to be accepted

    And there is no trim on the unit to be able to manually set the pH

    It's a bugger, I need a precise pH reading and no paper strips I have are sufficiently sensitive to give direct and replicable colour changes, even tho I have a pH 4-7 paper set

    Might take it into a pet store and test it with the aquarium pH probes

    Good news is I found a cheaper Hanna genuine probe. Bad news is it's still $130AUD. I don't want another unit, I want this one to work

  7. Posted

    ... and yeah, great, regulating companies which sell DNA sequences. That's gunna seriously stifle some research down the track. It's so dumb somebody will actually do it too because Think Of The Children

    Let's face it, this is highly unlikely to become a backyarders process within any realistic time frame, right up until the specific yeast ( which I'm not familiar with ) is available in kit form to idiot-proof the process entirely. And anyone benefiting from the workup is most unlikely to be interested in proliferating the cultures via kit, so that's self limiting for a couple of decades ( by which time other teks will be in the ascendancy )

    I haven't seen the paper, but normally ( and keep in mind research changes exponentially every year ) if you mod something there is a selection gene and a marker gene attached to the gene of interest, so that non-transformed cells don't live and outgrow the transformed cells.

    Theoretically that's a major stumbling block for backyarders, the dodgiest of whom will not understand the requirement and whose batches will fail to produce because of it. Or who won't be able to access the selection chemicals. Or who won't keep enough library stock back so their culture changes down the generations

    ( Edit: don't forget I was wrong yesterday in this field, so YMMV )

    Things which are likely to happen before this drugcopalypse:

    Law enforcement panics are perpetrated via the news cycle and suddenly you need End User Declarations and ID to procure anything which could be 'diverted' for anything sciencey, like pressure cookers or dextrose

    Dodgy bogus yeast cultures purporting to be of this strain are sold for $stupid on ebay and distributed on the grey market to people who won't know what they're doing and won't be able to work out why their sludge isn't getting them opiated.

    Law enforcement comes up with a brilliant plan to extort several million taxpayer $ to train three specialists in forensic prokaryote genetics and nobody will call them

    The panic will subside, but the legislation won't be repealed and you'll still need an EUD and a no-knock raid to buy a spatula

    PS I am most cynical. And I haven't read the paper yet

    • Like 3

  8. Posted

    the Smolke et al plan had an air of underpants-gnomes to it... phase one, create reticuline->morphine yeast; phase two,????; phase three, morphine!

    (*hilarity*) Underpants-gnome. Is that a Linux thing?

    Love the term. I've seen a few cross-discipline papers which use that method, everyone assumes some term and process is understood by the other specialists and the fuckup/ omission/ swiftie escapes scrutiny.

    I'm stealing your underpants gnomes

  9. Posted

    I have a lovely digital pH meter which has been a rock solid unit for 15 years. It's a corker

    But now the electrode is almost out of juice, and it isn't refillable ( I checked the manual )

    15 years is pretty good lifespan for an electrode ( I calibrate regularly ). But I priced an identical new electrode at $260 from the manufacturer

    pH meter has a BNC connector. There are a shit ton of aftermarket BNC connector pH electrodes on ebay for about $35

    Anyone had any experience with using an aftermarket cheapie electrode attached to their lovely branded desktop pH meter?

    I'll do a side by side with the old electrode and a regular calibrate of course

    But if most people's experiences of cheap aftermarket pH electrodes is bad, I won't even bother waiting the 2 weeks for one to arrive from overseas

  11. Posted

    IME GFP does not glow in the dark, not to the extent that it is visible with the naked eye, unless there have been some recent changes made.

    If that cactus has been modified with standard GFP, you probably won't see it glowing at all unless you flood it with a particular type of blue light seen through a particular screen of orange

    Chlorophyll successfully transformed with GFP glows *red* under that light regime, and it's only the really transparent cells, like root tip cells, which can be easily seen as glowy under a fluorescent microscope

    So unless you cut the cactus open and viewed a slice of it under a fluorescent microscope with those screens, you'd see fuck all glowy. Might be more visible if it were variegated or albino, no idea. Doubt it but. And you'd still need the blue light to see it

    Just leaving the cactus next to the bed at night in it's pot would give you nada

    OTOH I wonder, if it were transformed with that compound which makes glowy mushrooms glow- now *that* is visible without special visuals. CBF searching... it's a different compound. Can't remember

    And I could be wrong, it's not a field I follow. Teks change all the time and fast ( edit: yes there have been considerable advances in the number of fluorescent proteins available )

    If you were caught with one of these you would be in vast amounts of trouble unless it was specifically licensed for release by the Office of the Gene Technology Regulator. Federal. Expect major headlines, absolutely no mercy, expensive legal bills and complete shitstorms for anyone else trying to import anything at all

    And remember- I could be wrong. Teks change all the time. I have repeated this for emphasis

  12. Posted

    Any information on autoclaving coconut water vs filtering it/cheap ways to filter sterilise?

    Coconut water which is sold for TC is heated to denature the proteins then filtered, there are teks for it. And for standard TC use people will do this with coconuts they source raw ( there was a coconut water shortage back in the late 90s sometime and labs were preparing their own )

    But people can add whatever they want to TC media and prepare it however they want

    I can't imagine either raw, unprocessed coconut water or commercial TC coconut water making it through any filter which would sterilise it sufficiently to add to a TC media post autoclave ( removing the need to autoclave the CW and preserving some of it's raw qualities ) but perhaps if you added it to media and filtered the whole lot it might make it through the standard filters we use these days. Is the difference between filtering the 100-200ml of coconut water and adding it post-autoclave and filter sterilising the whole litre or more of media

    A solution of sterile gelling agent would need to be added post-filter to solidify as it too would not make it through a filter. You wouldn't need it if you were doing liquid culture, but you'd want really good tek because liquid culture is so prone to contamination and bugs love coconut water

    Interesting thought, but too impractical for use at a home level. I couldn't even do it here cheaply. I can sterilise 10ml of a reasonable solution for about $4 if I can source a syringe filter right for the job

    0.22uM is the standard filter for sterile technique at this point in time, gets out everything but viruses

    All of this is subject to change the minute someone invents something new and cool ( like a super cheap microfilter system which doesn't cost thousands and clog up a lot and take expensive filters ) which subverts the current standard of autoclaving for sterilisation

    Short answer: you wouldn't filter sterilise coconut water now at the concentrations generally used in TC. Coconut water sold for TC has already been filtered, but only to bump out precipitates- it's not sterile and is AFAIK always autoclaved with media

  13. Posted

    One question I have which I did not put very well earlier is this:

    Say I have induced shoot proliferation at the axial buds at an increased level with a hormone.

    Now, will I get a finished plant quicker, if I let these shoots grow longer attached to the main stem, with the rest of the shoots and the top

    and then separate them later, allow them to recover, grow and root.....

    Or, would I get them further along/faster by separating these newly proliferated shoots from the main plant when they have a few nodes,

    so that they grow into a separate plant on their own, starting as soon as possible, like when they have three nodes perhaps??

    I understand there is a recovery time after they are divided, but they would be divided eventually anyway...

    do I produce plants faster, by leaving the new branching out plants on the main stem,

    or, dividing them sooner, allowing them to recover and grow separately?

    Took me a while to work out what you meant, thanks for clarification

    This is a *suck it and see* question, individual to each species, media, growing conditions etc. These are the questions rarely specifically answered in scientific publications and need to be tailored by each lab for their own requirements.

    You could do a side-by-side experiment to see what works for you, but it would only be an important consideration if you had, say, a commercial contract for replication with a definitive and punitive time line that was, say, six- eight months away ( to give you time to run the experiment and change replication protocols )

    One other thing I have been wondering about...

    Is there some way to contaminate plants send out to market, in agar

    so they will be fine if 'deflasked' but cannot be used to produce functionally sterile cultures from/ would be contaminated 'in vitro'?

    Hee hee :) There was a lot of speculation on this in the late 90s- ways to maintain IP, mask TC media so plants were still saleable but the media wasn't suitable for analysis ( if it ever was )- addition of food colouring etc. Not sure where it got to, didn't keep up with it

    Not sure about your functional contam to maintain IP and commercial advantage. Short answer- I wouldn't try it. If there is a fart in an elevator's chance that whatever contam was in there could harm a worker ( spores, people with lower immune systems ) or an ecosystem ( including agriculture ) or a business I wouldn't touch it with a pole with a condom on the end. Nice theory tho

    If you have developed a distinct variety you want to maintain you could patent it and protect that patent by having the plant fully sequenced ( sequencing costs about $1k last I checked but prices dropping all the time ). Patenting is a few $k too, and last I checked a patent needed to be applied for in every country the plant is sold.Then you have to enforce the patent by maintaining intel on whatever it is coming out on the market, a full time job for someone

    I am in favour of patents on plant varieties which are very distinct and have been developed by the patent owner- particularly ornamental species. Some scummy companies were trying it on for older strains of food plants as a kind of astroturfing, which was a bastard act of the lowest kind. Patenting is an ethical issue so YMMV

    Thanks mate for keeping us up to date on your progress, you have raised many many good scientific and ethical questions which are relevant for both beginners and experienced people alike, and you're also helping my professional development as well :D

    I did check the TC email list too, on your advice, and ended up signing on again, the volume there has definitely shrunk since I was a member last time, it's down from about 40 messages a day to 4-5 a week. Most manageable, and the search function on the archives is still pretty OK

  14. Posted · Edited by Darklight

    Thanks again!

    I will post more/ask more soon, but wanted to reply to a question asked earler.

    Here are some bad photos of the rooting of micropropagated M. Speciosa plants obtained by adding honey and coconut water to the medium.

    Honey was from the grocery store, said to be pure raw honey but who knows?

    Sry for delay, I had to do a bit of background checking to confirm my memory was correct

    How much honey/ how much coconut water did you use per litre of media?

    Raw honey would be interesting, tho I suppose the autoclaving would knock the enzyme activity out as much as it would processed honey.

    All the reading I have done has suggested coconut water has a stronger cytokinin activity than auxin.

    Now the important factor with hormone interactions is not just how much of what hormone in which species, but also the ratio between the hormones you add in combination. So maybe the interplay is important with the coconut water. Or maybe it's some other aspect of coconut water which is beneficial with rooting. About to give it a go in some recalcitrant species, may have more info in a month or so

    Those biological indeterminates can be lovely but so elusive to pin down, but shonman your experience shows that addition may have some benefits for rooting as well

    Inspired me, so I'll try it anyhow :)

    Don't worry about the quality of your pics, they look pretty bloody good to me. Getting publication quality TC pics is super hard, carn, we all know that. Yours are fine!

  15. Posted

    Mate you totally have the touch when it comes to easy teks and getting good fruit out of this place

    This tek has never worked for me at my place, I have too many fungus gnats, but I'm glad it works for you

    But you put a lot of work into it too and you deserve every success

  16. Posted

    The difference between fresh Damiana and dried commercial Damiana is like the difference between chamomile you harvest yourself and the chamomile you get in tea bags. The latter tastes like carpet sweepings in comparison to the former. Much of the beauty is lost

    • Like 2

  17. Posted

    Do caapi take well to pruning, will she grow back bushier and stronger than before?

    Yes they do, and I reckon it will. Put it this way- if it doesn't, I shall be surprised.

    At least she didn't do it when it is any cooler here. Now is about as late as you can do it to really get Spring growth happening

    • Like 5

  18. Posted

    Gelrite is cheap as! Its clear and soft and you can see infection with ease

    It also is .3% not 3% so you need very little

    http://www.plantculture.com.au/GELRITE_50_Grams/p3017392_13618904.aspx Only $20 for 50grams enough for 150 odd liters of media

    Lol gelrite works out at 4x cost the amount for agar. I guess that's a matter of scale. I don't like it's capacity to cause hyperhydricity, but it does have it's uses

    Just a quick note- this is *not* a criticism, but where you are typing in decimal amounts for scientific ( or any other ) publication it greatly enhances clarity and translation if you put a 0 in front of the decimal point.

    0.3% is clearer visually than .3%, and while they mean the same things, the ease of visual identification the former gives is polite and makes for fewer 3am critical errors- critical where ingredients are toxic when they are increased by that order of magnitude

    Am mentioning this because it caught me out once too. I used to think it was trivial. Then a colleague ( a PhD, who should have known better- they ram that ritual into your head early in most science degrees ) scribbled me a media list on a post-it. The decimal point required for one critical ingredient was not visible ( it was listed as 1% ). I did question the note but she said it would be OK. I ended up killing a batch of rare explants

    Am a convert to scientific notation. It's a good habit

    • Like 1

  19. Posted · Edited by Darklight

     

    also, speaking of weird ingredients and wondering about just how they were obtained, thought up etc,,,

    .....has anyone tried female stuff, like salmon eggs in the medium, 'Juice' or estrogen, etc?

    Nup. That's not to say it doesn't happen, or I haven't thought the question through enough to link it to some of the commonly used additives. And don't forget, commercial micropropagation people rarely publish their unique findings- so it's entirely possible

    Closest would be coconut water, the liquid endosperm of coconut seed

    Ha! Knew there would be something on a rethink. Google Foetal Calf Serum and Foetal Horse serum ( hint- it's ugly ). Not used in plant TC too often, but used in TC regularly. There will be others

    Edit: Also add: banana powder, tomato powder, V8 juice. Fruity goodness.

    • Like 1

  20. Posted

    Has anyone propagated Mitragyna Speciosa from callus?

    I also found some info saying that tryptophan as a precursor, added to the plant, increases alkaloid content...

    in this case it was a cell suspension culture, and showed increased alkaloid content after six days

    Nup, never did it back when it was legal and I don't know of anyone who has tried it since

    Ax node proliferation was sufficient for the purpose of replicating the numbers of whole sale plants I needed- mucking round finding a suitable media ( there were no other protocols around back then that I could find ) would have been a waste of effort unless there had been an urgent need for more plants

    Plus suspension culture, like any liquid culture, is very prone to contamination and can be hard to keep clean, especially for large volume systems where you need to add sterile air

    • Like 1

  21. Posted

    Has there been success with TC/MP with genetics that fruit with age rather than being photoperiodic? Is it possible even, or do the clones carry the same hormone that induces fruiting with age?

    Or can it be successfully done taking clones while the plant is still in its juvenile growth stage and propagated without the plants fruiting in vitro?

    It took me a while to work out what you were talking about sry

    Bamboo would be a prime example, I have a few colleagues who have worked on it extensively in TC, one said something about flowering at some point but I wasn't paying attention. I'll ask her again next time she is in touch

    ( Non-photoperiod sensitive pot would be another candidate, but there isn't the same amount of data on it/ I don't know if the same mechanism is involved re. flowering )

    • Like 2

  22. Posted · Edited by Darklight

    Thanks!

    Dang it, did my Mitragyna Speciosa thread disappear from the forum, or is it just buried?

    Is buried, there are like 3 or 4 good micropropagation threads on here now

    What is it with sperm and micropropagation? Adding Endosprem, herring sperm.....who thinks up this stuff?

    "I know. lets add some HERRING SPERM to the medium, that ought to do it !!"

    Next thing you know, I might as well experiment with ancient witches formulas, you know, eye of newt, toad, etc

    maybe a few bats boiled in cows milk at the full moon, then bury the medium in a graveyard at Midnight for one lunar cycle, and stuff like that.

    lol yeah I know, I've often wondered stuff like that myself. Some scientific publications read like film scripts once you get used to the flow ( all it takes is practice and experience, you do not need a tertiary education to get to that point )

    I remember seeing warfarin added to one media fairly early on, and vegemite to another, and thinking ' poor bastards got desperate, threw in their lunch or the lab rat bait or what was in front of them and hey it worked '- but there is actually some good science behind the reasoning for it. Once something is published that works, no matter how weird, people start trying it themselves and the repetition finesses the known effects of that ingredient and it enters general use.

    But who in their right mind was the first person who thought ... yeah, salmon sperm, we need salmon sperm. We have salmon, let's go and wank one until it comes and store it in a jar in the fridge '

    Still, formal science publishing etiquette means they take out the bits where you can hear everyone screaming in rage and frustration and fear. Only sometimes will the echoes remain...

    Will love to hear of the honey experiments. Was it raw honey or commercial? Honey would certainly be a biological indeterminate, so variable

    • Like 1

  23. Posted

    ...and coconut milk (from a live green coconut is said to be best)

    Hehe, it is so so easy, given the infinite numbers of possible media formulations and hormone combinations, to forget that sometimes simple changes offer equally good chances of success

    I'd forgotten about coconut water as a possible hormone substitute/ elicitor for rooting, but I have some and I'm going to throw it into my next batch of media for trial rooting mix

    Ta for reminder mate

    • Like 1

  24. Posted · Edited by Darklight

    You really know your stuff!

    Please elaborate on gelrite?

    I wouldn't bother with gelrite for micropropagation unless you have very particular research needs. It's expensive, not really necessary for most commercial replication, and prone to causing hyperhydricity in many species ( cells swell, appear almost transluscent, and replication becomes unreliable )

    If you required absolute control over a media ( gelrite is more chemically consistent between batches, wheras agar is almost a biological indeterminate between agar batches of different origin ) then yeah, go the gelrite if you have a bulletproof protocol and enough material to keep back so hyperhydricity isn't an issue, then yeah, gelrite.

    Dammitt, I broke my Hannah instruments PH electrode taking it back and forth between hot and cold fluid, wont do that again!!

    I didn't know about that but in hindsight it's logical- ta for the warning!

    Do you think a plant like the coffee tree, or M. Speciosa, where allowed, could be poropagated from leaf/petiole?

    Theoretically yes, but I've never tried it

    ...sometimes I get overwhelmed, and leave cultures in medium too long.

    Me too. Just make a note of it. And keep enough material back so you don't lose the lot and can start again if needed

    1) How long can these plants stay in medium, before they will stop growing and or suffer in quality in some way?

    Until they do. There are few hard and fast rules. Most advice is informal from other TC people. I ran generations of Mitragyna til they ran out after about 5 years. Another media formulation could have made them last longer, or a different light and temp regime, or a different starting material. Running regen from generations of callus could have reduced the time or introduced variation and unreliability too

    2) How long does medium made in advance, keep in the refridgerator?

    The SDICN medium, says it is good for six weeks storage, and then use.

    If you've sterilised media in the fridge, the other problem is going to be risking contamination via temp changes, especially if your lids and containers are made from different material and you open the fridge door a lot. Domestic fridges don't have the same temp control as lab grade ones too. My advice, don't store autoclaved media in the fridge and expect it to be reliable

    Hormones in media, autoclaved or not, are another storage issue. Some like IAA degrade quickly. Others not so much. IDK the death curve, I have a mate with chemical knowledge I ask about this stuff when in doubt, and try not to store made up media too long at all

    3) also, if there is a phase where the plant adjusts itself after being divided,

    am I further ahead to let it grow while connected, then divide, or just divide right away?

    Eh? I don't understand

    4) what is the best way to make these grow quickly after deflasking?

    If they are fully acclimatised post deflask, then you need to talk to a nursery person. Sorry, no idea

    5) what are some profitable plants to produce via micropropagation?

    You keep asking this, you've already got a bunch of species you're working on which look good :D Go and talk to some local specialist nurseries to you.

    I would also add, that combining honey as a sugar source, and coconut milk (from a live green coconut is said to be best)

    into the medium seem to increase growth and rooting a bit

    I did not know that- thank you! Got any data to share for it? Have added coconut water occasionally, but not as a regular ingredient. Never tried honey, have some raw. How much do you add?

    • Like 1

  25. Posted

    I have been experimenting with micropropagation for a while now...it is quite interesting!!

    Much thanks again to those who have lit the path before me, helping guide my steps...

    (...Darklight, Carol @ Kitchen Culture Kits, Shaman Australis Forum, and so, Torstein & friends...and the book, 'Plants from Test Tubes.)

    Dividing and multiplying the plants is very effective.

    They seem to put out roots, in some cases, in 1`.2 strength medium...oir, medium after the nutrients run out.

    They can be slowed down bv temperature, I have one batch that has not been transferred for five month!! (at 60F)

    Now, I am thinking about growing plants to a certain size, so they can be deflasked by others.

    The bigger the better up to a certain point.

    What might be the best way to do this part quickly?

    How big, is big enough?

    Here is what I am thinking:

    Divide explants until a certain number is reached.

    Root.,...in agar...

    Then, transfer to final sales container (any suggestions on these containers would be helpful).

    The medium in this final container in which the plant will be sold, would be full strength.

    They would have roots when placed in there.

    I would keep temperatures maybe at 70-75, and increase light.

    Theory being, that the plant would grow faster with light and full strength nutrients.

    Any thoughts on this are appreciated!!

    Thanks!

    Bigger is not necessarily better with direct sales TC plants.

    What you want is a saleable plant which will survive deflasking. A plant with a large surface area is going to be at a disadvantage after a certain size, because it will need to feed so much more of itself and learn to survive contam risks.It is also more likely to damage during shipping

    Other points against raising larger plants in TC include- time on the lab shelf which could be occupied by faster growing products, giving you more sales per square foot of grow shelf space. If you leave plants for six months prior to sale and they are six inches tall, you are probably not going to recover that against sale price when compared to the fact that the same space could be used to produce six smallersale plants every two months

    Finally, there is a tradeoff between plant size, and the increased risk of contamination when transferring it. More surface area on the plant, more potential for contamination to fall on it, or it to be injured during handling, and longer times in containers which may not be 100% contam proof

    I'm a fan on 30ml tubes for individual plant sales. Max 120ml. Polycarbonate, for ease and cost of shipping.

    Ta on yr update about cooling plant growth to 60F to maintain longer shelf life. I've tried it in a few species too

    • Like 1
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