Saturday, June 24, 2006

Mycelial Colonies are collections of individual single celled organisms (by DaveScot)

DaveScot asks:

"Could you start a thread for Bob OH and I to continue arguing about whether mycelial colonies are, in the context of evolution, single multi-celled organisms or collections of individual single celled organisms. My position is that they are the latter and that evolution can take place within a single colony via various mechanisms including gene induction.

"I bring this up because I have resumed experimenting with a volvariella volvacea colony which had apparently (barring experimental error on my part) acquired the ability to break down high concentrations of hydrogen peroxide. I hypothesized that the ability was due to gene induction whereby (probably through methylation) a peroxide decomposing enzyme was inducted. I further hypothesized that the induction would be sticky and would persist across generations of mycelia even in the absence of high peroxide concentrations.

"After a long distraction I just recently recultured the h2o2 resistant colony. It has been living in a more or less dormant state on a non-peroxide agar plate (several plates actually) for about 7 weeks now. I recultured it onto fresh non-peroxide agar yesterday and it is already showing new growth so it's still a vibrant, normal colony on its usual PDA medium. I have also prepared agar plates with various concentrations of peroxide to reculture these onto as soon as they grow out (4-5 days). The original colony took 2 weeks to adapt to the peroxide. If my hypothesis is correct the colony I'm growing out now shouldn't have any lag time when recultured onto the new peroxide plates."

29 comments:

Alan Fox said...

I have to wonder, Dave, why you just couldn't let Bob O'H continue posting at UD?

Anyway there it is and I PM'd Bob a heads up.

Alan Fox said...

How are you going to prevent H2O2 oxidising any organic material present on your plate and breaking back down to water and oxygen? Will you verify your H2O2 concentrations over timeƉ

Alan Fox said...

Dave,

I get the impression Bob O'H is not interested in further discussion of the subject.

I don't really see the problem. The mycelium is a continuum of cytoplasm enclosed in a membrane with many nuclei, reproducing asexually. In a large mycelium one would expect to find all nuclei containing identical DNA (i. e. clones) and possibly, where a non-deleterious mutation had occurred, a group of nuclei containing the DNA with mutations. So it is what it is, and it is a failure of language, rather than a misunderstanding of the reality that has happened between you and Bob.

DaveScot said...

Alan

UD isn't neutral territory and droning on about fungi isn't really topical.

There is nothing in PDA that will cause peroxide to break down. Peroxide decomposing enzymes, which are found in most living cells to some extent, break down when exposed to autoclave temperatures. The agar has been autoclaved and the enzymes rendered inert. Nothing else in the agar will decompose peroxide at a rate significant in that timeframe. The peroxide was of course added after autoclaving as it will break down at autoclave temperature too.

I purchased a substantial amount of research on using peroxide to facilitate mushroom culture from Doctor Rush Wayne who's been pursuing it informally for quite a few years. I'm using a species he's never experimented with and the volvariella species is unique amongst mushrooms in a variety of ways. However, the ability of peroxide to remain stable in PDA for a long period of time is well established by Wayne. He's also conducted many experiments with various other organic and inorganic substrates to evaluate peroxide's efficacy. Unfortunately most organic substrates that haven't been exposed to high temperatures are not suitable. Where he found a niche is in wood decomposers grown on commercial stove pellets. The process of making the stove pellets exposes them to high heat so there's a convenient substrate that requires no additional heating in order to use it provided you moisten it with a peroxide solution. And of course it makes sterile tissue culture a lot easier because a peroxide agar resists contamination by airborne spores. I already developed an inexpensive ampicillin-agar formula that totally eliminates bacterial contamination but fungal contamination, especially from common household penicillium, remains an obstacle and I still use a HEPA laminar flow hood to do sterile transfers but I can be pretty sloppy about it when not having to worry about bacteria which are orders of magnitude smaller and more plentiful in the air than are fungal spores.

Unique features of v.volvacea:

First of all v.volvacea is unique in that it cannot survive temperatures below 45F. It has some unique enzymes that break down below that temperature and kill it. All other studied mushrooms can tolerate temperatures down to just above freezing. It also requires a rather high temperature for optimum growth: 90-95F. At room temperature it is almost dormant while most other mushrooms thrive at room temperature and below.

Secondly it is the most aggressive known mushroom when it comes to the speed at which it can engulf a food source. While most mushroom mycelia take weeks to entirely cover an agar plate this will do it in 4 days and after 6 days will have grown all over the surface of the glass looking for more food and about a half inch into the air from any surface. They're snow white when young and on the hunt and I affectionately refer to them as fluffballs. After about 10 days of not finding any food they start producing an asexual resting "spore" (I'm observing this under a microscope in another line of experiments) called a chlamydospore and the colony takes on an orange color and buckles down for a long period of dormancy awaiting a change in the environment. I'm trying to figure out to produce and isolate chlamydospores so commerical cultures can be stored for long periods of time without liquid nitrogen or repeated reculturing. Normal spores are not pure strains and not suitable for commercial production. Reculturing isn't a bad way to go for those without cryogenic facilities but senescence inevitably destroys vigor and fruiting capacity after some number of recultures.

Thirdly, it can digest both wood and non-woody substrates but is very slow at digesting wood as its lignin decomposing enzymes are not the same as other wood decomposers and not as efficient at it.

Like most mushrooms it will adapt itself over a period of weeks to a range of different substrates for optimum growth. Basically they each have a suite of enzymes that can digest different organic molecules but they don't produce all the enzymes all the time and through some mechanism (probably methylation) induct and/or increase production of the ones they need and turn off or slow down production of ones they don't need.

Anyhow, in Dr. Wayne's experience no species he's experimented with (about a dozen species) has ever demonstrated the capacity to break down the peroxide concentration I tried. Only one of a dozen colonies I used adapted and the one that managed it did it on the highest concentration while those on much lower concentrations did not adapt. Moreover, when other mushrooms species do adapt to lower concentrations, it takes them about 2 weeks to do it (the same as I observed) but even then they exhibit a slower growth rate than normal. The v.volvacea colony I isolated did not grow more slowly once it adapted.

There's a lot of room for experimental error. It has been suggested that the peroxide decomposed by itself over that time from exposure to light. However, had that happened, there should have been other things growing in it as it had been well exposed to unfiltered air. After I removed most of the mushroom mycelia I left the plate out at room temperature and after another 2 weeks in partial sunlight a very sickly penicillium colony sprung up that never grew larger than a matchhead. Even 8 weeks later nothing is growing on it except another puny penicillium colony so it appears the peroxide is still acting as a sterilant.

There was one other variable in that I was simultaneously experimenting with a synthetic hydrogel instead of agar. No one that I can find has experimented with using a hydrogel as a substitute for agar as the gelling medium. The hydrogel should be completely inert and be able to absorb and give up water & nutrients at a suitable rate but I saw inconsistency in that in the non-peroxide experiments with some thriving colonies and some duds with presumably the same environment. I set aside the hydrogels for now as they are a little too mushy compared to agar for culture work and when absorbing a nutrient solution instead of plain water their fluid holding capacity is vastly diminished which makes them less interesting as a larger scale substrate.

The peroxide response however was WAY interesting.

DaveScot said...

alan

Bob is adamant that evolution cannot take place in a mycelial colony. He says it is a single organism and cannot evolve until the organism reproduces.

You of course are quite correct in what you said. Each cell in mycelial colony has the capacity to start another colony and so in the normal course of asexual cell division within a single colony a heritable mutation can occur. If that cell or its progeny happens to be the original cell in a new colony then viola - a new strain is born.

Propogation of colonies by asexual means is an important way these organisms reproduce in nature. Think how many viable cells are distributed when a rainstorm produces a stream of water that washes over a colony breaking off cells and transporting them downstream where they can land and immediately begin vegetative growth of a new colony.

I can only presume that Bob refuses to concede not because he doesn't know he's wrong but because he is unwilling to concede to ME. How insecure is that?

Wonderpants said...
This comment has been removed by a blog administrator.
Wonderpants said...

Just to add my tuppence worth, I fail to see why DaveScot should be given a platform on this blog, Pandas Thumb, or After The Bar Closes.

If he won't give others the courtesy of having their views heard without endless editorialising, censorship, or banning (see any thread on UncommonlyDense), what good reason is there for us to give him this courtesy?


"I can only presume that Bob refuses to concede not because he doesn't know he's wrong but because he is unwilling to concede to ME. How insecure is that? "

Newsflash: Rank hypocrisy from the ringmaster in charge of the shyster Dembski's freakshow blog.

Alan Fox said...

There is nothing in PDA that will cause peroxide to break down.

This should be confirmed experimentally, otherwise your results will be meaningless.

Alan Fox said...
This comment has been removed by a blog administrator.
Alan Fox said...

Open means open to all.

I'm hoist by my own petard, Wonderpants.

Jeannot said...

Dave: I further hypothesized that the induction would be sticky and would persist across generations of mycelia even in the absence of high peroxide concentrations.
After a long distraction I just recently recultured the h2o2 resistant colony. It has been living in a more or less dormant state on a non-peroxide agar plate (several plates actually) for about 7 weeks now. I recultured it onto fresh non-peroxide agar yesterday and it is already showing new growth so it's still a vibrant, normal colony on its usual PDA medium

Do you have an estimation of the mean number of asexual generations (cell divisions?) that your colony has undergone during that time?

Epigenetic heritability is actively studied. If you think you found something valuable, you can reproduce you experiment under strictly controlled conditions and publish.
Gene induction by methylation should be reproducible.

Jeannot said...

...mycelial colonies are, in the context of evolution, single multi-celled organisms or collections of individual single celled organisms. My position is that they are the latter...
This just depends on the reproduction of mycelia.
If any cell can start a new autonomous colony in natural conditions, mycelia can be seen as collections of individual single celled organisms.
You didn't have to peform your experiment to show that.

DaveScot said...

ds -> There is nothing in PDA that will cause peroxide to break down.

af -> This should be confirmed experimentally, otherwise your results will be meaningless

Doctor Wayne's extensive research confirmed this. I said that already. I can't reprint his research results as it's copyrighted and only available by purchase. However, you're free to follow the link I left and read the bits he's made public.

In any case I confirmed it myself by purposely exposing the plates repeatedly to unfiltered room air both during initial inoculation and subsequently took them out of the incubator several times and removed the plate cover for about 10 seconds exposing them to room air. There was no contamination whatsoever except at the lowest peroxide concentrations and even then it was a diminished amount. If there had been no peroxide I would have had penicillium (common green bread mold) all over everything.

Alan Fox said...

Dave

I doubt "If there had been no peroxide I would have had penicillium (common green bread mold) all over everything." will cut it, if you want to convince anyone you are on to something. Here is a way of checking.

DaveScot said...

jeannot

It's gotta be in the millons of cell divisions. I took small wedges of the original culture and put them onto new plates. They grew out in all directions about 3 centimeters. Individual cells of the mycelium are about 10 times as long as they are wide and they are about 4 times as wide as an individual nucleus. Each cell is multinucleated with 2-4 visible depending on what stage of growth it's in. It has to take many millions of cells to cover the plate. I could give you a better answer if I had a reticule for my microscope with a measurement grid on it. I looked for one for sale somewhere and didn't find anything. It's a Swift M1000 and the eyepieces are common enough and it appears a reticule can screw into the bottom side of the eyepiece. Anyhow, each cell is connected on either end (except for the active growing tip) by a perforated division like a reel of paper tickets. The strands intertwine to form larger structures of various types including the fruiting body (mushroom) and chlamydospores. The cells separate easily at the perforations without damage and the detached cell can continue to divide and form a new colony or integrate itself into an existing genetically compatible colony. It's kind of fun exploring the 3D maze they make with a microscope that has a good fine adjustment controls in 3 axes. 400x is just about right for detailed viewing. Staining helps but isn't really necessary. A substage colored filter makes viewing a bit easier. I tried both overhead and substage halogen lighting. Substage is better but you have to work at getting a thin enough sample to be translucent.

Nothing I've done was intended to show that mycelial colonies of this type are still collections of individual organisms even if the cells are normally connected end to end by perforations. That's a given as far as I'm concerned. I really couldn't understand Bob's position that only the whole colony taken together could be considered an individual organism. Even worse, as I recall he said his PhD dissertation was something about fungi that parasitize trees in fruit orchards.

In any case, we'll know in a few days. The recultured ostensibly peroxide resistant colonies are now almost 3 days old and have just about completely covered the new plates. I wait until they're 5 days old before doing anything with them as at 4-5 days old the most common fungal contaminant spores will germinate and become visible. I've got my methods down now so I get virtually no contamination but I also unseal them at 5 days and replace the solid plate cover with a microporous filter otherwise they'll choke and die for lack of oxygen in another 10 days. The micropourous filter isn't 100% effective but after the colony has completely covered the agar surface any contaminant spores that make it through the filter can't get at the food and don't become much of a problem. The filter lets water vapor escape and a quarter inch thick layer of agar will dry up completely in about 8 weeks. The chlamydospores can survive that and I'm in the process of determining just how long they can survive it. Very little work has been done on propagating pure tissue cultures of this species. The literature is ambiguous with some people (like Stamets) reporting cultures kept at room temperature remaining viable for up to a year and others saying a few weeks at best. I've gotten some to survive for at least 6 months in a special refrigeration unit (I adapted one used for beer brewing) that keeps them at 50F but it's hit & miss and they take about 4-5 days to recover from the cold before growth resumes, if it resumes.

I think the problems other labs have encountered are due to not providing the proper conditions for chlamydospore formation. Because these grow so fast for healthy growth to the point of chlamydospore formation they need a lot of oxygen. More oxygen than you get in a regularly sealed petri dish and if you don't seal the dish it contaminates on the edges. So part of what I did was find a way to reliably grow pure tissue cultures to chlamydospore production. They seem to go dormant at that point and now I'm seeing how long they remain viable after the agar is dried up. Last week I lifted a few dried up cultures out like little frisbees and tossed them into an unsterilized mason jar for microscopic viewing at some later point and darned if the little buggers didn't sense something changed and they threw out a few exploratory aerial mycelial strands onto the glass to see if there any food out there. Finding none they stopped exploring. I swear these things have complex behaviors almost like animals of some sort.

DaveScot said...

Alan

What part of "Doctor Rush Wayne already confirmed that h2o2 persists in a PDA medium" didn't you understand?

I don't need to reinvent the wheel. Do you think I should get a spectroscopic analysis to prove that the potatoes I boiled for the PDA were really potatoes too?

It's exactly this kind of argument that gets you banned at Uncommon Descent time after time no matter what name you sneak in with. If something doesn't agree with your preconceived view of things nothing and no one can ever change what you think. And contrary to your inflated view of yourself your arguments are not well constructed.

Alan Fox said...

You need to know the concentration of peroxide ions. To convince anyone that your experiment is valid and worth repeating, you need to confirm presence and level of peroxide.

Sorry if that makes you feel defensive, but facing it now will avoid disappointment later.

Alan Fox said...

OK, the mass spectrometer was a bit tongue-in-cheek but what about this?

DaveScot said...

Alan

This isn't anything meant for publication in a journal. If it becomes more than an interesting hobby it'll be a commercial product such as tissue cultures and spawn that can be handled in the open air without risk of contamination and/or spore samples that produce a pure strain and can be airmailed in a flat envelope anywhere in the world and won't die in transit. The proof is mailing it out and it not being dead when the customer receives it.

DaveScot said...

Alan

Just for the heck of it I checked out the peroxide assay kit you linked to. It doesn't appear to be usable for an agar substrate. It's for liquids and lipids.

Let me tell you how I do measure peroxide concentration (I do this to the peroxide solution I buy at the drugstore to ensure it's the advertised 3% solution that I'm starting with as the concentration can vary a lot from that particular source).

I take a graduated cylinder, immerse it in a pan of water and bring it partially out with the bottom facing up such that most of it is out of the water but the opening is underwater and it is still filled with water. I then take a measured volume of peroxide solution in a separate closed vessel with a tube going from the peroxide vessel to the mouth of the graduated cylinder. I then inject a suitable peroxidase (liquified banana works well) into the closed vessel which causes the peroxide to decompose into water and oxygen. The graduated cylinder collects the o2 and the gas volume can be measured off the side of the cylinder. The amount of gas collected is in direct proportion to the amount of peroxide.

I can adapt this method easily to measure the concentration of peroxide at the surface of an agar plate if I wanted but for my purposes the proof is that the agar is not supporting the growth of any airborne contaminants while allowing healthy mycelial growth. I really fail to see why, when your development effort has acheived its end goal, you need to continue analyzing the intermediate steps taken to get there. That's just wasted effort in my book and we engineers don't like waste. We're goal oriented.

Biogeer said...

That's just wasted effort in my book and we engineers don't like waste. We're goal oriented.

You're an engineer? Where did you get your degree?

Colin said...

"I really fail to see why, when your development effort has acheived its end goal, you need to continue analyzing the intermediate steps taken to get there. That's just wasted effort in my book and we engineers don't like waste. We're goal oriented."

To paraphrase, "Once I've achieved my goal by reaching the desired conclusion, it would be a waste of time to examine the accuracy of that conclusion."

To an ID Creationist, the goal is not an accurate conclusion, just a ideologically correct one.

DaveScot said...

biogeer

I never finished a college degree. As I was working my way through college, just as I was about to enter my junior year and become a computer science major, I went on a job interview at a company that designed and marketed portable computers (circa 1980). The VP of engineering there interviewed me and he was so impressed with my knowledge of computer hardware he wanted me full time in a design engineering position and offered me a salary far in excess of what newly degreed engineers were getting. Southern California was a hot market then and the personal computer industry was created by non-degreed people like Bill Gates, Michael Dell, and Steve Job, it was moving so fast universities couldn't keep up with industry, so degrees weren't much of a qualification in any case. In 1982 one of my portable computer designs made the cover of Popular Science magazine along with Adam Osbourne's, the Kaypro, and a few others. There followed 20 years of job hopping at ever higher salaries until in 1993 I landed a senior engineering position at Dell Computer Corporation in laptop R&D. Our laptops became the gold standard in reliability and battery life, and I became financially independent there from stock incentives and retired in 2000 to pursue other interests.

DaveScot said...

Colin

The desired conclusion is a marketable product. Academics generally don't know when to stop investigating and start exploiting what they've learned or developed. But hey, if being stuck in a low paying job teaching children what you think you know turns you on, far be it from me to discourage you. To each their own.

Colin said...

Then I'll rephrase - ID Creationists don't seek accurate conclusions, but rather popular or profitable conclusions regardless of their accuracy. This would certainly explain Dembski's focus on book sales and speaking fees and abstention from any sort of scientific process.

DaveScot said...

Next data on Sunday. Yesterday I inoculated 2 series of 6 PDA plates with h2o2 concentrations ranging from 0/100 to 4/100 of a 3% h2o2 solution. One series was inoculated from the culture that was able to grow on a 5/100 h2o2 back in first part of May (transferred back to 0/100 h202 for six weeks) and the other series from a culture of the same age/strain that was never exposed to h202. It took the h202 tolerant colony 2 weeks to condition itself to the h202 back in May. The lag time on this run should be revealing.

Alan Fox said...

Dave

You should qualify concentrations as initial concentrations, unless you are going to assay your plates for residual peroxide concentration.

DaveScot said...

Damn! Ambiguous results!

The control colony (one never exposed to peroxide) evidently senesced on me because not only did it grow poorly or not at all on the peroxide agars it didn't grow with vigor on normal agar.

The colony exposed to high peroxide concentration many weeks ago grew vigorously (i.e. normally) on normal agar and with declining speed with increasing peroxide concentrations. However... it IS growing much faster that it did in the original h2o2 exposure. In the original series most of them died at higher h2o2 concentrations while in this series there's not a single fatality. So something did change and it was a sticky change across many generations. I want to reiterate that this is nothing new for mushroom mycelia. It's been observed for a long time that colonies optimize their enzyme production for most efficient digestion of the food available. The optimization can take days or weeks and will persist even when the food source is changed and none of the orginal cells in the colony are still alive. So it's hereditary but probably epigenetic and certainly not random. Lamarck is vindicated again.

At this point I'm only interested in one more related test and that is taking the same peroxide series of agar plates, exposing them to the open air for an hour, then sealing them up and timing how long it takes contaminants to bloom. In this last experiment I hope to find an optimum peroxide concentration for inhibiting contaminant growth while allowing an acceptable rate of growth for the mushroom mycelia.

The goal of this was never anything other than finding a way to reduce the overhead associated with growing these mushrooms.

Alan Fox said...

OT

How is your relationship developing with Janiebelle? Does Mrs Springer approve?