NEMO-Chemistry
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Bacteria suggestion for feeding
Hi I am after suggestions for a bacteria to grow and use, the purpose is to feed a amoeboid in a growth solution.
I cant find information on what is normally used, first a few needs the bacteria has to fill.
Must be a max of LV 1.5! I have Lvl 2 fumecupboard with ducted HEPA outlet, it also has a formalin sterilizer built in, and UV lights.
I have a couple of professional Gas incubators, 2 are Lvl2 and one is Lvl 1.
I have the equipment to handle LVl2 organisms, but for safety I would really like to stay the reasonable end of Lvl 2, hence lets call it 1.5
The bacteria needs to be able to flourish in mainly water, while it needs to be able to grow fairly rapidly, I dont want it out competing the
amoeboid.
Its got to be easy to get hold of.
It needs to be easy to grow and keep fairly pure.
The amoeboid in question is the flagellate stage of Physarum. I want to culture Physarum this way as its the best way to produce a large number
quickly.
I have sclerotium stage Physarum, a fair bit of it. I could just activate and grow it in the plasmodium state. But it takes too long (for my purpose),
to grow it to plasmodium and then dry it out and cut it up on filter paper.
So the idea is to activate some the scerotium as normal, and then gradually alter the environment so it breaks into the amoeboid stage.
Once I have some in that stage, I need to rapidly culture it for a constant supply in good quantity. The Physarum is actually going to be used to feed
something else!
I like culturing and studying the plasmodium stage, but I have never tried to culture it in solution. From the little I found, it looks like my best
option for food, is bacteria.
But i have no idea which bacteria to culture for it. So anyone got any ideas?
I forgot to mention the obvious, i dont have a problem using Lvl1 !! so lets say upto 1.5 max 
[Edited on 27-1-2018 by NEMO-Chemistry]
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Harristotle
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Can you not use something like a lactobacillus from yoghurt or kefir?
That is pretty much jam jar on the fridge level, and shouldn't present any biosafety issues.
L. acidophilus seems a good candidate - but need a bit more complex media than just salts and a C source.
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NEMO-Chemistry
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Quote: Originally posted by Harristotle  | Can you not use something like a lactobacillus from yoghurt or kefir?
That is pretty much jam jar on the fridge level, and shouldn't present any biosafety issues.
L. acidophilus seems a good candidate - but need a bit more complex media than just salts and a C source.
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My problem is Physarum in the amoeboid state, dosnt like much in its water. A build up of nutrients might not suite it.
I spose I could find some clean old puddles, take some samples and see what grows out. Do gram and acid tests, basically as much as I can to make sure
its pretty safe.
But that wont tell me for sure what I am growing. I will look into L. acidophilus, I dont intend growing in the same media as the physarum, so I could
filter out a bacterial culture with a syringe filter, then flush that into the physarum.
What I dont want is the bacteria liking the physarum conditions too much. So your suggestion might work, there is also a huge well known cheese maker
factory near me .
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aga
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It's funny, as i would like to attempt to grow my own cheek cells.
Got as far as buying some sterile petri dishes and agar-agar, also PID controllers off ebay.
The un-boxed PIDs are for a temperature controlled box, heated with a simple light bulb.
Still done absolutely Nothing with them, yet.
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NEMO-Chemistry
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| Quote: Originally posted by aga | It's funny, as i would like to attempt to grow my own cheek cells.
Got as far as buying some sterile petri dishes and agar-agar, also PID controllers off ebay.
The un-boxed PIDs are for a temperature controlled box, heated with a simple light bulb.
Still done absolutely Nothing with them, yet. |
You would likely be better with flasks for cells, petri dishes and agar (aga with agar is a head fuck) will likely grow bacteria not the cells.
Never done mammal cells but I will look through the books I have.
I totally forget the flask name now, but what your after is a flat flask, they have a special coating on the bottom of them, the cells stick to that.
The you use a special nutrient solution, I need to look up the protocol, my guess however is, cheek cells are likely to be exactly the same as mice
cell lines.
Agar I cant see working though, it would grow the bacteria from your mouth, not matter how much to try and sterilize first with ethanol lol.
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NEMO-Chemistry
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Just as I a start point the flasks you want are these, they have a special surface on the inside, you use a special liquid media.
https://www.ebay.co.uk/itm/Corning-cell-culture-flasks-150cm...
Here is a paper on mouse cheek cells, not a million miles off, but not what I was looking for. I will go switch on the pc, pretty sure mendely should
bring up some protocols for you.
https://sci-hub.tw/https://onlinelibrary.wiley.com/doi/10.11...
Now here is where the piss gets taken out of me........... Human cell work needs care, especially oral cells. Normally you have some bad dudes in the
mouth, they dont harm because numbers are small, but your culturing witch grows large amounts rapidly, its really important to be careful and use good
technique.
Sounds a bit old woman like, but some bacterial grown big enough to see on a plate, should be treated with a great deal of respect and caution.
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Harristotle
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There are a couple of ways to try this:
1) Could you feed them on freeze-dried bakers yeast? No culturing required!
2) Do you have a centrifuge? Maybe spin down some yoghurt and wash a couple of times with water (pond? rain?) that your SM likes.
(This is becoming quite meta - aga on agar and SM discussed on SM !)
Cheers,
H.
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NEMO-Chemistry
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Yes I have a couple of centrifuges, from a tiny fischer desktop one to a floor standing heat controlled monster, that sadly dosnt work (needs
bearings).
My tiny desktop one would be ideal for this, I hadnt thought of that! I did think of yeast and had a bad experience once with it.
I use yeast for alot of things, even in fresh water freeze dried stuff can be slightly active, you dont notice it mostly. But SM in water is real
sensitive and tiny amounts of ethanol would kill it. This is working on the principle that the ameboid behaves like most others, i have grow common
amoeba and used yeast, the yeast cultured ones died after 12 days.
For most pond dwellers of the microscopic size, i spin down yeast pellets from live batches, then wash and use. It works well in most cases, eulgena
seems to like it alot.
I have to build my diatom etal stock up again! I had a great collection of cultures, most died when we moved, mainly because we had several months
where I could get to them in boxs. I assumed (wrongly) I would cysts or spores, but most came never came back to life.
We have some great puddles around the house, always got some water in, maybe time to go puddle dipping again.
Ignore the new lab oven on top, The thing under it is the orbital incubator i got cheap, finally fixed it! does need a new belt as old one is about to
go. But works great!
Pond stuff will go in cultures in that, I am picky what I put in the CO2 incubators!!
I got a great book for aga, but the file share thing isnt set up properly yet.
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Reboot
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Mmm, it's been a long time since my cell culture labs, but as memory serves it's a challenge. Short term success tends to require growth media
enriched with things like fetal bovine blood serum (rich in growth factors.) A living vertebrate is a complex stew of regulatory and other
non-nutritive components that affect viability.
Long term establishment of a cell line is harder still, requiring mutations that turn the cells into something more cancer-like (immortalization.)
The first real success story was a cervical cell cancer line from Henrietta Lacks. (God only knows what happened to privacy rules that we know her
name and illness.) The cells were robust enough to grow in culture and became incredibly important for human biochemical research. Unfortunately
the cells were a little TOO robust; they've become a major pest in labs, contaminating and over-running many other cell lines in labs. Today, the
HeLa line is a novel life form of it's own, arguably a new species that evolved from human cells spiked with HPV (virus.)
https://en.wikipedia.org/wiki/HeLa
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NEMO-Chemistry
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Actually aga has hit the jackpot, call me selfish but waiting a couple of days before I spill the beans  .
HeLa is same kind of privacy thing with the little girl, the little African girl who's cells first made Interferon. That was back in the 1970's, I
dont remember much as I glanced passed the info, but it came up while reading about techniques.
Aga dont get Bovine serum....yet .
But you still need those flasks or you have to coat your own flasks, its alot to write in full, so put simply. You do some stuff to the flask and the
cells stick to the bottom (flask is on its back), then once you need free cells you do something else and the cells release.
The you need a centrifuge....
But culturing your own cells is well neat.
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SWIM
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| Quote: Originally posted by aga | It's funny, as i would like to attempt to grow my own cheek cells.
Got as far as buying some sterile petri dishes and agar-agar, also PID controllers off ebay.
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Okay, I think I see where this is going, and I'd like to propose an absolute limit on the number of agas allowed to register on this board.
Nothing personal, aga, but otherwise this is going to turn into a bad Red Dwarf episode.
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NEMO-Chemistry
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aga if you want to try this seriously let me know, i am working on how to get a 200mb book linked to you, and I have a free source of serum (temporary
its free). I will U2U you the details, but keep under the hat for a few more days.
I need it and the supply is fairly limited. Once I get my order excepted you can tell the world
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Tsjerk
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Why not start with something simple as E. coli? They are easy to isolate and grow, and they will not grow -but survive- in poor nutrient environments.
To isolate: anaerobic conditions with galactose (milk-sugar) as sole carbon source, this will isolate E. coli as long as you don't have anything
really strange in your start culture (just fill a small bottle too the top with galactose medium, making sure there is no bubble left in the cap).
Edit; what is level 1.5? I know 1, 2, 3 and 4 but I didn't know there are levels in between.
[Edited on 29-1-2018 by Tsjerk]
[Edited on 29-1-2018 by Tsjerk]
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NEMO-Chemistry
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| Quote: Originally posted by Tsjerk | Why not start with something simple as E. coli? They are easy to isolate and grow, and they will not grow -but survive- in poor nutrient environments.
To isolate: anaerobic conditions with galactose (milk-sugar) as sole carbon source, this will isolate E. coli as long as you don't have anything
really strange in your start culture (just fill a small bottle too the top with galactose medium, making sure there is no bubble left in the cap).
Edit; what is level 1.5? I know 1, 2, 3 and 4 but I didn't know there are levels in between.
[Edited on 29-1-2018 by Tsjerk]
[Edited on 29-1-2018 by Tsjerk] |
No levels in between, I did explain that. I have most the equipment to deal with level 2, but not the experience, so i was saying aim the suggestion
upto a maximum of.....
Some Level 2 are not that bad to work with, some are not that good, so your right 1.5 dosnt exist in real life, its just my way of asking for
something that isnt a PITA.
For example HIV is Lvl 2, I wouldnt want to work with it, but so is Staphylococcus aureus and I have worked with that.
Actually Staphylococcus aureus is a good example of what I would think of as 1.5. Yes you need all the stuff of Lvl 2, but you dont shit yourself
working with it.
As for the Bovine serum....
We got lucky, aga mentions cheek cells and fischer scientific think hmm, so aga wants to grow some, tell you what we will help you out.
https://www.thermofisher.com/uk/en/home/products-and-service...
Probably no voodoo in it, but a lucky break all the same, I have asked for some, so fingers crossed.
[Edited on 29-1-2018 by NEMO-Chemistry]
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Tsjerk
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With some exceptions E. coli is officially level 2, as they can be pathogenic, but personally and practically they are treated as level 1.
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NEMO-Chemistry
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| Quote: Originally posted by Tsjerk | | With some exceptions E. coli is officially level 2, as they can be pathogenic, but personally and practically they are treated as level 1.
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I have used known strains from a supplier alot, the only problem with it is knowing when its not the strain you thought it was.
But anaerobic growing then poor nutrient conditions might do the trick.
Just to utterly confuse me, I contacted someone who works with DVSM alot, I am meeting them at the big bang in Birmingham in march.
They have sent me some dried Physarum, but according to them they found DVSM (Dog Vomit Slime Mold), needs a rich medium in the flagella stage.
Which makes utterly no sense, in the wild apart from the normal puddle crap, i cant see too high a nutrient level..
I got enough DVSM to experiment with, so I will start with that, but it means going from one stage then trying to coax it into a slurry and then
finally getting it grow!
I got a small amount of time on my side, i can keep and grow enough the normal way, this should last me a couple of months before I need volume and
the smaller flagella stage.
So now my problem is culture bottles and flasks, I have 2 contacts who sell ex uni surplus stock cheap (ish), I will see what they got thats just gone
out of date.
I know this is mostly a chemistry loving forum, but this side of chemistry is what I love most. I got permission to borrow some content from here, i
will set the learning site up with some as a demo.
If it still dosnt float anyones boat, I will use the web space for documenting these sorts of experiments.
I might have landed two Nikon C mounts for scopes cheap , they should fit the
Nikon scope . Now that would be a cool write up!
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NEMO-Chemistry
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Oats is the answer.
Old Oats left in the cupboard for no idea how long. Same Oats I feed the plasmodium form on, seems in culture they have enough bacteria to suite the
need. Not sure what the bacteria yet, but 3 strains have popped up so far, one dies out real quick in media (i suspect the media is lacking
something), but the other two seem fine.
Quick look under scope and Gram test hasnt made the bum squeak. Having said that.....I cant think of many spiral bacteria!!! but one is a spiral, or a
Rod that is seriously out of shape .
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Tsjerk
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Can you make pictures? Maybe with your phone camera through the eye piece?
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NEMO-Chemistry
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I have tried that, but.....
Recently I got some old Nikon camera scope bits, these were from a Nikon film camera on a scope. I know this because the camera came with it .
I got a Nikon DSLR and a Nikon scope, but for the life of me I cant work out how it sets up!! It should just stick on the top, but dosnt.
The main tube with the C mount does fit my DSLR, but the tubes dont fit the scope????
If i post pics of the bits I got then someone can perhaps point me how to set it up. this is something I have wanted to do a very very long time.
With access to a Nikon D810 with 4K video and 38 meg frames...The pics would be stunning. My phone is a note2, none the phone things I have tried fit
it...
Besides I am pretty sure I must have just about all the proper bits, maybe a small thing missing, so I will post the bits I have up and someone with
microscopy knowledge could maybe guide me how the hell it sets up.
the scope is Binocular and not tri nocular.
I can do most stain tests including acid fast etc, so if you help set up the camera i can do some cool pics. I havnt tried to identify yet, all i was
bothered about was making sure it wasnt something seriously nasty.
And it appears it isnt, but the spiral bothers me.
[Edited on 17-2-2018 by NEMO-Chemistry]
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NEMO-Chemistry
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I have a fitting on order for the camera, i think this is a tube i am missing for the scope side. If not I will post up what i have and someone can
work out what i need .
The bacteria crash out really quickly, so its back to plating out and isolating from the oats. I have a feeling one of the bacteria is producing
acetic acid (smell mainly), but is different from the other bacteria i have used to make acetic acid, then again smell alone is not exactly a good
diagnostic .
I did rush the plating and isolating, I need to keep the physarum going. These are now plasmodium stage and ready for drying out into scalarum, can
store from that point.
I thought i found a wild type slime mold in the wood, it was on a branch ready to sporalate. No luck with it however, its a gamble at that stage, 3
chromosomes need to be compatible and combine for the physarum to divide like that.
I would pay to go on a walk with someone who collects them! I either dont have my eye in, or they are really rare in the wild around here. 12 months
of looking for wild types has me a bit despondent, in the local library i read the archive from the local paper.
Going way back there is reports from 30 years ago, mainly spring time but apparently it sounds like there was physarum all over the place.
People panicked a bit not knowing what it was, the paper speaks about creeping slime patches on the lawns in gardens. There is reports of a red type
thats pretty large, what seems to have bothered people was the fact it moved.
I doubt they saw it move, but i guess it was in one place and then a couple of hours later in another! No pictures in the archive unfortunately, but
sounds like physarum. So we have either made it extinct in this area ( that would be really unlikely), or i simply dont have the knack to spot it.
Bloody Lichen dosnt help much!! All over the place so finding some creeping up onto branches is not easy. Being nosy in the leaf litter dosnt throw up
much. What bugs me is the vast majority of it in schools and universities in the UK, are all pretty much the same genetically and all came from
Carolina Scientific originally.
Even our own Blades Biological got it from Carolina at the start. Why does this matter? Well I am a member of the Physarum collective (yeah really
geek like), a couple of people on there are real scientist's unlike myself. But they work in optics and imaging.
It seems the genetics are getting a bit scrambled and physarum you mainly get now, has suffered a shift in light tolerance. It still dosnt tolerate
much light, but the wavelength of light has shifted. I went hunting for proof in the literature, MOVE ALONG NOTHING TO SEE HERE!
So you get the impression its well talked about in corners, but no one mentions it out loud or wants to discuss it openly! Really odd, but then again
i think its only odd people who are into this Amoeboid.
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VictorMedvil
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I feed my Bacterial Plasmids, Sugar Water if Aquatic or Jello if needed in a liquid medium or Agar if needed in a solid medium, Any of those should do
as they like sugar Bacteria and such even Hybrid Organisms tend to like sugar like most lifeforms being their primary energy source. Agar is probably
the best feeding substance next to jello in my opinion.
Agar Ebay
[Edited on 16-2-2019 by VictorMedvil]
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Tsjerk
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Dude, you don't feed bacteria plasmids...
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VictorMedvil
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*feeds them anyways so they will replicate*
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