Sciencemadness Discussion Board - Plant tissue and cell cultures (anyone experienced?)

Plant tissue and cell cultures (anyone experienced?)

NEMO-Chemistry - 1-11-2017 at 01:17

No idea where to post this, I am about to do alot of experimentation with both plant tissue and plant cell culturing.

I was wondering who here has experience of doing this?

I have a mini Lab setup dedicated for this (its small but functional), I am now waiting for deliver of a class1 and a class 2 bio safety cabinet I have managed to pick up cheap.

I found some papers regarding media for the cultures, one describes the use of starch as a gelling agent, from the results it seems it gave better results than agar.

So I am starting my journey by looking at different growing media, anyone else interested in the topic? If there is I will post my experiments here and any papers etc I come across.

I wasnt sure if this came under Bio Chemistry or Misc chemistry... To me its more Bio Chem but feel free to move to a better section

.

I will be referencing all material so please dont move to beginners where it will get drowned!

For those who have done this before, did you use commercial medias and recipes or did you go down the self made route?

I have some commercial media, but I am interested in trying out some of the media mentioned in papers I found.

I am having trouble finding Glass petri dishes at a reasonable price, so for now I will be using single use plastic ones.

I will post the starch papers later, once I am back home on my main machine. But for the moment I do have this one I have on this laptop.

Attachment: henderson1988.pdf (306kB)
This file has been downloaded 758 times

Metacelsus - 1-11-2017 at 04:46

For the growth medium, I would recommend this: https://en.wikipedia.org/wiki/Murashige_and_Skoog_medium

I have a little bit of experience with plant cell culture, but I'm not an expert.

NEMO-Chemistry - 1-11-2017 at 07:07

Thx Metacelsus, Skoog is one of the mediums I want to asses, what sparked this off was finding several buried papers, these seemed to suggest that using a starch gelling medium instead of agar agar, gave better growth rates and survival etc.

That alone is nothing to get excited about, however the results in more than one paper, seemed to suggest the benefits were >40%. However reading some more modern texts on the subject, these methods hardly get a mention, I am baffled why agar is used instead of starch, if these results are indeed correct.

So my starting point is trying to see if I can replicate the different media experiments. I will post some of these later on, I am currently working on a different computer, i havnt managed to transfer all my files across yet.

Tsjerk - 1-11-2017 at 12:19

Cool!

I have a little experience as well, not much though.

I grew some Cannabis indica plants in a hydro culture with self-made medium, I don't know the details of my mixture, but it looked like Murashige and Skoog medium, but definitely with a lot less vitamins. I'm pretty sure the vitamins needed differ per species. What are you growing? My plants grew like crazy (5 cm a day sometimes).

I also grew Arabidopsis in sterile environment at university, these media contained glucose, and the new plants love it. Just make sure to use antibiotics against unwanted growth if you want to do this.

Main thing is finding out what your plant species needs to grow. Many species have been researched and described in literature, I think I used tomato plants' needs for my plants as for some reason Cannabis indica was not described anywhere.

Edit: I wouldn't be suprised if the agar/starch phenonomen was caused by a contaimination of sorts in the agar used in the lab where they found those results. Practically all labs on the planet grow on agar.

[Edited on 1-11-2017 by Tsjerk]

NEMO-Chemistry - 1-11-2017 at 12:31

Tomato and common nettle are model plants for cannabis (so i am told). Not sure which plants exactly yet, i already grow alot of stuff.

Alot of my gardening is now hydro and aqua based, as an extension of this I want to get into cell culture growing, and cloning. So at the moment I have several picked out for testing.

Strawberry, tomato, bean, lettuce and a couple of hardwood plants i have decided on, its likely one will be Orchid and maybe something like fuchsia.

I have done alot of digging (pun intended), found alot of really interesting stuff.

many of the older books and papers mention Mercuric Chloride to sterilize the explants!! Ultimately I want to grow from single cell cultures.

Tsjerk - 1-11-2017 at 12:58

Ok, single cell cultures, even cooler, I'm getting memories back from what I did. I actually did some protoplasting and transformations.

Then I guess you will need some sugar in the medium, as photosynthesis of single cells probably is not sufficient for them to grow on, and after the shock of skinning them from the cell wall, they can use a bit of sugar.

We sterilized the seeds with bleach, a lot nicer than mercury! In combination with some antibiotics this worked against bacterial growth.

I think I also found the reason for 3005 starch to have so much more growth; starch is far from clean starch, and hypothesized was (already in the 80's by other scientists) that 3005 has a nice and steady phosphorous release, compared to other starches.

Edit: Do you have an technique in mind you want to use for preparing single cells?

[Edited on 1-11-2017 by Tsjerk]

Regarding petri dishes;

Sulaiman - 1-11-2017 at 13:59

I have zero experience, but that should soon change,
last week I received these: http://www.ebay.co.uk/itm/10Pcs-Sterile-Polystyrene-Plastic-...
and today I received this: http://www.ebay.co.uk/itm/60mm-Glass-tissue-petri-dish-cultu...
your local eBay may have cheaper.

The polystyrene ones can be had for even less, I was impatient.
The glass one is borosilicate, nice quality - clear for photography.
The polystyrene ones stack, the glass ones would be quasi-stable.

I too am considering media, equipment, techiques ... dummies gude level.

[Edited on 1-11-2017 by Sulaiman]

NEMO-Chemistry - 2-11-2017 at 03:02

Quote: Originally posted by Tsjerk

Ok, single cell cultures, even cooler, I'm getting memories back from what I did. I actually did some protoplasting and transformations.

Then I guess you will need some sugar in the medium, as photosynthesis of single cells probably is not sufficient for them to grow on, and after the shock of skinning them from the cell wall, they can use a bit of sugar.

We sterilized the seeds with bleach, a lot nicer than mercury! In combination with some antibiotics this worked against bacterial growth.

I think I also found the reason for 3005 starch to have so much more growth; starch is far from clean starch, and hypothesized was (already in the 80's by other scientists) that 3005 has a nice and steady phosphorous release, compared to other starches.

Edit: Do you have an technique in mind you want to use for preparing single cells?

[Edited on 1-11-2017 by Tsjerk]

Still reading up on techniques, there is alot of info but not easy to choose. I guess Bleach would be the Chlorox I keep seeing mentioned!! Not sure if Chlorox is a brand or what, but its mentioned alot.

Bleach I can handle, Mercury Chloride I would rather avoid

. Protoplastng would not come to mind yesterday! I kept typing Chloroplasts, so thx for bumping the memory!!

I have a very good microscope and also a good dissecting one that can manage down to around 60X . Looks like I also now have a centrifuge I got cheap off facebook last night.

If you got suggestions for separating protoplasts i am all ears. I think I have found my niche finally, I love the mix of chemistry and biology. Next step make or buy some Led Lighting, add a lux detector and DAQ, so I can keep variables down.

The starch makes sense, especially for sugar, but Phosphate I hadnt considered.

Tsjerk - 2-11-2017 at 07:53

Usually enzyme mixes with for example cellulase are used for protoplasting are used, these are quite expensive though. Maybe I can look around for a cheaper protocol tonight.

NEMO-Chemistry - 2-11-2017 at 09:21

Cheers, I didnt have alot of luck. The mechanical ones I found are not good at all. Might be worth me trying to find a cheaper way to get enzymes.

NEMO-Chemistry - 2-11-2017 at 09:27

Quote: Originally posted by Sulaiman

I have zero experience, but that should soon change,
last week I received these: http://www.ebay.co.uk/itm/10Pcs-Sterile-Polystyrene-Plastic-...
and today I received this: http://www.ebay.co.uk/itm/60mm-Glass-tissue-petri-dish-cultu...
your local eBay may have cheaper.

The polystyrene ones can be had for even less, I was impatient.
The glass one is borosilicate, nice quality - clear for photography.
The polystyrene ones stack, the glass ones would be quasi-stable.

I too am considering media, equipment, techiques ... dummies gude level.

[Edited on 1-11-2017 by Sulaiman]

Thats cool, its always good to do something with someone else also trying. I will post what papers I have once sorted.

At the moment i am trying to find something that backs up the two starch papers. But I think TSJerk is right, i think maybe the experiment was a dud.

If you need it I got a couple of books on pdf also, i want to scan through them first though make sure they are relevant. The only other info i have is some msd sheets from a company that makes nutrient solutions for agar growing.

NEMO-Chemistry - 2-11-2017 at 09:31

Sometimes I am really thick!! I just worked out why i keep seeing Arabidopsis being used and mentioned!! DOH!

I guess I netter add in Arabidopsis thaliana as well

NEMO-Chemistry - 3-11-2017 at 16:10

After much reading, the protoplast will have to wait a while, its not that its difficult as such. But out of the various methods, enzymes are the realistic way to go, these are currently out of reach.

So I will concentrate on other methods of dell propagation etc for now. The advantage of doing this way, mostly gaining experience with the different Auxins etc. I am searching for ways to extract the enzymes needed, but looking at the cost of them, i dont imagine it can be easy.

Still this leaves a huge amount to study and experiment with anyway. I will post a list of useful reading material over the weekend, most of the files are too big to post and the links would not be right to post openly

NEMO-Chemistry - 3-11-2017 at 18:30

Deep Joy!!

I found a supplier of cellulase and pectanase, no idea if they will sell to me but price is good. Actually pectanase should be easy to get from brewing shops. But i found a industrial supplier, the product is marked as industrial quality and is around £1 gram and £1 ml respectively. They sell 10 gram amounts by the look of it. I will contact them monday

.

The book is
Davey, M. R. and Anthony, P. (2010) Plant Cell Culture: Essential Methods, Plant Cell Culture: Essential Methods. doi: 10.1002/9780470686522.

Around page 433 i think

We are not over the hurdle yet, i am tracking down some of the other stuff we need, but beats the £120gram i was finding

.

The enzymes were my main concern, the other stuff not so much so.

[Edited on 4-11-2017 by NEMO-Chemistry]

Mesa - 6-11-2017 at 04:10

I've had a fair amount of experience with plant based cultures in the past from projects me and my brother took on with varying success(He'd done a lot of lab work with plant cultures during his postgrad in molecular bio. lucky for me).

Mostly working with suspension cultures, though a few(failed) membrane cultures too.

The biggest challenge you run into doing it in the amateur setting is contamination. No matter how much effort, or time, you put into sterilization, you'll regularly get contamination issues, which for plant cultures, are an instant and unrecoverable fail.
Significantly more difficult than e.g. Psilocybe cultures from my experience.

NEMO-Chemistry - 6-11-2017 at 15:26

Quote: Originally posted by Mesa

I've had a fair amount of experience with plant based cultures in the past from projects me and my brother took on with varying success(He'd done a lot of lab work with plant cultures during his postgrad in molecular bio. lucky for me).

Mostly working with suspension cultures, though a few(failed) membrane cultures too.

The biggest challenge you run into doing it in the amateur setting is contamination. No matter how much effort, or time, you put into sterilization, you'll regularly get contamination issues, which for plant cultures, are an instant and unrecoverable fail.
Significantly more difficult than e.g. Psilocybe cultures from my experience.

I did manage to get hold of a class II cupboard from ebay cheap. so far just playing with plates (I do alot of biological stuff), the aseptic side is not too bad.

I might need to construct something out of a commercial fridge , one with a all glass front door. I will use this for incubation/growing or whatever.

Getting the explants sterile is really time consuming, so far i havnt begun to isolate protoplasts, I havnt got everything I need yet.

But general explant stuff I have been trying out. So far its 34 cultures no contamination and 7 with contamination. I am aware for the protoplast experiments I have to improve on this.

I am waiting to get the enzymes and some other bits, i have serilogical one use glass pipettes, these I can autoclave if I get low, what I lack at the moment is enough pipette fillers!

I have two and they are not big enough. Also disposable tips and the small centrifuge pots etc I could do with some more.

My work area is pretty good, it amounts to a sealed plastic tent inside my lab. I have 30W UV lamps and a autoclave, the cabinet I am using has good HEPA filters in, they were changed roughly 4 months ago I am told.

I could do wityh a better bunsen as well, mine is a bit big and fierce.

aga - 7-11-2017 at 15:03

Not quite sure what this thread is about.

Grow plant Cells or entire Plants ?

A couple of years back i did some work with the Bento Bucket (aka Dutch Bucket) system
and a nutrient made 100% from chemicals that was reverse-engineered from a tomato plant 'growth-promoter'.

Without a single grain of 'soil' the plants loved it and went crazy. We were bored with tomatoes by the end of summer.

A neighbour tried the same system with cannabis and was astounded by the growth rate, but not by the science (they thought it was not 'eco' enough to be good).

If anyone has any (non-googled) ideas about growing human tissue from, say, easily captured cheek cells, i would be very grateful.

Tsjerk - 8-11-2017 at 08:38

I just remembered an enzyme-less technique to produce protoplasts: with the help of cell wall synthesis inhibiting herbicides in an osmotic-protective environment.

I don't know whether this is established in plant protoplasting, but it is quite well described for the formation of L-forms of bacteria.

There are even L-form strains of certain species that don't turn back to their normal form upon discontinuation of the antibiotics due to random mutantions that are beneficial for the L-form but not viable without osmoprotection. Multiple genes, potentially interesting drug targets, involved in cell wall metabolism were identified this way.

NEMO-Chemistry - 8-11-2017 at 09:11

Quote: Originally posted by aga

Not quite sure what this thread is about.

Grow plant Cells or entire Plants ?

A couple of years back i did some work with the Bento Bucket (aka Dutch Bucket) system
and a nutrient made 100% from chemicals that was reverse-engineered from a tomato plant 'growth-promoter'.

Without a single grain of 'soil' the plants loved it and went crazy. We were bored with tomatoes by the end of summer.

A neighbour tried the same system with cannabis and was astounded by the growth rate, but not by the science (they thought it was not 'eco' enough to be good).

If anyone has any (non-googled) ideas about growing human tissue from, say, easily captured cheek cells, i would be very grateful.

At the moment its about growing plants in a culture medium, like say a 2mm sliver of Cauliflower, and using different Auxins at different times to get the explants you want.

Ultimately i want to make the cultures from plant cells (protoplasts).

Because of the nature of it i would therefore say its a kind of get used using Auxins etc, and a baby step approach to plant breeding/crossing using protoplasts.

Where it ends up depends which rabbit hole i choose as I go along. Its a huge topic, i love the biology of plant growth and production.

Sorry the thread is actually a real jumble at the moment, but its early days and while i have some the experiments running, i am still researching and trying to source OTC things for the cell cultures.

Maybe as it branches into more defines areas i will edit things into single posts.

As a side note

I have finally found persil biological powder contains a good amount of protease. But I also have a source of purer commercial grade stuff as well.

Aga I like your input into threads, but this particular one is likely to annoy you until its more organized. I would however like to hear what you used in your formula.

I was told you cant beat willow water for rooting, then found references to it in papers. I was expecting to discover exotic enzymes or amino acids responsible, turned out the magic ingredient is aspirin! Hence my aspirin extraction questions in another thread

.

I appear disjointed, but normally its because i write like I think. There is always a goal, its simply not always easy for others to know what that is.

aga - 8-11-2017 at 13:48

A wonderful publication is by Norman C. Deno, detailing exactly how to germinate almost any kind of seed you get your hands on, using stupidly simple methods.

It appears disjointed because it is disjointed

Nothing wrong with that so long as it leads somewhere ...

Some cuttings require a period in which the cut stem is exposed to air, such as Pitaya (aka Dragon Fruit), that needs about 3 days un-planted before it will successfully take root after planting.

All i did was take a tomato fertiliser recipe and work out the stoi for all the listed components, then re-work that to fit the locally available agricultural chemicals.

Then the other chemicals that a plant might encounter in the local soil and also some 'micronutrients' that i found for sale in a bag in the garden centre (trace elements in EDTA).

The local water is high in calcium carbonate so balancing the pH was done with phosphoric acid in small quantities, which is why i still have over 40 litres left - they don't sell agri-chem products on a small scale unfortunately.

NEMO-Chemistry - 8-11-2017 at 14:19

Some the stuff I am doing is seed based, but mainly I am interested in growing plants from cells, or things like parts of the root meristem.

this is normally done in non soil cultures, the kind I am interested in are more like agar based. The original question I asked about starch as a constituent on the media, was based around starch being a possible carbon source. So yes in some ways i am looking at fert recipes, but thats a small part.

I am interested in hydroponics, so starting with gel based media gives me control on the nutrients taken in. it also allows me to look the role of different Auxins.

CO2 also plays a part of what I am upto but thats another story.

At the risk of mockery.....eventual aim is to grow grass that has the ability to act like a legume and the root structure of hairy root virus. All in the name of helping nitrogen sensitive areas.

So now it seems more disjointed, but really its just part of a fairly long journey with some sight seeing on the way.

As for leading somewhere....No idea where it will end up, but if leading somewhere alludes to actually doing experiments, then yes its leading somewhere. i am doing loads of different experiments related to this already, many have failed or are failing, but a fail is just as good as a win!

For example I started by using vermiculite in boiling tubes as my growing media, i still do with some experiments. But because of some of the fails I have now turned my attention to cell culture growing, same kind of experiments, different media. Obviously swapping techniques is a whole series of experiments and different learning experience in its own right.

Its incredible fun getting a sliver of cauliflower 2mm in size, to actually grow into a full sized plant using chemicals. The fact it was in gel media made it better as you could watch the whole process. Everything from the top of the plant grow first, then adding Auxins to make the roots start to grow.

I would post some of these things up, but i doubt it interests that many people. Things like making complex organic reagents, get people excited, using small amounts of aspirin to stimulate root growth dosnt so much. So i mainly ask things that will help what I am doing, or things I am unsure of.

I tend not to just experiment blindly, i like to research things first. I like to hear different approaches and snips of info, purely selfish reasons as they often lead to me having new ideas. I notice in your own experiments, your more of a do it to find out person, while I am more of a see what i can find out before doing it person.

BUT i do the experiments and i do use the information, i dont ask simply to get information to store away for a quiz night.

Tsjerk - 8-11-2017 at 16:45

I have been reading a bit about plant "L-forms", and came to the conclusion that there is no stable L-form line for research purposes! With a bit of luck anyone could in theory make such a line and describe it! I think this would be quite a nice article.

The lack of a dedicaded line and a described procedure to produce more lines is a big miss for science, as they could be used to address the pourly described topic of natural cell-cell phusion in plants.

Compounds as idobenzoic, isoxaben, vulpunic acid, erythromycin, Epopromycins, coumarin, 2,6‐dichlorobenzonitrile are just a couple of potential candidates for inducing the genotype. They could also be used as a mix in order to combine different mechanisms of cell wall inhibition, as i read 2-by nicotiana dont become L-forms if treated with just 2,6-dichlorobenzonitrile

NEMO-Chemistry - 8-11-2017 at 18:19

Quote: Originally posted by Tsjerk

I have been reading a bit about plant "L-forms", and came to the conclusion that there is no stable L-form line for research purposes! With a bit of luck anyone could in theory make such a line and describe it! I think this would be quite a nice article.

The lack of a dedicaded line and a described procedure to produce more lines is a big miss for science, as they could be used to address the pourly described topic of natural cell-cell phusion in plants.

Compounds as idobenzoic, isoxaben, vulpunic acid, erythromycin, Epopromycins, coumarin, 2,6‐dichlorobenzonitrile are just a couple of potential candidates for inducing the genotype. They could also be used as a mix in order to combine different mechanisms of cell wall inhibition, as i read 2-by nicotiana dont become L-forms if treated with just 2,6-dichlorobenzonitrile

Seems a bit odd, the obvious candidate would be Arabidopsis thaliana.

I havnt got that far yet. Still mastering the whole cell culture thing at the moment. Not ready to try protoplasts but getting close.

What have you been reading relating to L Lines? is this via books or papers?

I have read alot of the books on plant cell cultures, but still looking for more friendly OTC methods. I have most the enzymes covered but now looking into a OTC substitute for tween20. My guess though is washing up liquid would do the job, maybe add some fatty acids from veg oils.

I am looking at sigmas msd for tween20. I might skip it altogether see how it goes.

For some reason tomato plant keeps calling me!! So i might start with that as the first protoplast culture I do. Which ironically means doing a seed grow first!! I had blight this year, so ditched my tomato plants and have stripped the poly tunnel and started to disinfect/fumigate it.

Lab side I didnt have any tomato growing in culture, so maybe a good time to start a fresh one via seed first. Obviously going to go the gel route with the seeds

.

[Edited on 9-11-2017 by NEMO-Chemistry]

Tsjerk - 8-11-2017 at 21:37

I studied /worked in a lab where some people were working with l-forms until last summer. I also did an internship in the lab of Jeff Erringthon, the guy who made them well known to the field.

If you are interested in L-forms, read Erringthon's papers, those are good. He is often highly overrated, but never becomes bad.

Too bad I don't work in a lab anymore, otherwise I would have send you some tween... Maybe you can send a nice email explaining what you are doing to a postdoc somewhere closeby with the question if you could have a couple of milliliters? I would have given it if someone would have asked me.

What step do you exactly need tween for? Only the seed sterilization right? If so I guess you can indeed use soap instead. I do think it would fail without any detergent, as bacterial /fungal spores can be quite hydropobic, making them resistant against bleach.

I used Nicotiana as example because there is this famous immortal 2By single cell line, comparable with Hela cells in oncology. Nicotiana used to be the model organism of choice before arabidopsis became popular iirc.

Let us know how you do with your experiments, I'm pretty sure there are more people here who find this interesting, or at least worthy of reading. Nice to see you have a flow-hood, that makes things easier.

I think you will be fine growing cultures, 34/41 is not bad without antibiotics! If you can, I would add a dash of antibiotics anyway just to be sure

What are the "not as OTC as you would like" points? I have lots of experience with getting these kind of experiments to work, I could help you think of more OTC alternatives.

[Edited on 9-11-2017 by Tsjerk]

[Edited on 9-11-2017 by Tsjerk]

[Edited on 9-11-2017 by Tsjerk]

NEMO-Chemistry - 9-11-2017 at 03:29

Most the procedures I have seen for protoplast culture all use tween, i got the feeling it is kind of used as a step in all initial culture set ups.

the OTC stuff i was mainly thinking of enzymes and antibiotics. But I think i found a OTC way. Its a bit stupid of me really. I had an issue getting a reasonable quantity of aspirin, in the UK your allowed a max of 32 pills or something like that per person per day. Obviously you can go to shop after shop and get more, but in a tiny place like where I live, there isnt that many shops.

Then someone pointed out that amazon sells them for dogs....in 100 pill bottles.
So I checked again and I can get some antibiotics fron Amazon, they are for animal use. Which is just a arse cover!

So the next question is then which antibiotic to use? Most books mention Cychlo something (i forget the name). Now I have a decent lam air hood, i should be able to cut right down on contamination. Biggest issue i have is fungi spores. My lab is in a cold and slightly damp room.

So if i leave a plate out for 6 hours I mostly get molds growing. I have several UV lights taken from pond UV sterilizers, each bulb rated at 30W. I have a proper autoclave (local uni threw it out), so now its just a question of collecting a few bits.

I have plastic petri dishes, i would like to swap these out for glass, conical flask wise I have 30 250ml ones, these are the short dumpy ones with a pretty wide mouth. Again these were donated from the uni and brand new when I got them.

I dont mind posting up the experiments, i just tend to assume most are more pure chemistry or pyro. I tend to be plant orientated, i like plant extractions and biochemistry with yeast etc. I have been lucky in the equipment from the UNI. I have a couple of 2 ltr bio reactors they gave me, with all the septums etc and those stainless steel condensers you get for them. Including a stirring motor as well!

I got the centrifuge real cheap from ebay, it was listed as non working, turns out all it was is the timed safety lock on the lid. They thought it was broken because the lid didnt open, but on my model, you have to wait 30-40 seconds after it stops before the lock releases.

Its fairly small, but has a good speed and 4 decent sized swinging holders in it. The holders for it are pretty cheap so I might get one that takes the small tubes.

I have a anti bacterial called PG80 thats used for cosmetics. But looking at the msd for tween 20, its mainly different fatty acids in it like lauric acid. But all the Indian university vids i seen, all use dish washing soap and ethanol.

Culturing plants with gels etc is a huge topic, enough experiments to last a life time lol. I even found a couple of those water uptake measuring things, again in a box of uni donations

. I cant wait for there next clear out

.

I might email a couple of unis, explain what I am doing and see if they got anything they can spare

. I will go look up those papers. Working in a lab doing cultures, must have been really interesting. The hardest thing i found so far, was learning to uncap/unscrew something with one hand, and still keep hold of it!! Took me ages to master that lol.

EDIT
I should have spotted this earlier! I make soap and body care stuff from natural ingredients normally, Tween 20 turns out to be Polysorbate 20, i have this anyway!! From cosmetic suppliers its pretty cheap. I am going to double check that the tween brand dosnt have anything special in.

But at first glance, Tween is just the brand name! The actual stuff is Polysorbate 20. That would make my life easier, 100ml of it is around £4

Also just found another source of antibiotics, no prescription needed!
[Edited on 9-11-2017 by NEMO-Chemistry]

[Edited on 9-11-2017 by NEMO-Chemistry]

Tsjerk - 9-11-2017 at 09:27

Good to hear such an well equipped amateur lab is forming!

Nystatin and cefotaxime are frequently used in plant cultures be careful though not to go too high with the nystatin, as it is still a bit toxic for plants.

Working on a lab is interesting indeed, but don't expect it to everyday heaven though, as with everything in life you get used to it way to fast.

[Edited on 9-11-2017 by Tsjerk]

NEMO-Chemistry - 9-11-2017 at 10:04

got my eye on a ebay flask swirling thingy. Looks super cheap no bids so far.

Electro fusion voltage

NEMO-Chemistry - 10-11-2017 at 09:52

I have been reading up on electro fusion of protoplasts, my issue is the voltages quoted.

I have seen everything from just DC voltage in the range of 5V, with bursts in us of 30V.

But also others are quoting voltages of 100V AC with DC bursts of 5KV for 5us

So it seems two schools, both agree in very short higher DC bursts and a steady voltage for X mins. The problem is the massive difference in voltages.

I would obviously prefer to use low voltage, but cant see how that works, when so many other papers quote voltages in the Kv range.

Anyone got any idea if the 30V range works? I think its roughly 5V DC for X mins then 30V DC in 10us bursts, two or three times.

NEMO-Chemistry - 10-11-2017 at 17:42

I ran out of agar, so rather than do nothing this weekend, i might try some gelatin media with starch. I might get away with it for seeds. I cant get agar locally, it is likely to be next Thursday by the time I get more Agar. Annoying seeing as I live right near a beach lol, but making my own agar seems doubtful. We got brown seaweed with gas bulb things on, but not sure its the same kind of seaweed I would need.

i know you can use the large brown kelp, but that is 20 miles away from here. i think our local beach has some kind of bladderwrack or something similar, i havnt found it in a book yet. I cant remember but i thought making agar from scratch was a fairly long process?

As for my question above, look what i found

https://www.ebay.co.uk/itm/BTX-Electro-Cell-Manipulator-600/...

only one on ebay and decent price!! Now that has to be a sign does it not lol

[Edited on 11-11-2017 by NEMO-Chemistry]

PHILOU Zrealone - 10-11-2017 at 23:28

Quote: Originally posted by NEMO-Chemistry

I ran out of agar, so rather than do nothing this weekend, i might try some gelatin media with starch. I might get away with it for seeds. I cant get agar locally, it is likely to be next Thursday by the time I get more Agar. Annoying seeing as I live right near a beach lol, but making my own agar seems doubtful. We got brown seaweed with gas bulb things on, but not sure its the same kind of seaweed I would need.

i know you can use the large brown kelp, but that is 20 miles away from here. i think our local beach has some kind of bladderwrack or something similar, i havnt found it in a book yet. I cant remember but i thought making agar from scratch was a fairly long process?

As for my question above, look what i found

https://www.ebay.co.uk/itm/BTX-Electro-Cell-Manipulator-600/...

only one on ebay and decent price!! Now that has to be a sign does it not lol

[Edited on 11-11-2017 by NEMO-Chemistry]

Gelatin and starch will loose their structure and integrity if there is fungal or bacterial contamination... agar remains stable towards those and that is why it is used as a media...

Maybe the use of a mineral or an artificial water scavenging polymer will do (like polyacrylate/polyacrylamide)

The device you speak about is based onto the principle of electroporation / nucelar transfection... it allows to enter DNA strain into bacterias or living cells but the later are very sensitive and the amount of killed cells is very high if the electric pulse, shape and intensity is not well masterized... the media is also important to reduce the cellular stress and increase efficiency...
It was discussed onto the forum... so do a search with the key words

Mesa - 11-11-2017 at 16:15

Regarding difficulties sourcing OTC reagents;

In my experience biotech suppliers have none of the qualms fine chem suppliers do selling stock to private citizens. We purchased the majority of our reagents from the same domestic(Aus) biotechnology companies my brother used when he was doing his doctorate.

NEMO-Chemistry - 11-11-2017 at 16:53

I will do a search and see whats been said on the fusion technique. opens up alot of new and interesting possibilities.

If I am messing with protoplasts then some fusing would be a great experiment, and that is not alot of money for a half decent machine.

Thx Mesa, i found some....not sure what they call them, but like a msd sheet but tells you exactly what they use in the buffers etc they make. Turns out some the exotic sounding stuff is just NaOH, or citric acid!!

I found a Biotech that supplies schools and unis, they replied to an email and are indeed happy to supply the public. they dont have a huge range, but its a good start and the prices are not so bad. its called blades scientific for people in the UK, website is being redone, but they have a PDF catalog you can download.

How cool if you could put a methogen plasmid into a yeast cell with electrofusion! Yeast that make Ethanol and fart methane!! I can see £££ applications for that

[Edited on 12-11-2017 by NEMO-Chemistry]

Vmedvil - 13-11-2017 at 00:33

Well, don't hurt yourself plants aren't very dangerous but remember when in doubt hit it with disinfectant, I remember once when merging about 10,000 types of bacteria, I had calculated together I got a drop on my hand of the bacteria and within 5 seconds it had made a 10mm spot where it was eating my hand, I disinfected and then burned the area and it disappeared. The scar always reminds me to not be careless when working with things such as that. Secondly, on bacteria never use antibiotics too much they will adapt antibiotic resistance. I have since named that the Cytotoxic Cellular Paste, and no I didn't realize how virulent it was until that day being one of my earlier experiments with bacteria. There are definitely some white blood cells that got weird antigens that day and odd antibodies on their surface even then they were getting slaughtered in this fight.

In any case, the Protoplast fusion of Bacteria is the same method for plants both having cell walls the enzyme used to dissolve their walls is cellulase which can be bought here Buy Cellulase or Cellulase Seller 2

But remember, that was the same substance used to dissolve my hand by the bacteria it is a mutagen having the warning symbol for it.

[Edited on 13-11-2017 by Vmedvil]

NEMO-Chemistry - 13-11-2017 at 01:52

Quote: Originally posted by Vmedvil

Well, don't hurt yourself plants aren't very dangerous but remember when in doubt hit it with disinfectant, I remember once when merging about 10,000 types of bacteria, I had calculated together I got a drop on my hand of the bacteria and within 5 seconds it had made a 10mm spot where it was eating my hand, I disinfected and then burned the area and it disappeared. The scar always reminds me to not be careless when working with things such as that. Secondly, on bacteria never use antibiotics too much they will adapt antibiotic resistance. I have since named that the Cytotoxic Cellular Paste, and no I didn't realize how virulent it was until that day being one of my earlier experiments with bacteria. There are definitely some white blood cells that got weird antigens that day and odd antibodies on their surface even then they were getting slaughtered in this fight.

In any case, the Protoplast fusion of Bacteria is the same method for plants both having cell walls the enzyme used to dissolve their walls is cellulase which can be bought here Buy Cellulase or Cellulase Seller 2

But remember, that was the same substance used to dissolve my hand by the bacteria.

[Edited on 13-11-2017 by Vmedvil]

I will put a big notice on the toilet door

. Since writing my last post I have done alot of reading into it, the obvious one i want to try is GFP.

But this kind of thing would be great for tomato plants and trying to breed in higher sugar content.

Loads of avenues to go down. I take Bio security and general safety seriously, while its not perfect I do have a proper class II hood for micros and bio related stuff.

I also have strong UV sources,bleaches and even small ozone generator. I have a small formaldehyde generator thing i use for the poly tunnel. So I try and keep the hood,incubator and fridge really clean.

Sounds like a nasty experience you had!! Lucky you didnt a pee with that on your hands

, I will post some papers later, still reading them and following a couple of things up.

There is several references to a low voltage method, looks interesting. Seems easier to get Biochemistry stuff than normal chemistry reagents!!

Spoke to a couple this morning who were really helpful.

NEMO-Chemistry - 16-11-2017 at 05:48

A couple of times i have come across information that implies, agar, while by far the most common substrate, is not always the best. A couple of papers have been mentioned, these apparently show in many cases agar actually retards the process or stops it.

These are often mentioned in papers dealing with adding starch as a substrate. If i have trouble tracking these down i will ask in refs.

Arnold SV, Ericksson T (1984) Effect of agar concentrations on growth and anatomy of adventitous shoots of Picea abies (L) Karst. Plant Cell Tissue Organ Culture 3:257-264
2.

Singha S (1980) Influence of two commercial agars on in vitro shoot proliferation of 'almey' crab apple and 'seckel' pear. Hort Sei 19:227-228

Debergh PC (1983) Effects of agar brand and concentration on the tissue culture media. Physiol Plantarum 59:270-276
4.

Kohlenbach H, Wernicke W (1978) Investigations on the inhibitory effect of agar and the function of active carbon in anther culture. Z Pflanzen Physiol 86:463-472

There are others but these are mentioned the most. So where am I at the moment? Well i am having trouble extracting viable protoplasts, not too sure what the problem is, I will order more chems when I get paid. Also i dropped my haemocytometer and blew the bulb in my microscope on the same day!! Might have a way to attach the camera to the scope though, so once the bulb is fixed (its ordered), i will try and take a pic. maybe someone can spot whats wrong with my cells or technique.

I have some seeds in culture (agar & gelatin) with and without starch. Nothing much to report yet, i might need to move them somewhere warmer, the temp here has dropped recently. Should really have them in an incubator, but my Chinese lights have not arrived yet.

Also been using Calcium Carbonate columns for chromatography, they give pretty good results, this came about after reading something in JCE.

Vmedvil - 17-11-2017 at 03:29

I have used Enriched Blood which is what you use for Animal Retrovirus/Lentivirus as a medium then again I was Trained formally as a A.S. Biotechnologist and B.S. Biophysicist, but stick with Agar or a medium to that nature until you master the techniques in them, where the higher level organisms/pathogens are not nearly as forgiving. Secondly, what is your problem with extracting the Protoplasts? If it is Fusing them then your method of electrolysis is not working, if it is extracting them then Filter them through a Nano-mesh Filter for water purification.

[Edited on 17-11-2017 by Vmedvil]

NEMO-Chemistry - 17-11-2017 at 03:43

Quote: Originally posted by Vmedvil

I have used Enriched Blood which is what you use for Animal Retrovirus/Lentivirus as a medium then again I was Trained formally as a A.S. Biotechnologist and B.S. Biophysicist, but stick with Agar or a medium to that nature until you master the techniques in them, where the higher level organisms/pathogens are not nearly as forgiving. Secondly, what is your problem with extracting the Protoplasts? If it is Fusing them then your method of electrolysis is not working, if it is extracting them then Filter them through a Nano-mesh Filter.

[Edited on 17-11-2017 by Vmedvil]

Thx
Yes i think its filtering, i have ordered some 75um syringe filters.
Not ready by a long way to mess with viruses

, but very interesting method.

I am using chemical fusion until my machine turns up, its got a long way o travel. I read your posts on the cat, really interesting stuff.

Makes me wonder if one day it could be used to identify it in feral populations, but i guess wouldnt be 100%.

Vmedvil - 17-11-2017 at 03:50

Actually, I am thinking too small a coffee filter @ 20 Microns, where a Water Filter is 5 microns if the coffee filter is a good one would probably work in this case, may save you a couple hundred bucks... Too late now but those are 75 microns.

[Edited on 17-11-2017 by Vmedvil]

NEMO-Chemistry - 17-11-2017 at 04:12

Ok off to try the coffee filter! Which is about the same as a number 4 glass frit isnt it?

Vmedvil - 17-11-2017 at 04:18

Quote: Originally posted by NEMO-Chemistry

Ok off to try the coffee filter! Which is about the same as a number 4 glass frit isnt it?

I dunno what a number 4 glass frit is, but I have used a coffee filter for Bacterial Protoplasts in the process and it worked when doing a experiment to see if I could do this for 10$ or less for a bioethics/bioterrorism paper........... but whatever, you are doing it on plants and don't seem like a Islamic Jihadist.

[Edited on 17-11-2017 by Vmedvil]

NEMO-Chemistry - 17-11-2017 at 04:23

Quote: Originally posted by Vmedvil

Quote: Originally posted by NEMO-Chemistry

Ok off to try the coffee filter! Which is about the same as a number 4 glass frit isnt it?

I dunno what a number 4 glass frit is, but I have used a coffee filter for Bacterial Protoplasts in the process and it worked when doing a experiment to see if I could do this for 10$ or less for a bioethics/terrorism paper........... but whatever, you are doing it on plants.

[Edited on 17-11-2017 by Vmedvil]

I am using plants because i am not allowed a cat!

. Or a mouse.....

Vmedvil - 17-11-2017 at 04:28

Lol, My poor guinea pig, Einstein the Pig never got experimented on because I felt bad, though one time he did get put in the microwave. Its the eyes man, Look into the pig eyes and Yes, it is a myth that if you put small furry animals into the microwave they explode as long as its not for like an hour or over like a couple minutes, actually it didn't seem phased at all.

Though, actually I have heard conflicting reports about this.
Cat put in Microwave for 5 seconds
Where I don't believe that was actually for 5 seconds which is how long I did it to the Pig which would only be 5,500 Joules assuming a standard 1100 watt microwave oven.

Q = MC_PΔT

C_P = 4185.5 J/(kg⋅K) for Water which Literally is most of a animal's body mass.

Where mass = 4 Kg for the cat.

Where 5500 Watts would have raised its temperature by 0.32851511169513797634691195795007 Degrees Celsius doing nothing to the animal being such a small change in net temperature.

[Edited on 17-11-2017 by Vmedvil]

NEMO-Chemistry - 17-11-2017 at 04:51

Chicken embryo's might be an option

. I couldnt do it to a pig.... just havnt got that killer instinct lmao. Yeah its the eyes, I would likely stick my hand in the oven instead of the pig.

NEMO-Chemistry - 17-11-2017 at 08:33

I found i am using too high a concentration of osmotic fluid. I have accumulated alot of papers, i will at some point put these in refs in case others are interested.

NEMO-Chemistry - 26-11-2017 at 17:52

Machine arrived on Saturday, it dosnt power up

, i have checked as much as i can and its nothing obvious. I own an oscilloscope, i have tried tracing the power rails but came up blank.

Without a schematic its near impossible to work out whats wrong. I am faced with sending it back to America....or doing a deal and keeping it. the guy isnt interested in giving me much of a discount for keeping it. But getting one in the UK even with what i would describe as OUTRAGEOUS!! Shipping and import costs, is still much cheaper and easier than sourcing one in the UK.

Currently its wrapped and packed, and tomorrow it will be sent to a friend in England. Hopefully they will be able to fix it...

On the protoplast front and isolation of them, i am starting to get usable results, i have no idea how many different combinations i have tried. But at least that now seems to be working ok.

I might try the chemical way of fusing while i wait for the machine

. I was speaking with someone from the village i havjnt seen for ages, he is an old guy and was a farmer, he asked what i was upto lately. When i told him what i was trying to do, i expected some piss taking. Actually he was really interested and asked me a question i cant answer.

Apparently at one time (80's i think) he grew and sold Strawberries locally, he asked if using this technique could produce a strawberry that tasted of a hint of pineapple.... I have no idea at the moment, but when i asked him why?

He told me that when he was selling large amounts of strawberries, a couple of shop keepers had spoken of a day in the future when you could buy pineapple tasting strawberries, apparently the consensus among st said grocers, was a strawberry with a hint of pineapple would be a world better fruit.

I have absolutely no idea how or why they even had this conversation, but apparently it was discussed a number of times, i also have absolutely no idea why they think/thought it would be a best seller! Sounds utterly rank to me lol.

Was kinda nice to talk to a guy in his late 70's, who thinks messing with plants is cool. He came round the house today to look at my Polly tunnel and lab, asked loads of questions and seemed really interested. He also gave me a shed load of information on growing strawberries!

Anyway i have wondered off topic. Sorry

Only other thing i have done this weekend is ditch any chemical i should be licensed for, having finally found out what happened to another member here, there is no way i would risk it Scotland, they are far harder on people than England, and i dont think its worth the risk.

Tsjerk - 6-12-2017 at 13:22

I haven't been online too much last weeks, but good to hear things are progressing. I'm still reading with interest what the developments are.

If you want to experiment on chicken embryos I could give you a protocol Haha, I had a practical on that back in uni. Quite interesting stuff of you stain the bones and have a look at them with a objective. I even found an embryo with anencephaly.

NEMO-Chemistry - 26-12-2017 at 17:45

Ok this isnt dead. I had to wait for some supplies which have now arrived. I also managed to get two incubators, one is a gas incubator with a multi compartment type arrangement, this got damaged unloading! I need to sort the electrics as they got soaked when I couldnt get the thing inside!

the other is a desktop shaking (rotary type) incubator, this was sold as broken, but in fact the fix is pretty easy. Mainly the issue is the mains switch and the gear mechanism needs greasing etc, another problem is its been overloaded and the weight light stays on. Both these problems should be an easy fix.

Lastly i got a couple of cheap centrifuges, one has cold and hot capability and although technically a desktop, its extremely large and heavy, this one goes insane speeds as well. I also got a free one thrown in with it, this is tiny and sits on a dinner plate!

The last one is just a normal small centrifuge, these were all picked up from someone i know via ebay, they do lab clearances. i have a deal with them, they keep the crap back for me and i pay peanuts and pallet price for delivery.

Finally got my microscope bulb from America!! I couldnt source one in the UK. Still dont have a good option for taking decent pics through the scope though. Also I need to know or find a way to introduce antibiotic resistance, otherwise i wont be able to separate the cells.

I need more agar again!! Its really expensive IMHO for what it is, but i have now got a good contact in poland, they can get adipic acid and other stuff cheaply, so making agars up myself is now possible. GB acid is another hard to find (here at least) i can get from him cheap.

Going to rearrange a few things tomorrow and hopefully get some pics. Once that is done I could do with some info, i have a book on procedures, but i am missing some info. Its back to basics for a little bit, my technique needs brushing up. Then finally in a couple of weeks i hope to ask my main questions, i have an end goal, it might not be possible! But lets get this other shit done first!

Tsjerk - 26-12-2017 at 21:35

What kind of microscope do you have?

NEMO-Chemistry - 12-1-2018 at 20:01

Quote: Originally posted by Tsjerk

What kind of microscope do you have?

I have several
Might be easier to take a pic later.

One is a Nikon binocular research grade, one a Nikon boom arm dissecting (again research grade). And next week should see a really neat fluorescence one arrive FOC from a uni.

I also have a cheap stereo one