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NEMO-Chemistry
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[*] posted on 1-11-2017 at 01:17
Plant tissue and cell cultures (anyone experienced?)


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 :D.

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.



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[*] posted on 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.




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[*] posted on 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.
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[*] posted on 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]
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[*] posted on 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.
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[*] posted on 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]
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[*] posted on 1-11-2017 at 13:59
Regarding petri dishes;



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]




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[*] posted on 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 :D. 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.
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[*] posted on 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.
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[*] posted on 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.
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[*] posted on 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.
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[*] posted on 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 :D.
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[*] posted on 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 ;).
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[*] posted on 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 :D.

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 :D.

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

[Edited on 4-11-2017 by NEMO-Chemistry]
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[*] posted on 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.
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[*] posted on 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.
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[*] posted on 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.




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[*] posted on 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.
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[*] posted on 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.
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[*] posted on 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.




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[*] posted on 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.
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[*] posted on 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
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[*] posted on 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 :D.

[Edited on 9-11-2017 by NEMO-Chemistry]
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[*] posted on 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]
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[*] posted on 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 :D. I cant wait for there next clear out :P.

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