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Author: Subject: Plant tissue and cell cultures (anyone experienced?)
Tsjerk
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[*] posted on 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]
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[*] posted on 9-11-2017 at 10:04


got my eye on a ebay flask swirling thingy. Looks super cheap no bids so far.
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[*] posted on 10-11-2017 at 09:52
Electro fusion voltage


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




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

[Edited on 12-11-2017 by NEMO-Chemistry]
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[*] posted on 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]
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[*] posted on 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.
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[*] posted on 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.
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[*] posted on 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]
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[*] posted on 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 :D, 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%.
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[*] posted on 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.

tools_filt101_1.gif - 11kB

[Edited on 17-11-2017 by Vmedvil]
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[*] posted on 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?
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[*] posted on 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]
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[*] posted on 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! :P. Or a mouse.....
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[*] posted on 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.


guinea-pig-tan.jpg - 72kB


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 = MCPΔT

CP = 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]
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[*] posted on 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.
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[*] posted on 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.
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[*] posted on 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 :D. 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.
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[*] posted on 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.
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[*] posted on 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!
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[*] posted on 26-12-2017 at 21:35


What kind of microscope do you have?

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[*] posted on 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
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