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Little_Ghost_again
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Methylene violet
Hi
I have checked ebay uk and no luck, apart from the criminals at sigma anyone know where I can get it for less than the cost of a house?
I need it for live cell counts
Dont ask me, I only know enough to be dangerous
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Etaoin Shrdlu
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Surely you're looking for methyl violet?
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Chemosynthesis
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Trypan blue is much cheaper if you reverse count with a hemocytometer or the like. Methylene blue as well (assuming you meant methyl violet). What are
you staining? Might make a difference.
[Edited on 17-11-2014 by Chemosynthesis]
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Little_Ghost_again
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Here is an extract from the paper
Viable cell numbers were assessed using the methylene violet staining method and light microscopy (400× magnification) with a
Neubauertype haemocytometer. Methylene violet staining is proposed as a better method for monitoring yeast cell viability compared to the
traditional methylene blue staining method (Smart et al. 1999). An equal volume of the sample was mixed with methylene violet solution (0.01% w/v in
2% sodium citrate solution) (Smart et al. 1999).
The reason is it crosses the membrane and live cells therefore metabolize it and show no colour change, I want it for something slightly different but
related, so I need to to metabolize.
Normally I would use methyl blue, maybe thats why its hard to find and very very expensive
[Edited on 17-11-2014 by Little_Ghost_again]
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Chemosynthesis
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Methylene blue functions identically in yeast. Trypan blue, for comparison, only stains permeable membranes of dead cells.
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Tsjerk
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As far as I know the difference between staining live and dead cells isn't because cells metabolize the dye, but because they kick or keep it out
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Chemosynthesis
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It gets complicated depending on what you stain, since the membranes/walls, transporters and channels vary. Some stains are metabolic, like formazans
(MTS/MTT assays), whereas others are on the basis of pumping or pure exclusion (Evan's blue can be either, depending, I believe.) Try pan is pure
exclusion. Mordants and counterstains complicate it too.
The good news is it sounds like you could probably use methylene blue, though you didn't say what you needed it for exactly. This is cheap in aquarium
and brewing circles.
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Tsjerk
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As far as I know the difference between staining live and dead cells isn't because cells metabolize the dye, but because they kick or keep it out
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Little_Ghost_again
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From the paper
Methylene
violet crosses the membrane of all cells but in dead
cells is unable to be metabolized, as a consequence
dead yeast cells stained violet. Viable cells are able
to metabolize methylene violet and as a result are
unstained under the microscope
I want it for both Yeast and a type of bacteria that is similar, I have tried some the other stains and they dont work the same way.
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aga
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Have you tried all of the stains you already have available ?
Perhaps one will serve your purpose.
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Chemosynthesis
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Methylene blue will be metabolized in live yeast, if that is the metric you want for viability as per the paper, however bacteria is too vague to
assist with.
Bacterial staining and counter staining depends heavily on the type of bacteria (acid fast, Gram positive, Gram negative, etc.). Too many dyes,
mordants, and counterstains to remember.
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Little_Ghost_again
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Quote: Originally posted by aga  | Have you tried all of the stains you already have available ?
Perhaps one will serve your purpose. |
Yeah I have tried them, but dont have that many. As for the bacteria one, thats much harder because at the moment I dont know enough to say anything
useful about it.
I dont have it at the moment so cant even Gram stain etc to start a profile. Its just a hunch at this point that the stain will work for both, the
other papers I have read that mention this stain all indicate metabolism as the route taken. I have one other company I can call for a price in the
morning, generally cheaper than Sigma but not so much stuff. Also they have a min order
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Chemosynthesis
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Just to clarify, do you know what the bacterium is? When you say work a profile, it makes me think you are interested in doing a differential
identification, which can require a bit of work.
Definitely an exercise within reach for an amateur lab, and fun, if you can tolerate possible odors, have access to a decent microscope, a couple
reagents, differential/selective media, and some stains. I am surprised I haven't seen more of that on scimad. Is that part of what you're up to?
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Little_Ghost_again
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| Quote: Originally posted by Chemosynthesis | Just to clarify, do you know what the bacterium is? When you say work a profile, it makes me think you are interested in doing a differential
identification, which can require a bit of work.
Definitely an exercise within reach for an amateur lab, and fun, if you can tolerate possible odors, have access to a decent microscope, a couple
reagents, differential/selective media, and some stains. I am surprised I haven't seen more of that on scimad. Is that part of what you're up to?
|
I dont have the organism yet but will soon, its likely it was miss identified as being an algae. But yeah your pretty much on the right track. Lab
wise I have access to dads home work lab, thats a decent biology lab, so scope wise I have access to some the best scope around.
If my hunch is correct (a few weeks and I should have a sample of the organism) then working up a profile and identifying it should be straight
forward. I also need to sequence the genes as I am looking for a particular gene.
If I am really lucky I might actually have some endospores! If so I can get a sample pretty quickly, I have the phillip harris complete fermentor kit
(got it free) so small scale is a option if I can get find some endospores, if not I have to wait until it comes back or I can order it
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Chemosynthesis
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Interesting. Consider yourself particularly lucky with the genome sequencing. I remember when 454 and ion torrent were a grand for a couple hundred
base pairs not long ago. Would have to ask what "at cost" is now.
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Little_Ghost_again
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I will ask as I dont know, but I do know the costs are now much cheaper.. Much the equipment we have here is connected to my dads research and work
for the uni, plus his 30 years as a biologist.
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Chemosynthesis
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Cool. We bought one because someone thought it was a good idea despite having a good history with a sequencer, and I don't think the powers that be
ever hired anybody that can use it. I moved on and never bothered keeping up with what was happening with it. I suspect the same person ultimately
responsible for that annoyance was the bone who thought having a bunch of unique equipment was such a benefit... despite having zero customer support
or trained personnel.
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Little_Ghost_again
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| Quote: Originally posted by Chemosynthesis | | Cool. We bought one because someone thought it was a good idea despite having a good history with a sequencer, and I don't think the powers that be
ever hired anybody that can use it. I moved on and never bothered keeping up with what was happening with it. I suspect the same person ultimately
responsible for that annoyance was the bone who thought having a bunch of unique equipment was such a benefit... despite having zero customer support
or trained personnel. |
Some of the latest systems are pretty much plug and play now.
Even things like GC's, The one I got a few days back comes with a complete pc and control unit, although you can set up on the front panel of the GC,
its just so much easier to do it via the pc and let the software work it all out. All I have to really do is set up the methods, make sure the correct
column is fitted and measure the gas flow at the FID with a bubble counter. Everything else is just chosen via total chrom software on a pc in drop
down box's!
Compared to the what my dad described as using when he was at uni (sharpening a pencil for a chart recorder!), its on another planet.
Hopefully the gas will be here soon and it will be up and running properly.
Very cool powerful system, of course SM members will be given mates rates for any samples they want run through it  .
SHAME LESS PLUG AHEAD
Polar column non polar column and a wax mid polar column, split/splitless FID etc etc. I will give column numbers tomorrow if anyone wants something
run through.
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nezza
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There are several alternatives available for supravital staining. In the lab I work at we use new methylene blue or brilliant cresyl blue. I don't
know if either of these are easier/cheaper to source than methylene violet.
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Little_Ghost_again
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| Quote: Originally posted by nezza | | There are several alternatives available for supravital staining. In the lab I work at we use new methylene blue or brilliant cresyl blue. I don't
know if either of these are easier/cheaper to source than methylene violet. |
Thanks I will have a look, I dont understand why its so expensive and hard to get hold of. I have also contacted some the people on the original paper
to ask why they chose that stain.
I have a few questions to ask them, I am not sure why they used that one if they were only looking at yeast. For me I need it for a bacterium, but
maybe brilliant cresyl blue might work. My dad read the paper and pointed out a few obvious mistakes, so he thinks maybe it was just a typo or they
used what they had.
He is adamant that yeast wont metabolize that stain so why it was mentioned the way it was confuses me. Everything else in the paper is pretty normal,
so he thinks they just used what they had or they got a few things wrong.
He also pointed out that the institute isnt exactly world class well known and the paper was full of spelling mistakes etc. but then again he is
pretty strict with papers! He also didnt have much faith in the institute name! You can imagine his reaction after he asked me what institute wrote
the paper and I replied
"Lovely School of Pharmaceutical Sciences, Lovely Professional University,"!
No I am not kidding, that is what its called, I tried to get more info out my dad but it was a bit hard with him ROFLHAO.
What I did get from him (eventually) was his normal cryptic type answer! " Think about what the yeast normally metabolizes and find a stain that binds
to it", now I know normally he throws in a curved ball so this time I am ahead, the obvious choice would be a stain that binds with sugar, but this is
my dad and he wouldnt make it that easy, so now I am thinking.............what does yeast use to break down sugar! Then I need a stain that binds to
that.
The only other clue he would give is yeast live in acid so dont think blue. WTF does that mean??? Just once I would like a normal persons dads
reaction, like ask a question and get the answer. I have NO IDEA why his students think he is the mutts nuts!
If I find one I will let you know, and I will try those above.
Any other institution name snobs? Or any other institutions that have odd names?
[Edited on 20-11-2014 by Little_Ghost_again]
In case some live in a bubble and have never heard of the institution here is a link
http://www.lpu.in/schools/pharmaceutical_sciences/highlights...
Looks ok to me
[Edited on 20-11-2014 by Little_Ghost_again]
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Mailinmypocket
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Just took a quick look in this book:
And the use of safranin is one of the dyes suggested for staining yeasts, spores etc. I'm not an expert in this area but the info might help.
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Little_Ghost_again
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| Quote: Originally posted by Mailinmypocket | Just took a quick look in this book:
And the use of safranin is one of the dyes suggested for staining yeasts, spores etc. I'm not an expert in this area but the info might help.
|
Thanks I will see if there is a online copy, or better yet a CHEAP paper copy on abe books, it looks like a useful text to have.
I need something that stains only live cells and dosnt actually kill or harm them (in an ideal world), although a test for just live cells even if it
then destroys them isnt that detrimental.
If I have to then I will sell my kidneys and buy from SIGMA at extortion prices
[Edited on 20-11-2014 by Little_Ghost_again]
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Dr.Bob
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The price for both methylene blue and violet is about the same everywhere I found it, that is pretty typical of a small dye molecule cost. What I
would suggest is to buy a kit of many dyes and stains, Sigma-Aldrich used to sell one, with a few mg (or ml of a solution) of each dye in it. Not
sure who sells them now, but Carolina Biological does(below). Search their catalog for methylene violet and you get a bunch of choice, including
solutions and kits that are much cheaper.
http://www.carolina.com/biological-media-stains-and-reagents...
IF you want to find several free manuals, just google "biological histology stain dye kits " or a similar term, I found about 5 handbooks that way.
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Mailinmypocket
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This reminds me, UK based eBay seller "Magnacol" sells all sorts of indicators and dyes in smaller quantities at decent prices. I have purchased from
them previously. A quick search in their eBay store for your dye in question yields results:
http://stores.ebay.ca/Magnacol/_i.html?_nkw=Violet&submi...
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Little_Ghost_again
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Thanks guys I will go look. some the stains I can make, but others are harder. Normally I only use gram stains and sometime methyl blue or malachite
green
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