Trouble with staining

[mrsonchus]

Hi, can anyone give me a bit of advice - I'm practicing with onion epidermis and am
having difficulty with staining....
I keep my specimens (the epidermis in this case) in 75% IPA. When moving through
a decreasing series of IPA/DI water mixes into aqueous methylene blue and rinses
everything goes very well and I have nicely stained cell-walls and especially nuclei
and nucleoli. I've quite successfully mounted from here into PVA and also into
glycerin - sealing with nail polish to finish. So far so good...
But, when I try to stain the epidermis from the 75% IPA with fast green in cellosolve
by dropping the FG into the alcohol containing the epidermis I get very little staining
if at all - rinsing briefly in alcohol and attempting to mount into nail polish gives
awful results in terms of staining - 'blobs' of oily residue everywhere and virtually zero
staining effect. The result is the same when mounting into glycerin although the actual
mount is very good (unlike the nail polish attempt) there is again very very little
staining at all! Similarly when attempting the same using safranin in cellosolve -
no staining effect.
I'm making some awfully basic mistake somewhere here - can anyone help a
confused and somewhat incompetent beginner with this? What stupid mistakes am I making here?
Is the cellosolve/alcohol clashing in some way (I believe the stain formulas also both
contain ETOH and clove oil)? The 'blobs' look a lot like oil - and the almost total lack of any
staining effect has me stumped - help!

regards

JB

[75RR]

Can't help as I have never done any staining.
Just wanted to say that I am impressed by how you have jumped in at the deep end!
What book are you using as a guide?

[Peter]

Hi JB,
What formulations are using when mixing your stain? For what amount of time are you staining, fast green should take between 10 and 20 minutes, safranin can take 1 to 3 days.
Peter.

[mrsonchus]

Can't help as I have never done any staining.
Just wanted to say that I am impressed by how you have jumped in at the deep end!
What book are you using as a guide?

I'm learning slowly and to say I'm enjoying it is an understatement! I only have the usual
college experience of (gulp) about 35 years ago. I'm using some really superb books and
websites (this forum definitely included!).

I use the microscopy-uk site constantly, I especially like the late Walter Dioni's articles. A
small book containing many of his articles was published shortly after his death and I use
it every single day - here's a link to it.... http://www.amazon.co.uk/MICROSCOPIC-TEC ... r-mr-title
Its a superb little book for the home enthusiast starting out like myself.

I also invested in (I use this term as this book is quite pricey) the absolutely excellent
book on microtechnique by S. Ruzin.... http://www.amazon.co.uk/Plant-Microtech ... r-mr-title

This book will explain an awful lot, but there's no substitute for doing - I'm using onion-epidermis as my
test subject (see W. Dioni's book above) as my interest is in botany and palynology. I've had a lot of good
results with pollen, it's really an rather easy subject to mount (simply in pre-stained glycerol jelly) but a
devilish subject to process/handle (mainly I suppose because it's so tiny) and quite difficult to image
well under the microscope.

Check out those books - they've been invaluable to me in my adventures!

regards

[mrsonchus]

Hi JB,
What formulations are using when mixing your stain? For what amount of time are you staining, fast green should take between 10 and 20 minutes, safranin can take 1 to 3 days.
Peter.

Hi Peter, thanks for helping. I'm using the formulations supplied pre-mixed from Brunel and they are in
cellosolve, which appears to include ETOH, clove oil etc.

I had dropped 3-4 drops straight from the bottle onto the epidermis in about 20ml of 75% IPA and the results
after leaving for about 5 minutes were pretty tragic.

I made a fresh attempt this afternoon - but using a much more disciplined protocol (my first attempt was
definitely rather sloppy I think)...

I've placed my epidermis pieces in about 5ml of 75% IPA and just dropped about 7 drops of the 'neat' stain
straight from the bottle as before giving a far stronger-looking solution. They were left for about 90mins this
time and then rinsed and taken through decreasing IPA concentrations down to were they are tonight - at 25%.

This time a quick look revealed significantly better results - both the fast green & the safranin samples had
pretty well-stained nuclei and slightly lighter stained cell walls, no 'blobs' anywhere this time! I sampled both inner
(upper) epidermis and outer (lower) epidermis this time around and was delighted to see my first stomata
on the outer sample!

I suspect you're onto the problem - more time in the stain and tighter procedure.

What do you think I may do to get the best from these stains with onion-epidermis?

Your advice would be greatly appreciated.

regards

[75RR]

Check out those books - they've been invaluable to me in my adventures!

Many thanks for the links.

[Peter]

Hi JB,
looking at the stated ingredients of your stains, these look like they may be to Johansen's formulation, if so, they are intended to be used with differentiating solutions. I do not know either of the books in your links, however, do neither they, nor the suppliers of the stains not to tell you how to use these stains?
Peter.

[mrsonchus]

Hi JB,
looking at the stated ingredients of your stains, these look like they may be to Johansen's formulation, if so, they are intended to be used with differentiating solutions. I do not know either of the books in your links, however, do neither they, nor the suppliers of the stains not to tell you how to use these stains?
Peter.

Not really, I know the 2 work together but perhaps I should ask Brunel for some help - they're really good at responding to enquiries.
It seems the formulation is a big factor - I'll have another look.

I left another batch for 3 hours to stain and got lightly stained FG results and somewhat overstained (messy) results with the
Safranin - but at least the rinsing down to water (for mounting in Glycerin) is getting rid of all 'blobs' - only masses of air
bubbles to conquer now. Previously I stained onion epidermis with Methylene Blue with very good results - especially the
nuclei. Having great time experimenting though.

I've posted a few of my sorry attempts for all to see.

Thanks for your input Peter.

John