In article <bucc0003.765611484 at gold>, bucc0003 at gold.tc.umn.edu (Paul A Bucciaglia) writes:
|> I've been staining tobacco microspores (about 3-5 days post tetrad) with
|> acetocarmine to follow microspore mitosis. Using the std. 1% carmine
|> in 45% acetic acid on both fixed (ethanol/acetic acid) and unfixed antehrs
|> gives me bright red cytoplasms that obscure any sign of a nucleus. i dug
|> up a reference from 1937 (Maheshwari, Stain Tech. 12#2) which suggested
|> a few drops of chloral hydrate to clear the cytoplasm. "ahh, an archaic
|> name for HCl" i thought so i added a drop of 1N HCl. This did help some
|> as the nucleus was just barely visible and the cytoplasm cleared somewhat.
|> So i checked the Sigma catolog and was surprised to find that chloral
|> hydrate is a nasty chlorinated hydrocarbon that is (was?) used to make DDT.
Chloral hydrate was also the active ingredient in the Mickey Finn (knockout drops
added to alcohol in all the old film noir mysteries).
|> So my question: is there a (much) less toxic compound to clear the micro-
|> spore cytoplasm and allow me to visualize the nucleus?
If you're worried about exposure to chlorinated hydrocarbons, don't add HCl to ethanol.
Don't substitute other inorganic acids either. Nitric in particular ... the combination forms
potentially explosive fulminates (if memory serves me correctly). NB this does not mean a
slide will blow up in your hand ... but a bottle on the shelf containing such a mixture might.
Given the above, why not relax and learn to use chloral hydrate safely?
Two other suggestions.
1) Replace carmine with orcein (same procedure) or hematoxylin (slightly different procedure,
see papers by Wittmann in 1962 and 1964 volumes of Stain Technology). Both have given much
more intense and better differentiated staining of various objects than carmine in my hands.
2) Try staining earlier microspore stages, preferably in an organized daily series from day 1
on. You may find that earlier stages stain fine, but as you get to your target point, the
staining reaction progressively gets worse. When I've done this, I've discovered that it was
the microsporangial wall, not the cytoplasm, that was the culprit.
|> Thanks for bearing with me,
Hope this helps!
|> paul bucciaglia
okellyc at bch.umontreal.ca