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acetocarmine staining of microspores

Paul A Bucciaglia bucc0003 at gold.tc.umn.edu
Fri Apr 8 09:19:29 EST 1994


In <2o0tek$i6r at taco.cc.ncsu.edu> calfor at unity.ncsu.edu (Constantine A Lapasha) writes:

>okellyc at megasun.BCH.UMontreal.CA (Charles J. O'Kelly) writes:

>>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).

>>Given the above, why not relax and learn to use chloral hydrate safely?

>>|> paul bucciaglia

>>Charley O'Kelly

>the other problem we ran into when checking into using chloral hydrate
>as an ingredient in a clearing solution was that chloral hydrate is a
>controlled substance -- the permits and paperwork involved are a giant
>hassel - to say nothing of the worries of storing a current "street drug"
>in the lab --- we decided to work around the problem rather than go through
>the federal and state controlled substance permit routine

>Kostya
>calfor at unity.ncsu.edu

Exactly!  That was my main concern although I failed to mention the status
of chloral hydrate.  How did you work around the problem, are there other
more easily obtained clearing agents?  Acetic acid seemed to help some
although it seemed to destain the nuclei also.

paul bucciaglia




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