[Histonet] Re: Eugenol questions

John Kiernan jkiernan <@t> uwo.ca
Fri Dec 5 12:14:27 CST 2003

The web site quoted in Mary Lou's email gives a
procedure for staining paraffin sections with 
safranine O followed by fast green FCF. There is 
no statement about fixation, expected results or 
what tissue to use it on. Probably the most
frequent use of this dye combination is as a
general oversight method for vascular plant 
tissues. It colours xylem and nuclei red, and 
cellulose & cytoplasm green. The same combination 
can be used (after chromic-osmium fixation) for 
chromosomes and other classical cytological 
demonstrations (see M.Gabe, "Histological 
Techniques," 1976 for discussion).

The method described in the web page resembles
the method of Sass (1958; cited from Ruzin's
1999 book, "Plant Microtechnique and Microscopy") 
but it has two unusual features:

1. Treatment with 1% CrO3 after staining with
safranine; said to improve retention of the
dye. (In Gray's "Formulary and Guide" there
are several methods in which chromic acid is
applied to sections before staining, but I
couldn't find one where it was used after.)

2. A strange and illogical procedure for
dehydrating and clearing the stained sections.
This involves two mixtures containing different
proportions of eugenol, xylene and ethanol.

Eugenol is the chief ingredient (85%) of clove
oil, which has many traditional uses in 
microscopy: as clearing agent, solvent for
dyes, differentiator etc. It was often used as
a clearing agent in earlier times when 100%
alcohol was not easily available, because it's
miscible with 90% ethanol (see Gray, p.56 etc).
Eugenol has a high refractive index (1.54), which
makes for good transparency in whole-mount
preparations (cf clove oil 1.53, xylene 1.50;
data from the Merck Index).

In the staining method on that web site, the
eugenol-xylene-alcohol mixtures are applied after
complete dehydration in "abs. EtOH," and are
followed by a 10:1 xylene-alcohol mixture and
final clearing in 2 lots of xylene. Thus, the
eugenol is not needed for dehydration (already
thoroughly done), and it is all throughly removed
from the section before mounting in permount (so
no possible effect on refractive index of the
final preparation).

This has the look of a method that has been passed
down through generations of people who were not
thinking about what they were doing! There are
plenty of sensible safranine-fast green methods.
All are slightly tricky because the second dye
can remove too much of the first.
A very simple procedure is described in great 
detail on pp.97-98 of Berlyn & Miksche, "Botanical 
Microtechnique and Cytochemistry" Ames: Iowa
State Univ Press (1976). This 326-page hardback
can be bought new for a very low price from the
publisher. Ruzin (1999) describes this method
more briefly, and also gives a more complicated
one, which I have not tried myself.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan <@t> uwo.ca
Mary Lou wrote:
> http://ntmain.utb.edu/lcabrera/4420/safranin-frastgreen_sllide_staining.html
> Dear Histonetters,
> I had never heard of eugenol before the above procedure. Could someone
> explain the importance of it please?  My search has not shown a histology
> connection, just use in perfume, as a dental analgesic, and insect attractant.
> Thanks,
> Mary Lou Norman
> College of Veterinary Medicine
> Cornell

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