Re: Twort's gram stain

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From:Victoria Baker <>
To:gary powers <>
Date:Sun, 12 Sep 1999 18:57:35 -0700 (PDT)
Content-Type:text/plain; charset=us-ascii

Ollett's Modification of Twort's Stain
pgs. 393-5, Culling, 3rd edition

Gram-negative Bacteria

"Gram-negative bacteria may usually be demonstrated by staining
sections with Leishman or Geimsa stain in addition to "control"
sections stained by Gram's stain. Organisms present in the Leishman
slide which are not Gram-positive are assumed to be Gram-negative. In
practice this method works well.

There are a number of other methods described for the differentiation
of Gram-positive bacteria, but of these only the following has given
consistent results.

Ollett's Modification of Twort's Stain

This method (Ollett, 1951) is simple and gives reasonable contrast
between Gram-positive bacteria, Gram-negative bacteria and tissues. The
light green in Twort's stain is replaced by fast green F.C.F to avoid

Special Reagent Required

Modified Twort's stain
0.2 per cent alcoholic neutral red (C.I. No. 825).....90ml
0.2 per cent alcoholic fast green F.C.F.......10ml

For use, dilute one volume of the above stock solution with three
volumes of distilled water.

The pH of this stain, like that of Twort's stain, is 4.9; it may depend
on the dye samples used, but uniformity can be secured by diluting the
stock solution in M/5 acetate buffer instead of distilled water.

The proportions of the dyes given are only approximate, and the optimum
formula will depend on the dye content of the samples used.  The stock
alcoholic solution should be of reddish-magenta tint; too much green
will weaken the red bacterial staining; the total dye concentration is
less critical.


1.  Fix material in 5 per cent formol saline, pass through the alcohols
and embed in paraffin.
2.  Cut sections at 3 microns in thickness.
3.  Bring sections to distilled water.
4.  Stain in aniline crystal violet for 3-5 minutes.
5.  Pour off stain and wash quickly in distilled water.
6.  Treat with Gram's iodine for 3 minutes
7.  Pour off the iodine, wash quickly in distilled water and blot dry.
8.  Decolorize with 2 per cent acetic acid in absolute alcohol until no
more colour comes away - the section should be a dirty straw colour at
this stage.
9.  Wash quickly in distilled water.
10. Counterstain in the modified Twort's neutral red-fast green stain,
diluted 1 part with 3 parts of distilled water, or pH 4.9 buffer, for 5
11. Wash quickly in distilled water.
12. Differentiate with 2 per cent acetic acid alcohol until no more red
stain (neutral red) comes away (15-30 seconds).
13. Clear in xylol and mount in D.P.X. or H.S.R


Cytoplasm......................................Light green
Red blood corpuscles...................Green
Gram-positive bacteria..................Dark blue
Gram-negative bacteria.................Pink"

Sorry to say, I'm not all that familiar with this stain.  When this
stain was developed Formol saline was considered a common fixative,
it's still used but only in specialized procedures.  Perhaps someone
else in this server might be able to give you better information.  If
you're working in a clinical lab, you may not have a lot of choice with
fixation and processing.  I don't know of a more update version on this
procedure.  The pH most likely plays a major part in this procedure
given the results.  

Best of luck.

--- gary powers  wrote:
> Would anyone have a procedure for Twort's gram
> stain?
> Thanks,
> Marian

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