On Sun, 12 Sep 1999, gary powers wrote:

> Would anyone have a procedure for Twort's gram stain?
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  Twort's mixture is actually the counterstain, for showing
  Gram-negative organisms (and also the tissue if you do it
  on sections). It's quite a nice general-purpose stain if
  you like red nuclei and green collagen.

Twort's stain was originally made by mixing saturated aqueous
solutions of neutral red and light green, collecting the resulting
precipitate, dissolving it in 80% alcohol to make a stock solution,
and diluting this before use with an equal volume of water. A
modification by Ollett (1951) is easier to make, and light green is
replaced by fast green FCF, which is less prone to fading. The working
solution contains red cations and green anions, which impart their
colours simultaneously to different components of the tissue. Usually
the neutral red staining is too strong, so it is differentiated in
acidified alcohol.

This method was originally devised as a counterstain for Gram-positive
bacteria that had been selectively stained with crystal violet. The
nucleic acids in Gram-negative bacteria and in nuclei of eukaryotic
cells take up the neutral red cations, and most of the background of
cytoplasm and collagen is green.

Solutions required.

  Ollett's modified Twort stain

   Stock solution

    Neutral red (C.I. 50040):  0.36 g
    Dissolve in 180 ml of 95% alcohol.
    Fast green FCF (C.I. 42053): 0.04 g
    Dissolve in 20 ml of 95% alcohol.

    Mix these two solutions and store in a screw-capped bottle. 
    It is stable for at least a year.

  Working solution

    To one volume of stock solution, add three volumes of either 
    distilled water or 0.2 M acetate buffer, pH 4.9. This mixture is
    used only on the day it is made.

  2% acetic acid-alcohol

    Absolute ethanol:     200 ml
    Glacial acetic acid:    4 ml

    Usually this is made up as needed, but it can be stored for several
    weeks without deterioration. It is used only once.

Procedure.

  1. Dewax and hydrate paraffin sections. Remove mercury deposits if 
     necessary.
  2. Immerse in the working solution of Twort's stain for 5-10 minutes.
  3. Wash quickly in distilled water.
  4. Immerse slides in 2% acetic acid-alcohol for about 15 seconds,
     with agitation.
  5. Complete the dehydration in two changes of 100% alcohol.
  6. Clear in two changes of xylene and examine. If there is too much
     red in the sections, repeat Step 4.
  7. Apply coverslips, using a resinous mounting medium.

 Result.

 Nuclei, mast cell granules and sites of high RNA concentration are
 red. Cytoplasm and collagen are green. Purple colours are seen in some
 sites, such as the matrix of cartilage and bone, that take up both
 dyes. The staining is influenced by the fixative and the thickness of
 the sections. Don't expect too much of a procedure that allows little
 visual control of the staining achieved by simultaneously applied dye
 ions.

  In a variant of this method (Monroe & Frommer, 1967) the sections are
  placed in 1% phosphotungstic acid (PTA) for 2-3 minutes and rinsed in
  water before staining, and the acetic acid-alcohol at Step 4 is
  replaced by 2-3 minutes in 70% alcohol. The PTA treatment introduces
  large negatively charged ions into collagen and most cytoplasms, so
  that these, like nuclei, are stained in shades of red and pink,
  whereas striated muscle, erythrocytes and keratin are green.

References.

Monroe, C.W. & Frommer, J. (1967). Neutral red-fast green FCF, a
  single stain for mammalian tissues. Stain Technol. 42: 262-264. 
Ollett, W.S. (1951). Further observations on the Gram-Twort stain.
  J. Path. Bact. 63: 166.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1