Prepared by
ROY ELLIS
IMVS Division of Pathology
The Queen Elizabeth Hospital
Woodville Road, Woodville, South Australia 5011
Principle
The three dyes Victoria blue 4R (CI 42563), new fuchsin (CI 42520), and crystal violet (CI 42555) are in the same group of aminotriarylmethanes dyes, and one may expect their mechanisms of action to be similar. The resorcin-fuchsin mechanism is thought to be by hydrogen bonding of the dye complex to the elastin fibrils. The ferric ion present is known to form black dye lakes with the aminotriarylmethanes dyes, and the complex lake is thought to have cationic dye properties, rather than a simple dye-mordant action.
Miller's method is the result of several modifications of the original Weigert's resorcin fuchsin method (1898). French (1929) modified Weigert's method by incorporating crystal violet into the resorcin fuchsin stain. The combination of dextrin with crystal violet is the Sheridan (1929) modification.
Full connective tissues are visualised by counterstaining with Van Gieson.
Technical Points
1. Steps 2 to 5 - The Mallory bleach sequence improves results, especially after acetic acid containing fixatives.
2. Step 7 - Differentiation is not required. Adequate staining of elastic fibres is normally within 1½ hrs, but better results are obtained after 3 hrs. The stain may be diluted with an equal volume of 95% alcohol for overnight staining.
3. Step 12 - The acidity of the counterstain is sufficient to differentiate the nuclear stain
Method
1. Bring sections to distilled water
2. Oxidise with potassium permanganate 5 mins
3. Rinse with distilled water
4. Decolourise with oxalic acid 1 mins
5. Wash with distilled water
6. Rinse in 95% alcohol
7. Stain in Miller's stain 1½ - 3hrs
8. Wash in 95% alcohol
9. Rinse with distilled water
10. Stain nuclei with Celestin Blue 5 mins
11. Rinse in distilled water
12. Stain in Haematoxylin 5 mins
13. Wash in running tap water 5 mins
14. Rinse with distilled water
15. Counterstain with Curtis’ stain 2 mins
16. Blot dry
17. Rapidly dehydrate, clear and mount.
Results
elastic fibres, Mast cell granules.....................jet black
collagen.................................................red
muscle, cytoplasm, fibrin.............................yellow
nuclei....................................................brownish green
erythrocytes............................................yellow
Reagent Formulae
1. Miller's elastic stain
Victoria blue 4R (CI 42563) 1.0 g
new fuchsin (CI 42520) 1.0 g
crystal violet (CI 42555) 1.0 g
Dissolve the dyes in 200 mls hot water, then add the following in order:
resorcin 4.0 g
dextrin 1.0 g
fresh 30% aq ferric chloride 50.0 ml
Boil the mixture for 5 mins, then filter while still hot. Return the precipitate plus the filter paper to the original vessel, and redissolve in 200mls of 95% alcohol. Boil on a hot plate, or on a water bath, for 15-20 mins. Filter, and restore the volume to 200 ml with 95% alcohol. Finally, add 2.0 ml conc hydrochloric acid, mix well and store.
Miller's stain has excellent keeping properties, and may be reused for many years.
2. Celestin Blue (see Trichrome for Connective Tissues)
3. Curtis’ stain
1% aq ponceau S (CI 27195) 10.0 ml
sat aq picric acid (CI 10305) 90.0 ml
glacial acetic acid 1.0 ml
Mix the solutions. The stain keeps well.
CARE - explosive
Picric acid is highly toxic, with a very bitter taste. Picric acid is also a percussion explosive when dry. Reagent bottles containing solutions of picric acid must be maintained scrupulously clean, taking especial care of any residue around the neck and stopper of the container bottle.
References
Weigert C,(1898),Zbl.allg.Path.path.Anat.,9,289
French RW,(1929),Stain Technol,4,11
Sheridan WF,(1929),J.tech.Meth.Bull.int.Ass.med.Mus.,12,123
Miller PJ, (1971),Med.Lab.Tech.,28