Movat's Pentachrome

From:Hermina Borgerink


Movat's Pentachrome

 

Jenny and Fred,

My former boss, mentor and life-long friend, Dr. Daria Haust, was one of Dr. Movat's grad students way back when.  Here is the original method as used in his lab.

Fixation:  10% formalin, Bouin's, or formol-sublimate-acetic acid

 

1.        Xylene, 2 changes, several minutes each.

2.       Absolute alcohol, 4 changes. (Formol-sublimate-acetic acid fixed tissue should be put through iodine and hypo).

3.       Rinse thoroughly in water.

4.       Alcian Blue solution for 15 - 30 minutes.

5.       Wash in running water for 3 minutes.

6.       Alkaline alcohol (pH over 8) for 2 hours.  Prepared by adding ammonium hydroxide to 95% alcohol.  This converts the alcian blue into the insoluble pigment monastral fast blue.

7.       Wash in running water for 10 minutes.

8.       Rinse in 70% alcohol.

9.       Weigert-Hart Resorcin solution for 16 hours.

10.    Wash in running water for 10 minutes.

11.     Rinse in distilled water.

12.    Weigert's hematoxylin for 15 minutes.

13.    Wash in running water for 10 minutes.

14.    Rinse in distilled water.

15.    Woodstain-Scarlet Fuchsin solution for 5 minutes.  Differentiation of nuclei will take place in this solution.

16.    Rinse in 0.5% aqueous glacial acetic acid.

17.    Differentiate in 5% aqueous phosphotungstic acid solution, 10 - 20 minutes until collagen is pale pink and the ground substance which is covered by the red, is bluish again.

18.    Rinse in 0.5% glacial acetic acid.

19.    Rinse thoroughly in 3 changes of absolute alcohol.  It is essential not to use low grade alcohol.  If low grade alcohol is used, the cytoplasmic stain are dissolved out and the tissue will not take on the collagen stain, which is made up in absolute alcohol.

20.   Alcoholic saffron, 5 - 15 minutes.  It is important to use an air-tight staining jar.

21.    Absolute alcohol, 4 changes.

22.   Xylene, several changes.  If the collagen is not sufficiently yellow, repeat the staining with saffron.

23.   Mount in permount.

 

RESULTS:

Nuclei - blue to black

Cytoplasm - red

Elastic fibers - dark purple to black

Collagen and reticulum fibers - yellow to greenish yellow

Ground substances and some reticulum fibers - blue to bluish-green

Fibrinoid - intense red

Reference:  Movat, Henry, Z.  AMA Archives of Pathology 55:  289 - 295, 1955

Note:  It is important for these staining methods to prepare solutions with HCl of specific gravity 1:124 (the "offizielle Salzaure") and not concentrated HCL as stated in some texts.  It is prepared by adding 100ml of concentrated HCl (specific gravity 1.19) to 50ml of distilled water.

 

Formal-Sublimate-Acetic

Mercuric chloride          - 4gm

37 - 40% formaldehyde - 20ml

Distilled water               - 80ml

Glacial acetic acid          - 5ml

 

Alcian Blue

Alcian Blue             - 1gm

Distilled water      - 100ml

Glacial acetic acid  - 1ml

 

Weigert's Resorcin Fuchsin

Basic Fuchsin   - 2gm

Resorcinol        - 4gm

Distilled water - 200ml

Mix in a porcelain dish.  Cook 1 minute after it comes to a boil, until scum forms.  Add 25ml of a 29% aqueous ferric chloride solution.  Cool, filter, and leave precipitate on filter paper until dry.  Cover overnight.  Return to enamel dish and add 200ml of 95% alcohol and boil very carefully (use electric hotplate).  Add 4cc of concentrated HCl.  Bring volume to 200ml with 95% alcohol.  This solution keeps well.

 

Weigert-Hart Resorcin-Fuchsin

70% Alcohol                                      - 94ml

Weigert's Resorcin-Fuchsin solution - 10ml

HCl, concentrated                             - 1ml

 

Woodstain Scarlet-Acid Fuchsin

Stock solution A

Woodstain Scarlet NS - 0.1gm

Distilled water             - 99.5ml

Glacial acetic acid        - 0.5ml

Stock solution B

Acid Fuchsin         - 0.1gm

Distilled water      - 99.5ml

Glacial acetic acid - 0.5ml

Mix 8 parts of solution A with 2 parts of solution B.

 

Alcoholic Saffron

Saffron              - 6gm

Absolute alcohol - 100ml

After preparing the solution, place in a tightly corked container to prevent hydration and place in an incubator at 58C for 4 hours before use.  This is done to extract the saffron.  Keep in an air-tight dark bottle.

 

 

 

Movat's Pentachrome II  (modified Pentachrome)

 

1.        Toluol 3X.

2.       Absolute alcohol 3X.

3.       Wash in water.

4.       Stain in Alcian Blue for 10 minutes.

5.       Rinse in Water.

6.       Placei n alkaline alcolhol for 1.5 to 2 hours.

7.       Wash well in water.  

8.       Rinse in 70% alcohol.

9.       Stain in Weigert-Hart overnight, or 3 -4 hours in a 55 - 60C oven.

10.    Rinse in 70% alcohol.

11.     Wash for 5 minutes.

12.    Rinse in distilled water. 

13.    Stain nuclei in Weigert hematoxylin for 15 minutes.

14.    Rinse in water for 10 minutes.

15.    Differentiate individually in 0.5% acid alcohol

16.    Wash for 5 minutes.

17.    Rinse in 1% acetic acid.

18.    Stain in 1% Orange G for 10 minutes.

19.    Rinse in 1% acetic acid.

20.   Stain in Woodstain Scarlet-acid Fuchsin for 5 minutes.

21.    Rinse in 1% acetic acid.

22.   Differentiate in 5% phosphomolybdic acid for 1 - 5 minutes.

23.   Rinse in 1% acetic acid.

24.   Stain in 2% Chlorantine Fast Red for 10 - 30 minutes.  If Orange G has not been added to this stain, for 5 - 10 minutes.  Examine every 5 minutes.

25.   Dehydrate in acetone, clear in toluol and mount.

 

 

 

 

RESULTS:

Nuclei - blue-black

Elastic fibers - dark purple to black

Ground substance -  blue to blueish-green

Cytoplasm - shades of red

Connective tissues -orange to yellow

 

 

Modifications are: 

         Prepare hematoxylin with conc. HCl as described by Weigert.

         1% acetic acid is to be used for the preparation of all staining solutions.

         The concentration of Woodstain Scarlet and Acid fuchsin is to be increased from 0.1% to 1% and the ratio changed from 8 : 2 to 5 : 5.

         Prolong the mordanting to twice its original timing.

         Prolong Chlorantine Fast Red staining to twice the time.

         Alcian Blue is made up as a 0.1% solution in 1% acetic acid.  A few crystals of thymol are added.  This solution can be kept for several months.

 

Alkaline alcohol

95% Alcohol               - 95ml

Ammonium hydroxide - 5ml

PH to 8.0

 

Weigert Iron Hematoxylin

Solution A:

95% Alcohol  - 100ml

Hematoxylin - 1gm

Solution B:

Distilled water         - 95ml

29% Ferric chloride - 4ml

Conc. HCl                  - 1ml

For use, mix equal parts.  Renew frequently.

 

Orange G

Orange G               - 1gm

Distilled water      - 99ml

Glacial acetic acid - 1ml

 

Resorcin Fuchsin (Stock)

Basic Fuchsin   - 2gm

Resorcinol        - 4gm

Distilled water - 200ml

Mix ingredients in a porcelain dish and bring to a boil.  Let boil for 1 minute and add 25 ml of 29% ferric chloride solution.  Cool, filter, and leave precipitate on filter paper until dry.  Cut the filter paper up into pieces and return to the porcelain dish, which should be dry but still contain whatever part of the precipitate remains adherent to it.  Add 200ml of 95% ethanol and heat carefully, dissolving all precipitate from the filter paper before discarding it.  Add 4ml of conc. HCl.

 

 

Resorcin Fuchsin (Working)

Stock resorcin fuchsin - 10ml

70% Ethanol                 - 90ml

Conc. HCl                      -  2ml     

 

Alternatively, Resorcin-Fuchsin may be made up as follows:

 

Weigert-Hart Resorcin Fuchsin

Stock.  Prepare a saturated solution of resorcin fuchsin by dissolving the powder in 110ml of 95% ethanol.  Mix 98ml of the supernatant with 2ml conc. HCl.  Filter and store in a dark bottle.  This stock solution keeps for several weeks.  Working solution:  add 5ml of the stock solution and 1ml of HCl to 94ml of 70% ethanol.  This stain should be made up fresh as soon as the elastic fibers do not stain intensely enough.

 

Chlorantine Fast Red

Chlorantine Fast Red - 2gm

Distilled water          - 100ml

Glacial acetic acid     - 1ml

Orange G *                - 0.5gm  *If Orange G is not good in this mixture, it may be left out.
Hermina M. Borgerink, BA, HTL, HT(ASCP)IHQ
Department of Pathology
Wake Forest University School of Medicine
Medical Center Blvd
Winston-Salem, NC 27157
Phone (336)716-1538
Fax (336)716-1515
hborgeri@wfubmc.edu
 



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