Special Stains (M-Z)

Question 1

Masson’s Trichrome Principle

Answer 1

detection of collagen fibers in tissues such as skin, heart, etc. on formalin-fixed, paraffin-embedded sections, and may be used for frozen sections as well.

Question 2

Masson’s Trichrome Method

Answer 2

1. Deparaffinize and rehydrate through 100% alcohol, 95% alcohol 70% alcohol.
2. Wash in distilled water.
3. For Formalin fixed tissue, re-fix in Bouin’s solution for 1 hour at 56 C to improve staining quality although this step is not absolutely necessary.
4. Rinse running tap water for 5-10 minutes to remove the yellow color.
5. Stain in Weigert’s iron hematoxylin working solution for 10 minutes.
6. Rinse in running warm tap water for 10 minutes.
7. Wash in distilled water.
8. Stain in Biebrich scarlet-acid fuchsin solution for 10-15 minutes. Solution can be saved for future use.
9. Wash in distilled water.
10. Differentiate in phosphomolybdic-phosphotungstic acid solution for 10-15 minutes or until collagen is not red.
11. Transfer sections directly (without rinse) to aniline blue solution and stain for 5-10 minutes. Rinse briefly in distilled water and differentiate in 1% acetic acid solution for 2-5 minutes.
12. Wash in distilled water.
13. Dehydrate very quickly through 95% ethyl alcohol, absolute ethyl alcohol (these step will wipe off Biebrich scarlet-acid fuchsin staining) and clear in xylene.
14. Mount with resinous mounting medium.

Question 3

Masson’s Trichrome Results

Answer 3

Collagen —————————————- blue

  Nuclei ------------------------------------------- black

  Muscle, cytoplasm, keratin ------------------- red

Question 4

PAS (Periodic Acid Schiff) Principle

Answer 4

detection of glycogen in tissues such as liver, cardiac and skeletal muscle on formalin-fixed, paraffin-embedded tissue sections, and may be used for frozen sections as well.

Question 5

PAS (Periodic Acid Schiff) Method

Answer 5

1. Deparaffinize and hydrate to water.
2. Oxidize in 0.5% periodic acid solution for 5 minutes.
3. Rinse in distilled water.
4. Place in Schiff reagent for 15 minutes (Sections become light pink color during this step).
5. Wash in lukewarm tap water for 5 minutes (Immediately sections turn dark pink color).
6. Counterstain in Mayer’s hematoxylin for 1 minute.
7. Wash in tap water for 5 minutes.
8. Dehydrate and coverslip using a synthetic mounting medium.

Question 6

PAS (Periodic Acid Schiff) Results

Answer 6

Glycogen, mucin and some basement membranes — red/purple

  Fungi ------------------------------------------------------ red/purple

  Background ----------------------------------------------- blue

Question 7

PAS Diastase Principle

Answer 7

Alpha-amylase, also known as diastase, is an enzyme commonly present in saliva. The action is to cleave the a-glucosidic 1,4 linkages of starch or glycogen to yield water soluble sugars

Question 8

PAS Diastase Method

Answer 8

1. Bring sections to distilled water. Note: Omit steps 2 and 3 for PAS stain only
2. Treat sections with amylase solution 20 mins
3. Wash well in running tap water
4. Treat with periodic acid .5 mins
5. Wash well in distilled water
6. Stain with Schiff’s reagent 10 mins
7. Wash in fast running tap water 3-5 mins
8. Stain nuclei with haematoxylin 1 min
9. Rinse in running tap water
10. Differentiate with acid alcohol
11. Wash in running tap water
12. Blue nuclei in Scott’s
13. Rinse in running tap water
14. Dehydrate, clear and mount.

Question 9

PAS Diastase Results

Answer 9

PAS positive materials………………..magenta

                    nuclei. .......................................blue
                    erythrocytes. ...............................pale pink

Question 10

Perls Prussian Blue Principle

Answer 10

Dilute mineral acid hydrolysis releases ferric ions from protein bound tissue deposits, which, in the presence of ferrocyanide ions, is precipitated as the highly coloured and highly water-insoluble complex, potassium ferric ferrocyanide, Prussian blue

Question 11

Perls Prussian Blue Method

Answer 11

1. Bring sections to distilled water.
2. On a rack, flood with equal parts mixture of ferrocyanide and hydrochloric acid for 10 min (asbestos bodies for 30 mins)
3. Wash well in distilled water, several changes 5 min
4. Counterstain with filtered neutral red stain 1 min
5. Rinse in distilled water
6. Rapidly dehydrate in absolute alcohol, clear and mount.

Question 12

Perls Prussian Blue Results

Answer 12

ferric salts…………………………………………….. deep blue

nuclei. …………………………………………………. red
erythrocytes. ………………………………………….. yellow

asbestos bodies……………………………………….. blue/black

Question 13

Sirius Red Principle

Answer 13

collagen

Question 14

Sirius Red method

Answer 14

1. De-wax and hydrate paraffin sections.
2. Stain nuclei with Weigert’s haematoxylin for 8 minutes, and then wash the slides for 10 minutes in running tap water).
3. Stain in picro-sirius red for one hour (This gives near-equilibrium staining, which does not increase with longer times. Shorter times should not be used, even if the colors look OK.)
4. Wash in two changes of acidified water.
5. Physically remove most of the water from the slides by vigorous shaking.
6. Dehydrate in three changes of 100% ethanol.
7. Clear in xylene and mount in a resinous medium.

Question 15

Sirius Results

Answer 15

collagen is red on a pale yellow background. (Nuclei, if stained, are ideally black but may often be grey or brown.

Question 16

Toluidine Blue Principle

Answer 16

Mast cells are found in the connective tissue and their cytoplasm contains granules (metachromatic) composed of heparin and histamine. Toluidine blue should stain mast cells red-purple (metachromatic staining) and the background blue (orthochromatic staining).

Question 17

Toluidine Blue Method

Answer 17

Deparaffinize and hydrate sections to distilled water.

2. Stain sections in toluidine blue working solution for 2-3 minutes.
3. Wash in distilled water, 3 changes.
4. Dehydrate quickly through 95% and 2 changes of 100% alcohol (10 dips each since stain fades quickly in alcohol).
5. Clear in xylene or xylene substitute, 2 changes, 3 minutes each.
6. Coverslip with resinous mounting medium.

Question 18

Toluidine Blue Results

Answer 18

Mast cells ————————— violet/red purple.

  Background ------------------------- blue.

Question 19

van Gieson Principle

Answer 19

Van Gieson is used to differentiate between collagen and smooth muscle in tumours and to demonstrate the increase of collagen in diseases.

Question 20

van Gieson Method

Answer 20

1 Bring sections to distilled water.

2 Stain nuclei with Celestin Blue 5 mins

3 Rinse in distilled water

4 Stain in haematoxylin 5 mins

5 Wash well in running tap water 5 mins

6 Flood with Curtis stain 5 mins

7 Blot.

8 Dehydrate rapidly in alcohols, clear and mount.

Question 21

van Gieson Results

Answer 21

Nuclei …………………………………………….Blue

Collagen ………………………………………….Bright red

Cytoplasm, muscle, fibrin and red blood cells ……Yellow

Question 22

von Kossa Principle

Answer 22

demonstrating deposits of calcium or calcium salt

Question 23

von Kossa Method

Answer 23

Deparaffinize paraffin sections and hydrate to water.

Rinse in several changes of distilled water.

Incubate sections with 1% silver nitrate solution in a clear glass coplin jar placed under ultraviolet light for 20 minutes (or in front of a 60-100 watt light bulb for 1 hour or longer). Note: If stain was weak or rinsed off in washing steps, it indicated the UV light was not strong enough. Longer staining is required for up to several hours.

Rinse in several changes of distilled water.

Remove un-reacted silver with 5% sodium thiosulfate for 5 minutes.

Rinse in distilled water.

Counterstain with nuclear fast red for 5 minutes.

Rinse in distilled water.

Dehydrate through graded alcohol and clear in xylene.

Coverslip using permanent mounting medium.

Question 24

von Kossa Results

Answer 24

Calcium salts ———————— black or brown-black

Nuclei ——————————– red

Cytoplasm ————————— pink

Question 25

Ziehl Neelsen Principle

Answer 25

Mycobacterial cell walls contain a waxy substance composed of mycolic acids. These are ß-hydroxy carboxylic acids with chain lengths of up to 90 carbon atoms. The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular species (Lyon H 1991). Basic fuchsin binds to negatively charged groups in bacteria. The mycolic acid (and other cell wall lipids) present a barrier to dye entry as well as elusion (washing out with solvent) and this is partly overcome by adding a lipophilic agent to a concentrated aqueous solution of basic fuchsin and partly by heating.

Question 26

Ziehl Neelsen Method

Answer 26

Place the working solution in a coplin jar and pre-heat in 58 -60oC waterbath 10 mins 2. Deparaffinise sections, bring to water. 3. Stain in the pre-heated working solution in the water bath 15 mins 4. Place the COPLIN JAR containing the slides into running cold tap water 2 mins 5. Remove the slides from the coplin jar and wash in running water 1 min 6. Differentiate in 3% hydrochloric acid in 95% ethyl alcohol until no more colour runs from the slide. 7. Wash briefly in water to remove the acid alcohol. 8. Counterstain with 0.25% methylene blue in 1% acetic acid 15 to 30 secs 9. Wash in water, dehydrate, clear and mount in DPX.

Question 27

Ziehl Neelsen Results

Answer 27

Acid fast bacilli .......................................... Red Nuclei ..................................................... Blue Other tissue constituents .............................. Blue