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ALCIAN BLUE-PERIODIC ACID-SCHIFF FOR MUCINS
Procedure 1. Stain in Alcian blue for 15 minutes.
2. Rinse in water.
3. Treat with 1% Periodic acid for 5 minutes.
4. Rinse with distilled water.
5. Treat with Schiff's reagent for 5 minutes.
6. Rinse in distilled water.
7. Put in running water for 5 minutes.
8. Counterstain with Harris haematoxylin for 2 minutes, differentiate and blue.
9. Dehydrate, clear and mount.
Results: Acid mucopolysaccharide blue PAS positive substance, neutral mucin magenta nuclei blue
Solutions: 3% alcian blue in 3% aqueous acetic acid - pH 2.5
Schiff's reagent Refer to protocol PAS
ALCIAN BLUE FOR ACIDIC MUCINS
Procedure 1. B.S.D.W.
2. Stain in Alcian blue for 10 minutes.
3. Rinse thoroughly in water.
4. Counterstain with 1% neutral red for 1 minute.
5. Rinse briefly in water.
6. Dehydrate, clear and mount.
Results: Acid mucopolysaccharide blue
pH 3.1 general acidic mucins
pH 2.5 carboxylated and sulphated mucins
pH 1 sulphated mucins only
pH 0.2 strongly sulphated mucins only
Solutions: pH 3. 1 Dissolve 1 g alcian blue in 100 ml 0.5% acetic acid
pH 2.5 Dissolve 1 g alcian blue in 100 ml 3.0% acetic acid
pH 1.0 Dissolve 1 g alcian blue in 100 ml N/10 HCl
pH 0.2 Dissolve 1 g alcian blue in 100 ml 10% sulphuric acid
Notes: Hyaluronidase digestion 1 mg type IV testicular hyaluronidase in 1 ml pH 6.7 phosphate
buffer for 3 hours at 37 °C (preheat before use)
KERATIN - MUCIN
Procedure: 1. Stain section with filtered alcian blue solution for 10 minutes.
2. Rinse with water.
3. Stain nuclei with Harris' Hx, differentiate and blue.
4. Stain with phloxine solution for 5 - 15 minutes, blot dry.
5. Differentiate in tartrazine until a yellow background with red keratin and RBC.
6. Rinse with 95% alcohol, rinse in abs. alcohol, clear in xylene and mount.
Results: Mucin Blue to green Keratin, RBC, Fibrin Red
Nuclei Bluish grey
Other structures Yellow
Solutions: Alcian blue solution:
o 1% Aq. alcian blue 100 ml
o 1% Aq. acetic acid 100 ml
Phloxine solution:
o Phloxine 0.5 gm Calcium chloride 0.5 gm (Anhydrous)
Tartrazine solution:
o Saturated solution of tartrazine in cellosolve (ethylene glycol monoethyl ether),
about 0.3%.
ALDEHYDE FUCHSIN FOR PANCREATIC CELL - HALMI
Procedure: 1. B.S.D.W.
2. Treat with 0.25% acidified potassium permanganate for 5 minutes.
3. Wash in tap water and bleach with 1% oxalic acid.
4. Wash well
5. Rinse in 70% alcohol and stain with aldehyde fuchsin for 5 minutes.
6. Rinse excess stain with 70% alcohol followed by washing with tap water.
7. Counterstain nuclei with Celestine blue-haematoxylin sequence.
8. Rinse with distilled water and stain in Orange G-light green for 1 minutes.
9. Rinse with 0.2% Acetic acid followed by 95% alcohol (no water).
10. Dehydrate, clear and mount.
Results: Alpha cell yellow
Beta cell deep purple-violet
D cell and collagen green
Elastic fibre and some mucopolysaccharide purple-violet
Solutions: Halmi’s or Orange G -light green Light green 0.2 g
Orange G 1 g Phosphotungstic acid 0.5 g
Glacial acetic acid 1 ml
Distilled water 100 ml
0.25% acidified potassium permanganate
ALDEHYDE FUCHSIN TECHNIQUE - GORMORI
Procedure: 1. B. S. D. W.
2. Treat with 0.25% Acidified potassium permanganate for 5 minutes.
3. Wash in tap water and bleach with 1% Oxalic acid.
4. Rinse in 70% alcohol and stain with Aldehyde fuchsin for 5 minutes.
5. Rinse quickly with 0.5% acid alcohol.
6. Check microscopically.
7. Dehydrate, clear and mount.
Results: HAA positive purplish
Background very pale purplish/colourless
Elastic fibres, sulphated mucin (include mast cells), beta cell of pancreas & pituitary, some
lipofuscins, gastric chief cells hypothalamic neurosecretory cells purple
Solutions: Aldehyde fuchsin Basic fuchsin 0.5 g
70% alcohol 100 ml
Concentrated HCl 1 ml
Paraldehyde 2 ml
Dissolve the dye in the alcohol. Filter and add the acid and paraldehyde. Leave at room
temp. for 72 hours until the stain has assumed a deep purple colour. The stain should be
stored in the refrigerator at 4oC.
BEST'S CARMINE METHOD-BEST, 1906 (BC)
Procedure 1. B. S. D. W.
2. Stain the nuclei with an iron haematoxylin stain. Differentiate and blue.
3. Stain with Carmine solution 5 - 15 minutes. The older the stock carmine used the longer the
staining time.
4. Wash well in either Best's differentiator.
5. Rinse in fresh alcohol.
6. Clear in xylene and mount as desired.
Results: Glycogen deep red Some mucin, fibrin weak red Nuclei blue Solutions:
a) Carmine Stock solution Add 2 g carmine, 1 g potassium carbonate and 5 g potassium
chloride to 60 cm3 distilled water. Boil gently for 5 minutes, using a large flask to avoid
spillage (the heating produces a carimine-metallic ion complex which is the active
staining ingredient). Cool and add 20cm3 concentrated ammonia. Filter and store in a
dark container at 40oC, where it should remain usable for several months.
b) Carmine working solution Stock solution 15 cm3 Concentrated ammonia 12.5 cm3
Methanol 12.5 cm3
c) Best's differentiator Methanol 40 cm3 Ethanol 80 cm3 Distilled water 100 cm3
Remarks: a. Failure to stain is usually due to either using an old deteriorated stock solution, or to
having washed the section in water instead of alcohol or differentiator, immediately
following staining with carmine.
b. An artefact encountered with this technique is stain precipitate on the section. To
minimise this, is it suggested that the following points are noted:
i. The working solution should be filtered before use.
ii. Staining should be carried out in a closed container (e.g. Coplin jar).
iii. The section should not be allowed to dry following staining with carmine, but
should be washed immediately in alcohol or differentiator.
ALIZARIN RED S FOR CALCIUM
Procedure 1. B.S.D.W.
2. Flood with Alizarin red solution for 1 minutes.
3. Rinse with 95% alcohol.
4. Dehydrate, clear and mount.
Results: Site of calcium deposits orange red Background yellowish
Solution: 2% aqueous solution of Alizarin red S
Adjust pH to 4.1-4.3 with dilute ammonium hydroxide using a glass electrode pH-meter. The
solution should be a deep iodine colour and keeps well at 4oC
BODIAN COPPER PROTARGOL METHOD FOR NERVE FIBRES (BODIAN)
Procedure 1. B.S.D.W.
2. Place in copper-Protargol solution at 37 °C for 48 hours.
3. Rinse with distilled water and reduce in hydroquinone solution for 10 min.
4. Wash in distilled water and tone in 0.2% Gold chloride solution for 5 min.
5. Intensify in 2% Oxalic acid until section appears purplish.
6. Rinse in tap water and fix in 2.5%,sodium thiosulphate solution for 3 min.
7. Dehydrate, clear and mount.
Results: Nerve fibres, nuclei Greyish black
Background Purplish
Notes: After step (6), section can be counterstained with PAS. No haematoxylin staining of the
nuclei is required.
Solutions: Copper protargol solution
Silver protargol 0.4 g
Distilled water 20 ml
Copper chips 1 g
Sprinkle the silver protargol powder onto the surface of distilled water and let it dissolve
without stirring. Add copper chips
Hydroquinone solution
Sodium sulphite 1 g
Hydroquinone 0.2 g
Distilled water 20 ml
CHROMOTROPE-ANILINE BLUE METHOD FOR COLLAGEN AND
MALLORY BODIES (CAB)
Procedure 1. B.S.D.W.
2. Stain nuclei with Weigert's iron haematoxylin. Rinse in distilled water.
3. Immerse in 1% phosphomolydbic acid for 1 - 3 min. Rinse well in distilled water.
4. Stain with CAB solution for 8 min. Rinse well in distilled water. Blot.
5. Dehydrate quickly, clear and mount.
Results: Collagen Blue Mallory bodies Blue or sometimes red Giant mitochondria Red
Solution: CAB solution 1.5 g aniline blue is dissolved in 6 ml HCl and 200 ml distilled water with gentle
heat, 6 g Chromotrope 2R is added. The pH should be 1.0.
Remark: As used at Mount Sinai Hospital, New York; modified from Roque (1953) and Churg and
Prado (1956).
BIEBRICH SCARLET-FAST GREEN TECHNIQUE - Guard 1959 (BSFG)
Procedure 1. B.S.D.W.
2. Treat with the diluted Harris haematoxylin solution.
3. Drain and transfer direct to the Biebrich scarlet -solution 2 min.
4. Rinse in 50% alcohol.
5. Place in a Coplin jar of fast green solution 12 - 24 hr (overnight at room temperature is
convenient.)
6. Rinse in water, dehydrate, clear and mount as desired.
Results: Nuclear chromatin & sex chromatin body dark green
Cell cytoplasm light green
Solutions: Harris Haematoxylin diluted 1 part with 5 parts distilled water.
Biebrich scarlet solution
Biebrich scarlet ws 1 gm
Phosphotungstic acid 0.3 gm
Acetic acid 0.5 ml 50% ethanol 100 ml
Fast green solution:
Fast green FCF -------------0.5 gm
Phosphomolybdic acid ------0.3 gm
Phosphotungstic acid --------0.3 gm
Glacial acetic acid -----------5 ml
50% ethanol ----------------100 ml
CARBOL FUCHSIN FOR SEX CHROMATIN - BARR BODIES (CF-B)
Procedure 1. Stain in working solution for 1-2 min.
2. Differentiate with 95% ethanol and check microscopically. (½ - 1 min.)
3. D.C.M.
Results: Sex chromatin bodies red (at rim of nuclear membrane)
Solutions: Stock solution Basic fuchsin 3 gm 70% Ethanol 100 ml
Working solution
Stock solution 10 ml 5%
Phenol in D.W. 90 ml
Glacial acetic acid 10 ml
37-40% Formaldehyde solution 10 ml (Stand for at least 24 hrs. Keep for approx. 1
month)
Remark: Medical laboratory technology & clinical pathology. Lynch p. 1280
CRESYL FAST VIOLET FOR NISSL SUBSTANCES
Procedure 1. Section at 8 mm.
2. Stain for 10 min. in cresyl fast violet solution. (It can be heat up to 56oC for rapid result)
3. Rinse in distilled water.
4. Differentiate in 95% alcohol until background is relatively clear.
5. D.C.M.
Results: Nissl substances, nucleoli and nuclei violet
Background colourless
Solutions: Cresyl fast violet 0.1%
cresyl fast violet 30 ml
10% glacial acetic acid 5 drops
CRESYL FAST VIOLET - FOR BARR BODY
Procedure 1. B.S.D.W.
2. Stain in Cresyl fast violet solution 10 min.
3. Rinse in 0.01N acetic acid.
4. Wash in 0.01N acetic acid.
5. Rinse in absolute ethyl alcohol.
6. Differentiate in absolute ethyl alcohol.
7. Control microscopically until only the sex chromatin body is deeply stained in contrast with
the rest of the nucleus.
8. Clear in xylene, mount in D.P.X.
Results: Sex chromatin body - deep blue
Nucleus - light blue
Notes: A simple method for the demonstration of the sex chromatin body.
Solution Cresyl fast violet 1 gm.
Acetic acid (0.01N), filter 100 ml
DIAZO
Procedure 1. B.S.D.W.
2. Treat with the solution for 1 min.
3. Rinse with D.W.
4. Counter stain with Hx.
5. D.C.M.
Result: 1. Argentaffin granules orange red
2. Nuclei blue
3. Background pale yellow
Solution: 4. 1% aqueous fast red salt B 5 cm3
5. Saturated aqueous lithium carbonate 2 cm3
6. Mix and pre cool at 4 °C before use.
CONGO RED FOR AMYLOID (CR)
Procedure 1. Stain nuclei with Harris' Haematoxylin, differentiate and blue.
2. Stain with Congo red solution for 5 minutes.
3. Wash in water and differentiate with alcoholic potassium hydroxide until the background is
clear.
4. Dehydrate, clear and mount..
Results: Amyloid, elastin & eosinophils orange red
Nuclei blue
Background clear
Solutions: 0.5% congo red in 50% alcohol
0.2% potassium hydroxide in 80% alcohol
FEULGEN FOR DNA
Procedure 1. B.S.D.W.
2. Treat section with 1 N HCl for 8 minutes at 60 °C.
3. Rinse in distilled water.
4. Stain in Schiff's reagent for 45 minutes.
5. Rinse in bisulphite solution.
6. Counterstain with light green for 1 minute.
7. D.C.M.
Results: DNA Magenta Other structure Shades of green
Solution: Light green: 1% light green in 1% acetic acid.
GIEMSA-DIFF Q
Procedure 1. For paraffin section, B.S.D.W. and allow section to dry.
2. For imprints, frozen sections, or smears perform step 3 directly.
3. Dip slide in Solution A 5 times, one sec. each time. Allow excess to drain.
4. Dip slide in Solution B 5 times, one sec. each time. Allow excess to drain.
5. Dip slide in Solution C 5 times, one sec. each time. Allow excess to drain.
6. Wash in tap water.
7. Allow section to dry.
8. Clear & Mount.
Results: Eosinophilic Granules - Red
Nuclei - Blue
Mast Cells - Blue
Cytoplasm - Pink
Bacteria - Blue
FOUCHET FOR BILE PlGMENT
Procedure 1. B.S.D.W.
2. Treat with Fouchet reagent 5 minutes.
3. Wash in running tap water 2-3 minutes.
4. Counterstain with Van Geison for 5 minutes.
5. D.C.M.
Results: Bile pigment greenish yellow
Collagen red
Other structures yellow
Solution: Fouchet reagent
25% aqueous trichloroacetic acid 10 ml 1
0% ferric chloride 10 ml
FEULGEN NUCLEAL REACTION FOR DNA - FEULGEN AND ROSSENMCKI
1924
Procedure 1. Bring all sections to water.
2. Rinse sections in HCl at room temperature 1 min
3. Place section in N-HCl at 60oC 8 min
4. Place sections in HCl at R.T. 1 min
5. Transfer sections to Schiff's reagent 45 min
6. Rinse sections in bisulphite solution. 2 min
7. Repeat wash in bisulphite solution. 2 min
8. Repeat wash in bisulphite solution. 2 min
9. Rinse well in distilled water.
10. Counterstain if required in 1% light green.
11. Wash in water.
12. Dehydrate through graded alcohols to xylene and mount.
Results: DNA Red-purple
Cytoplasm Green
Fixation: Not critical but not Bouin.
Reagents: a. N-hydrochloric acid Hydrochloric acid (conc.) 8.5 ml Distilled water 91.5 ml
b. Bisulphite solution 10% potassium metabisulphite 5 ml N-hydrochloric acid 5 ml Distilled
Water 90 ml
GRAM FOR BACTERIA
Procedure 1. B.S.D.W.
2. Stain in filtered crystal violet 30 sec.
3. Treat with Gram's iodine one minute.
4. Differentiate with equal parts of acetone and alcohol.
5. Rinse in water for 10-20 minutes or rinse in alcohol to remove the iodine.
6. Counterstain with neutral red.
7. Blot dry and transfer to absolute alcohol.
8. Clear and mount.
Results: Gram positive bacteria - Blue
Gram negative bacteria - Red
Nuclei - Blue
Other structures - Shades of pink
Note: Prolonged treatment with crystal violet will result in staining precipitate though the stain is
filtered. A control section should be stained at the same time.
Solutions: Crystal violet solution: Crystal violet 26 gm 95% alcohol 20 ml Ammonium oxalate 0.8 gm
Distilled water 80 ml
Gram's iodine: Iodine 1 gm Potassium iodide 2 gm Distilled water 300 ml
Neutral red: 1% aqueous solution
GRIMELIUS FOR ARGYROPHIL CELL
Procedure 1. Place the slides in the freshly prepared silver solution, for 5 hours at 60 °C.
2. The silver solution was drained off from the slides (not the section) and these were then
immersed in a freshly prepared reducing solution for 1 minute at 60 °C.
3. D.C.M.
Results: Argyrophil Granules : Brown / black
Note: 1. In case where the argyrophil islet cells appeared weak the staining reaction could be
improved by double impregnation. After staining step 2, the slides were immersed in an
aqueous solution of 5% sodium thiosulphate for 2 minutes. After rinsing in distilled water for
about 5 minutes the slides were placed in a further freshly prepared silver solution as in step
1 for 10 minutes at room temp. and then in a freshly prepared reducing solution as in step 2
for 1 minute at 40-45oC. 2. For step 1, the slides can be incubated at 37oC for overnight
Solutions: Silver solution: 0.1 M acetic acid-sodium acetate buffer pH 5.6 3.3 ml (0.2 M Acetic acid 19
ml, 0.2 M sodium acetate buffer 181 ml) Distilled water 30 ml Freshly prepared 10% silver
nitrate 0.1 ml
Reducing solution: Hydroquinone 0.4 gm Sodium sulphite 2 gm Distilled water 40 ml
GROCOTT METHENAMINE SILVER NITRATE METHOD WITH
MICROWAVE TECHNIQUE
Procedure 1. B.S.D.W.
2. Place section in 5% Chromic acid solution at 60 °C for 120 seconds (time at temp).
3. Wash section in tap water.
4. Treat section with 1% Sodium metabisulfite solution for 60 seconds.
5. Rinse section in tap water followed by several changes of distilled water.
6. Place section in Methenamine silver working solution and heat until the solution starts to
boil (time at temp at 100oC for 2 seconds).
7. Leave section in hot working solution until proper staining intensity is achieved.
8. Rinse section in distilled water. * If additional incubation is required, place section back in
hot working solution.
9. Tone section in 0.2% Gold chloride solution for 5 minutes.
10. Rinse section in distilled water.
11. Treat section with 2.5% Sodium thiosulphate solution for 2 minutes.
12. Wash section in tap water.
13. Counterstain section with 1% light green solution.
14. Dehydrate, clear and mount.
Results: Fungi Sharply outlined in black
Nuclei Grey
Background Pale green
Solutions: 3% Methenamine solution
o Methenamine 3 g
o Distilled water 100 ml
5% Aqueous silver nitrate solution
o Silver nitrate 5 g
o Distilled water 100 ml
GRIMELIUS METHOD WITH MICROWAVE TECHNIQUE
Procedure 1. Prepare silver nitrate and reducing solution.
2. B.S.D.W.
3. Incubate section in silver nitrate solution and heat to 100oC for 5 seconds.
4. Leave section in (3) for 1 minute.
5. Preheat the reducing solution at 100oC for 5 seconds.
6. Rinse the section with distilled water.
7. Reduce section in (5) until yellowish brown colour macroscopically.
8. Rinse section with distilled water.
9. Dehydrate, clear and mount.
Results: Argyrophil granules brown / black
Solutions: Silver nitrate solution
o silver nitrate 0.5 g
o Distilled water 45 ml
o Acetate buffer pH 5.6 5 ml
Reducing solution
o Sodium sulphite 2 g
o Hydroquinone 0.4 g
o Distilled Water 40 ml
GROCOTT'S METHENAMINE SILVER NITRATE METHOD FOR FUNGI
Procedure 1. B. S. D. W.
2. Oxidize in 5% Chromic acid for half an hour.
3. Wash in running tap water.
4. Rinse for 2 minutes in 1% Sodium metabisphite to remove residual chromic acid.
5. Wash in tap water, rinse thoroughly in distilled water.
6. Treat with Methenamine silver nitrate solution at 58oC for half an hour. (check
microscopically)
7. Rinse thoroughly with distilled water.
8. Tone in 0.1% Gold chloride for 5 minutes.
9. Rinse in distilled water.
10. Treat with 2.5% Sodium thiosulphate for 2 minutes.
11. Wash thoroughly with distilled water.
12. Light green counterstain.
13. Dehydrate, clear and mount.
Results: Fungi Sharply outlined in black Nuclei Grey Background Pale green
Solutions: 5% Chromic acid Chromic acid 5 g Distilled water 100 ml Methenamine silver nitrate solution
3% Methenamine(Hexamine) 10 ml 5% Silver nitrate 0.5 ml 5% Borax(std.borate) 0.8 ml
Distilled water 10 ml
Freshly prepared, filter before use.
5% Borax solution Borax 5 g Distilled water 100 ml
Methenamine silver stock solution 3% Methenamine solution 400 ml 5% Aqueous silver
nitrate solution 20 ml
Mix slowly, kept in brown bottle and store at 4oC.
Methenamine silver working solution Stock solution 20 ml 5% Borax solution 1.6 ml Distilled
water 20 ml
GOMORI'S TRICHROME - RAPID, ONE STEP METHOD
Procedure 1. Fix air dry frozen section in buffered formalin for 15 minutes.
2. Stain nuclei with an iron haematoxylin.
3. Differentiate and, blue as per the standard technique.
4. Wash well in tap water, then in distilled water.
5. Stain in Gomori solution for 5 to 20 minutes.
6. Rinse well in 0.2% glacial acetic acid solution.
7. Blot dry, dehydrate, clear and mount.
Results: Nuclei grey-blue Collagen green Muscle, cytoplasm, red blood cells, fibrin red Higher pH
Nemaline rods red Background blue-green
Solution: Gomori solution Chromotrope 2R 0.6 g Fast green FCF 0.3 g Phosphotungstic acid 0.6 g
Glacial acetic acid 1 ml Distilled water 100 ml
Note: For demonstrating nemaline rods, adjust pH of the solution to 3.4 using 1 M NaOH
GORDON AND SWEET'S FOR RETICULIN
Procedure 1. B.S.D.W.
2. Treat with 0.25% potassium permanganate solution 5 minutes.
3. Rinse in tap water.
4. Bleach in 1% oxalic acid solution 5 minutes.
5. Rinse in distilled water.
6. Treat with 2.5% iron alum for at least 15 minutes.
7. Rinse well with distilled water.
8. Treat with silver bath 20 seconds or above (determined by the age of the silver bath).
9. Rinse with distilled water.
10. Reduce with 10% formalin solution for 1 - 2 minutes.
11. Rinse in tap water.
12. Fix in sodium thiosulphate for 1 - 2 minutes.
13. D.C.M.
Results: Reticulin Black Collagen Golden brown Nuclei Black
Reagents: Silver bath 10% silver nitrate 5 ml. Titrate with ammonia dropwisely until solution is just
about to clear. Add in 5 ml 3% sodium hydroxide and titrate with ammonia until solution is
just about to clear. Make up to 50 ml with distilled water. Filter before use.
Notes: Rinse all the apparatus thoroughly in distilled water before use.
HAEMATOXYLIN-BASIC FUCHSIN-PICRIC ACID FOR MYOCARDIAL
INFARCTION
Procedure 1. B. S. D. W.
2. Stain nuclei with Harris' haematoxylin for seconds.
3. Wash in water, blue.
4. Rinse in distilled water.
5. Stain in 0.1% Basic Fuchsin for 3 minutes.
6. Rinse in distilled water, blot.
7. Rinse in acetone.
8. Differentiate in 0.1% Picric acid in acetone for 5 seconds.
9. Stop differentiation with rinse in acetone.
10. Clear in xylene and mount.
Results: Nuclei Blue Ischemic Muscle, RBCs Red Normal Muscle Yellow
Solutions: a) Harris' Haematoxylin.
b) 0.1% aqueous Basic Fuchsin (C.I. 42510).
c) Acetone.
d) 0.1% Picric Acid in Acetone.
GOMORI'S MODIFIED TRICHROME - ENGEL AND CUNNINGHAM
Procedure 1. Use air-dried, cryostat-cut sections (10mm).
2. Stain in Harris's haematoxylin for 3 min.
3. Rinse briefly in distilled water.
4. Decolorize in 0.5% acid alcohol.
5. Blue in Scott's tap water.
6. Wash in running tap water.
7. Stain in Gomori’s stain for 10 min. Check microscopically.
8. Rinse BRIEFLY in 0.2% acetic acid.
9. Dehydrate quickly, clear in xylene and mount with DPX.
Results: Intermyofibrillar material red Myofilaments green Nuclei reddish blue Connective tissue
green
Solutions: Gomeril’s stain Chromotrope 2R 0.3 gm Fast Green FCF 0.15 gm Phosphotungstic acid 0.3 gm
Glacial Acetic acid 0.5 ml
Add distilled water to make 50 ml. Adjust to pH 3.4 with 1N NaOH
Note: This stain must he freshly prepared and sections are stained after cutting.
Remarks: 1. Particularly helpful in identifying nemaline rods.
2. Lillie Page 701
HEIDENHAIN'S IRON HAEMATOXYLIN (H.I.H.)
Procedure 1. Bring section to water.
2. Place in iron alum mordant for 30 min.
3. Rinse in distilled water.
4. Place section in haematoxylin solution for 30 min.
5. Rinse well in tap water.
6. Differentiate in 2.5% iron alum solution until desired staining is achieved.
7. Wash in tap water for 10 min. to remove the iron alum.
8. Counterstain with sat. tartrazine in absolute cellosolve for 30 sec.
9. Rinse with 95% alcohol. D.C.M.
Results: Nuclei, muscle striations, RBC, keratin - Bluish Black Background - Yellowish
Solution: Iron Alum Mordant 5% aqueous ferric ammonium sulphate (iron alum)
Heidenhain's Haematoxylin Solution Haematoxylin 0.5 gm Absolute alcohol 10 ml Distilled
water 90 ml
Iron Alum Differentiater 2.5% aqueous iron alum solution.
HAEMATOXYLIN EOSIN (H&E)
Procedure 1. Section down to water.
2. Stain in Haematoxylin solution for 5 minutes.
3. Rinse well in tap water.
4. Differentiate in 0.5% acid alcohol.
5. Blue in 1% scott's solution for 1 minute.
6. Rinse briefly.
7. Stain in Eosin solution for 1 minute.
8. Rinse briefly in D.W.
9. D.C.M.
Results: Nuclei Blue Background Pinkish to orange red
Reagents: HARRIS HAEMATOXYLIN:
Haematoxylin 8 gm Absolute alcoholol 80 ml Ammonium alum or potassium alum 160 gm
Distilled water 1600 ml Glacial Acetic Acid 32 ml Mercuric oxide 2 gm
SCOTT'S TAP WATER:
Sodium Bicarbonate 7 gm Magnesium Sulphate 40 gm Dissolve in 2 litres of distilled water
Thymol is added to prevent mould formation
ACID ALCOHOL:
Conc. HCl 5 ml 70% Alcohol 995 ml
EOSIN SOLUTION:
10% Eosin 60 ml 1% Erythrosin 40 ml Distilled water 500 ml
LAQUEUR - ALCOHOLIC HYALINE (Laqueur)
Procedure 1. Stain in alum Hx for 5 minutes.
2. Stain in acid fuchsin solution and heat with a small flame to fuming. Let stand 5 minutes.
3. Wash, differentiate in a mixture of 7 parts 20% alcohol and 1 part sat. Alcoholic picric acid
until only hyaline and red corpuscles remain red, and collagen is faint grey or unstained.
5. Wash thoroughly.
6. Mordant 30 minutes in 1% phosphomolybdic acid.
7. Transfer directly to 1% light green SF in 1% acetic acid until collagen stains green.
8. D.C.M.
Results: Mallory's alcoholic hyaline Brilliant red Cytoplasm Pale brown Bile pigment, collagen Green
Haemosiderin and hemofuscin Unstained/yellowish brown
Solution: Acid fuchsin solution : Shake 2 ml of aniline with 100 ml D.W. Filter. Dissolve 20 g acid
fuchsin in aniline water prepared.
IRON HAEMATOXYLIN STAIN FOR AMOEBAE CHROMOSOME
STRUCTURE - CRAIG AND FAUST, CLINICAL PARASITOLOGY, P.864 (IH)
Procedure 1. Fix smears in Schaudinn's solution at 60oC for 2 minutes.
2. Remove Hg by 70% alcohol with iodine.
3. wash in water.
4. Mordant in 2% iron alum at 40oC for 2 minutes.
5. Wash in water for 3 minutes.
6. Stain in 0.5% aqueous Hx. 2 minutes.
7. Differentiate in iron alum until chromosomes are prominent.
8. Wash in water.
9. D.C.M.
Solution: Schaudinnls solutions Solution A:
Sat-aq. HgCl2 600 ml 95% ethyl alcohol 300 ml before use add 5 ml acetic acid to every 100
ml solution A
SOUTHGATE’S MUCICARMINE (MC)
Procedure 1. Stain nuclei with Harris Hx for 5 minutes, differentiate and blue.
2. Treat with Mucicarmine solution for 20 minutes.
3. Rinse in water.
4. Dehydrate, clear and mount.
Results: Epithelial mucin red Cryptococci red Nuclei blue
Soultion: Mucincarmine (stock) Carmine 1 g Aluminium hydroxide 1 g 50% alcohol 100 ml Aluminium
chloride 0.5 g (0.9 g if A1C13.6H20) is used.
After grading Carmine into paste with 100 ml 50% alcohol, then add 1 g Al (OH)3 and 0. 9 g
A1C13. 6H20. Mix and boil gently for 2.5 minutes, cool and filter, and store in the
refrigerator. When use, the stain is diluted 1:10 (The optimal dilution factor has to be tried
and varies from batch to batch.)
MANN’S METHYL BLUE - EOSIN FOR MINUTE STRUCTURE AND
INTESTINAL FLAGELLATE FLAGELLATE OF AMOEBAE( NW)
Procedure: 1. 1 Fix smears in Schaudinn's fluid 60oC 2 minutes.
2. Remove Hg pigment by 70% alcohol with iodine.
3. Stain for 4 - 12 hours in staining solution, as determined by trial.
4. Wash in distilled water thoroughly.
5. 5 Differentiate in 70% alcohol containing a little OG (a few drops to 100 ml.)
6. Wash in water.
7. D.C.M.
Solutions: 1% Methyl Blue 35 ml 1% Eosin 45 ml Distilled water 100 ml
Notes : Craig and Faustm, Clinical Parasitology, p.864
MARTIUS YELLOW - SCARLET RED, SOLUBLE BLUE FOR FIBRIN (MSB)
Procedures: 1. Stain nuclei with celestine blue - Haemalum sequence, 3 minutes each, differentiate and
blue.
2. Rinse in 95% alcohol and stain with 0.5% martius yellow for at least 2 minutes. Rinse in 95%
alcohol. Cheek that only RBC is stained yellow.
3. Stain in 1% brilliant crystal scarlet 1 minute, check microscopically.
4. Treat with 1% phosphotungstic acid for a short while and check that only muscle and fibrin
are stained red.
5. Counter stain with 0.5% soluble blue for 1 minute. (Be careful of overstain).
6. D.C.M.
Results: Fibrin and Muscle Red RBC Yellow. Nuclei Greyish blue Collagen Blue
Solutions: 0.5 % Martius yellow :
Martius yellow 0.5 gm Phosphotungstic acid 2 gm 95 % Alcohol 100 ml
1 % Brilliant crystal.scarlet : Brilliant crystal scarlet 6R 1 gm Glacial acetic acid 2.5 ml
Distilled water 97.5 ml
0.5 % Soluble blue
MASSON FONTANA (MF)
Procedures: 1. 1 B. S. D. W.
2. Rinse section thoroughly with distilled water.
3. Stain overnight in Fontana silver solution at dark in a closed jar.
4. Rinse in distilled water.
5. Fix in 0.5% sodium thiosulphate for 2 minutes.
6. Wash in running tap water for 2 minutes.
7. Counterstain with neutral red.
8. Dehydrate, clear and mount.
Results: Melanin black Argentaffin granule black nuclei red
Solutions: Fontana silver solution
Add strong ammonia to 20 ml of 10% aqueous silver nitrate, until the precipitate dissolves.
Add 20 ml of distilled water and filter before use.
TAEZER-UNNA ORCEIN METHOD (ORCEIN)
Procedure: 1. B. S. D. W.
2. Bleach with acidified KmnO4 for 5 minutes, then oxalic acid 1 min.
3. Take section to 70% alcohol.
4. Place in a closed jar of the orcein stain for 2 hours for HAA at room temp.
5. Differentiate with 1% acid alcohol.
6. Stain briefly in Hx for 10 sec.
7. Wash well in tap water for blueing.
8. D.C.M.
Results: Elastic fibers dark brown Nuclei blue HAA purple brown
Solutions: a) Orcein stain Orcein (synthetic) 1 g 80% alcohol 100 ml ( adjust to pH 1 - 2 with HCl)
conc. HCl 1 ml
b) 1% acid-alcohol
c) Methylene blue or alum haematoxylin
d) Acidified KMn04 : 0.25% KMn04 10 ml conc. H2S04 12 ml
MASSON TRICHR0ME
Procedures 1. Weigert’s haematoxylin 5 minutes (or Celestine Blue - Hx sequence, 5 min : min).
2. Cytoplasmic stain 5 minutes (optimum)
3. Differentiate with 1% phosphomolybdic acid, check under microscope.
4. Counterstain with 1% light green. Be careful of over staining.
5. . Rinse with 1% acetic acid and then 95% alcohol.
6. Dehydrate, clear and mount.
Results: Muscle, keratin, RBC red Nuclei bluish black Collagen, basement membranes, reticulin,
elastic fibres green
Solutions: Celestine Blue Celestine blue 0.5 g Ferric ammonium sulphate 5 g Glycerin 14 ml Distilled
water 100 ml
Dissolve the iron alum in the water without heat. Add the celestine blue and boil for 3
minutes. Filter when cool and add the glycerin. The stain can keep for at least 6 months.
Cytoplasmic stain 1% ponceau 2R in 1% acetic acid 2 parts 3% acid fuchsin in 1% acetic acid 1 parts
Fibre stain 2% Light green in 1% acetic acid Commercial Gomeri’s trichrome stain may also be used,
stain for 2-5 minutes, rinse in 1% acetic acid. result is the same for MT.
PALMGREN’S METHOD FOR NERVE FIBRES (PALMGREN)
Procedure 1. Cut paraffin section at 7 microns and celloidinize section.
2. Treat with acid bath for 5 min.
3. Rinse with 3 changes of distilled water for 5 min.
4. Stain with silver bath for 30 min. at room temp.
5. Drain off excess solution and shake continuously in reducing bath at 45oC for 1 min.
6. Rinse with 50% alcohol for 10 seconds.
7. Wash with distilled water, 3 changes in 5 min.
8. Intensify in 2% oxalic acid until section appears dark brown.
9. Treat with 2.5% sodium thiosulphate for 3 min.
10. D.C.M.
Results: Nerve fibres and neurofibrils Black Background Yellowish brown
Solutions : Acid Bath: 40% Formaldehyde 25 ml Distilled water 75 ml 1% Nitric acid 0.2 ml
Reducing Bath: Pyrogallol 1 gm Distilled water 45 ml Absolute alcohol 55 ml 1% Nitric Acid 0.2 ml
Silver Bath: 15% silver nitrate
OIL RED O METHOD FOR LIPIDS (ORO)
Procedure: 1. Cut frozen section at 6 mm.
2. Air dry at room temp. for 1 hour and rinse with 60% triethyl phosphate and fix in formalin
for 10 minutes.
3. Filter working solution onto section and leave in moist chamber for 15 minutes
(Alternatively, it can be stained in a coplin jar which contains 6 part dye and 4 part DW for 15
- 30 minutes).
4. Pour off excess stain and differentiate in 60% triethyl phosphate solution for a few seconds.
5. Bring section to water.
6. Counterstain nuclei with Harris Haematoxylin for 15 second.
7. Blue nuclei. Do not differentiate in acid alcohol.
8. Mount in aqueous mountant.
Results: Neutral lipids orange red
Nuclei blue
Note: Formalin fixed tissue block can be placed in gum sucrose solution overnight before the
frozen section. Use the free floating method or attach the section to albuminized slides.
Gum sucrose solution :
Gum acacia (gum arahic) 2 gm Sucrose 60 gm Distilled water 200 ml
Completely dissolve the gum acacia in water with stirring. Add sucrose and dissolve. Store at
4oC.
Reagents: Stock solution : sat. oil red 0 in triethyl phosphate.
PASM FOR BASEMENT MEMBRANE(PASM)
Procedure 1. B.S.D.W.
2. Oxidize in 1% Periodic acid for 15 minutes.
3. Oxidize in 5% Chromic acid for 15 minutes.
4. Wash in running water.
5. Rinse in 1% Sodium bisulphite to remove residual chromic acid.
6. Wash in tap water, rinse thoroughly in distilled water.
7. Treat with Methenamine silver nitrate solution at 58oC for half an hour. (check
microscopically)
8. Rinse thoroughly with distilled water.
9. Tone in 0.1% Gold chloride for 5 minutes.
10. Rinse in distilled water.
11. Treat with 2.5% Sodium thiosulphate for 2 minutes.
12. Rinse in distilled water.
13. Light green counterstain
14. Dehydrate, clear and mount.
Solutions: Methenamine silver solution
3% Methenamine 15 ml 5% Silver nitrate 0.75 ml 5% Borax 1.2 ml Freshly prepared, filter
before use.
Results: Basement membrane black Background green
PERIODIC ACID-SCHIFF REACTION (PAS)
Procedure 1. B.S.D.W.
2. Treat with 1% Periodic acid for 5 minutes.
3. Rinse with distilled water.
4. Treat with Schiff reagent for 5 minutes.
5. Rinse with distilled water.
6. Put in running water for at least 5 minutes.
7. Counterstain with Harris' haematoxylin for 2 minutes.
8. D. C. M.
Results: PAS positive substances magenta negative after digestion glycogen nuclei blue
Solutions: Basic fuchsin 1 g 0.15 M HCl 100 ml (1.275 ml of conc. HCl in 100 ml distilled water)
Sodium metabisulphite 2 g Dissolve Basic fuchsin in 0.15 M HCl with stirring. Add sodium
metabisulphite and stir for 8 hours until the solution appears straw colour. Add activated
charcoal (2 spoonful) to absorbs the colour. Filter and store aliquots at 4oC
Note: Treat section with amylase for 15 minutes at room temperature before step (2) for PAS with
digestion (PASD) method.
PHLOXINE TARTRAZINE (PT)
Procedure 1. B.S.D.W.
2. Stain nuclei with Harris Hx.
3. Stain in phloxine solution for 10 min.
4. Rinse in 95% alcohol.
5. Differentiate in tartrazine solution, controlling microscopically until a yellow background
with red keratin and RBC.
6. Rinse in 95% alcohol (never rinse in water).
7. D.C.M.
Results: Keratin, RBC, Paneth cell granules, fibrin, certain inclusion bodies red Nuclei greyish blue
Other structures yellow
Solutions: Phloxine solution: Phloxine 0.5 g Calcium chloride (anhydrous) 0.5 g Distilled water 100 ml
Tartrazine solution :
Saturated solution of tartrazine in cellosolve, about 0.3%.
PERLS METHOD FOR HAEMOSIDERIN (PERLS)
Procedure 1. B.S.D.W.
2. Rinse thoroughly in distilled water.
3. Stain in Perls' reagent for 25 min.
4. Rinse thoroughly with distilled water.
5. Counterstain with 1% Neutral red for 2 min.
6. Wash in 95% alcohol.
7. Dehydrate, clear and mount.
Results: Haemosiderin, Ferric ions Blue Nuclei Red
Solutions: Perls’ reagent
Equal parts of 2% potassium ferrocyanide and 2% hydrochloric acid 1% Neutral red
Neutral red 1 g Distilled water 100 ml
RUBEANIC ACID FOR COPPER (RA)
Procedure: 1. B. S. D. W.
2. Stain in rubeanic acid solution overnight at 37oC.
3. Place section in 70% alcohol for 15 minutes.
4. Counterstain with Van Gieson solution for 3 minutes.
5. Rinse in 95% alcohol.
6. D.C.M.
Results: Copper greenish black granules Collagen red Background yellowish
Solutions: Rubeanic acid solution
0.1% Rubeanic Acid in Absolute Alcohol 2.5 ml 10% Sodium Acetate 50 ml
In QAP, Rubeanic acid solution 0.1% rubeanic acid in 70% alcohol 1 parts 10% sodium
acetate 20 parts
MALLORY PTAH METHOD FOR MUSCLE STRIATIONS (PTAH)
Procedure: 1. B. S. D. W.
2. Treat section with 0.25% potassium permanganate minutes.
3. Bleach with 1% oxalic acid until section is clear.
4. Mordant with 2.5% iron alum for 1 hour.
5. Stain in PTAH solution overnight.
6. Rinse briefly with 95% alcohol.
7. D.C.M.
Results: Nuclei, Fibrin, Muscle Striations, Myofibrils, RBC, Astroglia Blue
Collagen, Bone and Cartilagenous Matrix Orange red
Solutions: PTAH solution
Haematein 1 gm Phosphotungstic Acid 10 gm Distilled water 1000 ml
Dissolve the haematein in one half of the water with a little of heat. The phosphotungstic
acid in the other half of water and then mix. Store in a tightly stoppered bottle and allow the
solution to ripen.
SCHMORL'S FERRICYANIDE (SF)
Procedures: 1. Wash section well in distilled water.
2. Stain in ferric ferricyanide solution for 10 minutes.
3. Rinse in 1% acetic acid.
4. Rinse in water.
5. Counterstain in Van Gieson solution for 1 minute.
6. Rinse with 95% alcohol.
7. D.C.M.
Results: Melanin, intestinal argentaffin lipofuscin, chromatin blue Cytoplasm, RBC, muscles yellow
Collagen red
Regents: Ferric ferricyanide solution :
1% aq. ferric chloride 30 ml 1% potassium ferricyanide 4 ml distilled water 6 ml
The solution has to be freshly prepared.
SOLOCHROME CYANIDE FOR MYELIN (SC-M)
Procedure 1. B. S. D. W.
2. Stain in solochrome cyanide solution for 10-20 minutes at R.T.
3. Wash in running water.
4. Differentiate in 10% iron alum until nuclei are scarely visible.
5. Wash in running tap water.
6. Counterstain if desired.
7. Dehydrate, clear and mount.
Results: Myelin sheaths bright blue RBC blue Nuclei pale blue to almost unstain
Solutions: Solchrome cyanide RS 0.2 gm D.W. 96 ml 10% iron alum 4 ml conc. H2S04 0.5 ml
TOLUIDINE BLUE - FOR. DEMONSTRATION OF METACHROMASIA IN
FROZEN SECTIONS (TB-F)
Procedure 1. Fix sections in an acetone: tetrahydrofuran mixture (1:1 by volume) at room temperature for
20 min.
2. Rinse in acetone.
3. Stain with toluidine blue solution for 2 min.
4. Rinse in acetone.
5. Clear in xylene and mount.
Results: Nnuclei blue Mucopolysaccharide red to purple
Solutions: Toluidine blue solution 0.5% toluidine blue in 25% acetone:
1% toluidine blue 5 ml acetone 2.5 ml D.W. 2.5 ml
VERHOEFF (VERHOEFF)
Procedure: 1. B.S.D.W.
2. Stain in Verhoeff’s reagent for at least 1 hour (a longer incubation time will give a better
result).
3. Rinse in water.
4. Differentiate in 2% ferric chloride, check under microscope until only elastic fibres and nuclei
are black.
5. Rinse in water or rinse in alcohol to remove.iodine.
6. Counterstain with Van Gieson for 1 minute.
7. Blot dry, rinse in 95% alcohol. D.C.M.
Results: Elastic fibres Black Collagen Red Nuclei Grey Other structures Yellow
Solutions: Verhoeff reagent:
5% alcoholic Hx 2 ml 10% ferric chloride 8 ml Iodine 8 ml
Verhoeff iodine :
Iodine 2 g Potassium Iodide 4 g Distilled Water 100 ml
5% alcoholic Hx:
Haematoxylin 5 g Absolute alcohol 100 ml
VICTORIA BLUE (VICTORIA)
Procedure 1. B.S.D.W.
2. Oxidize with 0.25% potassium permanganate for 10 minutes.
3. Reduce in 5% oxalic acid for 3 minutes.
4. Rinse well in distilled water.
5. Wash briefly in 95% alcohol and transfer to the Victoria Blue staining solution for 18 to 24
hours (overnight).
6. Wash in 95% alcohol until excess stain is removed.
7. Counterstain with VG for 1 minutes - Blot dry.
8. Clear and mount.
Results: Elastic fibres Blue-black Collagen Red Other tissues Yellow
Solutions: Victoria Blue staining solution
Victoria blue 4R (C.I. 42563) 1 g Ethyl violet (C.I. 42600) 1 g Dissolve in 200 ml of boiling
distilled water. Add, in the following order :
Resorcin 4 g Dextrin 0.5 g 30% ferric chloride (freshly prepared) 25 ml
Boil for 3 minutes, cool rapidly and filter. Wash precipitate well with distilled water. Transfer
precipitate plus filter paper to the original beaker. Add 100 ml 95% alcohol to precipitate
and boil gently in water bath for 15 minutes. Cool, filter and make up to 350 ml with 95%
alcohol. To this solution add 10 g phenol and 4 ml concentrated hydrochloric acid.
VAN GIESON (VG)
Procedure: 1. B. S. D. W.
2. Celestine blue 5 minutes, rinse in water, Harris Hx 5 minutes, rinse in water.
3. Differentiate and blue.
4. Stain in Van Gieson - stain 2-3 minutes.
5. Blot dry, rinse in 95% alcohol, D.C.M.
Results: Collagen red Nuclei grey Other structures yellow
Notes: Water removes the red colour, alcohol removes the yellow colour, so that dehydration
should be rapid. 2. Step 1 can be replaced by :
1) Weigert's hx Solu A : Solu B = 1:1 5 minutes
2) briefly differentiated by Solu B.
3) Rinse with water.
4) Van gieson stain.
Solutions: Van Gieson stain : 1% aqueous acid fuchsin 13 ml sat. aqueous picric acid 100 ml
WADE-FITE FOR M. LEPRAE (WF)
Procedure 1. Dewax section in a mixture of equal parts of liquid petrolatum and rectified turpentine
(Terpineol or xylene), blot until almost dry.
2. Wash in tap water, 5 minutes.
3. Stain in carbol-fuchsin solution for 30 minutes.
4. Wash in tap water, blot dry.
5. Decolorize in 3% sulphuric acid.
6. Wash in tap water.
7. Counterstain 0.1% methylene blue.
8. Wash in water, blot dry, xylene, mount.
Results: Positive - Magenta colour
Note: Bleach can be done for clearer background. (potassium permanganate and oxalic acid bleach
after step 6).
VON KOSSA (VK)
Procedure 1. B.S.D.W.
2. Rinse section well with distilled water.
3. Treat with 5% silver nitrate solution under strong light for 60 minutes.
4. Rinse well in distilled water.
5. Fix in 2.5% sodium thiosulphate for 1 minute.
6. Counterstain with neutral red.
7. D.C.M.
Results: Insoluble calcium salts (also urates) brown to black Other structures shades of pink
Notes: A negative control can be used by treating a parallel section with 2% HCl for 30 min before
staining in step (2).
ZIEHL-NEELSEN (ZN)
Procedure 1. B.S.D.W.
2. Flood section with carbol fuchsin stain 10 minutes.
3. Heat slide until steaming, stain for 10 minutes.
4. Rinse with water.
5. Decolourize with 1% acid alcohol until section is faintly pink.
6. Rinse thoroughly with tap water.
7. Bleach with 0.25% potassium permanganate, then 1% oxalic acid.
8. Rinse with water.
9. Flood slide with 0.25% methylene blue for ½ minute.
10. Dehydrate, clear and mount.
Results: Acid fast bacilli red Nuclei blue
Note: 1) A control should always be stained at the same time.
2) Methylene blue can be easily decolourised in alcohol water mixture.
3) Never place stained section in xylene for too long.
4) Long ZN : 3 hours 60oC
Reagents: Carbol fuchsin stain : Basic fuchsin 10 g Alcohol 100 ml 5% aqueous phenol 1000 m1
5% Aqueous phenol: phenol 50 g Distilled water 1000 ml
WARTHIN-STARRY
Procedure: 1. B. S. D. W.
2. Wash sections well in buffer solution (a).
3. Stain sections in silver solution (b) for 1 hour at 60oC.
4. Develop sections in developer solution (c) for about 1 minute at 60oC until sections appear
yellowish brown.
5. Rinse in tap water at 60oC.
6. D.C.M.
Results: Spirochaetes black Blackground : pale yellow to brown.
Solutions: a) Buffer solution Sodium acetate 8.2 g Acetic acid 12.5 g Distilled water 1000 ml
b) Silver solution Silver nitrate 0.2 g Buffer solution 20 ml
c) Developer solution Solution 1 Hydroquinone 0.06 g Buffer 2.0 ml
Solution 2 Gelatine 1.5 g D.W. 30 ml
Solution 3 Ag nitrate 0.12 g D.W. 6 ml
Notes: Solution 1 mix with solution 2 first, then add solution 3 immediately before use.
Celloidinization
When section is brought to absolute alcohol after deparaffinization, it is placed in 1%
celloidin in equal parts of ether and absolute alcohol for a few seconds. It is then withdrawn
slowly and steadily. The section is then air dried before being transferred to 70% alcohol to
complete the rest of the staining procedures.
LILLIE’S MAYER HAEMATOXYLIN (LILLIE)
Procedure: Dissolve alum in boiling and boil distilled water. Add haematoxylin and boil for 2 minutes.
Add sodium iodate and stir. Colour change to purplish. Cool and add glycerin and glacial
acetic acid. Filter before use.
Reagents Haematoxylin 10 gm Aluminum ammonium sulphate 100 ml Sodium iodate 0.6 gm Glycerin
600 gm Distilled water 1400 ml Glacial acetic acid 40 ml
WEIGERT’S IRON HAEMATOXYLIN
Solution A Haematoxylin 1 g
Ethyl alcohol 100 ml
Solution B 30% aq. Ferric chloride 4 ml
Conc. hydrochloric acid 1 ml
Distilled water 100 ml
Mix equal volumes of A and B prior to use.
MAYER’S EGG ALBUMIN - GLYCEROL
Solutions: Egg white 50 ml
Glycerol 50 ml
Distilled water 25 ml
Thymol 100 mg
Mix thoroughly, filter through several layers of gauze into bottle. Store at 4oC.
WHOLE PREPARATION OF WORKS (WORM)
Procedure: 1. Straighten the worm by shaking vigorously for a few seconds in normal saline.
2. Fix in 70% alcohol with 3% acetic acid.
3. Stain for 12 hours or longer in Kirkpatricks carmalum.
4. Differentiate in acid alcohol (may take several hours).
5. Dehydrate in ascending alcohol (50, 70, 95%) leaving for at least half an hour in each grade.
6. Complete dehydration in absolute alcohol, to overnight.
7. Clear in beechwood creosote (cedarwood oil). 8. Mount in balsam. Harden the mount on a
hot plate.
Solutions: Kirkpatricks carmalum: Carmine 5 g Glacial acetic acid 5 ml Potassium alum 5 g D.W. 200 ml
Notes: Grind the carmine in a morter, add acetic acid and 20 ml of water, allow to soak for 20
minutes, then boil gently for 1 hour. Dissolve alum in 100 ml of water and add it to the
cooled carmine solution. Boil gently for another hour. Cool and filter. Add 0.2 g salicylic acid
to inhibit growth of moulds. Stain keeps for at least 1 year.
ORCEIN FOR HEPATITIS B SURFACE ANTIGEN
Procedure: 1. B.S.D.W.
2. Incubate in 1% potassium permanganate for 5 minutes.
3. Wash in water.
4. Decolourise in 2.5% oxalic acid.
5. Stain in orcein solution for 2-4 h at room temperature.
6. Rinse in 70 % ethanol.
7. Rinse in water, then differentiate in 3% acid alcohol.
8. Dehydrate, clear and mount.
Results: HBSAg, copper-associated protein Brown and elastic fibres The method is suitable for
routine demonstration of HBsAg, though probably less sensitive than most
irnmunohistochemical methods.
Solutions: Acidified potassium permanganate
To 95 ml of 0.5% potassium permanganate add 5 ml of 3% sulphuric acid.
Orcein solution
Orcein 1.0 g 70% Ethanol 100 ml Concentrated Hcl 2.0 ml
The pH of the stain should be 1.0 to 2.0. The solution keeps for 1-4 weeks.
Different orcein vary in staining properties (Kirkpatrick 1982). Natural orcein from British
Drug Houses is suitable. Other orcein may require twice the concentration and twice the
amount of HCL (Poppe.personal communication).
GIEMSA STAIN (1 : 1000)
Solutions: 1. Giemsa Stock solution :
a. Giemsa Stain powder 4 g
b. Glycerol 250 ml
c. Methanol (pure) 250 ml
The powder is dissolved in the glycerol at 60oC with regular shaking. After the methanol was
added, the mixture well shaken, and then allowed to stand for 7 days. Filter before use.
2. Working Giemsa stain :
a. Giemsa stock solution 4 ml
b. Buffered distilled water (pH 6.8) 96 ml
Procedure: 1. Sections to water.
2. Rinse in buffered distilled water (pH 6.8).
3. Stain in working Giemsa stain overnight.
4. Rinse in distilled water.
5. Differentiate in 0.5% acetic acid until the section is pink.
6. Wash in tap water,rinsed in distilled water
7. Blot until almost dry.
8. Dehydrate very rapidly through alcohols, clear in xylene and mount in DPX.
Results: Eosinophil granules, R.B.C. orange-red Nuclei blue Background pink
Notes: To achieve a good colour balance, it is necessary to initially overstain with the Giemsa stain,
and then slightly over-differentiate in weak acetic acid until there is an overall pink cast to
the cells. -This is come to offset the loss of eosinophilia and gain in basophilia which result
upon eventual alcohol dehydration.
METHYL GREEN PYRONIN
Solutions: 2% Methyl Green (chloroform washed) 9 ml
2% Pyronin Y 4 ml
Acetate buffer pH 4.823 ml
Glycerol 14 ml
Mix well before use.
Procedure: 1. section to distilled water
2. stained with the solution for 30 mins.
3. washed in distilled water
4. differentiate with ethanol
5. cleared and then mounted
Results: RNA (plasma cell cytoplasm, nucleolus) Red DNA (nuclei) Green
Notes: Plasma cells are found in lymphoid tissue, spleen, bone marrow. The plasma cells have an
ovoid cytoplasm and eccentric nucleus with prominent radial chromatin. They are derived
from B lymphocytes and secrete immunoglobulins
This method demonstrated both nuclei acids - methyl green is a basic dye and combination
with the phosphoric groups of DNA and is generally held to be specific -pyronin staining of
RNA is not specific, as it stains some mucous cells and depolymerized DNA - the pH of the
staining solution is critical as well-as conc. of the 2 dyes
The differentiation is important Washing in water after staining is avoided Tissues that have
been decalcified in acids are unsuitable Loyez’s (1910)
HAEMATOXYLIN METHOD FOR MYELIN (AFTER ANDERSON, 1929)
Preparation of staining solutions: a. 4% aqueous iron alum
b. Loyez haematoxylin 10% alcohol haematoxylin 10 ml distilled water 90 ml saturated aqueous
lithium carbonate 4 ml
c. Final differentiator Sodium tetraborate (Borax) 2 g Potassium ferricyanide 2.5 g Distilled
water 200 ml
Method: 1. B.S.D.W.
2. Treated with iron alum solution at room temperature overnight
3. Washed in running water for several minutes
4. Stained in the haematoxylin solution at room temperature overnight
5. Washed in water
6. Differentiate excess background dye in the iron alum solution differentiate in the borax-
ferricyanide solution if necessary.
7. Washed in water
8. Dehydrate, clear and mount.
Results: Myelin - Blue-black
MARALAND, GLEEN AND ZRIKSON'S A METHOD FOR AXONS IN
PARAFFIN SECTIONS (1954)
Preparation of staining solutions: a. 20% aqueous silver nitrate
b. Ammoniacal silver 20 ml ethanol was added to 30 ml 20% aqueous silver nitrate.
Conc. ammonia was added until the formed precipitate was almost dissolved. Two more
drops of ammonia were added
Method: 1. B.S.D.W.
2. It was placed in the 20% aqueous silver nitrate solution at 37°C for 30 min
3. Treated with 10% formalin for 15 min
4. Drained the slide and treated with ammoniacal silver solution for 30 seconds
5. Washed with 10% formalin for 1 min
6. Washed in water
7. Toned in 0.2% gold chloride for 2 min
8. Washed in water
9. Fixed in 5% hypo for 5 min
10. Washed in water
11. D.C.M.
Results: Nerve fibres, Neurofibrils, Neurons, R.B.C. Black Backgroun Grey
Notes: Sections are better celloidinized using 1% celloidin to give a thick protective coat. This not
just to keep the. section on the slide, but also when the celloidin coat is removed at the
conclusion of the technique much of the background silver precipitate will be removed
Toning helps suppressing a heavy background, but a clear distinction between collagen and
nervous elements cannot be made Glass slides should be carefully cleaned before staining to
reduce non-specific precipitation and silvering
Repeated step (4) & (5) is possible if under-impregnation
O.P.G METHOD FOR CALLS OF ANTERIOR PITUITARY (SILDDARS
1961)
Preparation of staining solutions: a. Orange G Orange G 500 mg Phosphotungstic acid 2 g Absolute alcohol 95 ml Distilled
water 5 ml
b. Acid Fuchsin Acid fuchsin 500 mg Glacial acetic acid 0.5 ml Distilled water 99.5 ml
Method: 1. B.S.D.W.
2. Stained nuclei in celestine blue-haemalum for 5 min
3. Differentiate and blue
4. Washed in water,then 95% alcohol
5. Stained with orange G for 10 min
6. Rinsed in distilled water
7. Stained in acid fuchsin solution for 2 min
8. rinsed in distilled water
9. Treated with 1% aqueous P.T.A.
10. Rinsed in distilled water
12. Stained in light green for 1 min
13. Washed in water
14. D.C.M.
Results: Nuclei Blue-black Basophil cells Red-purple Acidophil cells, R.B.C. Yellow Connective tissue
Green
Notes: - the staining of the basophils by acid fuchsin is progressive and should stain until the cells
are a well defined red-purple
- this is one of the trichrome staining and the mechanism is similar to trichrome technique
- normally, in the pituitary over 50% of the cells are termed as chromophobe cells, that is
the unstainable cells and are thought to be the cells for secretion of adrenocorticotrophic
hormone (ATCH). 40% of the rest are called acidophils ((X) , they are usually clamped
together in the lateral wings of the anterior lobe. The remaining 10% are termed as
basophils (B) , they are usu. larger than acidophils and are divided into two groups,
basophil-S-cells and basophil-R-cells
TRIPAS ( PAS-ORANGZ G METHOD )
Preparation of staining solutions: a. 1% periodic acid
b. Schiff's reagent
c. 2% orange G in 5% phosphotungstic acid
Method: 1. B.S.D.W.
2. Oxidized in periodic acid for 5 min
3. Washed in running water for 5 min and rinsed in distilled water
4. Placed in Schiff's reagent for 5 min
5. Washed in running tap water for 5-10 min
6. Counterstained nuclei lightly with Hx
7. Differentiate and blue
8. Washed in water
9. Stained in orange G for 20 seconds
10. Differentiate in tap water until macroscopically the section is pale yellow. Check
microscopically that only the R.B.C.'s and acidophi cells are yellow
11. D.C.M.
Results: Basophil cells - Magenta Acidophil cells - yellow R.B.C. - Yellow Nuclei - Blue-black
Chromophobes - Pale blue-black Nuclei - Red
Notes: Other structures, e.g. collagen, will be PAS positive. If this obscures the delicate staining, the
oxidation and Schiff,s staining times should be reduced Nuclei - Red
Methyl violet/crystal violet method (after Jurgens 1875)
Preparation of staining solutions: a. 1% aqueous methyl or crystal violet solution
b. 0.5% acetic acid
Method: 1. B.S.D.W.
2. Stain in 1% aqueous methyl or crystal violet for 2-5 min.
3. Wash in water and examine with the microscope.
4. Differentiate in 0.5-1% acetic acid until amyloid is purplish-red or red in good contrast with
the blue-violet staining of nuclei and normal tissue.
5. Wash in running tap water for at least 5 min.
6. Drain the slide and while the section is still moist, mount with modified Apathy's gum syrup.
Ring coverslip with nail varnish.
Results: Amyloid purplish-red to red Nuclei, cytoplasm, and connective tissue shades of blue-violet