1. TISSUE FIXATIVE,MODES OF
FIXATION&HISTOCHEMICAL STAINING
TECHNIQUES FOR IDENTIFICATION OF
DIFFERENT ELEMENTS OF LESION
Submitted by-
RAHUL SINGH
M.V.Sc Scholar
ROLL-5455
MAJOR CREDIT SEMINAR
ON
2. TISSUE FIXATION-DEFINITION
A chemical process by which biological tissues preserved
from decay ,either through autolysis, putrefaction.
It terminates any on going biochemical reaction &may also
increase the mechanical strength or stability.
3. Inhibition of autolysis & putrefaction.
Preserve sample (tissue) as close to natural
state.
Disable intrinsic biomolecules - proteolytic
enzymes.
Protect sample from extrinsic damage.
Increase their mechanical strength& rigidity.
4. To aid in optical differentiation
To aid in the better staining property
Example-
Poor fixed tissueGood fixed tissue
6. FIXATIVES OR FIXATION SOLUTION
(Classification)
I Simple Fixatives
II Compound fixatives
7. Simple Fixatives
•Formalin – used as single reagent
•Mercuric chloride
•Picric acid
•Acetone
•Ethyl alcohol
•Osmic acid
•Osmium tetroxide : Used for fat
preservation
9. MODE OF ACTION OF FIXATIVES
I. Non coagulating cross-linking
II. Coagulating fixatives
10.
11.
12. 10% FORMALIN
• Formaldehyde is a gas sold as a solution in
water (approx. 40% in content ), in which form
it is known as formalin.
• Formaldehyde mainly preserves proteins.
(40%Formaldehyde ) formalin 10.0 m
Distilled water
90.0ml
15. • Long stored formalin produces formic acid .
• formic acid show deposition of brown colour
formic acid pigment in the section.
• To remove this formic acid pigment the
sections are treated with
16. 10% NEUTRAL BUFFERED FORMALIN
pH - 7.0
Formulation
40% formaldehyde: - 100 ml
Distilled water: - 900 ml
Sodium dihydrogen phosphate monohydrate: - 4 g
Disodium hydrogen phosphate anhydrous - 6.5 g
The solution should have a pH of -7.0
Fixation time: - 12 – 24 hours
most widely used formaldehyde-based fixative for routine
histopathology.
The buffer tends to
17. Formal saline
• Formulation
• 40% formaldehyde: 100 ml
• Sodium chloride: 9 g
• Distilled water: 900 ml
• Fixation time: 12 – 24 hours
• It often produces formalin pigment
18. BOUIN’S FLUID
Used for staining of glycogen & Masson’s
trichrome stain
Composition-
Sat.acq.soln. of picric
acid……………………….750ml
Formalin……………………………………
……250ml
Glacial acetic
acid………………………………...50ml
19. ZENKER’S FLUID
• Used for bone marrow & spleen fixation.
• Composition-
Mercuric chloride……………………….50g
Potassium dichromate…………………..25g
Sodium sulphate………………………..10g
Distill. Water
upto…………………….1000ml
Glacial acetic acid……………………..50ml
20. OSMIUM TETROXIDE
In solution, usually 1% aqueous solution, osmium
tetroxide.
Fats & other lipids reduce osmium tetroxide or
combine with it to form a dark compound
Osmium tetraoxide is used for fat preservation
21. • Ethyl alcohol hardens tissue but causes serious
shrinkage.
• It is strong cytoplasmic coagulant.
Precipitating (or denaturing) fixatives act by
reducing the solubility of protein molecules.
disrupting the hydrophobic interactions that
give many proteins their tertiary structure
22. • Picric acid is usually used in a saturated
aqueous solution , 0.9-1.2%
• It is an excellent protein coagulant.
• forming protein picrates having a strongly
affinity for acid dyes.
• It is recommended as a fixative for glycogen
23. Nuclear fixative-
Carnoy’s fluid-
This is used for biopsy of cancerous tissue.
Quickest fixative most of the cytoplasmic
details .
• Composition-
Absolute alcohol……………………………60ml
Chloroform…………………………………..30ml
Glacial acetic acid…………………………10ml
24. • Used for staining of enzymes, carbohydrate, fat,
minerals, nucleic acid, RNA, DNA, proteins etc.
• Examples-
1) Cold acetone
2) Absolute alcohol-used for glycogen & uric
acid crystals.
3) Formal saline
25. pH
Size of specimen
Volume of the fixative
Temperature
Duration
26. GOLDEN RULES
SIZE OF TISSUE- about 3mm.
VOLUME OF FIXATIVE-20x.
TIME OF FIXATION-24hour in formalin
28. By piercing the needle
By X ray
Testing the solution of HNO3 for calcium
29. Fat &GLYCOGEN
VON KOSSA'S METHOD – CALCIUM
PRUSSIAN BLUE REACTION – (FOR HAEMOSIDERIN)
DE GALANTHA'S METHOD FOR URATE CRYSTALS.
Congo Red Stain For Amyloid
30. 1. Oil Red O
2. Sudan
a. Sudan II
b. Sudan III
c. Sudan IV
d. Sudan Black B
3. Osmium Tetroxide
1. PAS
2. Best’s carmine
31. PRINCIPLE: Oil Red O is a lysochrome (fat-soluble dye) diazo dye used for
staining of neutral triglycerides and lipids on frozen sections
and some lipoproteins on paraffin sections.
Method
1. Cut frozen sections at 8 to10mm, air dry the sections to the slides
2. Fix in formalin, briefly wash with running tap water 1-10 mins
3. Rinse with 60% isopropanol
4. Stain with freshly prepared Oil Red O working solution 15 mins
5. Rinse with 60% isopropanol
6. Lightly stain nuclei with alum haematoxylin 5 dips
7. Rinse with distilled water
8. Mount in aqueous mountant or glycerine jelly.
Results:- Lipid......................................Red
Nuclei....................................Blue
32. Fatty change of liver Oil Red-O Stain.
Fatty change of liver HE Stain.
34. SUDAN BLACK STAIN FOR FAT
: Sudan Black is slightly basic dye and will combine with
acidic groups in compound lipids, thus staining phospholipids
:
1. Pick-up frozen sections on clean glass slides if fresh, albuminized slides if fixed.
2. Fix slides in 10% formalin if fresh.
3. Wash well it tap, rinse in distilled, drain off excess water.
4. Propylene glycol, two changes, 5 minutes each.
5. Sudan Black, 7 minutes, agitate.
6. 85% Propylene glycol, 3 minutes.
7. Rinse in distilled water.
8. Nuclear Fast Red, 3 minutes.
9. Wash in water.
10. Wash in tap water, rinse in distilled.
11. Mount with aqueous mounting media, Glycerin Jelly.
RESULTS: Fat ..…………….Black
35.
36.
37.
38. OSMIUMTETROXIDE STAINFOR FAT
Introduction : Osmium Tetroxide is a good fixative and excellent
stain for lipids in membranous structures and vesicles
Procedure
1. Before starting, dilute the 4% OsO4 to a final concentration of
0.1%.
2. Start with a fixed, sliced tissue sample.
3. Wash the sample three times with double distilled water.
4. Incubate the sample with 0.1% OsO4 for 30 minutes.
5. Wash four times with double distilled water.
RESULTS: Fat ..…………….Black
42. PRINCIPLE
Tissue containing vicinal glycol groups or their amino or alkylamino derivatives are
oxidized by periodic acid to form dialdehydes, which combine with Schiff's reagent
to form an insoluble magenta compound.
Procedure:
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.
5. Wash in lukewarm tap water for 5 minutes.
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.
RESULTS: Glycogen…………………….Magenta
Nuclei ………………………Blue
PAS STAIN for glycogen
47. This staining technique demonstrates glycogen by hydrogen bond
formation between OH groups on the glycogen, and H atoms of the
carminic acid. Fibrin and neutral mucin stain weakly with this method.
1. Dewax test + positive control sections and hydrate to water.
2. Duplicate sections may be treated with diastase if desired (See6.2).
3. Stain nuclei of all sections well using one of the iron haematoxylin
solutions eg. Weigerts iron haematoxylin (See 2.4)
4. Treat with carmine solution for 10 mins.
5. Transfer slides quickly to a Coplin jar of Differentiating solution.
6. Wash in alcohol (NOT water), clear in histoclear and mount.
RESULTS: Glycogen……………Bright red
Nuclei…………….....Blue
BEST'S CARMINE
50. VON KOSSA'S METHOD - CALCIUM
PRINCIPLE-Tissue sections are treated with silver nitrate solution, the
calcium is reduced by the strong light and replaced with silver deposits
,visualized as metallic silver.
REAGENTS:
5% Silver Nitrate Solution:
Silver nitrate 25.0 gm
Distilled water 500.0 ml
5% Sodium thiosulfate[hypo]
Nuclear fast red
FIXATION: 95% ethyl alcohol or 10% buffered neutral
formalin
51. Deparaffinize and hydrate to 95% alcohol and air dry the
slides.
Place slides on a staining rack and flood the sections with 5%
silver nitrate. Expose the slides to an ultraviolet lamp for 10
minutes.
Rinse with four changes of distilled water.
Sodium thiosulfate solution for 1 minute.
Rinse with four changes of distilled water.
Nuclear fast red solution for 3 minutes.
Rinse with three changes of distilled water.
Dehydrate through graded alcohols.
Mount with synthetic resin.
52. Calcium salts ------------------------------------------------------- black
Nuclei -------------------------------------------------------------- red
Cytoplasm ---------------------------------------------------------- light pink
DIAGNOSIS APPLICATION -Calcification in tuberculosis
-Atherosclerosis
-Myeloid tumours
-Dead(necrotic) or dying tissues
53. 1 &2black deposits in the walls of this lung that
was calcified . This lung was stained with VON
KOSSA STAIN
1
3
3- H&E Stain calcification tissue stain blue
colour
54. Thick calcified rings - that could be observed macroscopically - were found along
the aortas of animals with massive calcification. Von Kossa stained ...
55. PRINCIPLE:
FIXATIVE: 10%buffered neuttral formalin
TECHNIQUE: Cut paraffin sections 6μ
Pearl’s Prussian blue reaction is that potassium ferrocyanide will form ferric
ferrocyanide (Prussian blue) with reactive ferric salts in an acid solution. Dilute
hydrochloric acid liberates loosely bound ferric iron from protein.
56. •Deparaffinize and hydrate to distilled water.
•Place the slides in the hydrochloric acid-potassium ferrocyanide solution for 30
minutes at room temperature.
•Rinse with five changes of distilled water.
•Nuclear fast red solution for 3 minutes.
•Rinse with three changes of distilled water.
•Dehydrate in graded alcohols.
•Clear in xylene, three or four changes.
•Mount with synthetic resin.
57. RESULTS:
Iron (hemosiderin) - blue
Nuclei - red
Background - pink
Diagnostic Application:
Hemochromatosis, esp., liver. excess iron deposition is stained as blue
granules
Gastric ulcer 2nd to iron overdose:
58. Dog liver show overdose iron
Hemochomatosis
Gastric ulcer 2nd to iron overdose
59. Fixation: Absolute Alcohol. If specimen contains bone, decalcify in Nitric Acid
Alcohol (F-96). Wash in Absolute Alcohol for 24 hours.
Sections: Paraffin @ 8-10 microns.
Staining:
Xylene Absolute Alcohol, two changes each.
Expose sections in Silver Nitrate Solution to strong sunlight for 3 hours
(until urates are bright rose).
60. . Pour freshly prepared developing solution over slides until the urates turn
black and
the connective tissues are yellow. Prepare developing solution by mixing just
before use.
10.0 ml Gelatin, 3%
3.0 ml Silver Nitrate, 20%
2.0 ml Hydroquinone, 2%
Wash quickly in hot water (58oC).
Dehydrate in Absolute Alcohol, and clear in Xylene two changes each
Stain Results:
Urates Black
Background Yellow
61. .
. Section of liver from chick died
of group IV showing deposition of uric acid
crystals in hepatic parenchyma. H & E X
300
Section of kidney from chick
died of showing deposition of black
uric acid crystals in renal parenchyma.
Dgalantha's X 300e
.
62. Section of kidney from chick
died of showing marked deposition
of needle shaped uric acid crystals in renal
parenchyma.
. Section of kidney from chick
died of showing marked inter
tubular haemorrhages, deposition of uric
acid crystals degeneration and necrosis of
renal tubules. H & E X 300
63. PRINCIPLE-The Congo red dye is a linear molecule, and this configuration permits
hydrogen bonding of the azo and amine groups of the dye to similarly spaced hydroxyl
radicals of the amyloid.
Fixation:-10% buffered neutral Formalin
Reagents:
1.1% Congo red
Congo red : 1.0 gm
D.W. : 100.0 ml
2. 1% Sodium hydroxide
Sodium hydroxide: 1.0 gm
D.W. : 100.0 ml
3. Alkaline Alcohol Solution
1% Sodium hydroxide: 1.0 ml
50% Alcohol : 99 ml
4.Hematoxylin
64. Procedure
Deparaffinize and hydrate sections to water.
.Stain in Congo red solution for 1 hr.
.Rine in distilled water.
Differentitate quickly in alkaline alcohol solution.
Rinse in tap water for 5 minutes.
Counterstain with Hematoxylin for 5 minutes.
Rinse in tap water for 10 minutes.
Dehydrate through 95% alcohol ,2 changes of 100% alcohol.
Clear in xylene
Mounting
65. Results
Amyloid- under ordinary light pink to
red& polarized light a green birefringence
Nuclei- blue
66. section of renal glomerular amyloidosis in a dog. (A) Light eosinophilic amorphous
deposits partly effaced the glomerular architecture (H&E stain). The deposit stained light
red with Congo red stain (B) and exhibited green birefringence under polarized light (C).
The amyloid deposit failed to stain with Congo red (D) after pretreatment with Potassium
Permanganate solution, indicating that the amyloid is AA-amyloid
67. Kidney: Multiple Glomeruli:
Polarized light and Congo Red
stain- Apple green bi-refringence
= amyloid
Glomerulus with
multiple deposits of
amyloid. Congo red
staining
amyloidosis kidney HE stain
68. Histological features of skin of a dog with nodular cutaneous amyloidosis, which stained light
eosinophilic with haematoxylin-eosin (A) and red with Congo red stain (B). The amyloid
showed yellow-green birefringence (C) illuminated with polarized light, characteristic of
amyloid deposits
69.
70. Mallory staining
• Principle- Presence of phosphomolybdic acid on the fibres
(collagen) act as colourless acid dye. It act as dye excluder
with acid dye such as basic fuchin exclding them from
collagen. Then the aniline blue stains collagen exclusively
• Fixation- Mercuric chloride
1. Deparafinize and hydrate slides to water label
2. Stain in Mallory I:- 15 sec
3. Rinse in distill water:- 10 sec
4. Treat with phosphomolybdic acid:- 1-5min
71. 5. Rinse briefly in distill water
6. Stain in Mallory II:- 2 min
7. Rinse in distill water
8. Differentiate aniline blue in 90% ethyl alcohol
9. Dehydrate in absolute alcohol, clear and mount
• Result:
Nuclei- red
Muscle and some cytoplasmic elements- red to orange
Collagen- dark blue
Connective tissue and hyaline substance- blue
72. • Characteristics - cells and extracellular matrix (ECM)
• Distribution - lies between basal lamina of epithelia and external lamina of
muscle and nerve supporting cells
Epiglottis (Mallory-Azan stain)
74. • Purpose: Used to differentiate between collagen and
smooth muscle in tumours, and the increase of collagen in
diseases such as cirrhosis & hence routinely used stain for
liver and kidney biopsies.
• Principle: Three dyes are employed selectively staining
muscle, collagen fibers & fibrin. The general rule in
trichrome staining is that the less porous tissues are
coloured by the smallest dye molecule.
75. • Fixative: Bouin's fluid is preferred, 10% formalin.
1. Deparafinize and hydrate slides to water label
2. Mordant with iron alum: 5 min @40-50˚C
3. Wash in running water: 5 min
4. Stain in haematoxylene: 5 min @40-50˚C
5. Wash in running water: 5 min
6. Differentiate in saturated aquous picric acid
7. Wash thoroughly in running water: 10-20 min
8. Stain in Masson A: 5 min
9. Rinse in distill water
76. 10. Treat with Masson B: 10 min
11. Stain in Masson C: 2-5 min
12. Differentiate in acetic acid rinse: 1-2 min
13. Dehydrate quickly 70% & 95% alcohol
14. Dehydrate, absolute alcohol, 2 changes: 3 minutes each
15. Clear and mount
• Result:
Nuclei- black
Collagen, - blue or green
Cytoplasmic element, keratin, muscle- red
77. Chronic active hepatitis with
collapse in liver, trichrome stain.
77
Cerebral abscess in brain,
trichrome stain.
Scleroderma with fibrosis of submucosa in
stomach, trichrome stain.
81. • Tissue fixation is one of the most important
determinants of the quality of histological sections.
• Incomplete fixation can lead to unsatisfactory and
poorly reproducible results
• It follows that the best results often require a close
interaction between the investigator and Histology
Lab.
• Histopathological method as an aid in diagnosis of
diseases .
82. p 16 Cook H C, Manual of Histological Demonstration Techniques p 188
Bancroft JD & Stevens A, Theory and Practice of Histological Techniques,
2nd ed
Garvey, W. et al: Combined modified periodic acid-Schiff and batch
staining method. J. Histotechnol. 15:117-120, 1992.
McManus, J.F.A. and Mowry, R.W.: Staining Methods Histologic and
Histochemical, New York, Paul B. Hoeber, 1960, pp. 126-128.
Preece A, A manual for Histologic Technicians, 3rd Ed, 1972, Little,Brown
and Co, Boston
Crookham,J, Dapson,R, Hazardous Chemicals in the Histopathology
Laboratory, 2nd ED, 1991, Anatech
Luna, L.: Manual of Histologic Staining Methods of the Armed Forces
Institute of Pathology, 3rd edition,
Lillie, R.D.: Histopathologic Technic and Practical Histochemistry, 4th ed., New York,
McGraw-Hill, 1976, p. 507
