Académique Documents
Professionnel Documents
Culture Documents
I - INTRODUCTION:
IV – LABORATORY REQUIREMENTS:
a.) General glassware – pipettes, flasks, reagent bottles, etc.
b.) Specimen containers – various sizes.
c.) Couplin staining jars
d.) Microscope slides and coverslips
e.) Reagent bottles
f.) Fixatives
g.) Various organic solvents
h.) Decalcifying solutions
i.) Embedding materials
j.) Various staining solutions
k.) Various dilutions of ethyl alcohol
l.) Mounting media etc.
FIXATION:
Good fixation is the most important factor in the production
of satisfactory slides. If the tissue is well fixed one rarely has
difficulty with the later stages; if it is badly fixed, good sections
are absolutely impossible. The three essentials are: fresh tissue,
proper penetration of fixative and the correct choice of fixative.
Surgical specimens should be placed in fixative immediately after
they are taken and should be sent to the laboratory as soon as
possible so that the pathologist can supervise their proper
fixation. In postmortem cases, proper refrigeration of the body
will do much to retard autolysis and permit reasonably good
sections if delay is unavoidable. Even very early autolysis renders
tissue abnormally fragile and such tissues should be handled as
gently as possible to avoid artifact.
Inadequate penetration of the fixative is one of the
commonest causes of bad results. The maximum thickness that
can be penetrated is about 10mm for loose tissues and 5mm for
compact or cellular tissues (e.g. lymph nodes or spleen). For
dealing with specimens bigger than this the following methods
are recommended:
• Solid organs: Cut slices as big as necessary but not thicker than5mm.
• Hollow organs: Either open out or fill with fixative or pack lightly with wool
soaked in fixative.
• Large Specimens that require dissection: Inject fixative along vessels (or
bronchi, in case of lungs).
CHOICE OF FIXATIVE
The purposes of fixation are:
1. To inhibit autolytic enzymes and to kill the organism of
decompositions.
2. To preserve tissue as nearly as possible in its original form.
3. To protect the tissue against subsequent damage during
embedding.
4. To render the various constituents receptive to the proposed
stains.
The choice of fixative will depend on the nature of the tissue and
the type of staining to be employed.
2. Formaldehyde
Sold as formalin (a 40% w/w solution of gas in water). Used as
10% or better 15% of formalin in normal saline, or calcium
chloride solution. Penetrates rapidly but fixes slowly. Does not
precipitate protein but combine with NH2 groups to form an
insoluble gel. Preserves practically all elements including fats.
Renders phospholipids insoluble in fat solvents. Does not
shrink but allows shrinkage during embedding.
3. Mercuric Chloride
Used as saturated (about 70%) or half saturated aqueous
solution. Oxidizing agent. Penetrates rapidly. Penetrates
protein by combining with it. Causes no shrinkage by itself and
subsequent embedding causes only minimal shrinkage. Fixes
chromatin well and enhances its subsequent staining. Preserve
nearly all elements. Forms precipitates in tissues. Corrodes
metal containers. Rarely used along but it is valuable
constituent of many other fixatives.
4. Acetic Acid
Used as 1-5% aqueous solution. Precipitates and fixes
nucleoprotein but not cytoplasm and is, therefore, valuable for
nuclear fixation. Penetrates extremely rapidly. Tends to swell
tissue and does not harden. Destroys mitochondria and
cytoplasmic granules. Rarely used alone.
6. Chromic acid
Used either as a pure chemical or as a mixture of dichromate
and acetic acid (e.g. Zenker). An oxidizing agent and therefore,
incompatible with formalin or alcohol. Fixes by both
denaturing and precipitating proteins. Causes moderate
shrinkage but does not harden badly. Penetrates slowly.
Preserves most elements. Tends to weaken nuclear staining by
dissolving nucleoprotein.
7. Potassium dichromate
Used as 2-3% aqueous solution. A weak oxidizing agent and
cannot therefore, be kept with formalin for long. Fixes by
making proteins insoluble in water but without actually
precipitating them. Tends to dissolve chromatin and therefore
weakens nuclear staining. A good cytoplasmic and bad nuclear
fixative. Fixes lipids and mitochondria well. Causes no
shrinkage but allows no shrinkage during embedding. Gives
chromaffin reaction.
8. Osmium tetroxide
Used as 1% or 2% aqueous solution. Expensive and unstable
with an irritating vapor. Powerful oxidizing agent and
therefore incompatible with formalin or alcohol. Penetrates
very badly. Fixes by setting proteins in an insoluble gel
without precipitating them. Preserves fats and gives black
precipitate of osmium dioxide with unsaturated fats. Also
preserves very fine cell detail e.g. Golgi bodies. Used mainly
for electron microscopy.
(Store in dark bottle with 1 drop of saturated aqeous HgCl2 to
each 10cc to prevent oxidation).
1. Formol Saline
• Commercial formalin 15ml
• 0.85% aqueous solution of NaCl 85 ml
(Keep over marble chips or calcium hydroxide chalk to
absorb acid).
3. Formol-Mercury
(Formalin prevents protein flocculation caused by Hg).
• Mercuric chloride 70g (3.5%)
• Commercial formalin 350ml (17.5%)
• Water 2 liters
(We add Edicol pea green to aid recognition)
The color also helps keep track of tiny biopsies.
The original formula (Lendrum,1941) gave saturated mercuric chloride (7%
approximately) and 10% formalin. Our modification causes less hardening
and is cheaper.
This is probably the best routine fixative. It is rapidly acting and causes less
shrinkage and gives much more brilliant staining and sharper detail than
formol saline. It is equally useful for fresh biopsies or stable autopsies. It
permits all ordinary stains including the silver reticulin methods.
Time of fixation: 6-24 hours depending upon size.
Prolonged time does no harm.
Undecalcified bone
To distinguish between bone and osteoid the most reliable method
is to cut undecalcified sections and stain the calcium by Kossa’s
method. This is not unduly difficult and the distinction between
bone and osteoid.
• Fix in formol saline.
• Cut out a small piece of cancellous bone about 5 x 5 x 2 mm. (This
can be done with a sharp knife).
• Dehydrate in the ordinary way as far as absolute alcohol.
• Immerse in 3% nitrocellulose in methyl benzoate (8 hrs)
• Transfer to 6% nitrocellulose in methyl benzoate (8 hrs)
• Transfer to 12% nitrocellulose in methyl benzoate (8 hrs)
• Transfer to 20% nitrocellulose in methyl benzoate (overnight)
• Drain off the excess nitrocellulose and immerse the tissue with its
adherent nitrocellulose in Benzene (2 changes of ½ hour each).
This doubly embedded block can be cut on a sledge-type microtome using a
heavy razor without damaging the latter unduly but the sections break to pieces
in the process of cutting and the fragments can not be picked up. To prevent
this a strip of “Sellotape” is pressed on to the top of the block so that when the
next section is cut it remains stuck to the “Sellotape”. The strip of “Sellotape”
can be moved fprward with each cut until sufficient sections have been
obtained.
The individual sections are cut with scissors and attach to slides by pressing
them on to a warmed, albuminised slide (sticky side to glass), The tape can be
floated off by wetting it with wet blotting paper for 10 minutes. The adhesive can
then be dissolved off in warm benzene leaving the section of bone attached to
the slide.
• Stain the calcium by Von Kossa’s method and counterstain with Van
Gieson’s stain for 30 seconds only.
• Dehydrate, clear and mount.
FROZEN SECTIONS:
Gelatin 0.5g
Merthiolate 15mg (preservative)
Distilled water 100ml
• Warm to 56oC and dip clean slides in the solution to coat them
• Dry coated slides at room temperature under cover to avoid dust.
Store in boxes for use.
• Float frozen sections on to slide and flatten out.
• Blot carefully.
• Place slide in Couplin jar with a few ml of commercial formalin for 1-2
minutes for vapor to denature gelatin.
• Wash in tap water to remove formalin.
• Stain sections in the ordinary way.
MOUNTING MEDIA
The choice of a suitable mountant is important to avoid fading of
sections.
1. D.P.X.
Dibutyl phthalate 5ml
Xylene 35ml
Mix and add distrene 80
TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT
16
• D.P.X. can be bought ready made.
• A good general mountant. Does not cause fading as
badly as balsam. Shrinks a good deal. Must be in a
thin layer or it distorts the microscopic image. Dries
very rapidly. Used as a routine in this department.
2. Canada Balsam:
A very old mountant. Easily becomes acid and causes
fading of sections.
2. CELESTINE BLUE:
Celestine blue ( a synthetic oxazin dye) combines with iron
to form a lake which stains nuclei blue. If, however, sections
are stained for a few minutes in this lake and then restained in
haemalum the resulting stain is as resistant to subsequent
decolorization as an iron-hematoxylin stain. This method,
3. Erlich’s Haematoxylin:
2% alcoholic haematoxylin 100ml
Water 100ml
Glycerine 100ml
Glacial acetic acid 10ml
Potassium alum To excess
• Plug bottle with wool (not cork) and leave in the light
to ripen for a few weeks. The stain is usually fit for
use when it has darkened to a rich Burgundy color.
6. Delafield’s Haematoxylin:
Saturated aqueous ammonium alum 40ml
Haematoxylin 4g
Absolute alcohol 25ml
• Mix and ripen for a few days and then add 100ml of
glycerine and add 100ml of methyl alcohol. Ripen for
a further six weeks.
Note:
Liq. Ferri. Perchlor is an aqueous
solution of ferric chloride.
Iron ammonium alum is iron ammonium
sulphate.
Potassium alum is potassium aluminum
sulphate.
Ammonium alum is ammonium
aluminum sulphate.
7.Cole’s haematoxylin:
Haematoxylin 3g in 25ml of 75 OP spirit. Add this to 500ml
of warmed D.W., dissolve and 100ml of 1% alcoholic
iodine. Add 1,400ml of saturated aq. am. alum (mordant).
COUNTERSTAINS
Most pathologist use Eosin as a general counterstain, but some
prefer more complex mixtures such as phloxine-tartazine. Van
Gieson or even Masson’s trichrome. In this department, Eosin is
used as a routine and other counterstains are only employed to
demonstrate particular tissue elements.
Eosin is used as a 1% aqueous solution of the water soluble
yellowish variety. If staining is found to be weak, the dye can be
improved by adding 2% calcium chloride or a trace of acetic acid.
Too much acid causes precipitation. The dye will usually grow
molds but these are harmless and can be filtered off.
3. Carbol-Safranin:
• Melt 0.5g of phenol in a dry flask under the hot
tap.
• Mix in 0.1g of safranin to make dark red sludge.
• Grind together 0.25g of starch and 0.25g of
destrine.
• Add 50ml of water, with further grinding.
• Heat to 80oC. Cool, filter and dissolve the
carbolsafranin sludge in this filtrate.
• Stain for 10 minutes and differentiate in the
dehydrating alcohol.
Notes:
Scott’s tap water substitute:
Potassium bicarbonate 2g
Magnesium sulphate 20g
Distilled water 1 liter
FROZEN SECTIONS
Indications:
1. To stain fats.
2. Rapid diagnosis during an operation.
3. Some special stains (e.g. neurologia or histochemical tests).
4. To minimize artifacts.
5. To cut very thick sections.
Fixation:
• Formalin: All other fixatives make tissues too brittle.
For speed, small blocks may be fixed for 2 minutes in
formol-saline at 80oC, but sections obtained in this
way are of poor quality.
• Embedding in gelatin is useful for friable tissues.
Cutting:
• Set the microtome at 15μ and start with the knife
about 1mm (not less) from the surface of the chuck.
Results:
Nuclei - Black to dark brown
Collagen - Red
Other tissues - Yellow
Note:
• The fuchsin is removed by water and the
picric acid by alcohol. It is often better
to omit step 5 and dehydrate as quickly
as possible. Alkaline water removes
fuchsin very quickly.
• Picric acid decolorizes alum
haematoxylin, hence a more resistant
one must be used.
• In place of acid fuchsin in Van Gieson,
one may use poncean or sirdus red.
These are good for amyloid.
PICRO-MALLORY STAIN
Any fixative. Fix paraffin sections on albuminized slides.
1. Stain nuclei with iron haematoxylin or celestine blue
and haemalum.
2. Rinse in tap water.
3. Differentiate in picro-orange (1) until only nuclei are
stained (3-5 minutes).
4. Wash in water till only R.B.C. are yellow.
5. Stain in acid fuchsin mixture (2) till tissue is red (5-10
min.)
TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT
31
6. Rinse in 2% acetic and differentiate in red
differentiator (3) till only required elements are red
(up to 5 min.).
7. Wash in tap water (10 seconds).
8. Stain in aniline blue (4) till connective tissue is well
stained (10 minutes).
9. Rinse in 2% acetic.
10. Differentiate in blue differentiator (5) till only
connective tissue is stained blue (not less than 1-2
minutes).
11. Dehydrate rapidly, clear and mount in D.P.X.
SOLUTIONS:
1. Picro-orange:
80% alcohol saturated with picric acid 100ml
Orange G 0.25g
2. Acid- Fuchsin Mixture:
Acid fuchsin 0.5g
Ponceu Red 0.5g
1% acetic 100ml
3. Red differentiator:
Stock differentiator* 40ml
95% alcohol 40ml
Water 20ml
4. Aniline Blue solution:
• Aniline blue dissolve in 100 ml
boiling distilled water.
• Add 2.5 ml glacial acetic acid.
Cool and filter.
5. Blue differentiator:
Stock differentiator* 20ml
Water 80ml
*Stock differentiator: Absolute alcohol 100ml
HEIDENHAINS MODIFICATION OF
MALLORY’S ANILINE BLUE METHOD
Any fixative. Paraffin sections.
Staining Solution:
Stock solution 35ml
70% alcohol 30ml
(These amounts may have to be varied slightly
with each fresh batch of stain).
Technique of Use:
Any fixative. Paraffin sections.
1. Xylol
2. 2 changes of absolute alcohol.
3. Blot and dip in 0.5% colloidin (as this is a long
staining method)
4. Harden colloidin in 70% alcohol for 5 minutes.
5. Bring sections down to water.
6. Remove mercury (as described under routine
haemalum and eosin staining.
Result:
Elastic fibers - dark blue-black
Times of staining:
Elastic tissue 5 minutes
Pancreatic β-cells 15-30 mins.
Pituitary β- granules 30 mins to 2 hours
Mast cells granules 5-10 mins.
*Mallory’s stain:
Haematoxylin (or haematin) 0.1g
Phosphotungstic acid 2.0g
LENDRUM’S “PHOSTOX”
Any fixative. Paraffin sections.
1. Bring down to water.
2. Mordant in Lugol’s iodine for 1 hour.
3. Pour off and without washing treat with permanganete and
oxalic acid, as above.
4. Wash in distilled water.
5. Stain in Mallory’s stain till muscle is stained blue (a few
hours).
6. Dehydrate, clear, and mount as above.
PHLOXINE TARTRAZINE
Fix in formalin or F.M., avoid chromate or Bouin.
1. Bring down to water and remove mercury if necessary.
2. Stain nuclei with haemalum.
3. Differentiate in acid alcohol.
4. Wash in tap water.
5. Stain for ½ hour in phloxine.
*Phloxine solution:
Phloxine 0.5g
Calcium chloride 0.5g
Distilled water 100ml
**Tartrazine solution:
Saturated solution of tartrazine N.S., in cellosolve (ethylene
glycol monoethyl ether).
LENDRUM’S LISSAMINE FAST RED
FOR UNSTRIPED MUSCLE:
Any fixative. Paraffin sections.
1. Bring section down to water and remove mercury if
necessary.
2. Stain 5 minutes in celestine blue mixture.
3. Rinse in water.
Result:
Nuclei - blue
Muscle, red cells and some
cell granules - red
Background - yellow
Note:
The Lissamine fast red solution goes off in a matter of weeks.
Prolonged staining does not seem to help. The times given are
arbitrary and it may be desirable to use the phosphomolybdic
acid cold if the red comes out too quickly. A little further red
comes out in the tartrazine so do not push differentiation too
far and if necessary shorten the time in tartrazine.
Result:
• With fixation in Helly, the stain should be like ordinary
Romanowsky’s stain on films. Material fixed in
formalin alone will not take up enough red stain. This
may be partly corrected by adding a trace of eosin (alc.
sol.) to the alcohol used for dehydrating.
Note:
Buffer tablets at various pH can be bought G.T. Gurr.
Orange mixture:
1% Erythrocin 1 part
1% Orange G 2 parts
Distilled water 2 parts
Blue mixture:
1% methylene blue 2 parts
1% Toluidine blue 1 part
Distilled water 17 parts
Buffers:
PH 5.8 pH 6.4
M/15 KH2O4 45ml 34ml
M15 Na2HPO4 5ml 16ml
Note:
The staining mixtures should be made up freshly from the stock
solutions of 1% dyes. The two buffers can be tried because so
tissues vary in their reactions. A satisfactory result can usually be
obtained with one.
Method:
1. Bring paraffin sections to water. Cryostat sections,
briefly post-fixed in 5% acetic-ethanol should be rinsed
in distilled water.
2. Stain for 6 minutes in methyl green-pyronin*.
3. Blot gently with filter paper.
4. Dehydrate rapidly in absolute acetone.
5. Rinse in equal parts of acetone and xylene.
6. Rinse in 10% acetone in xylene.
7. Clear in two changes of clean xylene.
8. Mount in D.P.X.
Result :
DNA - green to bluish green (or purple)
RNA - red
FATTY SUBSTANCES:
Ordinary paraffin sections are useless for the recognition of fats.
Sometimes, as in liver, they suggest that fat is present but in most
tissues they give no hint. It is therefore, essential to cut frozen
sections if there is any question of fat being present.
Tests:
Result:
Fat - Orange-red
Nuclei - Blue
OIL RED STAIN FOR FAT
Several dyes of the oil red series can be used as fat stains. The
best in our hands has been oil red. It is used in a partly aqueous
solvent which helps to prevent solution of small fat droplets
during staining.
Stock Oil red O:
• Make up a saturated (0.5%) solution of Oil red O
in isopropyl alcohol.
• Stock staining solution:
• For use, dilute 6ml with 4ml distilled water and
allow to stand for 24 hours. Decant the
supernatant. This may be kept in a tightly
stoppered bottle for up to 6 months, filtered as
necessary through a No.42 Whatman paper,
directly on to the sections.
Method:
1. Cut formalin-fixed sections or use unfixed cryostat
sections mounted on coverslips.
2. Rinse briefly in water.
3. Rinse in60% isopropyl alcohol.
4. Stain in Oil red solution for 10 minutes.
5. Differentiate briefly in 62% isopropyl alcohol*.
6. Wash in water.
7. Counterstain and mount as for Sudan stain avoiding
ammonia at stage 8.
Method:
1. Rinse frozen sections in T.E.P.
2. stain in Oil red O in T.E.P. 10 minutes.
3. Rinse out excess stain in T.E.P.
4. Wash in distilled water.
5. Stain nuclei in haemalum.
6. Rinse in water.
7. Differentiate in T.E.P. containing 1% HCl.
8. Wash, blue, and mount in glycerine jelly.
(Fine fat droplets will not dissolve out in T.E.P. this being an
aqueous solvent).
SUDAN BLACK B
Sudan black B is a fat soluble dye like Sudan III and Sudan IV
(Scharlach R) and will stain all the fats that can be stainedby
them. A number of other lipid substances are rendered insoluble
by formalin fixation and can be stained in ordinary paraffin
sections by Sudan black (but not by Sudan III or IV). These lipids
include red cell envelops, lipofuscin, kerasin (Gaucher’s disease)
and myelin sheaths. For the latter, Sudan black is a rapid
alternative to the slower Weigert-Pal.
Sudan black is soluble in xylene and sections must be mounted in
a watery medium (glycerine jelly or Aparhy’s medium).
Result:
Lipids stain black
*Sudan black can also be used as saturated solution in 70%
alcohol and differentiated in 70% alcohol but staining takes
much longer.
COPPER PATHALOCYANIN IN
METHOD FOR PHOSPHOLIPIDS:
Fix tissues in formalin or preferably, in formol-calcium. Embed in
paraffin the usual way.
Method:
1. Bring sections to absolute alcohol.
2. Stain in 0.1% Methanol Fast Blue 2G* in 90-100% alcohol
at 60o for 8-16 hours.
3. Rinse in 70% alcohol and bring to water.
4. Differentiate in 0.05% aqueous lithium carbonate for ½ to 2
hours.
5. Rinse in water.
6. Counterstain in 1% aqueous neutral red, up to 30 minutes
(the neutral red forms a deep blue complex with the
phophalocyanin-phospholipid compound. This is very
insoluble and, if preferred, staining with neutral red may be
The following list gives the most useful methods and their
behavior with different mucins.
Mucin-carmine P.A.S. Alcian green Toluidine blue
Stomach ± or - + ± -
Duodenal mucosa + + + or ± -
Brunner’s glands - + - -
Colon + ± + -
Salivary gland + + ± -
Pancreatic duct ± + + -
Bile passages + + + or ± -
Bronchus + + + ±
Cervix + + + ±
Endometrium - + ± -
Bartholin’s gland + + + -
Prostate ± + + -
Seminal vesicles - + + -
Ovarian cyst + + + -
Mucicarmine solution:
Carmine 1g
Aluminum hydroxide 1g
• Add 100ml of 50% alcohol and then add 0.5g of
anhydrous aluminum chloride (beware of frothing
and use a 500ml flask).
• Boil for exactly 2 ½ minutes.
• Cool and filter.
Note:Cheap carmines seem to work better than purified ones. The
mixture frothes when being prepared.
Hotchkiss Method:
1. Bring section down to water and remove mercury.
2. Rinse in 70% alcohol or meth. Spirit.
3. Immerse in Periodic acid solution at room temperature.
4. Rinse in 70% alcohol or meth. Spirit.
5. Immerse in reducing solution* for 1 minute.
*Reducing bath:
Potassium iodine 1.0g
Sodium thiosulphate pentahydrate 1.0g
Abs. Ethyl alcohol 30.0ml
Distilled water 20.0ml
2N Hydrochloric acid 2.5ml
(use 20% of conc. HCl)
¾ (Ignore any sulphur deposit. Keep between
17-22oC. Lasts up to 14 days.)
**Schiff’s reagent:
CARBOHYDRATE SUBSTANCES
GLYCOGEN (BEST’S METHOD)
Glycogen is water soluble but is largely retained in tissues by
being trapped in the proteins by ordinary fixation. To detect small
PIGMENTS
The following colored materials may be encountered in sections:
• Haemosiderin
Golden to rusty brown granules. Gives positive iron reaction
with Berl’s stain.
• Haematodin (Bilirubin)
Reddish-brown crystalline deposits. Negative iron reaction.
See bilirubin below.
• Formalin pigment
Jet black granules present in stale tissues which in blood and
fixed in formalin. Can be removed by alcoholic picric acid
(Immerse slides for 2 hours).
• Melanin
Variable color from very pale brown to nearly black
negative iron reaction. Bleached out by acid permanganete.
Gives positive Masson’s silver bestand positive Schmori’s
stain.
• Carbon
Totally insoluble in anything. Jet black. Gives no reaction
with any test.
• Bilirubin
Greenish-brown granular masses. Negative iron reaction
Gmelin’s and Stein’s tests.
• Lipofuscins
Brown granules in parenchymous cells. Iron test may be
positive (due to associated iron). Positive satin for fat in
frozen sections. Stains by Schmorl’s method.
• Malaria pigment
Resembles formalin pigment and carbon. Soluble in conc.
sulphuric acid.
• Haemoglobin
Peroxydase stains with leuco patent blue.
TARIQ AZIZ / LAB APP OF HISTOPATHOLOGY DEPARTMENT
62
PERL’S REACTION FOR IRON
Any fixative but chrome salts better avoided.
1. Bring paraffin sections down to distilled water.
2. Place in slightly warmed mixture of equal parts of
20% hydrochloric acid and 20% potassium
ferrocyanide 5minutes.
3. Rinse in distilled water.
4. Counterstain in 1% aqueous neutral red, or other red
dye.
5. Wash rapidly in tap water.
6. Dehydrate, clear and mount.
Result:
Iron (ferric salts) - dark blue
Tissue - red
*Ferric-Ferricyanide solution
1% Ferric chloride 30ml}bothfreshly prepared
1% potassium ferricyanide 4ml }
Distilled water 6ml
Mix, fliter and use within 30 minutes.
MASSON’S METHOD:
Fix in any ordinary fixative, avoiding chromates.
1. Routine paraffin sections.
2. Bring to distilled water.
3. Leave overnight in Fontana’s ammoniacal silver* in
the dark in a covered jar.
4. Rinse in distilled water.
5. Fix in 5% “hypo”,1-2 minutes.
6. Counterstain with carbol-safranin.
7. Dehydrate, clear and mount.
Result:
Argentaffin coll granules - fiery orange red
Nuclei - dark blue
Cytoplasmic structures - yellow
MISCELLANEOUS SUBSTANCES
AMYLOID
Amyloid stains metachromatically with various violet dyes. It
stains red with congo red and the stained material is tropic.
It stains with Thioflavian T and the stained product fluoresces in
ultraviolet light.
It stains non-specifically with PAS (McManus)
HIGHMAN’S MODIFICATION OF
BENNHOLD’S CONGO RED METHOD
1. Stain in Congo red (0.5% in 50% alcohol) for 1-5
minutes.
2. Differentiate in 0.2% potassium hydroxide in 80%
alcohol for 1-3 minutes.
3. Rinse in water.
4. Counterstain with alum haematoxylin.
5. Wash inwater.
6. Dehydrate, clear and mount in D.P.X.
Result: Amyloid - red
Nuclei - blue
CALCIUM
Calcium deposits stain a dark blue black with haematoxylin but
the staining is due to trace of iron in the deposit and is not
specific. Von Kossa’s method stains calcium black but here the
reaction is due to the acid radical of the calcium deposits and is
also not strictly specific. It is, however, a very useful method.
Alizarin is a specific stain.
VON KOSSA’S STAIN
1. Any fixative that does not contain free acid.
2. Bring paraffin sections down to distilled water.
3. Place in 1.5% aqueous silver nitrate (freshly prepared
from a 10% stock solution) and leave in the light for 1
hour.
4. Wash in distilled water.
PARAFFIN SECTIONS:
Thin blocks of tissue should be fixed, according to Gomori’s
method (1946) in two or three changes of cold absolute acetone at
4oC for 24 hours. There are many subsequent ways in which the
tissues may be embedded in paraffin wax, most of them being
minor variations designed to minimize destruction of the enzyme.
As a routine procedure the following method gives good results.
FROZEN SECTIONS:
1. Cut sections 10-15μ thick and mount on clean glass
slides without any adhesive.
2. Dry in air at room temperature for 1-2 hours.
3. Incubate in the substrate solution for ½-4 hrs.
4. Wash in water treat with 2% cobalt solutions, wash
treat with dilute yellow ammonium sulphide.
5. Counterstain in 1% aqueous eosin 5 minutes
6. Wash in running water 5 mins.
Method:
1. Bring sections to water via absolute acetone after
removing the paraffin wax with light petroleum.
2. Cover with freshly made and filtered substrate-
diazonium salt mixture as above.
3. Incubate for 30 minutes to 4 hours (Salt 2), or for up to
2 hours (salt 7), or up to 12 hours (salt 9).
4. Wash inwater, counterstain as above and blue in
running water.
5. Mount in glycerine jelly.
• (The use of salt 9 is particularly
recommended).
Result:
¾ With salt 9, the sites of alkaline
phosphatase activity appear dark
reddish-brown localization is
excellent.
¾ Nuclei - blue
Solutions:
A. Pararosanilin HCl. Stock solution.
B. 4% sodium nitrite in distilled water.
C. Veronal-acetate stock solution
buffer pH 9.2
D. Dissolve 100mg Naphthol AS-TR
(or B.I.), phosphate in 10ml N,N-
dimethyl formamide.
Incubating medium:
Dilute 5ml of solution C with 12ml distilled water and
add 1ml of solution D. Mix 0.8ml each of solutions A
and B and add to the solutions. Adjust the pH of the
whole solution to pH 5.0 with N NaOH.
Method:
1. Sections to water.
2. Allow to dry.
3. Place in incubating medium for 30-90minutes at room
temperature.
Method:
1. Use either cold-formalin fixed frozen sections, without
washing out the formalin, or paraffin sections fixed in
cold acetone or in cold-formalin, and dehydrated with
cold acetone.
2. Incubate for 1-15 minutes at room temperature in the
following medium: Dissolve 20mg a-naphthol acetate
in 0.25ml acetone and add 20ml 0.1M phosphate
buffer (pH 7.4)*.
3. Wash in running water 2 minutes.
4. Counterstain in Mayer’s haemalum 4-6 minutes.
5. Wash in running water for at least 30 minutes.
6. Mount in glycerine jelly.
Result:
Esterase - black
Nuclei - dark-blue
GRAM’S STAIN:
1. Any fixative, paraffin sections.
2. Bring down to water.
3. Stain for 1 minute in Gram’s crystal violet*.
4. Rinse in water.
5. Mordant in Lugol’s iodine ½ minute.
6. Rinse.
7. Differentiate in acetone till no more clouds of stain
come out (about 3 seconds).
8. Rinse in water
9. Counterstain in neutral red-fuchsin.
10. Dehydrate quickly in alcohol (this takes some of the
counterstain out).
11. Clear and mount in D.P.X.
*Crystal violet:
Method:
*Carbol fuchsin:
Basic fuchsin 10g
Phenol crystals 50g
Alcohol 100ml
Distilled water 1000ml
Result:
Myco. leprae - red
Nuclei - blue