HISTOPATHOLOGY
Microtome
Microtome :- It is an instrument for cutting tissue section
of uniform thickness. A knov on the machine is use to adjest thickness section.
A knife is fixed is a clamp. Tissue block is clamped in to the microtome. If
the microtome is rocking time. That block is fixed a chunk of wood. That is
held in the microtome head.
Microtome are different types: -
1. Rotary Microtome: - The knife remains
fixed in the microtome clamp. Paraffin block is moved up and down with the help
of rotator. The tissue Advanced which each rotation.
2. Rocking Microtome: - Tissue block
remains fixed in the microtome knife is move to and fro to cut the section with
each rocking tissue is moved up to allow the section cutting.
3. Freezing Microtome: - The specimen is
kept frozen by a gassous coolant which is help in a tank and is blown through a
hose over the chunk. Holding the specimen which give the object frozen while
the knife is kept at R.T.
4. Cold Microtome: - Cold Microtome: - If
is also known as cryostat. It is a rotary microtome kept in cold chamber (-20˚c)
the knife in also kept cold. Specimen is kept frozen which provides needed
hardness to the tissue for section cutting.
5. Base sledge Microtome: - It is a
racking microtome used for very hard tissue or large block of brain and
heart.
Ø
Steps of
Microtomy.
1. Trimming of block with a raiser or blade
so as to have parallel site with a distance of 1-3 mm form of margin of the
tissue of the block face.
2. Attaching the paraffin block to
the metallic surface of chunk. Heat the chunk over a flam and fix the block
over heat thus block is fix to the chunk the chunk is fitted in to the object
carry of microtome.
3. Orienting the block with the
knife the orientation is done in such a way that top and bottom horizontal to
adage of knife at the movement of impact this is done with the help of
adjusting screw on the microtome.
4. Setting the till of knife: - Set
the gauze controlling thickness end of knife to trim the block when whole
surface is being cut set the gauze at 5 micron and move the knife to the
position where no scratch mark is visible on the surface of block.
5. Cutting the section: - Cool the
block surface with ice cube for a few minute move the microtome head with block
and start cutting section the cut section will appear on the knife maintain a
regular cutting rhythm until a ribbon is cut when cut section regular ribbon
remove the ribbon with the help of camle hair brush and transfer it tissue
flotation bath in which the water has a temperature of 45˚c this temperature help spread of
section is uniform all creases disappear the tissue section stands out very
well in the matrix of paraffin.
6. Attaching section to microscopic
slide: - A small drop of egg albumin adhesive.
Ø
Preparation of Egg Albumin
Equal volume of egg albumin (white) equal volume of glycerol and small
crystal of thymol.
Is smear over the slide with finger pulp dry the adhering the air deep
the slide in to the floatation bath. Slowly with draw the slide allowing if
surface of touch the edge of section when the section in near lay the slide
flat on the table at 40-50˚c for at least one hrsfor through fixing of section over
slide section is now ready of staining.
Staining
Ø Slide prepration: - Tissue embedded
paraffin has been in to a section of 5 micron thickness.
Ø This section is passed through xylene
to remove paraffin form the tissue.
Ø The slide is kept in xylene form 5-10
minutes until the section appearance transparent xylene is removed by passing
the slide through alcohol.
Ø 1st Absolute alcohol
followed by descending strength of alcohol (90% Alcohol, 80% Alcohol) (70%
Alcohol, 50% Alcohol) Two minutes ego.
Ø Finally section is passed in running
water.
Ø After 5 minutes in running water
tissue is passed in halmatoxylene stain ( nuclear stain) This is follower by
process of differentiation. It is done by 3-10 dips in acid alcohol mixture.
Ø After differntation the section
appearance light bule back ground. The tissue is examine under microscope to
know the degree of staining.
Ø Nuclei should appears light colour if
the staining is more , it is again put up in acid alcohol mixture to remave the
excess stain.
Ø If the nucliai are not dark enough
repeat the haimatoxylene stain.
Ø Finally the slide is dipped in
ammonia water or lithium carbonate solution.
Ø The causes changes of colour to blue
now atide is cooked in running water for 10-20 minutes.
Counter staining with eosin
Eosin staining for cytoplasm. Colour the stide is pink slide
is depped in eosin for 2 minutes . if eosin is prepared in alcohol the slide
should firstly coddled in 95% alcohol before the placed in eosin alide washed
in water if it has been stain with eosin slide prepare is it was alcohol eosin
slide prepare is it was alcohol eosin is not with washed water.
Note: - Haematoxylene is
Nuclear Stain
OG6:- (0range G for kainite 6)
E-A36 (Eosin – Azure 36 ) is for plasmic stain.
Composition of Haematoxylene Stain
Usually harris haematoxylene is used it has following
composition.
1. Haematoxylene crystal → 5 gm
2. Absolute alcohol → 50 ml
3. Ammonia or potassium alum → 100g (mordant)
4. Dlw → 1
hiter
5. Mercuricoxide (Red)
→
2.5gm (oxidizing Agent)
Composition of Eosin
1. Eosin – y (yellow) → 5ml
2. Eosin – B → 5ml
3. Erythrocin – B→ 1ml
4. Phlexin – B → 1ml
The four soln are kept in a tube and it is ready of used.
Preparation of Alcoholic Eosin
1. Eosin → 1gm
2. Dlw → 80ml
3. 95% Alcohol → 320ml
4. GAA → 0.4ml
Dissolve in Eosin in water at it to 95% Alcohol. 0.4 ml GAA is
added in last . it should appear transparent after preparatoion. If
transparency is less add more GAA.
Post Staining procedure
Place in 70% alcohol for 1 minute.
↓
90% Alcohol for 1 minute.
↓
Absolute Alcohol for 1 minutes
↓
Xylene for 1 minute.
↓
More unit the smear is fully transparent.
Mounting
Slide is taken out in xylene and is pressed on table one drop of
Dpx mounting material is added over the stain material. A coverslip is placed
on Dpx . So that it covers the whole tissue and protect tissue from
enviromnt Dpx stands for Dibotile
phthalate xylal.
Note: - what are other mounting media other that Dpx.
1. A pathu’s medium to demonstrate fat.
2. Glycerine jelly for frozen medium.
3. Gum orabic medium .
4. Karacorn serum medium for carbo wax embedding.
Rapid H.E staining (Haematoxylene Eosin
Staining)
This method is used for staining tissue cut by frozen microtome.
This process is a rapid process.
Ø Procedure
Haematoxylene
stain for 1 minute
↓
Rinse in
tap water.
↓
Differentiate
in 1 % acid alcohol (1 drop only).
↓
Rinse
in water.
↓
Blueing
is done by passing the section over ammonia vapour.
↓
Rinse
in tap water.
↓
Counter
satin is 1 % aoweous eosin for 5 minutes.
↓
Rinse
in tap water.
↓
Dehydration
95 % and absolute alcohol dip in each.
↓
Clearing
in xylene (1 drop)
↓
Mount
in DPX.
↓
Examine
under microscope.
Microscopic Examination of Stain Tissue
The purpose of microscopic
examination of tissue is to find out the type of disease. Most important is to
find out whether there is a tumour (Benign tumour or malignant tumour.) If it is not a tumour wher it is tuberculosis
, Amyloid or any other infection.
Malignancy is recognized by following features
of cells : -
Malignant cell is larger
than normal. It has bizarre out line, pleomorphic , large nucleus , increase
chromatin matter , prominent nucleoli , lobed & irregular nucleud , thick
nuclear membrasia , chromosomal abnormalities , anaplasia , loss of polarity ,
increase mitotic activity & abnormal mytosis.
Some Special Stain
1. Pas stain (periodic Acid Schiff stain) for giycogen.
2. Gordon and sweet rreticuline stain for reticuline fibres.
3. Silver nitrate stain for reticuline fibres.
4. Massions trichrome stain for collagen fibres nucleus.
5. Weigert’s haematoxyline stain for collagen.
6. Von geison stain for collagen.
7. Hella’s colloidal iron stain for muco poly saecharide.
8. Orcein stain for elastic fibres.
9. Mallory’s phosphotungetic acid haematoxyline stain for
connective tissue.
10.Cogal’s gold sublimat stain for glacial fibres.
11. Congo – Red stain for amyloid.
12. Prussion blue stain for iron.
13. ZN stain for mycobacterium tuberculosis.
14. Waste fits stain for mycobacterium leprae.
15. India ink negative stain for troponema-peridum.
16. Silver methane amine stain for fungus.
17. Geimsa stain for syphilis.
18. Alcian blue stain for acid muco polysaccharide.
19. Methyl green pyromine for plasma cell.
20. Schieff stein stain for negris body.
21. Toluidine blue stain for MP.
22.Warthine stain for spirochete.
23. Gridley’s stain for fungus.
24. Rhodium stain for copper.
25.Masson frontana stain for melanin.
26. Perl’s stain for sydrotic granules.
27. Luna’s stain for mast cells.
28. Best ca.rmine staIn for glyc.ogen.
29. Oilred – o stain for fat.
30. Verhoeff’s stain for elastic fibres.
Procedure of some special histopathological
stain
1. Oilred – o stain: - This is a
fat stain.
Tissue is a fix formaline and the frozen section is out at 10
micron in thickness.
↓
Cut section is collected in DLW.
↓
Tissue is passed through 70% alcohol for a few second.
↓
Place section on oilred – o solution for 5 minutes.
↓
Oilred – o solution is kept in closed container.
↓
After staining is oilred – o section is placed in 70% alcohol a
few minutes.
↓
It is followed by washing in water.
↓
Counter stain with haematoxiline for a few minutes. Followed by
washing in water.
↓
Now section is placed in ammonia water.
↓
If the section appears very dark.
↓
Place the section in 1% acitic acid until section is light blue.
↓
Wash the section in water and mount in glycerine jelly.
Microscopic finding: -
1. Fat appears → orange to bright red.
2. Nucleus appear to blue colour.
Other fat stain
1. Osmium tetra oxide stain → fat appears
block in yellow to brown back ground.
2. Sudan black stain → fat appears black
blue and nucleus red colour.
3. Sudan 3 – stain → fat appears
orange colour.
4. scarlet Red → fat appears bright
red colour.
5. Nile blue sulfate → Neutral fat red
fatly acid blue colour.
6. Marchi’s stain → It is stain for
myelin sheath appears black colour.
2. Vonkossa’s Stain:- This is
stain for
calcium
→ Tissue is fixed in alcohol / 10% of formaline → paraffin block is prepared
and section is cut of 5 µ
Staining Procedure:- Deparaffinsed the
section by passing through two changes of xylene.
↓
Absolute Alcohol → 95% Alcohol , 70%
Alcohol , 50% Alcohol → DLW.
↓
Place the tissue in 5% silver nitrate
solution for 60 minutes. Keep the jar exposed to sunlight UV lamp. Rinse the section of DLW.
↓
5% Thiosalphale for 3 minute.
↓
Wash in running water.
↓
Counte stain in nuclear fast red stain
for 5 minutes.
↓
Wash in DLW.
↓
Dehydrate with two changes of 95%
alcohol and absolute alcohol.
↓
Clear with two changes of xylene.
↓
Mount in DPX.
Result:-
1. Calcium salt → Black colour
2. Nucleus → Red colour
3. Cytoplasm → Pink colour
3. Mansson’s Trichrome Stain:-
This is stain for collagen tissue and
muscle fibres collagen tissue. Found mostly in tendon and aponeurosis. It is
also found arrround joint and bones. Tissue is fixed in boins fluid or
formalene is cut at 5 micron.
Staining Procedure:-
Xylene is two changes.
↓
Absolule alcohol → 95% alcohol → 70%
alcohol → 50% alcohol → DLW.
↓
Mordanting in 0.5% iodine in 80%
alcohol for 10 minutes and running tap water.
↓
5% sodium thiosuphale for 5 minute
↓
Rrunning water for 1 hours at 56˚ c
↓
Cool and wash in running water yellow
colour disappear.
↓
Weigert’s iron haematoxyene stain for
10 minutes.
↓
DLW
↓
Scarlet acid fuchisin solution for 5
mit.
↓
Aqueous phosphtungstic acid 5% for 5
minutes.
↓
Aniline blue solution for 10 mihules.
↓
DLW acetic water 1% for 5 minutes
↓
95% Alcohol.
↓
Absolute alcohol three changes.
↓
Xylene two changes.
↓
Mount in DPX.
Result:-
1. Collagen and muscle tissue → Biue
colour
2. Nucleus →
Black colour
3. Cytoplasm
& other tissue → Red colour
4.
Silver nitrale stain for reticuline fibres:-
Iesting
principle: - Reticuline
is a stain that contain silver in pregnation method. There is local reduction
and selective precipitionr of silver salt around reticuline fiber.
Stain procedure:-
Remove paraffin from the section by
passing through two changes of xylene → 95% alcohol → 70% alcohol → 50% alcohol
↓
Was in running tap water the section
is now stained with potassium permigrate 0.5% for 2 minutes.
↓
Bleach with oxaloacetic acid for 2
minute until a section becomes colour less. Wash in tap water.
↓
Stain with 4% free chloride four
minute.
↓
Wash in running tap water for 3 minute
wash in DLW.
↓
Stain with ammonicol silver nitrate
for 5 minutes.
↓
Rinse in DLW for half minute.
↓
Add formaline for 1 minutes.
↓
Tap water for 3 minutes.
↓
Auric chloride 10 minutes.
↓
DLW 2minute.
↓
Sodium thiosulphate 5 minutes.
↓
DLW 2minute.
↓
Dehydrate through 70% alcohol to 9%
alcohol and changes of absolute alcohol.
↓
Clear in xylene two changes.
↓
Monut with DPX.
Result:-
1. Reticuline fibres → Black colour.
2. Nuclear → Brown colour.
3. Collagen fibre → Brown colour.
5. Verhoeff’s stain for elastic tissue
For elastic tissue:-
Reagent Requirmet:-
1. Haematoxylene → solution “A”
2. Ferric chloride → (10g%) → solution
“B”
3. Iodine solution → (2g%) → solution “C”
4. Prepration of verhoeff’s solution:-
Solution A → 20ml
Solution B → 8ml
Solution C → 8ml
5. 2% ferric chloride solution.
6. 1% acid fuchsin solution.
7. Saturated picric acid solution.
8. Vongesion stain:-
1% acid fuchsin solution is dissolved in 1000ml of saturated picric
acid.
9. 5% sodium thiosulphate solution.
Testing procedure:-
Remove paraffin form section by
passing through two changes of xylene . hydrate by passing through absolute
alcohol → 90%, alcohol, 70% alcohol, 50% alcohol and the was the running tap
water.
↓
Stain the verhoeff’s stain for 45
minutes nutil the section of black.
↓
Wash in DLW.
↓
Treat with 2% ferric chloride solution
for 2 minutes.
↓
Place in 5% sodium thiosulphate
solution for 1 minute.
↓
Tap water for 5 mits.
↓
Counter stain with vongiesion stain
for 2 minutes.
↓
Differenciate in 95% alcohol.
↓
Two changes of absolute alcohol.
↓
Clear in xylene.
↓
Mount in DPX.
Result:-
1. Elastic fibre → Black colour
2. Nucleus → grey colour
3. Collagen → red colour
4. Muscle → yellow colour
Note:- Purpose of
this staining skin tumour , ligaments , lung and blood vessels.
6. Pas stain (periodic acid achiff
stain)
Testing principle:- periodic
acid reacts with aldehyde groups of carbohydrate aehiff reagent act on it
aldehyde groups to produces red or purple colour. This is done for
identification glycogen content of the tissue.
Reagent required:-
1. 0.5% periodic acid solution
2. Achiff reagent
2. Prepration Aehiff Reagent:-
1 gram of basic fuchsin dissolved in 100ml of water.
↓
Boil.
↓
Cool to 60˚c.
↓
Filter.
↓
Keep the solution for 48 hours.
↓
When solution becomes strow colour add
300mg optivated charcoal.
↓
Shake vigorously.
↓
Filter.
↓
Store at 4˚c
3. 1 NHcl.
4. o.1 gram of light gram
5. harri’s hematoxylin stainS
Testing procedure:-
Deparaffinised.
↓
Hydration.
↓
Rinse in DLW.
↓
Treat with periodic acid for 10
minutes
↓
Wash in running water for 2 minutes.
↓
Stain in achiff reagent for 15 minutes.
↓
Running water for 10 minutes.
↓
Until the red colour decelops.
↓
Conuter stain with haemaoxylene stain
for 3 mits.
↓
Dehydrate.
↓
Clear.
↓
Mount in DPX.
Result:-
1. Glycogen → magenta pink colour.
2. Nucleus → blue colour.
3. Other chemical containing
carbohydrate → purple colour.
Staining of AFB in tissue:-
Tissue is fixed in formaline paraffin
section is cut at 5 micron.
Staining procedure:-
Deparaffinised.
↓
Stain with carbel fuchsin solution for
10 minutes.
↓
Rinse well in tap water.
↓
Decolourised
with 1% acid alcohol until section is pale pink colour.
↓
Running
water for 8 minutes.
↓
Counter
stain with methylene blue colour dipping.
↓
Section
should appear pale blue colour if this is over stain.
↓
Decolourised
with acid alcohol until colour becomes pale blue colour.
↓
Wash with
rinning water dehydrate in 95% alcohol and absolute alcohol.
↓
Clear with
two changes in xylene.
↓
Mount in
DPX.
Result:-
1. AFB →
Bright Red colour.
2.
Erythrocyte → Yellowish orange colour.
3. Other
tissue → Pale blue colour.
Museum
Techniquic:-
Museum
specimen → They are specimen preserve for teaching programs. The student are
able to study the gross morphological feature of diseased organs are preserved
in sealed glass jar. This technique consist of following procedure →
1. Reception:- specimen
receved are number in undillable ink on a cloths which is shocked in paraffin
and tied to the specimen.
2.
Prepration of Specimen:- Specimen received is cut in to such a manner
that pathogical changes becomes visible on nacked eye examination.
3. Fixation
of specimen:- The specimen is fixed in a particular tape of fixative known as kaiserling
fluid which has following composition.
1.
Formaline → 400ml.
2.
Potassium Nitrate → 30 gram.
3.
Potassium Acetate → 60 gram.
4. Tap
water → 2 liter.
→ The
specimen is this for one week.
4.
Restoration of colour of specimen:- This is done by keeping the specimen
in 85% ethyle alcohol for 12- 14 hours. During this preoid two changes of
alcohol is required.
5.
Regeneration of Specimen in Mounting Media:- After the colour has been restored the
specimen is transferred to a fluid which has a following composition.
1.
Glycerine → 300ml.
2. Sodium
acetate → 100 gram.
3.
Formalene → 5ml
4. Tap
water → 1liter.
→This
solution is kept in jar known as Perspex box the fluid in filled in the box
keeping upper half inch of the box empty. This fluid is known as mounting
media. The specimen is suspended in this fluid through a glass rod 0.4%
solution sodium hydrosulphate is added before sealing the box. The sealing in
is done in such manner there is no air contact from out side.
Presentation:- A whole is drilled into tap conner the
lid is sealed on four sides by araldite. A light weight is applied for 2 hours
after removing the air bubble the whole is plug by Perspex plug and comented in
form the specimen is kept at a place where student can examine it with out
touching the specimen.
