1.

Histotecniques -- Introduction

BRANCHES OF PATHOLOGY

1. Histopathology --- Study of tissues

2. Cytology – Study of cells.It includes
 Exfoliative cytology,
 Fluid cytology,
 FNAC - Fine needle aspiration cytology.

3. Hematology – Study of blood

4. Clinical Pathology – Study of body secretions.
5. Immuno hematology—Study of antigen antibody reactions in blood.

HISTOPATHOLOGY LAB

Functions – Preparation of tissue sections for establishing histo pathological

diagnosis.

Samples are received from operation theatre either as small piece of tissue or as
whole organ.The role of histotechnician is handling and preparation of specimen
for gross and microscopic examination which is done by pathologist.

Steps involved in tissue preparation.

1. Fixation of tissue.
2. Grossing of specimen
3. Tissue processing
4. Embedding
5. Sectioning by microtomes.
6. Staining.
7. Mounting
8. Submission of slides.

A good histotechnician is

 able to prepare the good section.

  Able to recognize satisfactory preparation.
 Able to identify the cause and know the remedy when unsatisfactory
results are obtained.

Definition for basic terminology

1. Autolysis – self digestion and decomposition of tissue.

2. Fixation – The process by which the specimen is preserved as in live state.
Formalin is commonly used as histofixative.
3. Biopsy – Fragment of tissue is removed from the bodyof the living person
for examination.
4. Decalcification – A process of removing calcium from bone and hard
mineralized tissue to ease the cutting process. It is done by using acids
after fixation and before processing.
Example ( Nitric acid ,formic acid, HCL)

5. Rehydration – Taking the section to water to replace water insoluble wax

with water soluble stain.it is done prior to staining
6. Dehydration – Removing water from the tissues by putting it in increasing
grades of alcohol followed by treatment with clearing agent.
7. Impregnation – A process by which the embedding material enter into the
tissue and clearing agent diffuses out.
8. Tissue Block – Portion of tissue that is properly cut and trimmed for
processing.
9. Clearing agent – Substance that make the tissue more transparent for
microscopic examination. Eg. Xylene, chloroform.
10.Adhesion – The process of placing the section on the slide so that it is not
washed away during staining. Eg for adhesive – egg albumin.
11.Microtome—Instrument used for preparing thin slices of tissue. Eg-
rocking microtome, rotary microtome.
12.Microtomy – Process of section cutting.
13.Cryostat – Cold box containing microtome with a temperature below
freezing point -20 to -36c.
14.Freezing microtome – Device used to prepare section from frozen tissue.
15.Infiltration – Process by which clearing agent is eliminated from tissue and
making room for impregnation of embedding material.
 16.Mounting – Better visualization and improving the clarity of section using
mounting media like D.P.X for microscopic study.
17.Staining – The process of coloring of tissue in order to facilitate their
identification under the microscope.

INSTRUMENT: -

 Containers for holding specimen.

 Knife, forceps, scalpel, scissor,Bone cutting saw.
 Jars for processing,Embedding leukhard embedding moulds, oven.
 Glasswares: - Pipette ,beaker.
 Temperature water bath. Floating bath. Microtome, knife, knife sharpener
(hone, strope)
 Slides, cover slip, microscope,
 Coplin staining jars, slide tray (vertical horizontal) ,slide carrier.
 Accessories – labels, diamond pencil for marking glass slides, weighing
machine, electronic physical balance.
 Slide container – Cardboard box ,Plastic or wooden box, Metal filing cabinet
 Specimen container – Fixed tissue kept in heat sealed plastic bag.
 Block storage – wooden or plastic boxes at cool place.

Microtome—Types

 rotary, rocking – For paraffin embedding blocks

 Sliding – For cellodin embedding sections
 Freezing microtome – unfixed tissue freezed by using CO2
 Cryostat-Refrigerated cold microtome-

Microtome consists of three parts

1. Block holder to hold the tissue in position.

 2. knife carrier and knife.
3. Adjustment screws.

Care of microtome

1. Cleaning the dust with soft brush.

2. Lubricate the moving parts.
3. Cover it when not in use.
4. Prevent rust formation by cleaning with coconut oil
5. Metal parts are cleaned with xylene ( to remove paraffin.)

Paraffin oven

Temperature = 50 – 60c

USE

1. Formelting and storing molten paraffin

2. Infiltration of paraffin into tissues.
3. Drying of slides .
4. Warming of solution during reagent preparation.

Tissue floating bath

Temperature close to 43.c.

USE

1. To float and facilitate separation of paraffin embedded tissue section.

2. Fixing the section on the slide.

Automated tissue processor:

It automatically fix ,dehydrate, clear, infiltrate the tissues . It consists of

Time clock, circular super structure that contains bucket carrier, receptacle bucket
and receptacle. circular deck which holds reagent beaker and paraffin
bath.Tissuesare held in perforated capsule and put it into receptacle bucket.
Entire superstructure descends & immersing the bucket in the first solution and
sealing the other reagent beakers to prevent evaporation. To move the bucket
from one reagent to the other entire superstructure ascends and descends at
scheduled interval which is controlled by the time clock.

During immersing the bucket oscillates up and down the increase the speed of
penetration.

It takes 16 hours for routine processing .So it is set in the afternoon & processed
tissues are taken out in the next day morning for embedding.Solutions to be
changed regularly twice a week or if cloudy change it.

Slide warmer: - To removed paraffin from the sections.

REAGENTS: -

 Fixation – Formaldehyde, ethyl alcohol.

 Decalcification – Formic acid, HNO3, HCL
 Processing – ethyl alcohol, xylene, paraffin wax, acetone.
 Adhesive—Egg albumin.
 Mounting media – DPX, canada balsam.
 Stain – Hematoxylin, eosin for routine study.
 Various special stains.

Slide cleaning techniques: -

Old one – 1% acid alcohol – Distilled water – 95% alcohol – dry.

New one – 95% alcohol - dry

 2.Logging , Fixation& Decalcification

Logging of specimen: -

1. Specimen is put into fixative solution immediately.If not possible, the

specimen should be wrapped in saline moistened towel and kept in fridge.
For mailing the specimen it should be fixed before. Then sent.Large
specimen should be opened so that fixative can reach inside.
2. Check the identity.
3. Maintain a log book or register for specimen reception entry.
4. Give Biopsy identification number.
5. Grossing: - It is done by pathologist. Gross morphological description is
recorded by the assistant. Then portion of tissue is trimmed (Tissue block)
and give it to the technician for processing.Number it if more than one
block from single specimen is taken.Technician maintain a bit note which
included the detail of grossing date, specimen identity number, number of
blocks taken and their suitable sketch. It is helpful in tracking the specimen
in case of mixup.
6. Block is put in the capsule with a identification number marked with lead
pencil in a paper tag.
7. Now the capsule with block and tag is ready for processing.
8. Smaller specimen should be kept in tissue paper & soaked with eosin to
prevent tissue loss through perforated capsule and easy identification while
sectioning.
9.The size of the tissue block – should be within the size that can be
accomadated within slides. thickness – 3-5mm.

Fixation

The purpose of fixation is

 Maintain the cells as in live state and preserve the morphology.

 To harden them

It is done twice.
1. Immediatly after removing it from body.

2. After preparing the block & before processing.

Fixatives–

 Routine – 10% formalin

 Special – Zenker, Helly, bouins, Carnoys)

Routine fixatives --- Commonly used histo fixative is 10% formalin.

Preparation of fixative:

10% formalin = 100ml formalin + 1000ml – Tap water

Neutral formalin – to above add calcium carbonate.

After the fixation – excess formalin is removed by washing in water &Keep it in

70% alcohol.

Advantages:

 Inexpensive
 Easy to prepare
 Readily available
 penetrate the tissue well
 Compatible with most of the stains.

Disadvantages:

 allergy.
 Formation of acid slightly occurs in 10% formalin– that Interferes with
staining.It is avoided by using neutral formalin which is prepared by adding
calcium carbonate.
 Formation of Formalin pigment which is seen as dark brown crystals like
precipitate.

Removal of Formalin pigment from the section:

This pigment is removed by treating the section in Xylene – alcohol – water.Then
immerse in ammonium hydroxide solution (Ammonium hydroxide 2ml + 70%
alcohol 100ml ) for 1hour. Thoroughly wash in tap water to remove excess
ammonia. Then proceed for staining.

Special fixatives

Some tissues need special fixation for demonstration of special cellular detail .

Examples of special fixatives are zenkers,Hellys,Bouins, Carnoys fixative

 Zenkers fixative

Preparation

Stock solution –Mercuric chloride 60g

- Potassium dichromate 25gm

- Sodium sulphate 10gm
- Distilled water 1000ml.
Dissolve the above ingredients by stirring at room temperature.

Working solution – 95ml above solution+5ml glacial acetic acid.

Advantages – Use for bone marrow aspirate (acid decalcify bone spicules),

Spleen, Liver.

Excellent stain of nuclei and connective tissue

Disadvantages – Poor penetration. Tissue should be kept longer time for

hematoxylin stain .

 Hellys fixative

Preparation

Stock solution - Mercuric chloride 5gm.

- Potassium dichromate 5gms

- Sodium sulphate 10gms
 - Distilled water 1000ml.
Working solution – 5ml formalin + 95ml of stock solution

use – excellent cytomorphology

disadvantages – washing overnight in running tap water to remove yellow

staining of dichromate.

 Bouins fluid

Preparation

Stock solution -Picric acid -750ml

- Formalin – 250ml

-Glacial acetic acid – 50ml

Use - for hematologic and lymphoid tissue

Disadvantages – Time factor

 Fixation Time – 4 – 18 hours that depend on size of tissue.

 several changes wash in 50%, 70% alcohol – 4 – 8hours
 Yellow colour (picric acid) interface with staining reaction.

 Carnoys fluid

Preparation- alcohol -60ml

-Chloroform – 30ml

-Glacial acetic acid -10ml

Fixation time – 1-3 hour at room temperature &12-18hour in refrigerator .

No need of washing. Dehydration is already done during fixation

Use

 for small biopsy

  Quick acting fixative .
 For glaycogen demonstration

Since tissues are first fixed in formalin , de formalinization is necessary which is

followed by special fixation.

De formalinization & Secondary fixation

 Bring section to xylene – alcohol - water – Then to ammonia water (2ml

NH4OH in 100ml H2O) for 1 hour – wash in running tap water.
 Refix in Zenkers or Hellys fixatives – 1 hour – Then wash in running water
for 15 minutes. Residual mercuric chloride is further removed by
mordanting.

Mordanting –Process by which excess mercuric chloride is removed.

Treat the section with Xylene – alcohol – alcoholic iodine solution (0.5% iodine +
80% alcohol) for 5-10 minutes. wash in running water – Then treat with 5%
sodium thiosulphate ( hypo) for 2-5 minutes to bleach out iodine. Then wash in
water to remove hypo solution.

Decalcification

It is essential for bony and calcified tissues In order to facilitate easy cutting

First cut the tissue by hack saw or sharp knife – 4-6mm thickness.Then do fixation
– thoroughly washed it – proceed for decalcification

Methods – Ion exchange, chelation, electrical ionization

Acid method is most widely used.

Solutions used ad decalcifying agents are

 Nitric acid 5%
 Formic acid 5%
  Formic acid with HCL – Formic acid 10ml + HCL 8ml + Distilled water 82ml

All are corrosive –So be cautious in handling those chemicals.

 Procedure – Put the specimen with identy tag into acid (20times the
volume of the tissue).Change the fluid daily. Stirring – hasten decalcification
process.

Duration -- 1-2 days for cancellousr bone .2-4 days for long compact bone

Testing the adequacy of decalcification.

 Mechanical – Bending or piercing with sharp needle . It may damage the

tissue.
 Chemical Procedure of testing

Reagent – strong ammonia, Litmus paper,Saturated aqueous solution of

ammonium oxalate.

Take 5ml of decalcifying fluid in a test tube. Put litmus paper into it – turn red due
to acid. Then drop by drop add ammonia till litmus turns into blue colour. Look
for turbidity. If turbid calcium is present & continue decalcification process. If no
turbidity, add 0.5 ml of ammonium oxalate. If turbid ,continue decalcification
process. If there is no turbidity, decalcification process is complete.

Note – Atleast 5 hours should be allowed before repeating calcium test after
transferring tissue to fresh decalcifying solution.The decalcified tissue should be
thoroughly washed in running water overnight before processing.
 3. Tissue Processing
It is the process by which the tissues are hardened by infiltration of tissue spaces
by paraffin.For making thin microscopic section the tissues need to be hardened
by paraffin embedding. Since paraffin is not soluble in water, the water must be
removed from the tissue to allow paraffin wax to be infiltrated into the tissue
spaces.

Methods -- Manual & Automated

The steps involved in processing technique are

1. Dehydration
2. Clearing
3. Paraffin infiltration
4. Embedding

1.Dehydration

It is the process by which water from the tissue is removed.

Reagent used

 Ethyl alcohol – It is soluble in both water and xylene which is a solvent for
paraffin wax.
 Isopropyl alcohol -- It is the next alternative as dehydrating agent.

Procedure – The tissues are kept first in steel or plastic perforated capsules or
cassettes along with identity paper tag. Lid is closed well. If the specimens are tiny
and multiple ,it should be wrapped in tissue paper to avoid missing of specimen
while processing. Put the capsules into the containers containing different grades
of alcohol like 80%,90%, and three changes of absolute alcohol. The duration in
each container is 1hour but it varies depending on the size & type of the tissue. In
order to ensure complete dehydration , put anhydrous copper sulphate ( white
colour) in the last container containg absolute alcohol.If dehydration is not
complete white anhydrous copper sulphate turns into blue colour hydrated
copper sulphate.
Once dehyration is complete the tissue spaces are filled with alcohol.Now the
alcohol should be removed before wax infiltration since it is not soluble in wax.

2.Clearing

It is the process by which alcohol in the tissue spaces are replaced by xylene.

Reagent used

Xylene is commonly used as clearing agent. The advantages are

 It is soluble in both alcohol and wax.

 It removes alcohol from tissue spaces and make room for paraffin
infiltration.
 It makes the tissue more transparent
 It evaporates readily in paraffin oven.

The capsule with the tissues are transferred to the container containing xylene.

Duration – one hour. Two changes of xylene is desirable.

Prolonged treatment with xylene makes the tissue more brittle.

3.Infiltration & Impregnation

It is the process by which the xylene in tissue spaces are removed and replaced
with wax.

The capsule with the tissues are transferred to the container containing molten
wax which is kept in paraffin oven. Ideal temperature is 50-56C. Temperature is
important. If it is too hot, tissues will be cooked and if it is too low, incomplete
infiltration of tissues occurs.Paraffin wax diffuses & infiltrate into the tissue
spaces and provide support to cells and intercellular structures.After cooling, the
infiltrated paraffin gives firmness and support to the tissue.

Duration – one hour. Two changes of wax is ideal for complete infiltration.

Problems
  Inadequate impregnation – Drying and shrinkage of tissues leading to
cracks and crumbling while sectioning.
 Excessive exposure to high temperature wax --- Over hardening of tissues
leads to difficulty in sectioning.

Duration for Manual processing

Formalin fixation – 1 hour

Dehydration -- Alcohol 80% --1hour

Dehydration -- Alcohol 90% --1hour

Dehydration -- Alcohol 100% --1hour

Dehydration -- Alcohol 100% --1hour

Dehydration -- Alcohol 100% --1hour

Clearing -- Xylene -- 1hour

Clearing -- Xylene -- 1hour

Infiltration & Impregnation --- Paraffin -- 1hour

Infiltration & Impregnation --- Paraffin -- 1hour

Total duration for manual processing is ten hours.

Automated tissue processing

It is done by automated tissue processor. It consists of series of beakers arranged

in circle with a timing device. This containers hold reagents and paraffin wax.In
the top there is a circular rotating transfer arm which caries basket holder and
baskets for carrying tissue capsules. Automatic tissue processing is to be sheduled
at night. Set the timing lever at zero and the machine is started at 4.30PM.The top
rotating arm automatically rotates with a basket carrying capsule and takes bath
in different reagents in the beakers in that way dehydration, clearing and
infiltration is over. The final bath is in paraffin wax till the technician arrives in the
morning. The capsules are opened and the tissues are ready for embedding.

The duration time shedule for Automated tissue processing

4.30 -5.30 PM Formalin fixation – 1 hour

5.30 – 6.30 PM .Dehydration -- Alcohol 80% --1hour

6.30 – 7.30 PM Dehydration -- Alcohol 90% --1hour

7.30 – 8.30 PM Dehydration -- Alcohol 95% --1hour

8.30 –9.30 PM Dehydration -- Alcohol 100% --1hour

9.30 –10.30 PM Dehydration -- Alcohol 100% --1hour

10.30 –11.30 PM Dehydration -- Alcohol 100% --1hour

11.30 –12.30 AM Clearing -- Xylene -- 1hour

12.30 – 2.30 AM Clearing -- Xylene -- 2 hours

2.30 – 4.30 AM Infiltration & Impregnation --- Paraffin -- 2 hours

4.30 – 6.30 AM Infiltration & Impregnation --- Paraffin -- 2 hours

6.30 – 8.30 AM Infiltration & Impregnation --- Paraffin -- 2 hours

Total duration for automated tissue processing is sixteen hours.

Changing of Solutions

The dehydrating solutions,clearing agents tend to be contaminated with fluid

carried from the previous jars by the tissue. So more than one are kept like 100%
alcohol 3 times, xylene 2 times . Changing of solutions depend on tissue load.
Generally fluids to be changed once a week. In case of 100% alcohol,xylene where
more than one jar is there, the last one replaces previous one & first one to be
discarded

4. EMBEDDING
It is the process by which tissue block is converted into paraffin block by
incorporating the tissue into paraffin wax. It is otherwise called as casting or
blocking.

Embedding media – Paraffin wax is commonly used. Others are carbowax,

celloidin embedding.

Moulds used for embedding – Leuckhard L mould made of brass is commonly

used.Other one is plastic mould with a support to fit into microtome directly.

Procedure –L mould consists of one long and one short limb.L moulds are placed
over metal or glass plate.Two L moulds are joined together to form rectangular
box that act as the cast to make wax mould.

Make several boxes with L moulds on a enamel tray.The tissue is placed in the
bottom of the cavity formed by the 2 L moulds with the help of forceps.The
important point to remember during embedding is orientation of the tissue.It
should be kept in such a way that it will provide more information to the
pathologist. Use warm blunt forceps to fix the tissue firmly in place. Hold it flat
with forceps till it retains its position. Then pour the paraffin quickly upto the top
of the mould. Insert the identy tag into the side of wax mould adjacent to the
tissue. Make sure that the tag is securely placed and should not interfere with
knife while cutting.Then transfer the tray to refrigerator or immersed in cold
water for complete solidification and hardening of paraffin. This will take 15-30
minutes.Once it becomes hard the encased L blocks are removed from the base
plate and tapped them on the bench in order to separate it from the plate.Now
the hard paraffin block is ready for cutting.
 4. SECTION CUTTING

Tissues are cut into thin microscopic section from paraffin block by a specialised
instrument called Microtome.The process is called as Microtomy.

There are many types of Microtome namely Rotary microtome ,Rocking

microtome ,Freezing microtome,Cryostat. Fixed & processed tissues can be cut
by the first two microtome whereas fresh unfixed tissues can be cut by the last
two microtome. Commonly used one is Rotary microtome by which thin sections
can be cut.Sectioning efficiency comes with practice and mainly depends on the
knife.So technician should know the care of knife and microtome.

Care & Use of microtome Knife

Knife is a most important component of microtome and requires constant care

and maintenance. It is wedge shaped and made of high grade steel.The three
most important things that ruin the sharpness of microtome knife edge are

1. Trying to cut hard objects --- It is avoided by making the tissue soft by
decalcification in case of bony calcified tissue and treating with alkali (1N
NaOH ) in case of hard skin & epithelial tissues. It is also avoided by using
separate knife for cutting hard objects.
2. Careless storage – Improper storage leads to rust formation & corrosion of
knife. So it should be wiped dry using cloth & oiled lightly after using. Keep
the knife in its box and the storage box to be kept in airy place.
3. Improper handling during sharpening. – The knife should be sharp enough
for good sectioning. It is brought about by honing & stropping. Knife is
provided with a fitted back and handle for doing the procedures of honing
and stropping.

Sharpening of knife

There are two methods of sharpening.

 One is Manual mechanical method by honing & stropping .
 The other is automated method using automatic knife sharpner.
 Automatic method –Advantage - There is even grinding of edge,minimum
time requirement.
Disadvantage –Costly machine, Uncertainty of
electricity,Frequent repair
problem

Manual method -- It includes honing and stropping.

 Honing -- It is done to remove nicks and irregularities from the knife

edge. Grinding the edge of knife on a special stone called “hone”. It is a
rectangular yellow Belgian water stone with a coarse and smooth
side.When the knife has more nicks ,coarse side is used & If knife has
mild nicks, smooth side is used. Fit the back & handle to the knife. See
the knife edge under the microscope and note the irregularities.If the
knife is sharp thin bright line is seen & If broad beam of light is seen
knife has to be sharped.

Procedure –Place the hone on a bench of suitable height and keep a cloth under
the hone to prevent it from slipping during use.Place the knife at one end of hone
with the edge facing away from the operator.Then push the knife diagonally
forward with the cutting edge leading,so that whole edge is equally ground.The
pressure of the knife is just sufficient to maintain the edge in contact with the
surface of hone. Before reaching the other edge of stone, the knife is turned over
on its back without lifting it. Then pull back the knife with the cutting edge leading
towards the operator in a diagonal stroke. Continuously repeat the push & pull
movements .Inspect the knife edge time to time under microscope for the
progress in honing.

 Stropping – It is done to remove the wire edge caused by friction of

the steel on the stone during honing.The strop is made of leather or
linen.It can be flexible or rigid. In rigid type ,the leather strop is
stretched over a solid wooden block.It is commonly used & preferred
 than flexible type because it gives firm support to the knife during
stropping.

Procedure –Clean & dry the knife before stropping.Place the strop on the bench.
Keep the knife at one end of strop & push it forward diagonally with the cutting
edge trailing. Before reaching the other edge the knife is turned over on its back
with cutting edge trailing again and pull back the knife along the strop towards
the operator. Do it in a rhythmic steady flowing motion. 20 -30 double strokes are
sufficient.

Technique of section cutting

1.Trimming of paraffin block – Trim the paraffin block with a knife in such a way
that the sides are parallel & the tissue should be 1-3 mm from the edge on all the
sides.We can reuse the shaved waxes

And so return it to wax oven.The labeled wax blocks are stored in cardboard
boxes .

2.Attaching the paraffin block to the microtome – The paraffin block is attached
directly to microtome head or fixed to the wooden chuck that is clamped to
microtome head. Direct fitting of block can cause crumbling if clamped too
tightly.So better use wooden chuck. Melt the paraffin wax block base using
heated spatula & fix the block to the wooden chuck.Then wooden chuck is
clamped into microtome head without fear of crumbling of block.

3.Orienting the block – Orient the block in a way that top & bottom of the block
should be parallel and horizontal to the edge of knife. The block face should be
small and at least 1mm of paraffin should be present on all sides beyond the
tissue.In the beginning set the thickness to 15 mm and trim the face of the
block.When whole surface is trimmed ,now it is ready for sectioning.

4. Cutting the section – When block face is trimmed ,set the microtome for 5
micron thickness and start the cutting process.Set the angle of knife with a block
correctly. Cool the face of block with ice cube before cutting.In rotary microtome
,rotate the handle that gradually advances the microtome head with a block to
the knife and sections are cut which lie on the knife.Maintain a regular cutting
rhythm at a rate that is comfortable for the technician.Each time the block hits
the knife edge section is cut that will slide onto the knife pushing the previous one
ahead. Thus a ribbon of sections are produced.When the ribbon is 15 cm long
,grasp the first section with fine forceps in the left hand & brush away the last
section using camel hair paint brush in the right hand.Now the ribbon is
transferred to the water bath with a help of two hands.

Technique of attaching sections to the slide

Preparation of slides –Slides has to be prepared before attaching sections . Since

subsequent manipulation & movement of slides in & out of various fluids,stains ,
adhesives must be used to keep the sections firmly fixed to the slide .Otherwise
the sections will be washed away. Commonly used adhesive is Mayer’s glycerol-
egg albumin mixture. It is prepared by mixing equal parts of egg albumin &
glycerol.Add a small crystal of thymol to prevent the growth of fungi & bacteria.

 Slide is prepared by placing a small drop of above mixture over the

slide & smeared it over the slide with a finger.
 Another method is prepare a stock solution of mixture of gelatin 1% &
potassium dichromate 1% and add it to water bath ( 1ml of stock
solution to 500 ml of water).

Procedure – The purpose of putting sections to water bath is to remove wrinkles,

creases formed during section cutting. They should be flattened before attaching
to the slide.The temperature of waterbath is around 46C (10C below the melting
point of wax.).With the help of forceps & camel brush the ribbon is gradually
lowered into the waterbath.The ribbon will float and the wrinkling of sections
should flatten out. The technician inspect the sections and picks the section which
is flattened& fully expanded. Label the slide with the identification number.The
albumenized slides should be inserted obliquely into the water as close to the
section as possible.Slowly withdraw the slide allowing its surface to touch the
edge of section. Remove the slide from water. Adjust the section to suitable
position on the slide with the help of mounting needle.Drain off the excess water.
Keep the slide flat on the table. Then transfer the slide to incubator or hot plate
for 1 hour.That makes the section dry & fixes the section to the slide well. Now
the section is ready for staining.

Resealing of blocks

After sections have been cut, paraffin blocks has to be resealed to prevent tissue
drying & damage by insects. It is done by dipping the block face into the molten
paraffin.

Problems in section cutting

1. Failure of getting ribbon sections

Reasons –Dull knife
Knife is not parallel to the block & tilted too far towards the block
Hard paraffin wax
Too thick sections
Remedy – Unroll the first section with camel hair brush & hold it against the
knife. The ribbon will follow .
Chill the block face using ice cube.
2. Compressed ,wrinkled or jammed sections
Reasons – Dull knife
Too vertical knife tilt
Loose screws of the clamp holding the knife
3. Crumbling of section
Reason – Improper processing
4. Adherance of sections to knife
Reason -- Dull knife
Dirty knife edge
Too vertical knife tilt
5. Varying thickness of sections
Reason – Loose clamp
Insufficient tilt of knife that compress the tissue.
6. Lifting of sections from the knife on the upstroke
Reason – Dull knife
 Too vertical knife tilt
Too Soft paraffin
Too warm environment
7. Split ribbon or lengthwise scratches
Reason -- Nicks in knife edge
Dirty knife edge
Micro calcification in the tissue
8. Accumulation of air bubble under ribbon after it is spread on water bath
Remedy – Gently remove bubble by smooth teasing needle bent at right
angle in water bath.
Gently pull the ribbon along the slide kept below the section in
the water bath.
After the section is mounted on the slide, bubbles in the tissue is
removed by gentle brushing with a fine camel hair paint brush.
 5. STAINING

It means coloring the different components of tissue using

stains.Differentiation of cellular components by staining is necessary for
studying internal structure of tissues. It helps in studying the physical
characteristics and the relationship of tissues & cells.

Materials for Staining

 Coplin Jars – It holds 5-10 slides at a time & stain it.
 Staining baskets -- It holds 60 slides at a time & stain it.
 Staining rack by placing two glass rods about 5cm apart over the sink
and lay the slides flat on the rods. ( Eg. Leishman stain ,Gram stain )

Preparation for Staining( Pre staining procedure)

It includes removal of wax & hydration of section.

1. Drying – Sections are made dry by keeping it in oven. Then proceed for
dewaxing. If this step is not done ,sections will float away during staining
procedure.
2. Dewaxing or Deparaffinizing – It is done by placing sections in two
changes of xylene, 5 minutes in each.Gentle agitation hastens the
dissolving process.
3. Hydration ( Taking sections to water ) – It is done by placing sections in
decreasing grades of alcohol 90%,80%,70% then to distilled water.

Now the slide is ready for staining.

 Special pre staining treatment

A. If the tissue is fixed in chromate fixative, the precipitate must be removed
by treatment with iodine 1% & sodium thiosulphate 5% solution prior to
staining procedure.

1. Dry the section in the oven

 2. Deparaffinize the section in two changes of xylene

3. Hydrate the section in down graded alcohols.

4.Treat the section with 1% alcoholic iodine solution for 10 minutes.

5. Treat the section with 5% sodium thiosulphate solution for 5 minutes.

6. Wash with tap water .Then proceed for staining.

B. Formalin pigment formed by formalin fixative.

Some time due to acid formation in the formalin by insufficient fixative, it

produces fine brown granules in the section called “Formalin pigment”.

It is avoided by 1. Use large volume of fixative. 2. Use buffered formaldehyde as a

fixative.

Suppose this pigment is formed in the section ,It should be removed prior to
staining.

Procedure for removing formalin pigment

1. Dry the section in the oven

2. Deparaffinize the section in two changes of xylene

3. Hydrate the section in down graded alcohols.

4.Treat the section with saturated alcoholic picric acid solution for 1-
3hours

5.Wash well in running tap water. Then proceed for staining.

Another method for removing formalin pigment using hydrogen peroxide

bleaching solution.

( 3%Hydrogen peroxide 25ml + Acetone 25ml + Ammonium hydroxide 1

drop )

. 1. Dry the section in the oven

2. Deparaffinize the section in two changes of xylene

3. Hydrate the section in down graded alcohols.

4.Treat the section with Bleaching solution for 5-10minutes.

5.Wash well in running tap water. Then proceed for staining.

 Post staining procedure

It includes dehydration ,clearing & mounting.

1.Dehydration – It is done by placing sections in ascending grades of
alcohol 79%,95%,absolute alcohol.
2.Clearing – It is done by dipping sections in two changes of xylene.
3.Mounting –Placing mounting media & coverslip over the section is
called as Mounting.

Methods –
a. Place the slide with stained section over the coverslip with mounting
media.
b. Place the mounting media on coverslip which is gently placed over
the section.
c. Place the mounting media on the section & coverslip is gently placed
over it.

 ROUTINE STAINING REAGENTS

Haematoxylin & Eosin is a commonly used staining reagents.
Haematoxylin – It is a natural dye used to stain the nucleus &
cytoplasmic inclusions.
Alum hematoxylin – Here Alum acts as a mordant
Iron hematoxylin -- Here Iron acts as a mordant.

Preparation of Reagents
 Harri’s haematoxylin

Composition – Haematoxylin crystals – 1g

95% Alcohol --10ml
Ammonium or Potassium alum -- 20g
Distilled water --- 200ml
Mercuric oxide --- 0.5g
1. Dissolve the haematoxylin in alcohol in a mortar with a pestle
2. Dissolve the alum in water using heat
3. Mix the haematoxylin solution with alum solution while the latter
is hot.
4. Bring to the boil as quickly as possible. Then add 0.5g mercuric
oxide. The solution turns into dark purple colour.
5. Remove from flame. Cool as rapidly as possible. Filter it. Transfer
to suitable storage bottle. Record the date & label. The solution
stay stable for years.

 Eosin solution
Two types - Alcoholic eosin ,Aqueous eosin based on the solvent
used. Commonly used one is Aqueous eosin Y.
It is used as a counterstain which colors the cytoplasm rose
colored.

 Other reagents used for staining

Dilute aqueous Hydrochloric acid – 0.5%
Dilute Ammonia water --- Strong ammonia 1.5ml +Distilled water
500ml
Or
Saturated Lithium carbonate solution -- Lithium carbonate 5g +
Distilled water 500ml
 STAINING PROCEDURE

1. Deparaffinize the section by placing in two changes of xylene 5

minutes each.
2. Hydrate the sections by treating it with downgraded alcohol
(90%.80%,70%) for 30-60 seconds each.
3. Wash in tap water. Rinse in distilled water.Drain well.
4. Stain in Harris haematoxylin for 5-20 minutes depends on the
batch & age of the stain.
5. Wash in running tap water.
6. Dip in diluted acid solution. It changes the nucleus as dark
purple and the rest as pale called “Differentiation”.Check the
differentiation under microscope in wet stage.If everything is
too dark ,again dip in acid for differentiation. If everything is
pale, again go for haematoxylin staining. If good differentiation
of dark nucleus & pale cytoplasm is seen then proceed further
staining process.
7. Wash in tap water. Then dip in ammonia water or Lithium
carbonate solution for “Blueing”. This will change the section
to a blue colour.
8. Counterstain in eosin solution for 30-60 seconds. Drain it.
9. Dehydration in upgraded alcohols ( 70% ,90%, absolute
alcohol)
10.Clear in xylene with two changes 30-60 seconds each.
11.Drain excess xylene & Mount the section on DPX or Canada
Balsm with a coverslip.

NOTE – Each dip is equal to 10 seconds

 6.SPECIAL STAINS

A. PAS Stain (Periodic acid Schiff stain)

This stain is a useful indicator of presence of carbohydrate particularly

glycogen in the tissues like liver, heart, muscle ,glands.

PRINCIPLE

The periodic acid will bring out oxidative cleavage of carbon to carbon bond to
form dialdehydes. This aldehyde react with Fuschin sulphurous acid which
combine with basic para rosaniline to form magenta colored compound.

Carbohydrate ----------Aldehyde --------------Magenta colour

Other oxidants used are Chromic acid ,Potassium permanganate, Lead tetra
acetate, Sodium bismuthate.

REAGENTS

1.Periodic acid solution 0.5 to 1 % ( Periodic acid 1g + Distilled water 100ml)

2.Schiffs reagent -- It is prepared by

. Boil 200 ml of water. Remove it. Add 1g basic fuschin.

.Cool it to 50C.Add 2g potassium metabisulphite

.Cool to room temperature.Add 2ml of concentrated charcoal.

.Leave it overnight in the dark at room temperature.

.Filter through No 1 Watmann paper.

.Store in a dark container at 4C.

3.Harris hematoxylin

PROCEDURE
1.Dewax the sections on xylene & bring it to distilled water.

2. Treat it with 0.5% periodic acid --- 5minutes.

3.Wash well with several changes of distilled water.

4.Cover the section with Schiff reagent for 15 minutes.

5.Wash in running tap water for 5-10minutes.

6.Stain the nuclei with Harris hematoxylin.Then differentiate in acid alcohol

7.Wash in water.Rinse in alcohol.

8.Clear in xylene.Mount it.

RESULTS

Glycogen and other carbohydrate --- Magenta colour

Nuclei -- Blue

NOTE--- The solution of Schiff is colorless or pale yellow.

Potency of it is checked by adding few drops of reagent to 10%

formalin.If it turns to red purple color we can use the Schiff reagent.If the color is
blue or slow in developing color , it is better to change the Schiff reagent.

USE

1.It is used to demonstrate glycogen in heart,muscle,liver.

2. It is used to demonstrate mucin (Glycoprotein) in gastro intestinal glands.

3.It is used to demonstrate glycoprotein component of basement

membrane,reticulin fibers,hyaline deposits,colloid droplets and amyloid.
B. RETICULIN STAIN

Reticular fibers are fibrillary extra cellular connective tissue. They have affinity for
silver stains.They provide the supporting framework for cellular tissues like
liver,spleen,lymphnode.

PRINCIPLE

Reticular fibers have low natural affinity for silver salts.The affinity is increased by
pre treatment.Subsequent silver treatment makes the tissue to take more silver in
reduced form.Further treatment with reducing agent converts unreduced silver to
metallic silver which is deposited in sensitized sites.Remaining unreduced silver is
removed by sodium thiosulphate.

REAGENTS ( GORDON METHOD)

1. Silver solution ---.It is prepared by

.Take 5 ml of 10% aqueous silver nitrate and add concentrated ammonia
drop by drop until the formed precipitate is dissolved.Take care not to add
excess ammonia.
.Add 5ml of 3% sodium hydroxide solution.
Redissolve the precipitate by adding concentrated ammonia drop by drop
until the solutions retains traces of opalescence.
2. 1% Potassium permanganate
3. 1% Oxalic acid
4. 2.5% Ferric ammonium sulphate (Iron alum)
5. 10% Aqueous formalin
6. 5% Sodium thiosulphate
7. 0.2% Gold chloride
8. Eosin

PROCEDURE

1.Dewax the sections on xylene & bring it to distilled water.

2.Pretreatment
 .Add 1% potassium permanganate solution ----wait for 5minutes.Rinse
in tap water

. Bleech in 1% oxalic acid. Rinse in tap water.

.Treat with 2.5% Iron alum for 15 minutes. Wash with several changes
of distilled

Water.

3.Silver treatment for 2minutes --- Rinse in several changes of distilled

water.

4.Reduction with 10% formalin for 2minutes.Then rinse in tap water.

5.Removal of unreduced silver by treatment with 5% sodium

thiosulphate for 3 minutes.Rinse in tap water.

6.Toning by treatment with 0.2% gold chloride solution for

3minutes.Rinse in water.

7.Counterstain with eosin.

8. Dehydrate. Clear. Mount it with DPX.

RESULTS

Reticuar fibers ---- Black

Nuclei -- Black or unstained

Others --- Pink color

USE

1.It is used to demonstrate the pattern of arrangement of reticular fibers which

give clue for diagnosis in case of undifferentiated tumors.

2.It is used to demonstrate fibrosis in tissues.

C.PERLS STAIN ( PRUSSIAN BLUE)

PRINCIPLE

Unmasking of ferric iron in the tissue to reactive ferric oxide form by Hydrochloric
acid in acid ferrocyanide solution. That ferric oxide reacts with potassium
ferrocyanide solution to form insoluble blue color ferric ferrocyanide.

REAGENTS

Stock solution

1. 2% Potassium ferrocyanide solution ( Potassium ferrocyanide 2g + 100 ml

water)
2. 2% HCL ( Hydrochloric acid 2ml +100 ml distilled water)

Working solution

2% Potassium ferrocyanide 25 ml + 2 % HCL 25 ml.

Freshly prepare the above solution by mixing it.

3. Counterstain --- Eosin or 0.5% neutral red stain. ( 500 mg of Neutral red +
100ml water)

PROCEDURE

1.Dewax the section & bring it to water

2.Immerse the slide in freshly prepared acid ferrocynide solution for 10-
20minutes.

3.Wah well in distilled water.

4.Dip the section in eosin or neutral red. Wash in water.

5. Dehydrate. Clear. Mount it.

RESULTS

Iron pigment like hemosiderin --- Blue color

Nucleus --- Red

USE

 It is used to demonstrate iron pigment deposition in the tissues

(Eg.Hemochromatosis)
 It is used to demonstrate iron storage in the bone marrow

D. VON KOSSA STAIN

PRINCIPLE

Silver is substituted for calcium and form metallic salt.

REAGENTS

1. 5% Silver nitrate solution.

2. 5 % Sodium thio sulphate or hypo solution
3. Counterstain --- Eosin or Neutral red or Light green

PROCEDURE

1.Dewax the section in xylene. Rinse in distilled water.

2. Add 5% Silver nitrate solution and expose it to strong sunlight for 30 minutes.

3. Distilled water wash.

4. Place it in 5% sodium thiosulphate solution for 3 minutes. Tap water wash.

5.Counterstain it

6. Dehydrate. Clear it. Mount the section in DPX.

RESULTS

Calcium deposits --- Black

Background --- Color of counter stain used.

USE

It is used to demonstrate calcium deposition in the tissues.

E. STAIN FOOR AMYLOID –Congo red

Amyloid is a glycoprotein deposited in tissues excessively in some pathological

conditions.It is demonstrated by Congo red, Touldine blue,and crystal violet
stain.It shows metachromacia ( different color staining ).So counter stain is to be
avoided.

PRINCIPLE

Amyloid is a metachromatic tissue that takes up the metachromatic stain and give
red color

REAGENTS

1. Mayers Hematoxylin
2. Saturated sodium chloride in 80% alcohol ( 1g sodium chloride in 100ml
80% alcohol) –Stock satble solution
3. 1% aqueous sodium hydroxide solution
4. Alkaline alcohol-sodium chloride solution ( 50 ml of ethanol sodium
chloride + 0.5ml of 1% sodium chloride). Prepare just before use.
5. Alkaline congo red solution.
Stock solution - Congo red 500mg +300 ml ethanol sodium chloride
solution).Stir well and allowed to stand for 24 hours before use.Filter and
keep in a tightly stoppered container.It is stable for several months.
Working solution – Stock congo red 50ml + 0.5ml 1% sodium
hydroxide.Filter it and use it within 15 minutes.
PROCEDURE

1.Dewax the section in xylene. Rinse in distilled water.

2. Stain the nuclei with Hematoxylin for 10 minutes.

3.Blue in warm running water for 5-10minutes.

4. Distilled water rinse three times.

5.Treat the section with alkaline-alcohol-sodium chloride solution or 20 minutes.

6. Dehydrate in three changes of absolute alcohol. Clear it with xylene. Mount the
section in DPX.

RESULTS

Amyloid --- Deep Pink or Red

Nuclei -Blue.

USE

It is used to demonstrate amyloid in tissues.

Note – Stained section should be seen immediately as mounting media reverse

the metachromacia.

F. TOLUDINE BLUE STAIN

Orthochromatic staining --- Staining of tissue with the same color of dye used

Metachromatic staining --- Staining of tissue with the different color of dye used

Metachromasia ---Tissue elements staining a different color from the dye

solution. It is due to the pH, dye concentration and temperature of the basic dye.

Blue or violet dyes will show a red color shift, and red dyes will show a yellow
color shift with metachromatic tissue elements.
Toludine blue is one of the Metachromatic stain that stains the metachromatic
tissue in different color( Red).

REAGENTS:

Toludine Blue 0.1%

Toludine Blue 100mg + Distilled water 100ml.

Or

Modified Toludine Blue solution

Toludine blue, 1g + 20 ml of 95% alcohol + 80 ml of Distilled water + 1ml of glacial

acetic acid

PROCEDURE:

1. Deparaffinize the section and hydrate to distilled water.

2. Treat the section with Toludine blue - 1-2 minutes.

3. Rinse in distilled water - 3 changes.

4. Dehydrate quickly through 95% and absolute alcohols.

5. Clear in xylene and coverslip it.

RESULTS:

Amyloid, Mast cell granules --- Pink

Background ---- Blue

USE

 It is used as metachromatic stain for demonstrating amyloid and tissue

basophil.

G.FAT STAIN
Fat is one of the normal constituent of cells either as free form, or as
combined form like glycolipids,lipoproteins,phospholipids.Fat is soluble in
alcohol ,so routine processing will wash out the lipids. Routine fixatives like
formalin also will not preserve lipids well.So unfixed frozen section is ideal
for Fat demonstration.

Stains – Sudan IV , Oil red O,Sudan black B

Sudan IV stain
Principle
Oil soluble colorants stains the simple as well as complex fat in tissues
Reagents
Sudan IV stain – Sudan IV - 1g + Acetone 50ml + 70% alcohol 50ml.
Harris hematoxylin
Glycerine jelly

Procedure
Take the frozen section to 70% alcohol rapidly
Stain with Sudan IV for 5 minutes
Place it in 70% alcohol briefly.
Counter stain with harris hemtoxylin for 3 minutes
Wah in tap water. Mount in glycrine jelly.

Result
Fat globules – Bright red
Nucleus-Blue

Use
It is used for demonstration of fat in tissues – Lipma,Fat embolism in vessels
 7.STAINING FOR MICRO-ORGANISM

A. GRAM STAIN

PRINCIPLE

Bacterial (Both positive and negative) cell wall is composed

of peptidoglycan,(the gram-positive has a thicker wall) and both will take

up the crystal violet. The gram-negative bacteria has a layer of

lipopolysaccharide external to the peptidoglycan wall, which is disrupted

in the acetone rinse, allowing the crystal violet to be differentiated out.

This allows the gram-negative bacteria to take up the basic fuchsine

stain.

REAGENTS:

1. 1% Crystal Violet:

Crystal violet 1.0 gm

Distilled water 100.0 ml

Filter into a dropper bottle. Stable for 1 year.

2. Lugol's Iodine:

Stock solution is prepared by mixing the following well

Iodine 1g

Potassium iodide 2g

Distilled water 10ml

Working Solution –
For Gram iodine -- Add 300 ml of distilled water to stock solution

For Lugols iodine -- Add 100 ml of distilled water to stock solution

3.Acetone:

4.Counterstain – 1% Neutral red.

PROCEDURE:

1. Deparaffinize the section and hydrate to distilled water.

2. Place slides on staining rack, drop crystal violet stain onto tissue section, stain
for 2 minutes.

3. Wash in tap water.

4.Treat with Lugol's iodine for 2 minutes.

5. Wash in tap water.

6. Blot sections dry, breath on section then quickly pour acetone over section
until no color runs off.

7. Wash in tap water.

8. Counter stain with Neutral red for 3 minutes.

13. Air dry. Dehydrate. Clear by dip into xylene, and mount it with coverslip.

RESULTS:

Gram-positive bacteria -- blue

Gram-negative bacteria -- red

Nuclei --- red

USE
  For demonstrating gram-negative and gram-positive bacteria in
tissue.

B. ZIEHL-NEELSEN STAIN FOR AFB

Mycobacteria possess a capsule containing fatty acid ( Mycolic acid) which resist
the penetration of acid and alcohol. So it is very difficult to demonstrate it by
Gram stain.It needs AFB staining.

PRINCIPLE:

Phenolic acid and heat are used to reduce the surface tension, increasing porosity
and forcing the penetration of the dye into the capsule & stain the bacteria.

As the causative agents for Leprosy (Mycobacterium leprae) and Nocardiasis

(Nocardia asteroides) are much less acid and alcohol fast than Mycobacterium
tuberculosis bacilli, a more gentle dewaxing and minimal exposure to organic
solvents is required for adequate staining.

REAGENTS:

(1) CARBOL FUCHSIN

Basic Fuchsin 1 gm

Absolute alcohol 10 ml

Add the Basic fuchsin to the alcohol in a 100 ml flask and mix, on a magnetic
stirrer for 30 minutes.

Add 100 ml of 5% aqueous phenol and mix till dissolved. Filter and store in a
brown glass bottle.

(2) STOCK METHYLENE BLUE

Methylene Blue 2 gm

Using a magnetic stirrer, dissolve it in 100 ml distilled water

Then add absolute alcohol 100 ml

(3) WORKING ACIDIFIED METHYLENE BLUE

Stock Methylene Blue 40 ml

Distilled water 60 ml

Glacial acetic acid 0.5 ml

(4) 0.5% ACID ALCOHOL

Distiller water 700 ml

Absolute alcohol 300 ml

Hydrochloric acid 5 ml

PROCEDURE:

(1) Heat the slides in slide dryer to facilitate dewaxing or bring section to xylene.

(2) Hydrate through down graded alcohols.Take sections to water.

(3) Drain slide and Flood the section with Carbol Fuchsin.

(4). Heat it to steaming for 15 minutes OR Keep it in a oven at a temperature of

50-60C for 30 minutes.

(5) Wash well in running water, wipe away excess stain .

(6) Differentiate with 0.5% Acid Alcohol, for 10 minutes till sections are pale pink

(7) Wash well in water, for 5 min

(8) Counter stain with methylene blue for 30 seconds.

(9) Wash with tap water.

(10) Drain and air dry thoroughly.

(11) Dip slides in xylene and mount in DPX.

RESULTS:

Acid Fast Bacilli ---- RED (Leprae bacilli are short rods)

Nocardia ---RED (Nocardia organisms are long, thin, and filamentous)

Erythrocytes --- PALE PINK

Background --- BLUE

USE

 For demonstration of Mycobacteria

C. WADE-FITE FARACO STAIN --- FOR LEPRA BACILLI.

The procedure is same as AFB staining with little changes. In this method oil
mixture is used prior to carbol fuschin treatment for removing wax.

PROCEDURE:

(1) Warm the section & dewax it by oil treatment using 1 part of groundnut oil & 2
parts of xylene for 10 minutes.

(2) Take sections to water.

(3) Drain slide and Flood the section with Carbol Fuchsin at room temperature for
30 minutes.

(4). Wash well in running water, Blot dry.

(5) Decolorize in 10% Sulphuric acid. Wash well in water.

(8) Counter stain with methylene blue for 30 seconds.

(9) Wash with tap water.

(10) Drain and air dry thoroughly.

(11) Dip slides in xylene and mount in DPX.

RESULTS:

Acid Fast Lepra Bacilli ---- RED short rods

Background & Nuclei --- BLUE

D. GMS - GROCOTT'S, METHENAMINE SILVER STAIN FOR FUNGI

PRINCIPLE: The mucopolysaccharide components of the fungal cell wall

are oxidized to release aldehyde groups. The aldehyde groups then react

with the silver nitrate, reducing it to a metallic silver, rendering them

visible as black structure.

REAGENTS:

1. 5% Borax:

Sodium borate 5.0 gm

Distilled water 100.0 ml

Solution is stable for 3 months.

2. Methenamine Silver

Stock Solution:

3% Methenamine(hexa methylene tetramine)100.0 ml

5% Silver nitrate 5.0 ml

Mix slowly till the precipitate first formed dissolves well. Pour it into an acid
cleaned brown bottle. Store in the refrigerator. Solution is stable for 3 months.
Working Solution:

It is prepared by adding 5 ml of Borax solution + 25 ml of Methanamine silver

solution + 25 ml of Distilled water.All solutions are pre heated to 56C before
mixing to avoid degeneration of Silver nitrate.

3. 5% Chromic Acid or 1% Periodic acid

4. 1% Sodium Metabisulfite:

Sodium metabisulfite 5.0 gm

Distilled water 500.0 ml

Mix it and the solution is stable for 6 months.

5.0.1% Gold Chloride:

Gold chloride 0.1 gm

Distilled water 100.0 ml

6.Hypo or Sodium thiosulphate 2%

7.Counter stain 0.2% Light Green:

Light green SF yellow. 0.2 gm

Distilled water 100ml

PROCEDURE:

1. Deparaffinize the section and hydrate to distilled water.

2. Oxidize with 5% Chromic acid --- 10minutes.

3. Wash in tap water.

4. Rinse in 1% Sodium metabisulfite, to remove traces of chromic acid.

5. Wash in tap water, rinse in distilled water 3 changes.

6.Stain with Working methenamine silver solution in oven (58-60C) for 30-
60minutes. Tissue should be yellow brown color.

7. Rinse in distilled water, 2 changes.

8. Tone in 0.1% Gold chloride, 2 minutes or until gray.

9. Wash in distilled water.

10.Remove unreduced silver by treating with 2% Hypo, for 3 minutes.

11. Wash in tap water.

12.Counter stain with Light green for 1 minute.

13. Rinse in distilled water.

14. Dehydrate, clear, and coverslip.

RESULTS:

Fungi ---- black

Background ---- green

USE

 For demonstration of Fungus in the tissues.

E. GEIMSA - HARLECO RAPID - FOR HELICOBACTER

The combination of the azure dyes ( methylene blue and

Eosin) are also known as Romanowsky stains, that give a wide color range.

REAGENTS:
 1. Stock Solution:

Geimsa powder 4g + Glycerol 250ml +Methyl alcohol 250ml

Dissolve the powder in glycerol at 60C with regular shaking. Then add
methanol.Shake well. Allow it to stand for 7 days. Filter before use.

Working Solution:

Stock solution 1.5ml + Methanol 1.5ml + Distilled water 50ml

2. 0.5% Acetic Acid:

Acetic acid 1.0 ml +Distilled water 200 ml.Mix well, stable for 1 year.

PROCEDURE:

1. Deparaffinize the section and hydrate to distilled water.

2. Stain with Geimsa overnight.

3. Rinse in distilled water, 2 dips, 1 second each.

4. 0.5% acetic acid rinse till section is pink.

5. Tap water wash.

6. Dehydrate. Clear in xylene and coverslip it.

RESULTS:

Helicobacter & Some Parasites --- Blue

Background Tissue elements --- shades of blue and pink

Nuclei --- Blue.

USE:

 It is used to demonstrate Campylobacter pylori & some parasites

 It is used to Differentiates cells present in hematopoietic tissue
(lymph nodes), used in blood smears.
 8.Frozen Section
Definition – It is a technique by which sections are cut within few minutes from
fresh tissue without fixation using freezing microtome.

Advantages:

1. For Rapid diagnosis

2. For Intra operative surgical consultation
3. For histochemical study to demonstrate certain substances which may be
lost in routine paraffin sectioning.

Disadvantages:

1.Structural details are distorted due to lack of embedding

2.Serial sections can not be taken

3.Staining is not so satisfactory as fixed tissue.

4.Freezing artifact can occur.

5.Special stains can not be used.

Steps involved in Frozen sectioning:

1.Tissue preparation

 Tissue block is cut & trimmed to suitable size with 5mm thickness.
 It can be fixed by keeping it in a test tube or beaker with fixative and boil it
for 30-60 seconds.Then washed in distilled water.
 Unfixed tissue can be used for special staining procedure

2.Freezing of tissue

 Keep the tissue block with few drops of water over the freezing stage of
microtome with cutting surface facing up & parallel to the knife edge.
 Hold the block with finger or tissue holder and turn on the carbon dioxide
gas slightly.
  Once the block is fixed to the object disc, release more carbon dioxide till
the tissue is frozen.
 Once it is frozen ,object disc is inserted into the microtome object clamp.
 Knife edge,tissue block position, antiroll plate is adjusted and tighten the
clamp on object holder securely.
 Section thickness is adjusted to 10-15 microns.

3.Section cutting (Freezing microtomy)

 Pull the stage holding knife forward to allow 5mm clearance between block
and knife edge.
 Drive wheel lock is released and line up the block & knife parallel & 1-2mm
distance.
 When cutting surface is in contact with knife, release the ratchet from
micrometer wheel.
 Turn the wheel with the hand to move the tissue till knife begins to cut
sections.
 It cuts individual sections and no ribbon formation.
 Section will glide smoothly and flat beneath the anti roll plate. Use camel
hair brush to take the section.

Note :

1.If freezing is more (hard)– Sections may shatter

2.If freezing is less (soft) – Sections may scatter or fracture

3.Section thickness is 10-15microns.

4.Mounting of Frozen section

 Sections should be handled carefully. Anti roll plate is flipped back after
section is cut.
 One edge of slide is rested on the knife surface 1 inch beyond the section
and the other end is lowered till it is 0.5-1mm from the knife face.
 Sections will be automatically transferred from cold knife to warm slide.
  Never press the slide over the section.
 Frost mark on the knife surface is cleaned with gauze, dry and reposition
the anti roll plate for cutting another section.

Note:

No adhesive is needed for holding the sections of unfixed tissue (fixed tissue need
albuminized slides).Air drying for 30-60 seconds is sufficient to hold the sections.

5.Staining of frozen section

 Fix the air dried section in pure acetone for 15-20 seconds or in formol-
alcohol for 30-60 seconds
 Place it in water until no longer greasy or cloud.
 Place the section in Harris hematoxylin for 1-2 minutes
 Water wash with agitation for 5-10seconds
 Dip in 0.5% sodium borate till blue
 Place it in 70% alcohol for 5 seconds
 Counterstain with 1% alcoholic eosin -1-2 dips
 Wash well in running water
 Dehydrate through graded alcohol (80%,90%.100% )
 Clear it and mount the secion in DPX.

Alternate method:

 Place the section in Harris hematoxylin for 3-10 minutes

 Water wash in running tap water
 Differentiate in acid alcohol(1ml Hydrochloric acid +100ml 70% alcohol)
 Dip in ammonia water till blue
 Counterstain with 1% alcoholic eosin -1-2 dips
 Dehydrate through graded alcohol (80%,90%.100% )
 Clear it with xylene and mount the secion in DPX.
 Museum technique
It means preparation & preservation of the rare Interesting specimen and
mounting it in museum jars in such a way that pathological lesions are clearly
visualized for student learning purposes.
Materials required
Mounting jars of different sizes made of glass or Acrylic, Acrylic mounting plates,
Hand Driller machine, needles, suturing material- Nylon or Linen, Scissor,
Anobond pasting gum.
Reagents -Keiserlings solution – Museum mounting solution
Preparation of Keiserlings solution
 Glycerol -1.5L
 Formalin -15ml
 Sodium meta bisulphate – 500mg
 Tap water - 50L
Procedure:
1. Select the Pathological specimen to be mounted.
2. Cut & Trim it in such a way that pathological lesion is clearly visualized
without much disturbing the organ identity of the specimen.
3. Select the suitable size museum jars to keep the specimen inside it.
4. Cut the acrylic plate into suitable size for that jar & specimen.
5. Orient the specimen over the plate & mark the sites to be sutured over the
acrylic plate
6. Make holes in the acrylic plate using driller.
7. Fix the specimen with proper orientation of tissue over the plate by
suturing the tissue with the acrylic plate through the holes.
Note- Pathological lesion should not be disturbed by suturing material.
8. Keep the plate inside the jar & fill the jar with museum mounting solution.
9. Close the jar with lid.Pinch of thymol or camphor my be added to prevent
fungal growth and clarity of solution.
10.Label the jar with appropriate museum number, and tissue diagnosis
11.Then museum jar is kept in museum rack which is arranged as system wise
pathological specimen.
12.Filing of the slides of corresponding specimen in museum slides rack and
updating the museum specimen in system also for statistics & identity.