MADE BY:

DR. KALPAJYOTIBHATTACHARJEE

COUNTERSTAIN

DEFINATIONSEXAMPLES OF COUNTERSTAINUSES OF COUNTERSTAINH&E STAINVERHOEFF’S HAEMATOXYLINORANGE GGRAM’S STAINZIEHL-NEELSEN STAINGIMENEZ STAINNEUTRAL REDIHC COUNTERSTAIN

CONTENTS

STAINS : Are substances used to impart color to

tissues or cells, to facilitate microscopic study & identification.

STAINING : It is the artificial coloration of a

substance to facilitate examination of tissues ,microorganisms, or other cells under the microscope.

COUNTERSTAIN :

A stain applied to render the effects of another stain more discernable.

1) EOSIN counterstain to haemotoxyllin in H&E stain.

2) Malachite green counterstain to the fuschine stain in Gimenez staining technique.

3) In Gram’s staining- safranin counterstain is applied which stains all cells even following the identification of gram –ve bacteria as well.

4) PAS- Orange G

EXAMPLES OF COUNTERSTAIN

5) Ziehl- Neelson technique- Methylene blue

6) Neutral Red.

7) Verhoeff’s stain

8) IHC counterstain- chromogenic fluorescent

..

Used to differentiate one component or cellular structure from another or to differentiate an entity from another in a specimen.

It does so by coloring a portion of a specimen that remained uncolored following the 1st staining using a dye of different color.

USES OF COUNTERSTAIN

Most widely used stain. It clearly demonstrates an enormous

number of different tissue structures Haematoxylin : Stains cell nuclei blue – black Good intra nuclear detail Eosin : Stains cell cytoplasm & most connective

tissue fibers.

Haematoxylin & Eosin stain :

HAEMATOXYLIN

Extracted from : Heartwood (logwood) of the tree Haematoxylon campechianum that originated in the Mexican State of Campeche.

It is extracted from logwood with hot water & then precipitated out from the aqueous solution using urea.

Haematoxylin itself is not a stain Its major oxidation product is hematein- natural dye

responsible for the color properties. Hematein is produced in two ways a)Natural oxidation- exposure to light & air. slow process- 3-4 months. E.g- Ehrlich’s and Delafield’s hematoxylin. oxidation

b)Chemical oxidation- using sodium nitrate (Mayer’s hematoxylin) or mercuric oxide (Harris heatoxyllin).

ready for use immediately after preparation shorter life than natural oxidation one.

Mordents used- salts of aluminum, iron and tungsten.

TYPES OF HEMATOXYLIN: Alum hematoxylins Iron hematoxylins Tungsten hematoxylins Molybdenum hematoxylins Lead hematoxylins Hematoxylin without mordant

Counter stain EOSIN-Most suitable stain to combine with an

alum hematoxylin.It is a xanthene dye Variants: a) Eosin B- (eosin bluish, erythrosin B)

dibromodinitro derivative of flurorescein b) Eosin Y- (eosin yellowish, eosin water-

soluble) tetrabromo derivative of fluorescein.

c) Ethyl Eosin- (Eosin S, eosin alcohol soluble)

• Eosin Y is most widely used, satisfactorily soluble in alcohol- “water and alcohol soluble”.

• As a counterstain- 0.5 - 1% aqueous solution water – soluble solution eosin is generally preferred.

• Differentiation of eosin staining occurs in the subsequent tap water wash, and a little differentiation occurs during the dehydration through the alcohol.

• Ethyl eosin and eosin B are rarely used. E.g.- Harris stain for negri bodies.

USES OF EOSIN:Its ability with proper differentiation, to

distinguish between the cytoplasm of different types of cell, and between the different types of connective tissue fibers and matrices, by staining them differing shades of red and pink.

Counterstain to haemotoxylin in H&E staining- cytoplasm stains pink-orange & nuclei stained darkly blue-purple.

Stains RBC intensely red.

Method :

1) Remove paraffin wax with xylene, 5 minutes.

2) Treat with absolute alcohol, two changes, 1 minute each.

3) Wash in water.

4) Stain in haematoxylin, 10 minutes.

5) Wash in water.

6) If regressive stain is used, differentiate in acid / alcohol until only nuclei remain blue.

7) Wash in water.

8) Blue in running water - 10 minutes.

9) Rinse in water.

10) Stain in 1 % aqueous eosin for 10-15 seconds.

11) Wash in running water for 30 seconds.

12) Dehydrate in 3changes absolute alcohol for 1 minute.

13)Clear in 2changes xylene.

14)Mount in suitable synthetic resin.

Results :

NucleiRNA rich cytoplasm :Blue calciumMuscle, fibrin , keratin : Bright

redCollagen : PinkRed blood cells :

Orange/ red

Useful stain to demonstrate elastic fibers.PURPOSE: This stain is useful in demonstrating atrophy

of elastic tissue in cases of emphysema, and the thinning and loss of elastic fibers in arteriosclerosis, and other vascular diseases.

With increasing age, changes such as splitting and fragmentation occur, these changes are most obvious in the skin which becomes wrinkled and rather 'loose-fitting'

VERHOEFF’S HAEMATOXYLIN:

PRINCIPLE: The tissue is stained with a regressive

hematoxylin, consisting of ferric chloride and iodine. The differentiating is accomplished by using excess

mordant (ferric chloride) to break the tissue-mordant dye complex.

The dye will be attracted to the larger amount of mordant in the differentiating solution and will be removed from the tissue.

The elastic tissue has the strongest affinity of the iron hematoxylin complex and will retain the dye longer than the other tissue elements.

CONTROL: Artery or skin.

Reagent : 5 % haematoxylin in absolute alcohol : 20

ml 10% ferric chloride : 8

ml Verhoeff’s iodine : 8

ml

Method :

1) Bring sections to water.

2) Stain in Verhoeff’s iron haematoxylin for 15-30 mins until sections are jet-black.

3) Differentiate in 2% ferric chloride.

4) Wash in water, then in 95% alcohol to remove iodine coloration.

5) Wash in water for 5 mins6) counterstain in Van Gieson’s stain for 3 mins.7) Dehydrate rapidly, clear and mount in

synthetic resin.

Results : Elastic fibers : Black Collagen : Red Muscle : Yellow Nuclei : Black

Result: Elastic fibres - black; collagen - red; cytoplasm and muscle - yellow

The universal stain for cytological preparation is the Papanicolaou stain.

Pap staining is used to differentiate cells in smear preparations of various bodily secretions.

REAGENTS:Harris’s hematoxylinOrange G6 Counterstain (OG5 OG8)EA 50(Eosin Azure) Counterstain

ORANGE G

ORANGE G:The main use of Orange G is in the OG-6

 Papanicolaou stain, to stain keratinIt is also a major component of the Alexander

test for pollen staining.It is often combined with other yellow dyes

and used to stain erythrocytes in the trichrome methods.

EOSIN AZURE:EA (Eosin Azure) counterstain, comprising

three dyes; the number denotes the proportion of the dyes, e.g. EA-36, EA-50, EA-65.

a) Eosin Yb) Light Green SFc) Bismarck brown Y.

The Gram staining method is named after the Danish bacteriologist Hans Christian Gram (1853 –1938) who originally devised it in 1882.

It is a differential staining method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls.

Gram’s staining

 Principle:Gram staining is a differential staining technique that

differentiates bacteria into  two  groups: gram-positives and gram-negatives.

The procedure is based on the ability of microorganisms to retain  color of the stains  used during the  gram stain reaction.

Gram-negative bacteria are decolorized by the alcohol,  losing  the  color of  the primary stain, purple.

Gram-positive  bacteria  are  not  decolorized by alcohol and will remain as purple.

After  decolorization step, a counterstain is used to impart a pink color to the decolorized gram-negative organisms.

Gram-positive bacteria have a thick mesh-like cell wall which is made up of peptidoglycan (50-90% of cell wall), which stains purple.

  Peptidoglycan is mainly a polysaccharide composed of two subunits called N-acetyl glucosamine and N-acetyl muramic acid.

As adjacent layers of peptidoglycan are formed, they are cross linked by short chains of peptides by means of a transpeptidase enzyme, resulting in the shape and rigidity of the cell wall.

The thick peptidoglycan layer of Gram-positive organisms allows these organisms to retain the crystal violet-iodine complex and stains the cells as  purple.

Lipoteichoic acid (LTA) is another major constituent of the cell wall of Gram-positive bacteria which is embedded in the peptidoglycan layer.   It acts as regulator of autolytic wall enzymes.

Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall) and lose the crystal violet-iodine complex during decolorization with the alcohol rinse, but retain the counter stain Safranin, thus appearing reddish or pink.

They also have an additional outer membrane which contains lipids,  which is separated from the cell wall by means of  periplasmic space.

Gram staining consists of four components:

Primary stain (Crystal violet, methyl violet or Gentian violet)

Mordant (Gram's Iodine) Decolourizer (ethyl alcohol, acetone or 1:1

ethanol- acetone mixture) Counterstain (Dilute carbol fuchsin, safranin

or neutral red)

Procedure: 1)The smear on a glass slide is covered with

few drops of one of the primary stains. Gentian violet is a mixture of methyl violet and crystal violet. The primary stain renders all the bacteria uniformly violet.

2) After a minute of exposure to the staining solution, the slide is washed in water.

3)The smear is treated with few drop of Gram's Iodine and allowed to act for a minute. Gram's iodine serves as a mordant.

4) The slide is again washed in water

5) decolorized in absolute ethyl alcohol or acetone. A

mixture of ecetone-ethyl alcohol (1:1) can also be used for decolorization.

6) After the smear is decolorized, it is washed in water without any delay.

7) The smear is finally treated with few drops of counterstain such as dilute carbol fuchsin, neutral red or safranin.

8)The slide is washed in water; excess water is removed using a blotting paper, dried in air and heat

fixed before observing under microscope.

Gram-positive bacteria Gram-negative bacteria

Safranin (also Safranin O or basic red 2) a biological stain used

in histology and cytology Use: a) as a counterstain in some staining

protocols, colouring all cell nuclei red. b) This is the classic counterstain in

both Gram stains, and endospore staining. c) It can also be used for the detection

of cartilage, mucin and mast cell granules

Carbol fuchsin:Mixture of phenol and basic fuchsin.

USES:in bacterial staining procedures.Component of Ziehi- Neelson Stain.Used as a dye to detect acid fast bacteria

because it is more soluble in the cell wall lipids than in the acid alcohol.

Topical antiseptic.

The Ziehl–Neelsen stain, also known as the acid-fast stain

PURPOSE: Used in the demonstration of acid-fast

bacteria belonging to the genus 'mycobacterium', which include the causative agent for tuberculosis.

Ziehl–Neelsen stain

PRINCIPLE: The lipoid capsule of the acid-fast organism

takes up carbol fuchsin and resists decolorization with a dilute acid rinse. The lipoid capsule of the mycobacteria is of such high molecular weight that it is waxy at room temperature and successful penetration by the aqueous based staining solutions (such as Gram's) is prevented.

CONTROL: Any tissue containing acid-fast organisms

FIXATIVE: 10% formalin

ReagentsCarbol fuschin (basic dye)Mordant (heat)20% sulphuric acid (decolorizer)Methylene blue (counter stain) or Malachite

green

ProcedureFix the smear of the specimen over the glass

slide, either by heating or alcohol fixation.Pour carbol fuschin over smear and heat

gently until fumes appear. Do not overheat and allow it to stand for 5 minutes, then wash it off with water.

Pour 20% sulphuric acid, wait for one minute and keep on repeating this step until the slide appears light pink in color. Wash off with water.

Pour methylene blue, wait for two minutes, again wash with water

Allow it to air dry and examine under oil immersion lens.

ResultAcid fast bacilli - pink, straight or slightly

curved rods, at times having beaded appearance.

Background - blue due to methylene blue.

Methylene blue heterocyclic aromatic chemical compound molecular formula C16H18 N3SClAt room temperature- solid, odourless, dark

green powder that yields a blue solution when dissolved in water.

USES:redox indicator in analytical chemistry-

indicates the presence or absence of oxygen.Intravital and supravital staining of nerve

fibersAntidote to potassium cyanide poisonongTreatment of methemoglobinemiaplacebo

Gimenez staining technique uses biological stains to detect and identify bacterial infections in tissue samples.

Basic fuchsin stain in aqueous solution with phenol and ethanol colours many bacteria (both gram positive and Gram negative) red, magenta, or pink.

A malachite green counterstain gives a blue-green background cast to the surrounding tissue.

Gimenez stain

MechanismGimenez stain is made up of basic fuchsin,ethanol

and phenol,sodium Di-hydrogen phosphate and Di-sodium hydrogen phosphate.

Gimenez stain has a property to stain both type of bacteria that is Gram positive as well as Gram negative bacteria.

Basic fuchsin stains negatively charged cytoplasm and stains cytoplasm in red colour.

Where as when we treat smear with Malachite green and as we know malachite green is a basic dye it has a strong affinity towards host cell material as compared to cytoplasm and so it stains host cell material that is background of cell in bluish green colour.

METHOD1. Sections to water.2. Filter on carbol fuchsin solution for 1 minute.3. Wash well in water.4. Stain with 1% malachite green for up to 45

seconds.5. Wash well in water.6. Repeat step 4 until sections appear blue/green.7. Wash, blot dry, and dry in air.8. Mount sections in DPX

RESULTSBacteria- red/magentaBackground- blue/green

Malachite green:Malachite green is an organic

compound that is used as a dyestuffFirst prepared by Fischer in 1877USES:used as a dye parasiticide and antibacterial.used in endospore stainingMG has frequently been used to catch thieves

is a dye used for staining in histology.It stains lysosomes red.It is used as a general stain in histology, as

a counterstain in combination with other dyes, and for many staining methods.

Neutral red 

PRINCIPLE :The neutral red (NR) assay procedure is a cell

survival/viability assay based on the ability of viable cells to incorporate and bind neutral red within lysosomes.

It is generally performed on adherent cells. NR is a weak cationic dye that readily penetrates the

cell membrane and accumulates intracellularly in lysosomes (lysosomal pH < cytoplasmic pH), where it binds with anionic sites to the lysosomal matrix .

It is thus possible to distinguish between viable, damaged, or dead cells, which is the basis of the assay.

USES:used as a general stain in histology, as

a counterstain in combination with other dyes, and for many staining methods

Stains Golgi apparatus in cells and Nissl granules in neurons.

Added to some growth media for bacterial and cell cultures.

acts as a pH indicator, changing from red to yellow between pH 6.8 and 8.0.

  as a chloride salt

After immunohistochemical staining the target antigen, a second stain is often applied to provide contast that helps the primary stain stand out.

IHC COUNTERSTAIN

Mayers Hematoxylin is one of the most common dyes used in diagnostic histology, as well as a common nuclear counterstain in IHC.

Nuclear fast red, also called Kernechtrot dye, is also a nuclear stain. Differences between this dye and hematoxylin, though, are that Nuclear fast red dyes nucleic acids, results in red nuclear staining and only takes 5 minutes to stain, instead of an hour with hematoxylin.

Methyl green is also a nucleic acid dye that rapidly stains the nuclei green.

Chromogenic Counterstains

Methyl green

From blue to pink – Although not so often used as a counterstain, another common blue dye is toluidine blue.

This is a strong basic dye that stains nuclei a deep blue colour; however, it will also stain polysaccharadies a pink/red colour.

This colour shift is called metachromasia, a term used when a dye stains a tissue component a different colour to the dye solution.

Eosin can also be used as a counterstain when an antibody localized to the nucleus is used.

Eosin is an anionic dye and works best in acidic conditions.

As nearly all proteins contain these two amino acids, eosin is bound by the majority of structures in any tissue.

A ‘golden standard’ for fluorescence: DAPI.

DAPI (4', 6-diamidino-2-phenylindole) and Hoechst are common nuclear dyes used for fluorescent IHC because they intercalate into the DNA to give a strong blue color under UV excitation.

Propidium iodide is another nucleic acid dye that is frequently used to dye the nucleus red.

Fluorescent Counterstains

References :Culling.C.F.A ,Allison .R.T &

Barr.W.T,Cellular Pathology Technique; Butterworths; Fourth ed, Page no.211 -312.

Bancroft.J.D & Gamble .M, Theory & practice of histological techniques; Elsevier,China; Sixth ed, Page no 121-183.