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Histochemical stains-2 Histochemical stains-2

MR G.P. TIWARIMR G.P. TIWARI Tata Memorial HospitalTata Memorial Hospital

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Outline of the lectureOutline of the lecture

Technical aspects of histochemical stains Technical aspects of histochemical stains and its applicationand its application

Stain principleStain principle Control source Control source Procedures and preparationProcedures and preparation Its results Its results basic troubleshootingbasic troubleshooting

AFBAFB

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The Ziehl-Neelsen stain, also known as the acid-fast stain, was first described by two German doctors; Franz Ziehl a bacteriologist and Friedrich Neelsen a pathologist. IN 1882

ApplicationIT IS USED TO DEMONSTRATION

OF ACID FAST BACILLI BELONGS

TO GENUS MYCOBACTERIUM

TUBERCULOSIS AND M. LAPRAE.

CONTROL: AFB CONTAINING

TISSUE

PrinciplePrincipleMycobacterium( mycolic acid and lipids)Mycobacterium( mycolic acid and lipids)

These are ß-hydroxy carboxylic acids These are ß-hydroxy carboxylic acids

it has a chain lengths of 90 carbon atomit has a chain lengths of 90 carbon atom

The acid-fast ness depend on The acid-fast ness depend on

ability of mycobacterium to retain dyeability of mycobacterium to retain dye

acid solutionacid solution

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Preparation of reagentsPreparation of reagents1) Zeil Neelson carbol fuchsin solution1) Zeil Neelson carbol fuchsin solution

Basic fuchsin--- 1 gmBasic fuchsin--- 1 gm

Absolute alcohol-10mlAbsolute alcohol-10ml

5% phenol solution-100ml (5 grams phenol dissolved in 5% phenol solution-100ml (5 grams phenol dissolved in 100ml of distilled water100ml of distilled water Mix well. Filter into brown Mix well. Filter into brown

color bottle.color bottle.

2)1% acid alcohol2)1% acid alcohol

3)1% Methylene blue3)1% Methylene blue

PROCEDUREPROCEDURE

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1) Bring section to water. 2) Filter ZNCF solution over the sections for 30 minutes. 3) Wash with water for 10 minutes. 4) Differentiate with1% acid alcohol till decolorized. 5) Wash well under running water 6) Counterstain with 1% Methylene blue-1 dip 7) Wash, dehydrate, clear and mount in DPX.

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ResultsResults

Acid fast bacilli-bright redAcid fast bacilli-bright red

Background-light blueBackground-light blue

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Improper decolorization

It will lead to false positive results to overcome this problem proper decorization must carried out

FITE AND FARACO FITE AND FARACO MODIFICATIONMODIFICATION

IT WAS IN YEAR 1940 FITE AND IT WAS IN YEAR 1940 FITE AND FARACO HAVE MODIFIED AFB FARACO HAVE MODIFIED AFB TECHNIQUE FOR DELICATE TECHNIQUE FOR DELICATE

MYCOBACTERIUM LEPRAY BACILLI MYCOBACTERIUM LEPRAY BACILLI

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MODIFICATIONMODIFICATION

       MYCOBACTERIUM LAPREY BACILL IS MYCOBACTERIUM LAPREY BACILL IS LESS ACID FAST THEN AFBLESS ACID FAST THEN AFB

DEPARAFFINIZATION WITH MINERAL DEPARAFFINIZATION WITH MINERAL OIL( 2PART OIL 1PART XYLENE)OIL( 2PART OIL 1PART XYLENE)

5% H2SO4 SOLUTION (DECOLORIZATION)5% H2SO4 SOLUTION (DECOLORIZATION)

RemarkRemark

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We must filter the ZNCF stain before staining with watman filter paper

Proper decorization must be carried outAfter this thorough washing is required to

remove acid.We must filter methylene blue with watman

filter paper during counterstaineCounterstaining should be light otherwise it

will mask the bacilli.This procedure can be performed either using

heat or without using heat

GMSGMS

It was first described It was first described by Gomori in1937 by Gomori in1937

later on it was later on it was modified by Grocotts modified by Grocotts

in 1955in 195504/11/2304/11/23 1313

APPLICATIONAPPLICATION

This technique is This technique is mainly used to mainly used to

identify fungi in the identify fungi in the sections sections

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PrinciplePrinciple

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The mucopolysaccharide component of fungal cell wall is oxidized to release aldehyde groups. The aldehyde groups react with silver nitrate and silver stain precipitates along the fungal walls. CONTROL: Any tissue containing fungus.

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The oxidation of aldehyde may be carried out by The oxidation of aldehyde may be carried out by 5% chromic acid or by 0.5% periodic acid5% chromic acid or by 0.5% periodic acid

The aldehyde group of reticulum is reduced the The aldehyde group of reticulum is reduced the colorless silver complex , to dark brown ,form colorless silver complex , to dark brown ,form lower oxides of silverlower oxides of silver

This lower silver oxide is reduced to black This lower silver oxide is reduced to black metallic silver by 10% formalinmetallic silver by 10% formalin

The unreduced silver is removed by sodium The unreduced silver is removed by sodium thiosulphate thiosulphate

Toning gives pale grey back ground which is Toning gives pale grey back ground which is better for photography and counter stainbetter for photography and counter stain

SILVER INPREGNATIONSILVER INPREGNATION

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PrinciplePrinciple

Fungal cell wall (mucopolysaccharide)Fungal cell wall (mucopolysaccharide)

Aldehyde groupsAldehyde groups ReductionReduction

Silver stainSilver stain

Black precipitate along fungal cell wallBlack precipitate along fungal cell wall

Oxidation Chromic acid

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Preparation of reagentsPreparation of reagents

Stock solution – silver methanamine soln Stock solution – silver methanamine soln 5ml 5% silver nitrate + 100ml 3% methenamine 5ml 5% silver nitrate + 100ml 3% methenamine

(Hexamethelene tetramine)(Hexamethelene tetramine) Store in RefrigeratorStore in Refrigerator

Working solutionWorking solution 25ml stock solution + 25ml distilled water + 3ml 5% 25ml stock solution + 25ml distilled water + 3ml 5%

borax solution. borax solution.

Always prepare fresh working solutionAlways prepare fresh working solution

    

Gomori/ Grocotts Modification Gomori/ Grocotts Modification Oxidize with chromic acid at room temperature for 1 hour or by Oxidize with chromic acid at room temperature for 1 hour or by

periodic acid for 10 minperiodic acid for 10 min

2% sodium metabisulphite for 2 min( to remove chromic acid)2% sodium metabisulphite for 2 min( to remove chromic acid)

(Reduction)(Reduction)

silver methanamine sol. ( 24 hrs at R.T, / 1hrs at 60’c)silver methanamine sol. ( 24 hrs at R.T, / 1hrs at 60’c)

Gold Chloride 2 min( toning)Gold Chloride 2 min( toning)

Sodium thiosulphate 2 min( remove excess silver stain)Sodium thiosulphate 2 min( remove excess silver stain)

counter stain Light Green 30 seccounter stain Light Green 30 sec

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Results-Results-

Fungus-blackFungus-black Background-light greenBackground-light green

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RemarksRemarks

If chromic acid turns brown, prepare fresh If chromic acid turns brown, prepare fresh solutionsolution

It is mandatory to use fresh working It is mandatory to use fresh working solution each time.solution each time.

Avoid higher temperature as this causes Avoid higher temperature as this causes background staining.background staining.

After every step, rinse the slide in distilled After every step, rinse the slide in distilled waterwater

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Light staining if incomplete reduction

TroubleshootingTroubleshooting

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TroubleshootingTroubleshooting

Dark staining if over-reduction

Prussian BluePrussian Blue

It was first described by perls in 1867 It was first described by perls in 1867 it demonstrated hemosiderin it demonstrated hemosiderin

pigments in the tissue. it is a golden pigments in the tissue. it is a golden brown pigment it arise when brown pigment it arise when

hemoglobin broken down in the tissuehemoglobin broken down in the tissue

ControlControl :A known positive control :A known positive control tissue of hemorrhagic lesiontissue of hemorrhagic lesion

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applicationapplication

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1. Bone marrow – Iron deficiency anemia, Myelodysplastic syndrome, Refractory anaemias. 2. Hemosiderosis-due to excess therapeutic iron or blood transfusion, demonstratedin spleen, bone marrow, liver. 3. Hemachromatosis

04/11/2304/11/23 1% EOSINE1% EOSINE

PrinciplePrinciple Ferric iron

of the hemoglobin

2% ferrocyanide in2% hcl

Ferric ferrocyanide

+

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ProcedureProcedure

Wash thoroughly with distilled water

1% Eosin

2% conc. HCL2%Potassium ferocyanide

30 min

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Prussian BluePrussian Blue Demonstration of hemosiderin pigment.Demonstration of hemosiderin pigment.

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Alizarin Red S MethodAlizarin Red S Method

Demonstrations of calcium pigment

Alizarin Red S MethodAlizarin Red S Method

It was first introduce by It was first introduce by mc.gee –Russell in1958 mc.gee –Russell in1958

it is used to it is used to demonstrated calcium demonstrated calcium

salts in the tissuesalts in the tissue

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Principle Principle

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Calcium forms an Alizarin red S- calcium complex in a chelating process. The orangered precipitate formed is birefringent

Control:

Known calcium containing tissue section.

applicationapplication

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This method is useful in identification and detection of small amounts of calcium like in hypercalcinosis in kidney, in hyperthyroidism necrosis, in some myeloma and schwamoma body

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PrinciplePrincipleCalcium

Alizarine red S

Alizerine red S calcium complex (orange red is formed)

Birefringent in polarized light

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ProcedureProcedure

Air dry section

Alizarine red S (5min)PH-4.2

Dry section with acetone

D.C.M.

GIEMSAGIEMSA

It was first deIt was first described scribed byby Gustav Giemsa, ,

an early malariologist an early malariologist in 1902in 1902

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Giemsa stainGiemsa stain

Introduction-Introduction- Differentiation Differentiation of cells of hemopoietic of cells of hemopoietic

tissue tissue demonstrates some microorganisms like demonstrates some microorganisms like

h-pylori , trypanosoma ,and parasites h-pylori , trypanosoma ,and parasites malaria, in peripheral blood smears . And malaria, in peripheral blood smears . And for bone marrow and chromosomes for bone marrow and chromosomes structurestructure

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PrinciplePrinciple

Giemsa-Giemsa-1.Methylene blue-stains 1.Methylene blue-stains acidic cell componentsacidic cell components

2.Eosin-stains basic cell 2.Eosin-stains basic cell componentscomponents

Preparation of reagentsPreparation of reagents1 Gms Giemsa powder 1 Gms Giemsa powder +54 ml Glycerin +54 ml Glycerin preheated 60 preheated 60 0 0 C.+ add C.+ add 84ml Methanol.84ml Methanol. Mix well Mix well and filter the stainand filter the stain

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Preparation of reagents contPreparation of reagents cont Buffer solution Buffer solution Potassium dihydrogen phosphate—2.72 gms Potassium dihydrogen phosphate—2.72 gms Distilled water— 100 ml Distilled water— 100 ml Sodium hydroxide – 0.8 gm Sodium hydroxide – 0.8 gm Distilled water – 100mlDistilled water – 100ml 50 ml of Potassium dihydrogen phosphate is 50 ml of Potassium dihydrogen phosphate is

mixed with 23.6 ml of NaOH. The pH of the mixed with 23.6 ml of NaOH. The pH of the solution is adjusted to 6.8. solution is adjusted to 6.8.

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ProcedureProcedure

Giemsa stain must be diluted with buffer Giemsa stain must be diluted with buffer solution in1:9 ratio (Working) 20-30 solution in1:9 ratio (Working) 20-30 minuets.minuets.

Differentiate with 0.2 % acetic acid quickly Differentiate with 0.2 % acetic acid quickly and dip in tap water briefly and dip in tap water briefly

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Procedure for bone marrow Procedure for bone marrow imprints &smearsimprints &smears

Smears are fixed in methanol for 30 min.Smears are fixed in methanol for 30 min. Stain in working Giemsa solution 30 Stain in working Giemsa solution 30

minutes.minutes. Wash in water for 5 minutes.Wash in water for 5 minutes. Air dry smears.Air dry smears. Clear in Xylene , mount in DPX. Clear in Xylene , mount in DPX.

Result – Bone Marrow ImprintResult – Bone Marrow Imprint

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RESULTSRESULTS

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ResultsResults

H. Pylori – pinkish blueH. Pylori – pinkish blue Mast cells—Magenta pinkMast cells—Magenta pink Tissue elements—Shades of blue to pinkTissue elements—Shades of blue to pink

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Gram StainingGram Staining

The method is named after its The method is named after its inventor, the inventor, the Danish scientist scientist

Hans Christian Gram who who developed the technique in developed the technique in

1884 1884

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applicationapplication

The purpose is to The purpose is to demonstrate Gram negative demonstrate Gram negative & Gram Positive organisms & Gram Positive organisms in the tissue.in the tissue.

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Principle OF Grams stainPrinciple OF Grams stain

Crystal violet acetone Basic fuschin

Gram +ve

Gram -ve

Gram +ve

Gram -ve

pepidoglycan

Lipo poly saccharide

Insoluble lake barrier

Legols iodine

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Crystal violet (1 min)

Lugol’s iodine (1min)

Acetone (until no colour)

BROWN AND BRENNBROWN AND BRENN

Basic fuschin (3min)

Acetone (2dip)

Picric acid- acetone

Dry section with acetone

BROWN AND BRENNBROWN AND BRENN

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ORAL FLORA

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Melanin Melanin

It was first described It was first described by Massons in 1912 by Massons in 1912 later it is modified by later it is modified by Fontana in1914 Fontana in1914

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applicationapplication

For the detection of Melanin For the detection of Melanin pigments and Argentaffin pigments and Argentaffin granules can be demonstrated by granules can be demonstrated by this method. Melanin is an this method. Melanin is an intracellular pigment it is usually intracellular pigment it is usually located in skin retina substantia located in skin retina substantia nigra of brain and in hairnigra of brain and in hair..

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Principle Principle

MelaninMelanin

silver Solution at 60’C

Black coloured metallic silver

reduction

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ProcedureProcedure

        SSilver solution 60ilver solution 6000CC for 1 hour. for 1 hour.

0.2% Gold chloride0.2% Gold chloride

2% Sodium thiosulphate2% Sodium thiosulphate

1% Eosin1% Eosin

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Prepration of silver nitratePrepration of silver nitrate

5ml 10% Silver nitrate5ml 10% Silver nitrate

++ 45 ml distilled water (1 hr at 6045 ml distilled water (1 hr at 6000c )c )

RESULTSRESULTS

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Melanin Pigment In Basal Layer Of Skin

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Melanin bleach methodMelanin bleach method

Procedure-Procedure-

0.5 Potassium permanganate0.5 Potassium permanganate

2% oxallic acid2% oxallic acid

Routine H&E stainingRoutine H&E staining

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PrecautionsPrecautions

Check under microscope, if required Check under microscope, if required repeatrepeat

Bleaching stepBleaching step Preferably use Poly-L- Lysine coated Preferably use Poly-L- Lysine coated

slides. slides.

MELANIN BLEACHMELANIN BLEACH

04/11/2304/11/23 5858PIGMENTED MELANOMA

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Chloroacetate esterase Chloroacetate esterase Pararosaniline methodPararosaniline method

Introduction :Introduction :

Chloroacetate esterase is the only enzyme Chloroacetate esterase is the only enzyme which can be demonstrated in paraffin which can be demonstrated in paraffin section.section.

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Principle Principle

Leukocyte esteraseLeukocyte esterase

hydrolyses

Diazonium salt

Brightly coloured ppt at the site of enzyme activity

+Derivative of Naphthalene

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Preparation of reagentsPreparation of reagents

1) substrate solution1) substrate solution : : Naphthol As. D. Chloroacetate- 10 ml Naphthol As. D. Chloroacetate- 10 ml

N-N dimethyl formamide –1 mlN-N dimethyl formamide –1 ml: : Pararosanilin– 0.4gms, distilled water – 0.4ml, conc. Pararosanilin– 0.4gms, distilled water – 0.4ml, conc.

HCl – 1.6ml.HCl – 1.6ml. sodium nitrite – 0.4gms, distilled water – 10ml.sodium nitrite – 0.4gms, distilled water – 10ml. Concentrated HCl – 8.35 ml ,distill water 91.65 ml.Concentrated HCl – 8.35 ml ,distill water 91.65 ml. Sodium barbital powder – 1.03gm, distilled water – Sodium barbital powder – 1.03gm, distilled water –

100ml100ml..

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Procedure Procedure

          14.6ml of 0.1N HCL and 15.4ml of sodium barbital.14.6ml of 0.1N HCL and 15.4ml of sodium barbital.

          prepare substrate solution and keep aside. Drop prepare substrate solution and keep aside. Drop of Pararosaniline and add drop of sodium nitrite.of Pararosaniline and add drop of sodium nitrite.

Add all solutions adjust pH 6.3Add all solutions adjust pH 6.3

Filer and keep for 3hrFiler and keep for 3hr

counter stain with hematoxylin. counter stain with hematoxylin.

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ResultResult

Reactive lymph node -neutrophilsReactive lymph node -neutrophils

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