AP prac test final

affinity

movement of a dye from the dye bath onto a section

Electrovalent bonding 

Oppositely charged groups 

Covalent bonding 

• Strong bond 

• Sharing of electrons between atoms 

Hydrogen bonding 

Bonds between hydrogen atom and oxygen (+) or nitrogen (-) 

Van der waals forces 

Electrostatic interactions that exist between electrons of one atom and the nucleus of another 

Hydrophobic bonding  

Movement of hydrophobic dyes from surrounding water to hydrophobic tissue groups 

Chromophore

imparts colour by ability to absorb light in the visible part of spectrum 

Auxochrome

involved in attaching to the tissue component (charged groups) 

Cationic dyes (+) / basic

• Positive charge, binds to negatively charged tissue groups 

• Tissue components referred to as basophillic 

Anionic dyes (-) / acid

• Negative charge, bind to positively charged tissue groups 

• Tissue components are acidophilic  

Neutral dyes 

• Combination of acid/anionic and basic/cationic dye 

Direct dyeing 

• Direct attachment of dye to tissue component by ionic bonding 

• Opposite charge 

Indirect dyeing  

• Attachment of dye to tissue using an intermediate substance called a mordant 

• Mordant: metal, has affinity for both the dye and tissue 

• Dye + mordant = dye lake 

Metachromasia 

Certain dyes which when attached to particular tissue groups, produce a colour different from the original dye

Dyeing by permeation  

Difference in permeabilty of tissye structures and uses dyes of different colour and molecular size 

Differential staining 

Two or more of the above methods 

Histochemical methods 

Reagents used react with specific tissue components to produce a colour insoluble product

Metallic impregnation

• Particular chemical groups in tissue have the ability to bind or reduce silver salts 

• Metal precipitates in or on particular tissue constituents  

Argentaffin reaction

causes silver to be deposit without using a chemical reducing agent (black) 

Argyrophilic reaction

based on ability of certain substances to attract and be impregnated with silver salts, visualisation when silver is reduced to metallic silver using a reducing agent – can't do on its own 

Elective solubility 

• More soluble in the element to be demonstrated than in the solvent in which it is  

Immunostaining 

• Identifying cell or tissue constituents by means of antigen-antibody interactions 

Trichrome staining 

• Use of two or more acid dyes of contrasting colour to selectively colour different basic tissue components 

• Masson trichrome (multistep): acid dyes and polyacid applied sequentially 

• VVG (one step): acid dyes all in one solution 

• Both use acid-resistant haematoxylin: wiegert's haematoxylin 

Type I collagen

• Collagen fibres  

• Abundant, widespread distribution 

• Tensile strength 

Type III  collagen

• Reticular fibres 

• Fine, delicate fibres, connected to type I fibres 

Neutral mucin 

No acidic reactive groups

Acid mucin 

Can have a negative charge  

macroscopic description

1. Specimen labelled _ 

2. Describe specimen 

   1. Shape 

   2. Size, dimensions 

   3. orientation 

3. Describe lesion of visible 

   1. location 

   2. Size, Dimension 

   3. Colour 

   4. Contour 

   5. Borders 

   6. Distance closest to margin  

4. All processed in one block 

factors effecting fixation (6)

• Temperature 

• Time 

• 20 :1 ratio of fixative to tissue  

• 10% formalin 

• Take note of collection time, how long has it been fixed? 

• Dense, fatty 

frozen sections for? (3)

• Splitting between labs 

• Frozen 

• Immunofluorescence  

small biopsy fixation time

2-3 hours

larger derm samples fixation time

>6h

large specimens fixation time

overnight

fatty tissue processing

• Defatting step: 50/50 xylene and ethanol mixture between the alcohols and xylene  

• Add alcoholic formalin step 

issues with tissue processing (5)

• Dry out tissue before fixation or processing 

• Losing tissue: need biopsy pad 

• Wrong schedule  

• Contaminated reagents, or expired 

• Cassettes overloaded with tissue 

• Greasy = xylene retained 

• Short cycle 

• Varying consistency  

Retained xylene + alcohol evaporates tissue, shrinking down  

over-dehydration = brittle, chatter = dull blade

embedding considerations (3)

• 3d orientation 

• Enough tissue, adequate 

• Supporting wax  

embedding quality practices (4)

• One at a time 

• Ensure tissue type and number matches with macroscopy 

• Record who has done it 

• Cleaning forceps, warm  

microtomy considerations (3)

• Block must be cold  

• Sharp blade  

• Clean and warm water bath  

microtomy quality practices (3)

• One at a time 

• Label slides  

• Clean water bath between blocks 

microtomy issues and fixes (6)

• Problem blocks: reprocess 

• Brittle tissue: soften with tween (detergent), water 

• Surface decalcification: blocks have calcium, acid penetrate superficially  

• Floating off tissue: lower temp, move quickly  

• Chatter and knife lines: sharp blade, cut slowly and gently 

• Compression: cold block, optimise knife angle  

folds

knife lines

• Must be 42-47 degrees 

• Poorly processed 

• Long time in bath 

• Temp too high  

• Contamination 

• < kidney 

• > stomach  

 

• Squamous cells from fingers 

• Strain precipitate: haematoxylin not filtered  

 

appraisal

• What is staining 

• Quality of staining 

  • Adequate? 

  • Poor? 

  • Expected? 

• Background staining  

 

H&E progressive

1. Mayer’s Haematoxylin: Binds to negatively charged structures (DNA, RNA)

2. Scott's tap water: Blueing alkaline solution converts haematoxylin from red to blue 

3. eosin: Acidic dye that binds to positively charged proteins 

H&E components stain

Nuclei = blue-black 

Cytoplasm = shades of pink  

Muscle fibres = deep pinky red 

Red blood cells = orange/red 

Fibrin = deep pink 

regressive H&E

1. Mayer’s Haematoxylin: Haematoxylin and Scott's over staining (over stained nuclei with background staining)

2. Scott's water  

3. 1% Acid alcohol: Differentiate in acid alcohol, leaving nuclei stained, removed others 

4. eosin: Acidic dye that binds to positively charged proteins 

PAS

1. Periodic acid: Oxidises vicinal diols in carbohydrates to aldehyde 

2. Schiff's reagent: Colourless, reacts with aldehydes = magenta 

3. Haematoxylin: For nuclei 

4. Scott's tap water: Blueing 

PAS components stain

Glycogen and other carbohydrates = magenta 

Nuclei = blue 

PAS/D

diastase occurs before periodic acid  

removes glycogen into maltose and glucose

Alcian blue

1. Acetic acid: Cationic dye, forms electrostatic bonds with tissue polyanions 

   1. Polyanions: have carboxyl or sulphate group found in tissue 

2. Alcian blue: Sets pH, controls which acidic groups bind dye 

3. Nuclear fast red: Counterstain for nuclei 

alcian blue components stain

Acid mucins = blue 

Nuclei = red 

AB-PAS

1. Acetic acid: Cationic dye, forms electrostatic bonds with tissue polyanions 

   1. Polyanions: have carboxyl or sulphate group found in tissue 

2. Alcian blue: Sets pH, controls which acidic groups bind dye 

3. Periodic acid: Oxidises vicinal diols in carbohydrates to aldehyde 

4. Schiff's reagent: Colourless, reacts with aldehydes = magenta 

5. Mayer’s Haematoxylin: For nuclei 

6. Scott's tap water: Blueing 

AB-PAS components stain

Acid mucins = blue 

Neutral mucins = magenta 

Nuclei = pale blue 

Mixture of mucins = depends on dominant mucin 

colloidal iron

1. Acetic acid: Acidic pH (low pH) 

2. Working colloidal iron solution: Forms a chelate with carboxyl and sulphate radicals of acid mucins

3. Acetic acid: Removes unbound ferric ions  

4. Perls' regent: Reacts with ferric iron to form ferric ferrocyanide (prussian blue)

5. Nuclear fast red: Background stain  

colloidal iron components stained

Acid mucins = blue 
nuclei = red 

colloidal iron clinical relevance

Intestinal metaplasia  

Ziehl-Neelsen

1. Kinyoun's carbon fuchsin: Penetrates waxy mycolic acid coat, dyes everything 

2. Acid alcohol: Decolorizes non-acid fast organisms, Acid fast bacilli resist due to lipid coat  

3. Methylene blue: Counterstain for background  

Ziehl-Neelsen components stained

Acid fast bacilli = red 

Other components = blue 

Grocott Methenamine Silver

1. 5% aqueous chromic acid: Oxidises carbohydrates in fungal walls to aldehydes 

2. 1% aqueous sodium metabisulphite 

3. Silver bath: Aldehydes reduce silver to metallic silver ions, black deposits 

4. 0.1% gold chloride: Enhances contrast 

5. 2% sodium thiosulphate: Removes unreduced silver 

6. Light green: Counterstain 

Grocott Methenamine Silver components stained

Fungi = black 
background = green 

Perl’s

1. Hydrochloric acid and potassium ferrocyanide: Ferric iron reacts to form insoluble ferric ferrocyanide, Prussian blue 

2. Nuclear fast red: Counterstain

Perl’s components stained

Haemosiderin = dark blue 

Nuclei = red 

congo red

1. Congo red stain: Binds beta-pleated sheet amyloid fibrils   

2. Mayer's haematoxylin: Counterstain for nuclei 

3. Scott's tapwater: Blueing 

congo red components stained

Amyloid = red, apple green bifringence under polarised light 

Nuclei = blue 

Gordon and Sweet's reticulin 

1. Acified potassium permanganate: Oxidises, formation of binding sites 

2. 1% oxalid acid

3. 2% ferric ammonium sulphate: Sensitiser - ferric ions attach to binding sites  

4. Wilder's solution: Impregnate: ammoniacal silver > silver diamine complex attaches to reticular fibres 

5. 10% formalin: Reduces silver to visible metallic silver ions  

6. 5% sodium thiosulphate: Removes unreacted silver 

Gordon and Sweet's reticulin components stained

Reticulin = black 

Collagen = brown 

Masson Trichrome 

1. Bouins fluid: mordant, improves staining by increasing acidophilia by increasing reactive sites 

   1. As formaldehyde fixation turns off reactive sites, detrimental effect to anionic dyes  

2. Weigert's haematoxylin: Nuclei stains black, resists acid, mordant and oxidiser  

3. 1% brillian crocein in 1% phosphotungstic acid: Stains cytoplasm, muscle and keratin red, including collagen 

4. 1% phosphotungstic acid: phosphotungstic acid removes dye from collagen 

   1. larger molecule, cannot penetrate dense muscle fibres/cytoplasm 

5. 1% aniline blue in 1% acetic acid: Stains collagen fibers blue 

6. 1% acetic acid: Sharpens colour contrast 

7. 100% ethanol  

Masson Trichrome components stained

Nuclei = black 

Collagen = blue 

Muscle = red 

Gomori's Aldehyde Fuchsin (GAF) 

1. Aldehyde-fuchsin: Stains elastic fibres, beta cells and mucins purple 

2. 95% alcohol: Diffrentiates

3. Indigo carmine-picric acid: Counterstain for collagen and muscle  

4. 95% alcohol 

Gomori's Aldehyde Fuchsin (GAF) components stained

Elastic fibres and mucin = deep purple 
beta cells of pancreas = deep purple 
collagen fibres = blue-green 
muscle = yellow  

orcein

1. Acidified potassium permanganate: Oxidise, enhance binding sites 

2. Oxacilic acid: Bleach

3. Ethanol: Differentiates 

4. Orcein solution at 60 degrees: Binds to elastic fibres  

5. ethanol 

orcein components stained

Elastic fibres = dark brown 

Cytoplasm of hepatocytes containing hep B surface antigen = dark brown 

Copper deposits = red/brown  

VVG Verhoeff–Van Gieson

1. Verhoeff's solution: haematoxylin, ferric chloride, lugol's iodine - Binds strongly to elastic fibres, nuclei, forms complex, overstaining 

2. Water: Removes excess haematoxylin 

3. Aqueous ferric chloride: Selectively removes stain, elastic fibres retain longer due to affinity, differentiation  

4. 95% alcohol 

5. Van Gieson: Background stain 

6. Ethanol  

VVG Verhoeff–Van Gieson components stained

Elastic fibres = intense black 

Chromatin = black 

Collagen fibres = red 

Other elements = yellow/brown  

schmorl’s

1. Ferric ferricyanide: Melanin reduces ferric ferricyanide to ferric ferrocyanide (prussian blue) 

2. Nuclear fast red: Background stain  

schmorl’s components stained

Reducing substances (bile, melanin, argentaffin cells, thyroid colloid) = dark blue 
nuclei = red