11: Staining I

Stains

Chemical substances used to enhance visibility for microscopic studies of a prepared tissue

Staining

Treating tissue/cells with a reagent or series of reagent so that it acquires colour

The purpose of staining is to outline the tissue and cellular components:

To _ tissue

To observe __

To Demonstrate _

To Demonstrate specific __

To Demonstrate _

To establish the presence or absence of __ (pathologic process).

To enhance _

identify, general morphology, microorganisms, tissue structures, pigments, disease processes, visibility

Commonly used stains for histopathology

Routine Hematoxylin and Eosin (H&E)

Commonly used stains for microbiology

Gram’s Method, Ziehl-Neelson’s Method

Commonly used stains for haematolofy

Romanowsky stain

Commonly used stains for cytopathology

Papanicolau stain (PAP)

Physical staining theory

Solubility, dye is soluble to form a homogenous solution of the solid in the solution it is carried in

Chemical staining theory

A chemical reaction takes place between dye and tissue resulting in a colour reaction

Physical theory depends on…

osmosis, solubility, absorption, adsorption, permeability of tissue

Chemical theory depends on…

pH, amphoteric

Dyes appear coloured due to…

their ability to absorb light (we see the ‘unabsorbed’ colours)

Dyes are essentially ___ compounds or derivatives that posses the twin properties of ____

aromatic benzene ring, colour and ability to bind to tissue

Basic dye

Possess a negative charge

Acidic dye

Possess a negative charge

Natural dyes/stains

From natural sources

Artificial dyes/stains

From chemical reactions

Natural example

Carmine, orcein, hematoxylin

Carmine is sourced from…

female cochineal bug

Orcein is sourced from…

lichen

Hematoxylin is sourced from…

H. Campechianum tree bark

Types of staining

Simple (shapes and arrangements), differential (regressive), special (capsule, flagella, spores)

Direct staining involves…

ionic bonding

Indirect staining includes stains which need a…

mordant to link the dye molecule with the tissue component

Stains stay in tissue because of ____ between the dye molecules and specific _ of the tissue. These interactions allow the stain to bind or be __, making different structures visible under the microscope.

chemical and physical interactions, components, retained selectively

Mordant

Substance that binds a dye molecule to a tissue substrate

Ligand

Ion or molecule which combines with metal ion (mordant)

Chelate

Compound containing a ligand bonded to a central metal atom at two or more points

Recipe of haematoxylin: Digested in… then…, washed in water, … in sun, develops brown colour and sold. _ to become active dye haematin, _ staining achieved using…

alcohol, distilled, dried, Oxidised, nuclear, mordant

Progressive H&E

Tissues stained until desired colour is reached

Regressive H&E

Tissues are over stained then dye is selectively removed until desired level is reached (differentiation)

Differentiation

Removal of excess dye

H&E differentiation

0.5–1% HCl in 70% alc

Gram stain differentiation

Acetone

Bluing, done in __. Process of shifting __ to blue which provides better _ against pink background. Nuclei will be stained _.

alkaline water, colour range, contrast, purple

H&E is _-based

charge

Haematoxylin stains _ structures shades of blue or purple

acidic

Eosin stains _ structures shades of pink

basic

Counterstain is application of a different colour to…

provide contrast and background to enhance primary stain

Practicalities of staining sections: When applying a stain, you should carefully __ the previously added _ unless __ .

wash off, reagent, otherwise stated

Practicalities of staining sections: Do not let reagents _ on slides, gently rock the slide to __ and ensure no _ occurs.

stagnate, facilitate penetration, drying

Practicalities of staining sections: Fully cover the __ with the _.

section/slide, reagent

Practicalities of staining sections: _ control the staining method if needed.

Microscopically

Practicalities of staining sections: Always work in a clean, concise and safe manner when staining → minimize __

cross contamination

Considerations when staining paraffin and frozen sections

Availability/adequacy of test and control sections and reagents and stains, safety hazards, special requirements, availability of equipment, SOP, expert resources/personnel

Mounting media

Aqueous, buffer-based, xylol-based (DPX)

Basophilic

Entities stainable with basic dye, possess positive charge, usually acidic in nature e.g. nucleus

Acidophilic

Entities stainable with acidic dye, possess negative charge, usually basic in nature e.g. cytoplasm

Argyrophilic

Entities stainable with silver nitrate solution

Argentaffin

Entities stainable with silver nitrate solutions without chemical reduction procedures

Metachromatic

Entities will stain in a colour or hue different from that of staining solution itself