S T A I N I N G

DWI WINARNIDepartemen Biologi

Fakultas Sains dan Teknologi

Universitas Airlangga Surabaya

Unstained cells/

tissues

Stained cells/

tissues

depend on affinity of tissue component for

a certain dyes

PURPOSE of staining is to enhance contrast.

This is accomplished in two ways:

1) Different colors

2) Coloring to different intensities

Chemistry of Dyes

Basic Dyes = carry positive charge, attracted to acidic components of cells

Acidic Dyes = carry negative charge, attracted to basic components of cells

Neutral Stains = anion (-) and cation (+) provide different colors

GENERAL CATEGORIES OF DYES:

1. Acid-Base Combinations

Most sections are stained with both acidic and basic dyes to enhance contrast by providing different colors.

The most common combination is Hematoxylin and Eosin (H &E).

Hematoxylin = basic dye, stains nuclear structures blueEosin = acidic dye, stains cytoplasmic and intercellular structures pink

2. Trichrome Methods

provides 3 colors, allows differentiation between cytoplasmic and intercellular components

Van Gieson techniques for collagen (alum hematoxylin, celestine blue B and acif fuchsin)= nuclei, blue black; collagen, red; other tissues, yellow.

HEMATOXYLIN – EOSIN STAIN

3. Dyes for Specific staining stain certain structures or molecules specifically

Iron hematoxylin = useful in distinguishing finer cytologic details (e.g., subcellular organelles)

Mallory-Azan = trichrome method; stains collagen fibers and mucus blue, stains nuclei and cytoplasmic components red

Mason = trichrome method; collagen fibers stain green,cytoplasmic components stain purplish-red

Periodic-Acid Schiff = selectively stainscarbohydrate-containing molecules/substances red (e.g., glycogen,muco- and glycoproteins, glycosaminoglycans)

Silver Impregnation = selectively outlines reticular and neural fibers, Orcein, Resorcin-Fuchsin = selectively stains elastic fibers

Sudan Black B = specifically stains fat

PAS

• The term mordant comes from the Latin word, "mordere", to bite

• A mordant is a substance used to set dyes on tissue sections by forming a coordination complex with the dye which then attaches to the tissue. It may be used for intensifying stains in cell or tissue preparations.

• A mordant is often a polyvalen metalion. The resulting coordination complex of dye and ion is colloidal and can be either acidic or alkaline

• Mordants include tannic acid, alum,  sodium chloride, & certain salt of aluminium, chromium, copper, iron, iodine, potassium, and sodium  

Hematoxylin …………………………………….. 5 g

Etanol 96% ……………………………………... 50 ml

Potassium alum ………………………………… 100 g

Akuades …………………………………………. 950 ml

Merkuri oksida …………………………………… 2.5 g

Asam asetat glasial …………………………….. 40 ml

Metachromasia

1. the different coloration of different tissues produced by the same stain.2. change of color produced by staining

 toluidine blue becomes pink when bound to cartilage.Mast cell granules red to purple

A counterstain is a stain with color contrasting to the principal stain, making the stained structure more easily visible.

the malachite green counterstain to the fuchsine stain in the Gimenez staining technique. eosin counterstain to haematoxylin in the H&E stain

The Gimenez staining technique uses biological stains to detect and identify bacterial infections in tissue samples. Although largely superseded by techniques like Giemsa staining, the Gimenez technique may be valuable for detecting certain slow-growing bacteria.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

Rickettsia conorii detected in hemolymph from infected Rhipicephalus sanguineus adult ticks using Gimenez staining.

Shrinkage = separation of portions of tissue that weren't separated in life; gives appearance of empty spaces

Folds and Wrinkles = these may occur during cutting or mounting and will appear excessively darkly stained relative to the remainder of the section

Nicks = result from defects in microtome knife; appear as pale, straight lines across the section

Degeneration = occurs if tissue not removed immediately or not fixed immediately upon removal; results in inferior quality preparation

A R T I F A C T S= imperfections in tissue preparation

Shrinkage

Folds

washing in alkaline water after eosin staining which has no red since alkalinity hinders eosin staining.

washed in Adelaide tap water after staining in buffered eosin and it is just right.

washed in acid water after eosin staining which is too red.

Tergantung pada bahan perekat

(mounting media) yang digunakan