CHAPTER 7: STAINING OF TISSUES

Stains

The only way to render the tissue visible under the light microscope is to provide color to the cell components through

Differential staining

This enables microscopic evaluation of all areas of the tissue.

hematoxylin and eosin (H&E) method.

most common "routine” staining

"Special stains"

Other alternative staining techniques employed when the H& E cannot visualize certain tissue components under consideration.

Hematoxylin

This stain has a deep blue-purple color and stains nucleic acids.

Incompletely understool

Hematoxylin stains by a complex, ____ ____ reaction

Eosin

stain that is pink and stains proteins non-specifically.

blue; pink

In a typical H&E stained section, nuclei appear _____ while the cytoplasm and extracellular matrix show different shades of _____.

Nuclear chromatin activity

this influences the varying depth of hematoxylin stain.

eosin; blue-tinged

Nucleoli stain with _____. Ribosome-rich cytoplasm appears _______

perinuclear absent

A prominent Golgi zone shows _______ ______ staining.

complete removal of paraffin

Staining always begins with the _________ in fixed sections on slides for the water-soluble stain solutions to act on the tissue.

dry storage; wet; coverslip

Mounted tissue samples are stable in ______ until the wax is removed; after which, the tissue must remain ______ until staining ends and the slide is mounted with a ______.

60° C; 30 minutes

Dewaxing starts with heating the slides in ______ oven for at least _____ to soften the wax.

couple; xylene

The slides are then immersed in a _____ of changes of clean _____

descending; distilled water

The slides are taken through _____ concentrations of alcohol to _______.

Lipid stain that absorb the fat

Example of pure physical stain

Chemical stain

Most stains fall under the this type of stain, in which the dye is adsorbed by the cell components.

van der Waals
ionic or electrostatic

covalent

hydrogen

It is then bound to the tissue through ______ forces, _______ or ______ bonds, ______ bonds, or _____ bonds.

basophilic

Tissue elements attracting basic dyes are

acidophilic

Those attracting acidic dyes are

Acidic

Anionic

Negatively charged

Nuclei are generally 1) 2) 3)

Basic

Cationic

Positively charged

Nuclei stain with ___ dyes

Dye-salt

Nuclei stains by forming _____ unions

Basophilic

The reaction in nuclear staining is termed

dyes-metal mordants

Nuclei that are positively charged, decalcified, or contain only basic nucleoproteins stain with _______ combination.

chemical forces

a complex interplay of _______operates on the second mechanism of nuclear staining

amino acid side chains

Cytoplasmic staining is brought about by proteins or charged groups on the _________ of proteins.

amphoteric; pH

Proteins are ______ because they can have either a net positive or negative ionic charge depending on the ____ of the solution.

isoelectric point ; pH 6

The _____ of proteins, in which the positive and negative charges are equal and with no electrical field migration, is approximately _____.

positive; anionic or negatively charged; eosin

In a solution below the IEP or below pH 6, the net charge on the cytoplasmic proteins is ______ and the affinity is for an ______ or _______ dye such as ____.

negative; cationic dye

Above the IEP or above pH 6, the net charge is _____ and the attraction is for a ______.

too low

If the pH of the solution is _____, eosin is uncharged and stains the tissue non-specifically

net negative

Above pH 6, proteins will have a _____ charge and will no longer attract eosin.

carmine

orcein

saffron

hematoxylin.

Most important natural dyes

organic

coal tar

benzene

All dyes are _____ compounds, mostly derivatives of _____ or _____.

A group that confers the property of color is called a chromophore, which is the basis for the general classification of dyes. A compound containing chromophoric groups is called a chromogen.

Chromogens

These substances may or may not be able to act as a dye or combine with cell components to be colored.

auxochrome

To enable the dye to link' to the tissue, an ionizing group called an ____ is required.

Amino group (NH2)

The auxochrome in the 'basic' dyes is the

aniline

_____ contains amino group, and many of the dyes are ____ derivatives.

sulfonic acid (SO2OH) group

The usual auxochrome in the 'acid' dyes is the ______, ionized to produce negative charges;

Carboxyl (COOH) groups

Hydroxyl (OH) groups

Two other acidic auxochromes are the ____ and ____ groups

positive charge

Basic dyes, properly called cationic dyes, are those dyes with a ______

negative charge

Acid dyes, or anionic dyes, are those with a _____.

chloride salts

Cationic dyes most frequently are

crystal violet

safranin

Examples of cationic dyes

sodium salts

anionic dyes most frequently are ___

orange G

picric acid

Example of anionic dyes

pH 3 to 9

Most dyes remain cationic or anionic throughout the pH range in which most staining occurs

cationic

Some dyes are amphoteric; they are ____ at pH below the IEP

Anionic

Some dyes are amphoteric; they are ____ at pH above the IEP

Hematein; 6.6

It is an example of an amphoteric dye having an IEP of about pH _____

Lipid stains

These are non-ionic compounds incapable of electrolytic dissociation.

False

Lipids are dyes, as they have been classified as neutral dyes

organic solvents; water

Lipid dyes are soluble in some ______ but not in ____

dissolving

Lipid stains 'color' certain tissue elements by _____ in them (physical staining).

pH of the solution

The tissue element and the dye molecule must have complementary charges in order to bind.

Increase in temperature

This swells the tissue and facilitates diffusion of dye molecules for effective dye action and rapid staining time.

Increased concentration of dye molecules

The difference in gradient propels tissue-dye binding.

Salt ions

_____ in the solution and dye ions probably compete for the same binding sites, altering the intensity of dye binding.

Presence of other salts

competition for the same binding sites, altering the intensity of dye binding.

type of fixative

the ____ alters the staining character of the tissue by reacting with certain chemical groups in the tissue and making them unavailable for dye binding,

Formalin

____ reacts with the tissue amino (NH2 group)the primary eosin binder, lessening the eosin tissue uptake.

potassium dichromate

This fixatives take up less hematoxylin than eosin

Zenker

Bouin

unbuffered formalin

A tissue overexposed to acidic fixatives such as ___, ___, or ____ will lose its basophilic (nuclear) staining properties

Progressive staining

it is a staining method in which the reaction proceeds 'forward and is stopped once the desired intensity of color is achieved.

Regressive staining

the tissue is overstained and then differentiated and then decolorized, until only the desired element remains stained

mordant

This is substance is usually a metal, that links the dye to the tissue.

Basic

Mordant combines with the dye to form a "lake" that is usually _____ in action.

progressively

Many dyes, including counterstains (cytoplasmic stains) and even mordant dyes, are used ____.

Regressively

Mordant dyes are more effective when used _____ to achieve differential staining

weak acid

Basic (cationic) dyes are differentiated by ____ solutions

weak alkaline

acidic (anionic) dyes are differentiated by ____ solutions.

Aluminum hematoxylins

dilute HCl solution

Eosin

Dilute ammonium hydroxide solution

Alcohol

If the differentiating solutions are prepared in ____ instead of water, better control of decolorization is possible.

tissue-mordant dye

Excess mordant in the differentiating solution will dissolve the ______

True

The most dye-bound tissue elements will decolorize last.

Oxidizing differentiators

are substances added to the dye that oxidize it to a colorless substance, in effect differentiating it as well.

Potassium permanganate

this chemical is used as an oxidizing differentiator

Hematoxylin

most widely used nuclear stain

Haematoxylon campechiamum

Hematoxylin is extracted naturally from the log wood called ___ although synthetic equivalents are available

True

Hematoxylin per se is not a dye

hematein; anionine

it is the oxidation product of hematoxylin, is a weak _____ dye.

ripening

In the process termed _____, oxidation is achieved either by exposing the solution to air or by mixing oxidizers.

false

Oxidation to hematein is not needed.

pH

Alkaline
acidic

The oxidation rate is ______ dependent, as hematein forms rapidly in ____ solutions but slowly in ____solutions.

sodium iodate

potassium permanganate.

Oxidizers mixed with haematein to oxidize

Harris hematoxylin

This is the hematoxylin commonly used in routine H&E

Periodic Acid-Schiff (PAS)

selected special stains such where harris hematoxylin is used

mercuric oxide

sodium iodate

There are ready-to-use preparations of harris hematoxylin available in the market. The original formula uses _____ (toxic) but is now replaced by ______.

hematoxylin

Absolute ethyl alcohol

Harris hematoxylin preparation:

1. Dissolve ____ in ____

ammonium aluminum sulfate

Distilled water

Harris hematoxylin preparation:

2. dissolve _____ in ______ with heat.

Sodium iodate (oxidizer)

Harris hematoxylin preparation:

3. Mix the 2 solutions and bring rapidly to a boil. Remove it from heat and slowly add_____.

Dark purple

Harris hematoxylin preparation:

Reheat it to boiling until the solution becomes ___ ____. Remove it from heat, and immediately place the vessel into a basin of ice.

metallic sheen

The harris hematoxylin stain is ready for use after cooling or when there is a _______ on the surface of the solution.

glacial acetic acid

4mL

96 mL

Filter the harris hematoxylin before use, and add ______ to give a final concentration of 4% (_____ of glacial acetic acid for every _____ of hematoxylin).

progressive stain

Regressive stain

Harris solution is used best as a _____(as originally intended), but may also be used as a _______.

1g

How many grams of hematoxylin is present in mayer hematoxylin

1000mL

How many mL of distilled water is present in mayer hematoxylin