STAINS AND STAINING SOLUTIONS Flashcards by Kyra Catedral

• Stains are prepared from two categories of dyes:

Natural dyes
Synthetic dyes

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Natural Dyes

Hematoxylin
Cochineal Dyes
Orcein
Saffron

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Synthetic Dyes

Aniline
Coal Tar dyes

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are those obtained from plants and animals, previously utilized for dyeing of wool and cotton.

Natural dyes

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Hematoxylin is a natural dye derived by extraction from the core or the heartwood of a Mexican tree known as

“Hematoxylin Campechianum”.

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It is by far the most valuable staining reagent used by the cytologist due to its powerful nuclear and chromatin staining capacity, and its striking polychrome properties which may be produced with proper differentiation.

It may be used after almost any fixative and is a permanent stain.

Hematoxylin

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Hematoxylin itself is not a true basic dye.

The active coloring agent is____, which is formed by the oxidation of hematoxylin, a process known as “____.”

hematin

ripening

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This is usually accomplished by exposing the substance to_____ or _____, thereby oxidizing hematoxylin (natural ripening).

air and sunlight

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Ripening

Such a process is slow and takes as long as______, but it can be accelerated by adding strong oxidizing agents such as (5) which converts hematoxylin to hematin almost instantaneously by chemical oxidation (artificial ripening), so that the staining solution is ready for use immediately after preparation.

3-4 months

hydrogen peroxide
mercuric oxide
potassium permanganate
sodium perborate
sodium iodate

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Ripening

It is essential that the____ be used in correct amount, since excessive oxidation (over-ripening) leads to production of other useless compounds.

Using the least amount of ____ will result in satisfactory staining and longer life of the stain.

oxidant

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Ripened hematoxylin is seldom used alone due to its inherent low affinity for the tissue itself.

It is most frequently used in combination with (4), which act as mordants catalyzing or forming links between the hematin stain and the tissue.

alum
iron
chromium
copper salts

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….stains are recommended for progressive staining of tissues, and are usually counterstained with Eosin, Congo Red and Safranin.

Alum hematoxylin stains

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Both the Ehrlich’s solution and the Harris’ solution contain…

Alum Hematoxylin

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Rapid ripening of:

Ehrlich’s reagent, however, is brought about by the addition of______;

Harris solution is ripened with_____.

Sodium Iodate

Mercuric Chloride

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are used only for differential or regressive staining, using Acid-Alcohol as a differentiating agent.

Iron hematoxylin compounds

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An example of an Iron Hematoxylin compound is_____ Stain using Iron (Ferric) Chloride

Weigert’s

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…solutions are utilized for the study of spermatogenesis.

Copper hematoxylin

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In a skillfully made H & E preparation the red blood cells are almost____, and collagen and cytoplasm (especially muscle) acquire different shades of___

orange

pink

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is an old histologic dye extracted from the female cochineal bug (_____), which is treated with____ to produce the dye,_____.

Cochineal dye

Coccus Cacti

alum

carmine

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It is widely used as a powerful chromatin and nuclear stain for fresh material and smear preparations.

Cochineal dye

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Cochineal dye

When combined with picric acid (______), it is extensively used in neuropathological studies;

and when combined with aluminum chloride (______), it is used for the demonstration of glycogen.

Picrocarmine

Best’s carmine stain

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is a vegetable dye extracted from certain lichens which are normally colorless, but which, when treated with ammonia and exposed to air, produce blue or violet colors.

Orcein

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Color of orcein

Blue to violet

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It is a weak acid, is soluble in alkali, and is mainly used for staining elastic fibers.

Orcein

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is also obtained from lichens, treated with lime and soda, and exposed to ammonia and air. It is, however, not used as a cytological stain because of its poor staining property. It is instead, used mainly as an indicator.

Litmus

Synthetic dyes are sometimes known as "______" since they were originally manufactured from substances that have been taken from coal tar.

Coal Tar Dyes

Coal Tar Dyes/ Synthetic dyes They are derived from the hydro-carbon benzene (C6 H6 ), and are collectively known as

Aniline Dyes

are substances with definite atomic groupings and are capable of producing visible colors

Chromophores

Simple benzene compounds which contain such substances (chromophore) are known as

chromogens

These are different from the dyes in that any color that they impart to the tissue is not permanent and can, therefore, be easily removed. Before a chromogen can properly be called a dye, it must have the property of retaining its color in the tissue. This property is acquired by the addition of an________, an auxiliary radical or substance which imparts to the compound the property of electrolytic dissociation, thereby altering the shade of the dye, enabling it to form salts with another compound, and ultimately retaining its color.

auxochrome

A synthetic dye, therefore, should consist of a_____ + ______ attached to a________

chromophore and an auxochrome group hydrocarbon benzene ring

Synthetic dye The coloring property is attributed to the_______, and the dyeing property to the salt-forming______.

chromophore auxochrome

Depending on where the coloring substance (chromophore) is found, dyes may be classified into three groups:

Acid Basic Neutral

- where the active coloring substance is found in the acid component, and the inactive base, e.g. acid fuchsin, is usually the sodium salt of a sulfonate of rosaniline.

Acid Dyes

One example of such a dye is picric acid, which has the ability to form salt with an alkali.

Acid dyes

is outstanding in the sense that it is the only substance so far that can fix, differentiate and stain tissue all by itself. It may be employed as a counterstain to basic cytoplasmic stains, to acid fuchsin in Van Gieson's connective tissue staining, or to crystal violet for the microscopic study of fungi.

Picric acid

It may also be used as a fixative, as a decalcifying agent, or as a tissue softener.

Picric acid

Trichloracetic acid, picric acid and chromium-fixed tissues usually take in acidic dyes more readily. Basic cell structures (collagen, eosinophilic granules of leukocytes, etc.) have an affinity for the acid dye ions and are regarded as______

acidophilic

- where the active coloring substance is found in a basic component that combines with the acid radical (usually taken from sulfuric, acetic or hydrochloric acid).

Basic Dyes

An example of a basic nuclear stain is____, which may be used both as an indicator and as a dye. It is very widely used in microbiology for bacterial staining

methylene blue

Tissues fixed with mercuric chloride and formaldehyde usually favor staining with basic dyes. Acidic cell structures (chromatin, mucus, cartilage matrix etc.) have an affinity for basic dye ions and are therefore regarded as basophilic. Tissues fixed with mercuric chloride and formaldehyde usually favor staining with basic dyes. Acidic cell structures (chromatin, mucus, cartilage matrix etc.) have an affinity for basic dye ions and are therefore regarded as______.

basophilic

- are formed by combining aqueous solutions of acid and basic dyes, capable of staining cytoplasm and nucleus simultaneously and differentially.

Neutral Dyes

Because they are made up of large molecular complexes,_____ dyes are insoluble or barely soluble in water, but they are usually soluble in alcohol

neutral

T or F Ethyl alcohol or acetic acid-fixed tissues, on the other hand, readily take in both basic and acidic dyes

True

Examples of neutral dyes are: _____dyes used in hematology, ______and ______for leukocyte differentiation

Romanowsky Giemsa's stain, and Irishman's stain

The two main alum hematoxylin solutions employed are

Ehrlich's hematoxylin Harris hematoxylin solutions

Rapid ripening of Ehrlich's reagent is brought about by the addition of______; while Harris solution is ripened with______.

sodium iodate mercuric chloride

Scott's Tap Water Substitute (T.W.S.) consisting of_____. NaHCO4 and____ MgS04

33.5 gm 20 grams

Blueing with (3) Substitute has more rapid action (about 15, 30 and 60 seconds respectively), compared to the 5 to 15 minutes required for warm tap water to "blue" hematoxylin.

ammonia lithium carbonate Scott's Tap Water

Hematoxylin For regressive staining

Ehrlich's

Ehrlich's Hematoxylin For regressive staining Cartilage and cement lines of bones:___ Staining time:___

Blue 15-40 mins

T or F Ehrlich's hematoxylin is not an ideal stain for frozen sections.

True

Hematoxylin Routine nuclear staining

Harris

Harris Hematoxylin Staining time:

5-20 mins

T or F Harris hematoxylin is a good regressive stain

True

Routine nuclear staining Exfoliative cytology Sex chromosomes

Harris Hematoxylin

is another alum hematoxylin solution recommended for routine purposes, especially used in sequence with Celestine blue. This alum hematoxylin is artificially ripened with an alcoholic iodine solution.

Cole's hematoxylin

Hematoxylin Routine Purposes Used in sequence with Celestine Blue Staining Time:

Coles Hematoxylin 10 mins

Like any alum hematoxylin, it can be used as a regressive stain, but it is also useful as a progressive stain. It is used as a nuclear counterstain to demonstrate the presence of cytoplasmic glycogen by special stain

Mayer’s hematoxylin

Hematoxylin Mucopolysaccharide - Nuclear staining

Mayer's Hematoxylin

One disadvantage of ____is that it can be stored only for 3 to 6 months at the most.

Mayer's hematoxylin

IRON HEMATOXYLIN

1. Weigert's Hematoxylin Solutions 2. Heidehain's Hematoxylin 3. Phosphotungstic Acid Hematoxylin (PTAH)

Demonstrating muscle fibers and connective tissue Recommended when the preceding stains contain acid

Weigert's Hematoxylin Solutions

Cytological stain recommended for regressive staining of thin sections - Demonstration of chromatin, chromosomes, nucleoli, centrosomes, and mitochondria Voluntary muscle striation and myelin

Heidehain's Hematoxylin

- Nuclei, fibrin, muscle striations, myofibrils, fibroglia: Blue Collagen, bone, cartilage: Orange-Red or Brownish Red to Deep Red Stain Demonstrates structure in paraffin, celloidin, and frozen section Staining Time: 12-24 hrs

Phosphotungstic Acid Hematoxylin (PTAH)

Phosphotungstic Acid Hematoxylin (PTAH) Nuclei, fibrin, muscle striations, myofibrils, fibroglia:_____ Collagen, bone, cartilage:_________ Demonstrates structure in paraffin, celloidin, and frozen section Staining Time:_____

Blue Orange-Red or Brownish Red to Deep Red Stain 12-24 hrs

Staining connective tissue and cytoplasm differentially - Counterstain after hematoxylin and before Methylene Blue

EOSIN

Eosin - deeper red color - green yellow fluorescence

• Bluish (Eosin B) • Yellowish (Eosin Y)

Two main iron hematoxylin solutions are employed for routine work in the laboratory: _______, using ferric ammonium chloride _______, using ferric ammonium sulfate (iron alum) as mordants.

Weigert's Solution Heidenhain's solution

The dye lake obtained when ferric salts are used as mordants is an intense _______one.

blue-black

It is a popular cytological stain, especially for the study of mitosis. It can be used after almost any fixative.

Heidenhain's Hematoxylin

is one of the most valuable stains used for differentially staining connective tissues and cytoplasm.

Eosin

It is a red general cytoplasmic stain that combines with hemoglobin to give an orange color.

Eosin