STAINS AND STAINING SOLUTIONS Flashcards
• Stains are prepared from two categories of dyes:
Natural dyes
Synthetic dyes
Natural Dyes
Hematoxylin
Cochineal Dyes
Orcein
Saffron
Synthetic Dyes
Aniline
Coal Tar dyes
are those obtained from plants and animals, previously utilized for dyeing of wool and cotton.
Natural dyes
Hematoxylin is a natural dye derived by extraction from the core or the heartwood of a Mexican tree known as
“Hematoxylin Campechianum”.
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
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
This is usually accomplished by exposing the substance to_____ or _____, thereby oxidizing hematoxylin (natural ripening).
air and sunlight
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
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
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
….stains are recommended for progressive staining of tissues, and are usually counterstained with Eosin, Congo Red and Safranin.
Alum hematoxylin stains
Both the Ehrlich’s solution and the Harris’ solution contain…
Alum Hematoxylin
Rapid ripening of:
Ehrlich’s reagent, however, is brought about by the addition of______;
Harris solution is ripened with_____.
Sodium Iodate
Mercuric Chloride
are used only for differential or regressive staining, using Acid-Alcohol as a differentiating agent.
Iron hematoxylin compounds
An example of an Iron Hematoxylin compound is_____ Stain using Iron (Ferric) Chloride
Weigert’s
…solutions are utilized for the study of spermatogenesis.
Copper hematoxylin
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
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
It is widely used as a powerful chromatin and nuclear stain for fresh material and smear preparations.
Cochineal dye
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
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
Color of orcein
Blue to violet
It is a weak acid, is soluble in alkali, and is mainly used for staining elastic fibers.
Orcein
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
- Weigert’s Hematoxylin Solutions
- Heidehain’s Hematoxylin
- 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
