Unstained sections appear transparent when seen under the light microscopy as they transmit light. Dyeing or staining is the process where the cell or tissue components are colored using dyes so that the cell constituents, their physical characteristics and relationships of tissue can be studied
What is the difference between staining and impregnation
Staining is the process where the cell or tissue components combine with active coloring agent. No particulate dye is seen. Tissue remains transparent
Impregnation is process where heavy metals are precipitated with selectivity on specific cellular or tissue components. It consists of particulate precipitate. It is used specifically in nervous tissue. Most commonly used impregnation agent is Silver nitrate
what is the difference between primary and secondary staining
Staining the tissue sections by dye is called primary stain
Counter stain with contrast color used to highlight primary stain is called secondary stain.
What is direct and indirect staining
Direct staining is the process where the tissue are stained by simple dye solution
Indirect staining is the process where the dye is intensified by mordants
What is the chemical basis of color in dye stuff
They consist of certain groupings known as chromophores and quinoid groups which selectively absorb the light rays and cause color to appear
Introduction of chromophoric group into an uncolored molecule will cause it to be colored which is then “chromogen” but not dye.
For a chromogen to be a dye it must be composed of acid and base and should have salt forming properties
What is “Leuco forms”
Reduction of colored dye yields a colorless “Leuco” forms owing to hydrogenation of its chromophore
Classification of dyes
Natural dyes – Hematoxylin, Orcein, Carmine and Saffron
Basic stains – these substances stain the acidic structures like nuclei eg.
Basic Fuchsin (Chloride salt of base Rosaniline)
Cresyl violet
Crystal violet
Azocarmine G
Celetsine blue
Methylene blue
Neutral red
Safranine O
Toluidine blue O
Thionine
Acid stain – These substances stain the basic part of the cell eg.
Acid Fuchsin consisting of sodium salt of sulfonate of Rosaniline
Aniline blue
Congo red
Eosin y
Methyl blue
Orange G
Picric aid
Phloxine
Neutral dyes – They contain mixture of both acidic and basic dyes eg. Romanowsky dyes
What is progressive and regressive staining
Progressive staining – tissue section is stained continuously until desired intensity of coloring of various tissue elements is obtained
Regressive staining – Tissue sections are overstained and then excess dye is removed selectively until the desired intensity is obtained
What is metachromasia
Dye or stain usually stain the tissue to their own color. Certain dyes stain the tissues in different hue or color. This property is called metachromasia
Name some metachromatic dyes
Dyes having metachromatic properties are
Thionine
New methylene blue
Azure A
Azure B
Azure C
Toluidine blue
Crystal violet
Methyl violet
Safranine
What is the cause of metachromasia
The property of metachromasia depends upon the property of dye which are cationic
Primary color of the dye is the result of monomeric forms (single molecule) in the solution and metachromasia results as these molecules polymerize
For example in Toluidine blue, the color of simple molecular solution is blue and as dimers and trimers increase in number it becomes violet. As the basic dye molecules polymerize it gives red metachromatic color.
Tissue that exhibit metachromasia are made up of large anionic molecules containing carboxylic acid radicals, sulfate or phosphate group in abundance. These acidic polysaccharides combine with basic dyes as salts. These acidic groups are close enough to permit secondary bonding between dye molecules so that polymerization occurs.
What are the types of metachromasia
There are 3 types of metachromasia
Alpha – type – Color is orthochromatic i.e., similar to that of monomeric form of dye
Beta type – color is intermediate owing to the mixture of monomeric and polymeric forms eg. Violet color in Toluidine blue staining
Gamma type – color is fully metachromatic due to completely polymerized surface bound dyes (Eg red color in Toluidine blue staining)
How to prevent or reverse metachromasia
Methylation abolishes metachromasia
Sulfation induces metachromasia and it can be effected by placing the hydrated sections in concentrated sulfuric acid for 1 minute. Then the sections are washed and then stained in 0.01% azure A in30% ethanol
What is the origin of hematoxylin stain
Hematoxylin is a natural dye extracted from the core or log wood of Haematoxylon campechianum.
This tree was originally found in State of Campeche in Mexico
10 years old trees are chopped, the bark and sap wood or outer layers are stripped and core or heartwood is cut into logs of 3 feet from which stain is extracted hence term “log wood” is used
Who established its use in histology
Waldeyer established its use in histology in 1862.
Before this dye was used in textile industry for dyeing silks and wool.
How is the active dye prepared from log wood
Natural extract obtained from logwood is called Hematoxylon which is not active dye
Hematoxylon should be oxidized to active coloring compound “Hematein”. During this process hematoxylin loses two hydrogen atoms and gets a quinoid arrangement in one of its rings
What is “ripening”
Process of oxidation of Hematoxylon to Hematein is called “ripening”
Ripening can be done by oxidizing with a chemical agent or by air and sunlight
Chemical oxidants are – mercuric oxide, sodium iodate, Potassium permanganate, hydrogen peroxide and calcium hypochlorite
Natural oxidation by air and sunlight may take several days for ripening where as chemical oxidation takes shorter time
What are the advantages and disadvantages of ripening by natural and chemical oxidation
Ripening by natural oxidation is a slow process and takes 3 to 4 months but it retains staining ability for longer time
Ripening by chemical oxidation is achieved in shorter time and also have shorter staining ability
What is the disadvantage of over ripening
Over oxidation or over ripening renders nuclear staining weak
What is mordant
Mordant are substances which aid in attaching a dye or stain to the tissues
Mordants combine as hydroxides with dye by displacing a hydrogen atom from it and their remaining valencies serve to attach or bind the dye mordant complex to the tissue components especially to the phosphate groups of nucleic acids
What substances are used as mordants
Salts of aluminum, iron, chromium, copper, molybdenum and Vanadium can be used as mordants
Alums which are double salts of sodium or potassium or ammonium sulfate crystallized with one molecule of the sulfate salt of trivalent metals like Potash alum (Potassium aluminium sulfate), Iron alum (Ferric ammonium sulfate) and ammonium alum (Aluminum ammonium sulfate)
What is “lake”
Complex of stain and mordant is called a lake
What are accentuators
Accentuators are chemical substances which increases the color intensity, crispness, and selectivity of stain
They are different from mordants in that they do not link or bind the dye to tissues
Examples – phenol in carbolthionine, aniline used in Gentian violet and potassium hydroxide used in Loffler’s methylene blue
What is differentiation in staining
In regressive staining method excess dye is removed until the right intensity is obtained.
This means when excess is being removed, it is cleared from certain cell constituents and some of the constituents retain the stain. This process of selective removal of dye is called differentiation
Example – Acid alcohol bath in routine Harris hematoxylin method
If the dye is basic then the differentiation is carried by acid solution and for the acidic dye, alkaline medium is used.
What are the types of hematoxylins
Harris hematoxylin
Ehrlich’s hematoxylin
Mayer’s hematoxylin
Weigerts iron hematoxylin
Mallory’s phosphotungstic acid hematoxylin
Which type of hematoxylin is routinely used and what is staining method
Harris hematoxylin is routinely used as the preparation is easy and has less ripening time
Staining method used is regressive staining
What is blueing and what agents are used for blueing
Blueing neutralizes the excess acid where red soluble haemalum is converted to blue color insoluble form and this sharpens nuclei blue.
Alum (eg potassium aluminium sulfate) in watery solutions dissociate such that aluminium combines with –OH of the water forming Aluminium hydroxide.
Free hydrogen from water tends to form sulfuric acid by uniting with the sulfate from the alum
If excess of acid is present then the aluminium hydroxide cannot form and in such situation, in an alum hematoxylin dye, the insoluble dye lake cannot form because of lack of hydroxyl ions.
Acid solutions of alum hematoxylin is reddish in color where as aluminium lake of hematein is blue
In blueing, the sections which have been stained by an alum hematoxylin, the alkaline solution is used for blueing which neutralizes the free acid and makes hydroxyl group available to form insoluble blue aluminium-hematein-tissue lake.
What agents can be used for blueing
Tap water can be used as it is alkaline
Other blueing agents are
1% of aqueous Lithium carbonate
1% of alcohol ammonia
Scotts tap water ( Sodium or potassium bicarbonate, 2 to 3.5gms, and magnesium sulfate 20gms, dissolved separately and then combined to make 1000ml with distilled water)
During the routine staining when the sections are dipped in acid acohol, pH of sections become acidic and look pink. By placing the sections in Lithium carbonate or tap water which is alkaline, pH will be neutralized and sections appear blue.
How do we prepare Harris hematoxylin
This is used as progressive and regressive stain
Reagents required
Hematoxylin powder – 2.5g
Mercuric oxide – 1.25g
Ammonium alum – 50g
Absolute ethyl alcohol – 50ml
Distilled water – 500ml
Dissolve hematoxylin powder in absolute alcohol
Cover it and keep it at the temperature of 600C for 30 minutes by placing in oven. Stir the solution intermittently so that the powder is dissolved completely in solution.
Dissolve the ammonium alum in hot water separately
Mix the two solutions and heat to boiling
To this add Mercuric oxide powder and put off flame bring it to cool water immediately to avoid the formation of bubbles
Mix well and then boil for 1 to2 minutes
Later cool the solution and close the container with cotton wool and keep it overnight
Next day filter the solution and store in amber colored bottle at cool dry place at room temperature
Filter the solution with Whatman filter paper no.1 before using
How do you check the quality of hematoxylin
Add few drops of hematoxylin to water. The colour of the drop should be purple to blue.
If the color of the drop added in water turns red, then the hematoxylin solution can be discarded
Which stain is used as counter staining
Eosin is used as counter stain which is acid xanthene or phthalein dyes
What are the different types Eosins used
Eosin Y (yellowish – water soluble)
Eosin B (Eosin blue)
Eosin S (Ethyl Eosin)
Phloxine
Erythrosine
How to prepare 1% Eosin stock solution prepared
1% Eosin stock solution
Eosin Y – 1g
Distilled water – 20ml
95% ethyl alcohol – 80ml
Working Eosin solution
Eosin stock solution – 1 part
Ethyl alcohol 80% – 3 parts
15 ml of Glacial acetic acid is added to 100 ml of stain which gives deeper shades
How is Aqueous Eosin solution prepared
Eosin Y – 5g
Distilled water – 100ml
Dissolve Eosin in water and if deeper tone is required add 5cc of Glacial acetic acid to 100cc of stain solution
What are the reagents used in preparing Mayer’s haematoxylin
This is used in progressive staining method
Reagents
Hematoxylin -1g
Ammonium or Potassium alum – 50g
Distilled water – 1000ml
Sodium iodate – 0.2g
Citric acid -1g
Chloral hydrate – 50g
Preparation of stain –
Hematoxylin is dissolved in distilled water by heating gently
Add alum and dissolve it by shaking
Add sodium iodate and citric acid
Later add chloral hydrate
Stain should be used with in 1 to 2 weeks
Stain the slides for 1 to 5 minutes and then wash thoroughly in tap water for blueing
What is the composition of Ehrlich’s haematoxylin
This is used as regressive stain
Reagents
Haematoxylin – 2 g
Absolute alcohol – 100ml
Distilled water – 100ml
Glycerol – 100ml
Glacial acetic acid – 10ml
Potassium alum in excess – approximately 15g
Stain is ripened by either natural method or by adding sodium iodate.
Why is glycerol added to hematoxylin
Glycerol acts as stabilizer, prevents over oxidation and reduces evoparation
How do you prepare 1% of acid alcohol
1ml of Hydrochloric acid is mixed in 99ml of 70% alcohol
What are the steps in routine Haematoxylin and Eosin staining
This is regressive method of staining where haematoxylin stains nuclei and eosin stains cytoplasm
Steps of staining
Immerse the sections in Xylene – 2 – 3min
Immerse in second xylene bath – 2-3min
Immerse in the absolute ethyl alcohol- 2minutes
Immerse in the absolute ethyl alcohol- 2minutes
Rinse the section in running tap water – 1minute
Stain in Harris hematoxylin – 4 to 8 minutes
Differentiate by dipping 3 to 4 times in 1% acid alcohol – 3 – 10 seconds
Blueing in running tap water -5 minutes
Place it in blueing agent (Scotts tap water or 1% Lithium carbonate)- 2- 3 minutes
Rinse in running tap water – 1 minute
Dehydrate by passing through 3 to 4 baths absolute ethanol – 10-20 seconds (in each bath)
Pass through two baths of xylene – 10 – 20 seconds (in each bath)
Mount with DPX
Result
Nuclei – blue
Cytoplasm – pink
Bone and calcium – Blue
Erythrocytes, muscle, eosinophil granules – bright red
Proteins in edema fluid –pale pink
How do you restain the slides
Slides are dipped in xylene for 1 -2 days to remove the cover slip
After removing the coverslip, slides are rinsed in fresh xylene till the mounting media is removed
Tissue sections are dipped in 1%acid alcohol until it is colorless
Slides are washed in running tap water for 30 minutes to remove acid alcohol
Slides are stained routinely
What is the composition of Weigert’s iron hematoxylin
This is progressive stain
Solution I
Haematoxylin – 1g
Absolute alcohol – 100ml
Solution II
30% aqueous solution of ferric chloride – 4 ml
Distilled water – 100ml
Hydrochloric acid – 1ml
Put the solution I in test tube then add equal parts of solution II and mix before use
Stain for 2 to 10 minutes and then rinse in distilled water
It can used in conjunction with Van Gieson Stain as counter stain
It is useful demonstrating connective tissue elements and Entamoeba histolytica in sections
This is the best nuclear stain
What is the composition of Mallory’s Phosphotungstic acid hematoxylin (PTAH)
Phosphotungstic acid 10% – 20ml
Haematoxylin 10% alcoholic – 1ml
Distilled water – 80ml
Preparation of stain – Mix them and add 5ml of 1% of potassium permanganate for ripening
Results –
Nuclei, mitochondria, fibrin, alcoholic hyaline, contractile portions of heart muscle and striated muscle, neuroglial fibrils – blue
Bone, ground substance of cartilage, reticulum and elastin – yellow –orange to brownish red
Which type of haematoxylin is used demonstrating striations of cardiac and voluntary muscle
Mallory’s Phosphotungistic acid haematoxylin
Which substance can be used as substitute for haematoxylin
Celestin blue
Mayer haemalum
Iron Alizarine blue
Reference
Christopher Layton, John D. Bancrofti, S Kim Suvarna. Fixation of Tissues. In: Theory and practice of histological techniques by John D. Bancrofti . 8th edition.
Cullings. Histotecniques. In: Lynch Medical Laboratory technology by Mathew J. Lynch, Stanely S. Raphael. Saunders publication 1983