Microtechnique I Presented by Dr: Safia Mohammed Hassan What is Cellular ?

Cell staining is a technique that can be used to better • visualize cells and cell components under a microscope, one can preferentially stain certain cell components, such as a nucleus or a cell wall, or the entire cell. Most stains can be used on fixed, or non- living cells, while only some can be used on living cells; some stains can be used on either living or non- living cells. Why Stain Cells

Biological tissue has very little variation in colures/shades when viewed using either an ordinary light (optical) microscope or an electron microscope. So staining biological tissues is done to both: increase the contrast of the tissue also highlight some specific features of interest Depending on the type of tissue and the stain used Structure and Colour

The components involved in histological staining are and proteins. * The fundamental process involved is the chemical bonding between the carboxyl groups of one and the amino groups of the other. * The commonest bonds involved are ionic bonds, although there are exceptions especially in the case of nuclear staining of DNA. & The use of colour to identify individual components of tissue sections. Why dyes are coloured

Colour in dyes is invariably explained as a • consequence of the presence of aChromophore. Since, by definition, dyes are aromatic compounds their structure includes aryl rings which have delocalised electron systems. These are responsible for the absorption of electromagnetic radiation of varying wavelengths, depending on the energy of the electron clouds. An aryl group is a functional group derived from a simple aromatic ring compound where one hydrogen atom is removed from the ring

–C=C– –C=N– –C=O–

–N=N– –NO2 Quinoid rings Auxochrome;

Auxochromes are groups which attach to non • ionising compounds yet retain their ability to ionise

–NH3 –COOH –HSO3 –OH While this definition is largely correct, • it is also inadequate. This is because it • restricts the definition of the auxochrome to ionization, and does not comment on the effect of auxochromes on the absorbance of the resulting compound. • The word auxochrome is derived from two roots. The prefix auxo is from auxein, and means increased. The second part, chrome means colour, so the basic meaning of the word auxochrome is colour increaser. This word was coined because it was noted originally that the addition of ionizing groups resulted in a deepening and intensifying of the colour of compounds Colour enhancing by an auxochrome

To the left is naphthalene, a colourless compound.

The addition of a single hydroxyl group to naphthalene produces 1-naphthol which is also a colourless compound, but one which can ionise.

If instead of a hydroxyl group we add the nitro group, which is a chromophore, we get the compound 2,4- dinitronaphthalene. The addition of this chromophore has caused it to become pale yellow.

If instead of a hydroxyl or nitro groups, both a hydroxyl and nitro groups are added, we get the deep yellow dye, martius yellow.

The addition of both an auxochrome and a chromophore results in a much stronger alteration of the absorption maximum of the compound. The hydroxyl group must have deepened the colour, showing that auxochromes are also chromophores. Auxochromes are of two types. They may have a positive charge as the amino group and its substituted variants. Or they may be negatively charged as the carboxyl and hydroxyl groups, and the sulphonic group. This last is commonly used to convert basic dyes to acid dyes. Both negatively charged and positively charged auxochromes may be present on a single molecule Mordants 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 When mordant is necessary called indirect • stain if not direct stain Used in histology AL &Fe • A mordant is a metal with a valence of at least two. * The two commonest metals used in histotechnology are aluminum and ferric iron, both with valences of three The attachment of mordants to dyes is by means of a covalent and a coordinate bond This is otherwise known as chelation, and is a relatively common phenomenon The chelate formed from a mordant dye and a metal is called a lake. –:A lake can be defined as A coordination complex formed between a polyvalent metal ion and certain dyes he three methods used for mordanting are: • Pre-mordanting (onchrome): The substrate is • treated with the mordant and then dyed. Meta-mordanting (metachrome): The • mordant is added in the dye bath itself. Post-mordanting (afterchrome): The dyed • material is treated with a mordant. Nuclear stains Routine staining The most routine staining procedure in histopathology employs the standard “Hematoxylin and eosin” This procedure visualize nuclei (blue) and cytoplasm pink regardless the type of fixative used

How staining occurs

Staining occur after in vitro oxidation of hematoxylin (hematien)

Hematoxylin can form a complex with metals such as aluminum ,iron, chromium this complex is known as “hemalum” and carries positive charge which enables it to combine with acid groups in tissue (DNA,RNA, acidic polysaccharides) Eosin is a dye molecule which carries a –ve charge combines with other constitutions in cytoplasm which have a basic nature

Other stains for nuclei & cytoplasm Substitute dyes may be used to demonstrate nuclei such as (alizarin blue s, Celestin blue b and gallocyanin) For both nuclei & cytoplasm( methyl green pyronine giemsa & azure-eosin method) Feulgen reaction specific for nuclear DNA Types of HEMALUM solutions A- mayers hemalum 1-Very selective for nuclear chromatin and requires no differentiation Progressive stain 2-Stained section with clearly defined nuclei and completely colorless back ground 3- parasites and encapsulated organisms may not stained as desired Harris hemalum 1- requires acid diffrentiation to obtain desired intensity 2- greatest advantage in the superior staining with experienced technician 3- difficult to obtain identical results from one specimen stained at different times even if done by same technician Comment on harries H&E procedure 1- hemalum solution act with basic dye so they bind tightly to acid tissue group( nuclear DNA) 2-hemalum dye-mordant complex act as indicator which turns to red at acid pH levels 3- eosin synthetic dye has is acid dye with large size & highly diffusible 4-penetrate rapidly compact tissue like red blood cells & less rapidly with more permeable although basic collagens 5-less soluble in alcohols than in water 6- alcohols slow rate of differentiation Methyl green-pyronin 1- Demonstrates nuclei (DNA) and cytoplasm (RNA) in blue green and red respectively 2- fixation with zenker or carnoys preferred for preservation of nucleic acid 10% formalin or buffered formalin Comment on Methyl green-pyronin

1- Methyl green & pyronin is only a semi-chemical stains nuclei under certain conditions 2- Methyl green & pyronin both basic aniline dyes not chemical specific for nucleic acids which depends most on dye patch fixation & embedding 3- pure Methyl green solutions stain mucus in intestine probably not through its DNA but through ferric acid esters Cytoplasm red Nuclei blue green to blue mucus green to blue Feulgen reaction

Discovered by robert feulgen 1924 Used in histology to identify chromosomal material or DNA Depend on acid hydrolysis of DNA Fixation using strong acids must be avoided This reaction takes advantage of the hydrochloric acid to convert DNA fixed to an aldehyde which identified using shiffs reagent Comment on Fulgen reaction

1-This reaction is a specific test for DNA 2- it depends on treatment of tissue sections by mild hydrolysis 3- hydrochloric acid does not hydrolyze nucleic acids instantly therefore prolonged gives more intense reaction Magenta color • CLASSIFICATION OF SPECIAL STAIN

• Specific stains for connective tissue • Specific stain for particular tissue substances • Specific stain for microorganismes Specific stain of connective tissue

• Collagen stain • Reticuiin stain • Elastic fiber stain Collagen stain

• Masson’s trichrome • Weigert-van Gieson stain Masson's Protocol for Collagen Fibers

• Description: This method is used for the detection of collagen fibers in tissues such as skin, heart, etc. on formalin-fixed, paraffin- embedded sections, and may be used for frozen sections as well. The collagen fibers will be stained blue and the nuclei will be stained black and the background is stained red. • Fixation: 10% formalin or Bouin's solution • Section: paraffin sections at 5 um. Procedure:

• 1. Deparaffinize and rehydrate through 100% alcohol, 95% alcohol 70% alcohol. • 2. Wash in distilled water. • 3. For Formalin fixed tissue, re-fix in Bouin's solution for 1 hour at 56 C to improve staining quality although this step is not absolutely necessary. • 4. Rinse running tap water for 5-10 minutes to remove the yellow color. • 3. Stain in Weigert's iron hematoxylin working solution for 10 minutes. 4. Rinse in running warm tap water for 10 minutes. • • 5. Wash in distilled water. • 6. Stain in Biebrich scarlet-acid fuchsin solution for 10-15 minutes. Solution can be saved for future use. • 7. Wash in distilled water. • 8. Differentiate in phosphomolybdic- phosphotungstic acid solution for 10-15 minutes or until collagen is not red. • 9. Transfer sections directly (without rinse) to aniline blue solution and stain for 5-10 minutes. Rinse briefly in distilled water and differentiate in 1% solution for 2-5 minutes. • 10. Wash in distilled water. • 11. Dehydrate very quickly through 95% ethyl alcohol, absolute ethyl alcohol (these step will wipe off Biebrich scarlet-acid fuchsin staining) and clear in xylene. • 12. Mount with resinous mounting medium. • Results:

Collagen ------blue Nuclei ------black Muscle, cytoplasm, keratin ------red

Positive Controls: Skin, lung, stomach, intestine.

• Sliver impregnation method • Van gieson stain Elastic fiber stain

• Verhoeff’ stain • Weigert’s resorcin – fuchsin stain carbohydrate

• 1- PERIODIC ACID SCHIFF'S (PAS )- • Principle: periodic acid oxidizes the carbon to carbon bond forming aldehydes which react to the fuchsin-sulfurous acid which form the magenta color.

(Periodic Acid cleaves sugars into aldehyde groups. Aldehydes react with Schiff Reagent- RED) • ,BM,cartilage,cellulose,cerebrosides,epithelial mucins,fungi,glycogen,hyaline membrane fetal lung,lipochrome pigment,mucoid cells of ant lobe of pituitary,pancreatic zymogen granules,starch,thyroid colloid FAT - SUDAN BLACK B

• PURPOSE: For the demonstration of fat. • PRINCIPLE: Sudan Black is slightly basic dye and will combine with • acidic groups in compound lipids, thus staining phospholipids also. An • alternative stain to the Sudan Black B stain. Stain for particular substances

• Stains for carbohydrates Periodic acid schiff stain Stain for fat Sudan stain Stain for amyloid Toludine blue and methyl vilot Stain for iron perl’s stain Stains for microorganism

• • Zeihl Neelsen stain Gram Stain: It is the most important differential stain used in bacteriology because it classified into two major groups:

Gram positive: (a b) Gram negative:

Appears violet after Appears red after Gram’s Gram’s stain stain

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