Staining Techniques in Microscopy

• Staining is an auxiliary technique used in microscopy to enhance image contrast.
• Dyes and stains are substances used in biology and medicine to highlight structures in biological tissues for observation with different microscopes.
• Dyes can increase definition and examine large tissue sections, such as highlighting muscle or connective tissue, cell populations like blood cells, and organelles within individual cells.
• In biochemistry, specific dyes are added to substrates to qualify or quantify the presence of certain compounds like DNA, proteins, lipids, or carbohydrates
• Staining and fluorescence marking serve similar purposes.
• Biological stains are used to mark cells in flow cytometry, proteins, or nucleic acids in gel electrophoresis.
• Most dyes are organic compounds with specific affinities for cellular materials.
• Many commonly used dyes are positively charged molecules (cations) that strongly bind to negatively charged cellular components (anions), such as nucleic acids and acidic polysaccharides.

Dye Electronegativity

• Cationic dyes: Methylene Blue, Crystal Violet and Safranin
• Anionic dyes: Eosin, Acid Fuchsin and Congo Red

Stains in Connective Tissue

• Collagen Stains: Masson's Trichrome, Gomori and Van Gieson
• Reticulin Stains: Ammoniacal Silver, like Snook, Wilder and Manuel methods
• Elastin Stains: Ferric Hematoxylin like Verhoeff method, Resorcin Fuchsin like Hart method, and Aldehyde Fuchsin like Gomori method

Specific Staining Methods

• Masson's Trichrome stains collagen
• Van Gieson stains collagen, muscle, and nuclei
• Verhoeff method stains elastic fibers
• Snook method stains reticulin fibers

Stains for Carbohydrates

• Glycogen is found in the liver, heart, and skeletal muscle
• Neutral mucins are in gastric cells and Brunner's duodenal glands.
• Salivary mucins are in salivary glands, intestinal goblet cells, and gastric mucosa.
• Acidic mucins are in intestinal goblet cells.
• Chondroitin sulfate and heparin are in mast cells, the aorta, and cardiac connective tissue.
• Hyaluronic acid is in the umbilical cord and connective tissue of the dermis.

Specific Staining Techniques

• Periodic Acid-Schiff (PAS) stains neutral mucins; lipid stains are frequently done on frozen sections or fixed tissues
• Oil Red O stains lipids

Stains for Pigments and Minerals

• Pigments are substances with inherent color, commonly brown or black in tissues.
• Common methods include Warthin-Starry for melanin, Hall's method for bilirubin, and Perls' method for iron.
• Von Kossa method stains mineral salts, and the Rhodanine method is for copper localization.
• Warthin-Starry stains melanocytic reactions
• Von Kossa stains cartilage.
• Peris stains iron

Stains for Bacteria, Fungi, and Microorganisms

• Demonstrating infectious organisms in paraffin sections and smears through staining techniques is invaluable for diagnosis.
• Gram staining can differentiate between gram-negative and gram-positive bacteria

Specific Staining Techniques

• Brown and Hopps, and Brown Brenn stainings are commonly used.
• Gram-negative bacteria: Helicobacter Pylori
• Gram-Positive Bacteria: Streptococcus sp (blue) and Brucella sp (red)

Acid-Fast Staining

• Kinyoun stain identifies Mycobacteria.
• Auramine O and Rhodamine B are used for fluorescent staining of Mycobacterium.

Fungi Staining

• Common stains include Gomori methenamine silver (GMS) for Aspergillus fumigatus, PAS for Candida Albicans, Giemsa for Dermatophytes, Gridley fungus for Blastomycetes and Mucicarmine for Cryptococcus neoformans.
• PAS stains hyphae of Aspergillus spp
• Mucicarmine stains Cryptococcus

Other Staining Methods for Microorganisms

• Kinyoun stains Cryptosporidium
• PAS stains Actinomyces sp and Histoplasmosis,
• Fluorescent stains highlight certain microorganisms Fluorescent stains highlight certain microorganisms

Parasite Staining

• Parasite staining includes various organisms, from unicellular protozoa to visible helminths
• Protozoa of leishmania are seen using Giemsa stain.
• Pneumocystis Carinii, an opportunistic organism in immunocompromised patients' lungs, can be observed with Giemsa, Papanicolaou, and fluorescence stains.

Staining Applications

• Giemsa stains leishmaniasis.
• Papanicolaou and Fluorescent stains stain Pneumocystis Carinii.
• Giemsa is also used for cytology specimens, such as cervicovaginal smears.
• Histohemical methods react chemical reagents with cellular and tissue components to demonstrate them through colored products visible under a microscope, which can be classified in three types depending on what they stain: substances, microorganisms, or special components.