1. S T A I N I N G DWI WINARNI Departemen Biologi Fakultas Sains dan Teknologi Universitas Airlangga Surabaya

2. Unstained cells/ tissues Stained cells/ tissues depend on affinity of tissue component for a certain dyes

3. PURPOSE of staining is to enhance contrast. This is accomplished in two ways: 1) Different colors 2) Coloring to different intensities

4. Chemistry of Dyes Basic Dyes = carry positive charge, attracted to acidic components of cells Acidic Dyes = carry negative charge, attracted to basic components of cells Neutral Stains = anion (-) and cation (+) provide different colors

5. GENERAL CATEGORIES OF DYES: 1. Acid-Base Combinations Most sections are stained with both acidic and basic dyes to enhance contrast by providing different colors. The most common combination is Hematoxylin and Eosin (H &E). Hematoxylin = basic dye, stains nuclear structures blue Eosin = acidic dye, stains cytoplasmic and intercellular structures pink 2. Trichrome Methods provides 3 colors, allows differentiation between cytoplasmic and intercellular components Van Gieson techniques for collagen (alum hematoxylin, celestine blue B and acif fuchsin)= nuclei, blue black; collagen, red; other tissues, yellow.

6. HEMATOXYLIN – EOSIN STAIN

7. 3. Dyes for Specific staining stain certain structures or molecules specifically Iron hematoxylin = useful in distinguishing finer cytologic details (e.g., subcellular organelles) Mallory-Azan = trichrome method; stains collagen fibers and mucus blue, stains nuclei and cytoplasmic components red Mason = trichrome method; collagen fibers stain green,cytoplasmic components stain purplish-red Periodic-Acid Schiff = selectively stains carbohydrate-containing molecules/substances red (e.g., glycogen, muco- and glycoproteins, glycosaminoglycans) Silver Impregnation = selectively outlines reticular and neural fibers, Orcein, Resorcin-Fuchsin = selectively stains elastic fibers Sudan Black B = specifically stains fat

10. PAS

11. The term mordant comes from the Latin word, "mordere", to bite A 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. It may be used for intensifying stains in cell or tissue preparations. A mordant is often a polyvalen metalion. The resulting coordination complex of dye and ion is colloidal and can be either acidic or alkaline Mordants include tannic acid, alum, sodium chloride, & certain salt of aluminium, chromium, copper, iron, iodine, potassium, and sodium

12. Hematoxylin …………………………………….. 5 g Etanol 96% ……………………………………... 50 ml Potassium alum ………………………………… 100 g Akuades …………………………………………. 950 ml Merkuri oksida …………………………………… 2.5 g Asam asetat glasial …………………………….. 40 ml

13. Metachromasia 1. the different coloration of different tissues produced by the same stain. 2. change of color produced by staining toluidine blue becomes pink when bound to cartilage.toluidine bluecartilage Mast cell granules  red to purple

14. A counterstain is a stain with color contrasting to the principal stain, making the stained structure more easily visible.  the malachite green counterstain to the fuchsine stain in the Gimenez staining technique.  eosin counterstain to haematoxylin in the H&E stain

15. The Gimenez staining technique uses biological stains to detect and identify bacterial infections in tissue samples. Although largely superseded by techniques like Giemsa staining, the Gimenez technique may be valuable for detecting certain slow-growing bacteria. Basic fuchsin stain in aqueous solution with phenol and ethanol colours many bacteria (both gram positive and Gram negative) red, magenta, or pink. A malachite green counterstain gives a blue-green background cast to the surrounding tissue Rickettsia conorii detected in hemolymph from infected Rhipicephalus sanguineus adult ticks using Gimenez staining.

16. Shrinkage = separation of portions of tissue that weren't separated in life; gives appearance of empty spaces Folds and Wrinkles = these may occur during cutting or mounting and will appear excessively darkly stained relative to the remainder of the section Nicks = result from defects in microtome knife; appear as pale, straight lines across the section Degeneration = occurs if tissue not removed immediately or not fixed immediately upon removal; results in inferior quality preparation A R T I F A C T S = imperfections in tissue preparation

17. Shrinkage

18. Folds

19. washing in alkaline water after eosin staining which has no red since alkalinity hinders eosin staining. washed in Adelaide tap water after staining in buffered eosin and it is just right. washed in acid water after eosin staining which is too red.

20. Tergantung pada bahan perekat (mounting media) yang digunakan