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1. GROUP 6 MUTHUSI JOSHUA BMLS/024J/2014 ODHIAMBO GEOFFREY BMLS/032J/201 KOECH DENNIS BMLS/017J/2014 ODAWO SELPHER BMLS/030J/2014 MASAKA W. BRIAN BMLS/021J/2014

2. HISTOLOGICAL STAINING. Staining; loosely defined as treating tissue or cells with reagent or series of reagents so that it acquires a colour .usually no particles of dye are seen and the stained element is transparent Stains; Are chemical substances used to achieve visible colour contrast in the microscopic feature of a prepared tissue. The hematoxylin and Eosin stains are the most widely used histological stains They are comparatively simple with ability to demonstrate enormous number of different tissue structures. Hematoxylin stains cell nuclei blue/black Eosin stains cell cytoplasm and most connective tissue fibers

3. Theories of Staining Physical theories S imple solubility; Fat stains are effective because the stain is more soluble in fat than 70% alcohol absorption; Property by which a large body attracts to itself minute particles from a surrounding medium. Chemical theories; Generally acid dyes stain basic elements

4. Types of stains Hematoxylin Eosin Mallory’s Trichrome stain Masson’s Trichrome stain Toluidine blue Wright stain Silver stain Weigert’s stain PAS

5. Hematoxylin and Eosin Used for general staining. Principle; H and E stains for demonstration of nucleus and cytoplasm. The cell differentiation is achieved by treating the tissue with acid solution . The counter staining is performed using Eosin which imparts colour to cytoplasm Hematoxylin binds to acidic structures(Nucleic acids) and stains them blue/black Eosin binds to and stains basic structures(cytoplasm, muscle and connective tissue, RBC) pink

6. PROCEDURE Deparaffinize the section: Flame the stain and burner and place it in the xylene Hydration : Hydrate the tissue section by passing through decreasing concentration of alcohol baths and water (100%-70%) Stain in hematoxylin for 3-5minutes Wash in running tap water until section ‘blue’ for 5 minutes Differentiate in 1% acid alcohol for 5mins Wash in running tap water until sections are again blue by dipping in alkaline solution followed by tap water wash Stain in 1% Eosin 10mins wash in tap water for 5min Dehydrate in decreasing concentration of alcohols and clear in Xylene Mount in mounting cc media and observe under microscope

7. RESULTS Nucleus; blue /black Cytoplasm; pink Muscle fiber; deep red RBC; Orange Fibrin; deep pink

8. Masson Trichrome Stain Evolved from Claude L. Pierre Masson 1929 Three colour staining protocol Used to stain connective tissues: distinguishes between muscle and collagen tissue Stains muscles, erythrocyte and cytoplasm red, collagen blue and nuclei black Used in identification of fibrosis Principles; Trichrome produce three dyes Sections is first stained with acid dyes such as biebrich scarlet Acidophilic elements; cytoplasm, muscle and collagen will bind with acid dyes

9. RESULTS Muscle, Erythrocyte- Red Cytoplasm - Red Collagen – blue Nucleus- Black

10. PROCEDURE Deparaffinize and rehydrate through 100%-70% alcohol Wash in distilled water Rinse in running tap water for 5-10mins to remove the yellow colour Stain in weigert’s ion hematoxylin working solution for 10 mins Rinse in running warm tap water for 10minutes and wash in distilled water Stain in Biebrich scarlet acid fuchsin solution for 10mins Wash in distilled water Differentiate in phosphomolybbdic-phosphotungstic acid solution 10-15mins Transfer sections directly without rinsing to aniline blue solution and stain for 5-15mins. Rinse briefly in distilled water and differentiate in 1% acetic acid solution for 2-5mins Wash in distilled water Dehydrate very quickly through 95% ethyl alcohol (These step will wipe off B iebrich scarlet acid fuchsin solution) and clear xylene Mount in Resinous mounting media

11. MALLORY’S TRICHROME STAIN Reveals different macromolecules that make up the cell Uses three stains aniline blue, acid fuchsin and orange G Stains connective tissue. REAGENTS Solution A Acid fuchsin Distilled water Solution C Orange G Oxalic acid Methylene blue Distilled water Solution B Phosphomolybdic acid Distilled water

12. PROCEDURE Bring sections to water via Xylene and Ethanol Place into Solution A for 2mins Rinse with distilled water Place into solution B for 2 mins Rings quickly with distilled water Place into solution C for 15 minutes Wash with distilled water Dehydrate and differentiate with Ethanol Clear with Xylene and mount with a resinous medium

13. RESULTS Nucleus red Erythrocytes Orange Muscle Red Collagen Blue

14. SILVER STAINS Used to show Melanin and reticular fibers (argyrphilic tissue has an affinity for silver salts hence silver salts will be seen argyriphilic tissue REAGENTS Redundant - 200mg sodium thiosulfate Silver stain -2g silver nitrate Developer - 30 g sodium Carbonate Stop sol - 5% acetic acid in water water

15. Procedure Fix a tissue in a gel using a fixative for 20 minutes Wash the tissue 3 times in a wash solution Reduce in redundant for 1 minute Wash again 3 times in water (about 30 sec each) Stain with silver stains for 20 minutes Wash 3 times in water Develop in Developer solution until bands become visible Stop using a stop solution Observe under a microscope.

16. Expected results Reticular fibers-brown/black Nerve fibers-brown/black.

17. WRIGHT STAINS Based on blend of dyes such as methylene blue derivatives and acids dyes such as eosin. Used for staining blood smears ,urine smears and bone marrow smears. Named after James Wright.

18. Procedures Place 1 ml of liquid wright on the blood film for 1-9 minutes depending upon its behavior Add 2ml of phosphate buffer solution adjusted to a ph 6.5 Allow the mixture to react until the thin portions of the stained film are pink Stand the slide on end to air dry or blot carefully. Make observations

19. Expected results Eosinophil-red /orange Basophil –deep purple or violet Platelet-red /purple Neutrophil –purple/pink

20. weigert’s stain It is used to stain elastic fibers Is a sequence of 3 solutions; ferric chloride in dilute HCL,hematoxylin in 95% ethanol and potassium ferricyanide solution Solutions/reagents Weigert’s iron hematoxylin Van Gieson’s picrofuchsin Ethanol and distilled water PROCEDURE Bring sections to water via xylene and ethanol Place into Weigert’s solution for 20mins Wash with 95% Ethanol to remove excess solution Differentiate with 1% acid alcohol Wash in water Counterstain with iron hematoxylen and van Gieson Dehydrate with ethanol, clear with xylene and mount with Resinous medium

21. RESULTS Elastic fiber -blue black Collagen- red

22. TOLUIDINE Used to stain mast cells Is a thiazine metachromatic compound, usually an organic compound Solution and reagents Toluidine blue Sodium chloride Distilled water Procedure Deparaffinize and hydrate the sections to distilled water Stain sections in toluidine blue for 3mins Wash in distilled water Dehydrate through 95% alcohol Clear in xylene for 3mins Mount in resinous mounting medium

23. EXPECTED RESULTS Mast cell -purple

24. PAS Periodic Acid Schiff (PAS) staining is one of the most commonly performed special staining technique in histopathology laboratory which is used to highlight molecules with high percentage of carbohydrate content such as; mucin , glycogen, fungi and basement membrane in skin.

25. Procedure of PAS Staining 1. Bring sections to distilled water . 2. Treat with periodic acid for 5 minutes. 3 . Rinse well in distilled water. 4. Cover with Schiff ’ s reagent for 5-15 minutes. 5 . Wash in running tap water for 5-10 minutes 6. Counter stain with Herri’s hematoxylin for approximately 15 seconds . 7. Differentiate (if necessary) with acid alcohol and bluing as usual. 8. Wash in tap water. 9. Rinse in increasing concentration of alcohol (70,80, 95 and 100%) 10. Clear in xylene and mount as usual

26. . Result Formation of insoluble magenta colored complex denotes positive result . PAS staining can be used to assist in the diagnosis of several medical conditions such as :   Paget’s disease of breast Alveolar soft part sarcoma Whipple’s disease of immature RBCs