Special staining techniques for microorganisms

cells and microorganisms that we observe under the microscope lack color and contrast. This makes it difficult, if not impossible, to detect important cellular structures and their distinguishing characteristics without artificially treating specimens. We have already alluded to certain techniques involving stains and fluorescent dyes, and in this section we will discuss specific techniques for sample preparation in greater detail. Indeed, numerous methods have been developed to identify specific microbes, cellular structures, DNA sequences, or indicators of infection in tissue samples, under the microscope.

1. SPECIAL STAINING TECHNIQUES FOR MICROORGANISMS

2. BACTERIA Gram stain Brown- hopps modified gram stain Acid fast stain Ziehl - Neelsen Fite’s stain Fite’s new fuchsin formaldehyde technic Wade’s modification of fite’s new fuchsin formaldehyde Auramin - rhodamine fluorescent technic

3. BACTERIA 1) Gram stain Differentiate into gram positive and gram negative. Gram positive fungal filaments like Nocardia and Actinomyces can also be shown.

4. Gram’s staining

5. BACTERIA 2 ) Brown- hopps modified stain Gram positive – blue or blue violet Gram negative – red Background - yellow

6. BACTERIA 3) Acid fast techniques

7. ACID FAST STAIN

8. BACTERIA 4) Ziehl - Neelsen stain Acid fast bacilli – bright red Red blood cells- yellow orange Other tissue elements- blue Mycobacterium avium -complex, Ziehl-Neelsen acid fast stain .

9. BACTERIA 5) Fite’s stain Acid fast bacilli- red Background- blue Histioid leprosy – fite stain

10. BACTERIA CAPSULE STAINING Capsules are colourless and have low refractive index Difficult to observe without special staining Negative staining techniques can be used to demonstrate the capsule

11. SPIROCHETES Warthin - starry stain technic Steiner and sterner’s method

12. SPIROCHETES Treponema and leptospira - silver impregnation technique. Borrelia - Wright or Giemsa technics Staining tissue samples levaditi’s classic method Warthin -Starry technic Spirochetes are argyrophilic . 2% silver nitrate is used. Reducing agent in dye is hydroquinone.

13. SPIROCHETES WARTHIN- STARRY TECHNIC Results Spirochetes- black Background – pale yellow to light brown. Warthin -starry technic

14. SPIROCHETES MODIFICATION OF DIETERIE’S SPIROCHETE 5% uranyl nitrate is used. Organisms-black to dark brown Background – pale yellow

15. SPIROCHETES STEINER AND STEINER METHOD 1% uranyl nitrate 1 %silver nitrate Results organism-black background –yellow to brown   Treponema pallidum in a Skin-Biopsy Specimen (Steiner Silver Stain).

16. RICKETTSIAS & CHLAMYDIAS Giemsa Wright’s stain for rickettsia Pinkerton’s method for rickettsia

17. RICKETTSIAS & CHLAMYDIAS WRIGHT’S STAIN Results rickettsial cytoplasm- reddish pink nucleus – blue Wright stain of peripheral blood smears with Ehrlichia

18. RICKETTSIAS & CHLAMYDIAS PINKERTONS’S METHOD FOR RICKETTSIAS Results : rickettsia – bright red red blood cells- red Nuclei- blue

19. FUNGI Hotchkiss- mcmanus PAS technic Grocott’s modification of Gomori’s methenamine silver method Rapid grocott’s methenamine silver nitrate method for fungi Grideley’s stain

20. FUNGI fungus stain Actinomyces bovis Grocott and grams stain Aspergillus fumigatus PAS Blastomyces dermatitidis PAS Candida albicans Gridley, PAS,grocott stain Coccidiodies immites Grocotts stain Cryptococcus neoformans Grocotts stain,PAS Histoplasma capsulatum Grocotts stain

21. FUNGI PERIODIC ACID-SCHIFF (PAS) This method can be used to reveal fungal elements Fungi stain red Tissue material except glycogen and mucin fails to take up the colour

22. FUNGI HOTCHKISS- MAC MANUS PAS TECHNIC 1%periodic acid oxidizes the polysaccrides in fungal cell wall release aldehyde + schiff reagent magenta colour. Background is green colour .

23. FUNGI LACTOPHENOL COTTON BLUE Stains the fungal cytoplasm and provides a light blue background phenol serves as fungicidal agent Lactic acid – clearing agent Cotton blue – stains the cytoplasm of the fungus Glycerine- semi permanent preparation

24. FUNGI GOMORI METHENAMINE SILVER STAIN Fungus stains black Actinomyces israelii  stains with Gomori methenamine silver (GMS). (×250

25. VIRAL INCLUSION BODIES Orcein method for demonstration of HBsAg Aldehyde fuchsin method for HBsAg Lendrum phloxine - tartrazine method Parson’s stain for Negri bodies Schleifstein’s method for negri bodies

26. VIRAL INCLUSION BODIES Orcein method for demonstration of HBsAg oxidization: potassium permanganate 2%oxalic acid orcein stain(4hrs) Result : HBsAg : dark brown

27. picture

28. VIRAL INCLUSION BODIES Lendrum’s phloxine tartarazine method nuclear staining – mayer’s hematoxylin staining. Cytoplasmic staining- phloxine Results : inclusion bodies –red Nuclei – blue Background-yellow

29. VIRAL INCLUSION BODIES Parson’s stain for negri bodies Ethyl eosin Methylene blue Acetic acid Results – negri bodies : red nuclei : blue

30. THANK YOU SUBMITTED BY Dr Ranjini Manuel