SPECIAL BACTERIAL STAINING

SPECIAL BACTERIAL STAINING

1. SPECIAL STAINING METHODS IN BACTERIOLOGY Dr.P.B.PRAVEENKUMAR FIRST YEAR POST GRADUATE DEPARTMENT OF MICROBIOLOGY THANJAVUR MEDICAL COLLEGE

2. ACID FAST STAINING First discovered by PAUL EHRLICH (1882) Modified by ZIEHL-NEELSON EHRLICH’S METHOD : Staining was done using aniline-gentian violet followed by strong nitric acid As the ordinary aniline dyes do not readily penetrate the tubercle bacillus this method was modified Most widely useful staining for acid fast bacilli is the ZIEHL-NEELSON technique

3. PRINCIPLE Acid fastness is attributed to the presence of large quantities of unsaponifiable wax fraction called mycolic acid in the cell wall . Degree of acid fastness varies in different bacteria which depends on the content of mycolic acid In this method application of heat helps the dye to penetrate the tubercle bacillus Once stained the tubercle bacilli resist decolourizing action of acid-alcohol Other organisms that get decolourized are considered non acid fast

4. COMPONENTS OF AFS Primary and mordant Staining : strong carbol fuchsin Decolourize with acid alcohol: 3% HCL & 25%H2SO4 or 95% ETHANOL Counter stain : methylene blue/Malachite green Acid fast cells-red Non acid fast cells-blue

5. PROCEDURE (HOT METHOD) Heat fix the smear by gently passing over the flame(coagulation of proteinaceous material) Cover with strong carbol fuchsin ( carbol fuchsin + phenol) for 5 mins Allow it to stain for 5 mins . During this period heat the slide intermittently , don’t allow to dry Rinse with water Cover with 25% H2SO4 for 10 minutes for decolourization Wash the slide with water Cover the slide with methylene blue for 15-20secs Wash ,air dry & examine

6. INTERPRETATION Mycobacterium TB appears as long slender, straight/beaded red color acid fast bacilli Other non acid fast organisms and background take up counter stain and appear blue

7. MODIFICATIONS KINYOUN’S modification: Cold method where the intermittent heating is not required. Acid alcohol can be used as decolourizer Malachite green can be used as counter stain Concentration of H2SO4 depends on the acid fastness of the organism Brucella abortus – weakly acid fast (0.5% acetic acid – decolourizer)

8. FLUROCHROME STAINING Stain containing a fluorescent dye and examined under fluorescent microscope after staining AURAMINE – RODAMINE is an example where AFB appear yellow against a black background More rapid, sensitive & accurate compared to ZIEHL-NEELSON method in identifying AFB , because of higher cost & technical complexity it is less feasible

9. PROCEDURE Thin sputum smear that is dried & heat fixed is taken Smear is covered with Auramine phenol for 10mins Wash off with water ,add 1% acid alcohol and leave to decolorize for 5 minutes Wash with water, cover the smear with 0.1% potassium permanganate counter stain for 15secs Wash, air dry examine by fluorescence microscopy. Tubercle bacilli appears as yellow luminous rods in dark field

10. STAINING OF VOLUTIN CONTAINING GRANULES Volutin granules also called babes ernst / metachromatic granules / polar bodies are well developed granules within the bacterial cytoplasm They appear as round refractile bodies in unstained wet preparations They are made up of polyphospate PRINCIPLE: Granules exhibit metachromatic property whereas the remaining bacteria take up the contrasting counter stain STAINS USED : ALBERT’S STAIN , NEISSER’S OR PUCH’S STAIN, PONDER’S STAIN.

11. STAINING OF VOLUTIN CONTAINING GRANULES (cont.) With toludine blue/ methylene blue the granules stain metachromatically and appear reddish purple ALBERT’S STAIN : Granules in diphtheria bacilli exhibit metachromasia and hence appear bluish black when stained with toluidine blue present in ALBERTS STAIN I REAGENT ,while the diphtheria bacillus appears green due to methyl green in the reagent ALBERT-LAYBOURN METHOD: (Instead of methyl green, malachite green used)

12. ALBERT STAIN PROCEDURE : Prepare the film, air dry, heat fix Cover the film with ALBERT’S STAIN I for 3-5 mins Wash with water and blot dry Add ALBERT’S STAIN II ( ALBERTS IODINE) for 1 min Wash and blot dry GRANULES-BLUE BLACK BACILLI-GREEN

13. SPORE STAINING Spores are highly resistant and metabolically inactive forms Morphology of bacterial endospores is best observed in unstained wet films under phase contrast microscope where they appear as large, refractile , oval bodies Different staining methods are available for staining spores Schaeffer & fulton method- malachite green Modified Ziehl-Neelson stain -0.25% H2SO4 (appears red with blue bacilli), lipid granules also appear red Toluidine blue stain Grams stain - Appears as a clear area within stained bacilli Moeller stain

14. SCHAEFFER FULTON METHOD MODIFIED BY ASHBY Films are air dried and heat fixed Place the slide with the smear over a beaker of boiling water, resting it on the rim with bacterial film on the upper side When within several seconds large droplets have condensed on the underside of the slide ,flood the smear with 5% aqueous solution of malachite green and allow to act for 1min while the water continuous to boil Wash the slide with cool water Then cover it with 0.5% safranine or 0.05% basic fuschin for 30 seconds Rinse it with water and observe under microscope

15. SPORE STAINING (cont.) Spores take up malachite green where as the decolourized vegetative cell takes up the counter stain and appears red. Lipid granules remain unstained

16. CAPSULE STAINING Capsules are the protective substances secreted by the bacteria around the cell which prevents them against penetration by toxins Capsule staining is more difficult than other types of differential staining because capsular materials are water soluble and may be removed easily by vigorous washing Also called as SLIME LAYER/LOOSE SLIME Bacterial smears should not be heated as resultant cell shrinkage may create a clear zone around the organism that may be mistaken for a capsule

17. CAPSULE STAINING (cont.) Capsule is non ionic , so that the dyes commonly used will not bind to it . Two dyes, one acidic and one basic are used to stain the background and cell wall respectively METHODS: POSITIVE STAINING : Capsule is stained NEGATIVE STAINING : background is stained McFadyean’s reaction The best method for staining capsules on bacteria from cultures is the wet film India ink method

18. POSITIVE STAINING Prepare a smear from capsulated bacteria and allow it to air dry. SHOULD NEVER BE HEAT FIXED Flood the smear with crystal violet . Allow it to stand for 5-7 minutes Wash the smear with 20%CuSO4 solution and blot dry Observation : capsule is seen as light blue in contrast to the deep purple color of cell

19. NEGATIVE STAINING (WET FILM METHOD) Take a clean glass slide Place a large loopful of undiluted India ink on the slide Then add a small quantity of liquid bacterial culture to India ink & emulsify Take a cover slip and place on the India ink & press it down Observe under high power Capsule appears as clear halo around the organism against black background

20. NEGATIVE STAINING (DRY FILM METHOD) Nigrosin , modification of India ink 2% mercurochrome can be used Take a clean glass slide Place a small drop of nigrosin close to one end of the slide Using a sterile loop, loopful of broth culture is mixed with nigrosin With the edge of second slide make a thin smear Air dry and observe Capsule appears as clear halo around the organism against black background Occasionally shrinkage spaces may form & give false appearance as capsules

21. DEMONSTRATION OF FLAGELLA Flagella are best demonstrated with electron microscope /metal shadowed films/films made with phosphotungstic acid because of their extreme thinness Special staining methods are used by which flagella is thickened ten folds by superficial deposition of stain produced by action of mordant Easier and most reliable method for staining flagella in wet mounts was described by Heimbrook et al (1989)

22. WET MOUNT FLAGELLAR STAIN PROCEDURE Grow bacteria for 16-24 hrs on a non-inhibitory medium, eg : tryptic soy agar/blood agar Touch a loopful of water onto the edge of a colony and let motile bacteria swim into it. Then transfer the loopful into a loopful of water on a slide and cover with cover slip After 5-10 minutes apply two drops of Ryu’s stain and leave it to diffuse into the film Examine after 5-15mins

23. RYU’S STAIN Ryu’s stain: Crystal violet in saturated ethanol solution Mordant solution(tannic acid, 5%phenol, Aluminium phosphate)

24. STAINING OF LIPIDS BURDON’S METHOD: Prepare a film, air dry & heat fix Cover the entire slide with SUDAN BLACK stain for 15mins Drain excess stain & air dry Rinse thoroughly with xylene and blot dry Counter stain with 0.5% safranine or dilute carbol fuschin for 5-10 seconds, rinse with water & air dry Lipid granules-blue black Bacterial cytoplasm-light pink

25. LIPID STAINING (cont.) HOLBROOK & ANDERSON METHOD: (Mainly for bacillus species) Differential staining technique which combines BURDONS stain for lipid & ASHBY’S stain for spores Prepare a film, air dry & heat fix Stain with 5% malachite green for 2 minutes with the slide placed over boiling water, wash, blot dry. Cover the entire slide with SUDAN BLACK stain for 15mins Drain excess stain & air dry Rinse thoroughly with xylene for 5 secs and blot dry Counterstain with 0.5% safranine or dilute carbol fuchsin for 5-10 seconds, rinse with water & air dry Lipid granules-blue black Spores- green Bacterial cytoplasm-red

26. STAINING OF SPIROCHAETES Larger spirochaetes,eg ., Borreliae can be seen by ordinary methods like gram’s stain, leishman’s , giemsa’s stain Smaller spirochetes are too thin hence best observed in unstained wet films under dark ground microscopy Silver impregnation techniques help for permanent preparation from film/sections FONTANA’S METHOD(film), LEVADITI (tissue sections )

27. FONTANA’S METHOD Treat the film 3 times, 30 seconds each time with fixative (acetic acid, formalin, distilled water) Wash the fixative with absolute alcohol & allow the alcohol to act for 3 minutes Drain the excess alcohol and burn off the remainder till it becomes dry Pour on the mordant ( phenol,tannic acid,distilled water) heat till steam arises ,allow to stand for 30 seconds Wash with distilled water and air dry Treat with ammoniated silver nitrate heat till steam arises , allow to stand for 30 seconds till the film turns brown Mount under cover slip & examine Spirochetes- brown black on brown yellow background

28. ROMANOWSKY STAINING Neutral stains Original stain was made by dissolving the compound formed by interaction of watery solutions of eosin and zinc free methylene blue in methyl alcohol IMPARTS REDDISH PURPLE color to the chromatin of malaria Various modifications were formed later which are easy to use Giemsa’s stain, Leishman’s , Wrights, Jenners stain Each modifications of romanowsky stain varies according to the ripening and relative proportion of eosin and methylene blue

29. GIEMSA STAIN (TO SEE SPIROCHAETES) Consists of Azur I, Azur II and Azur II-eosin. ( Lillie’ preparation ) Fix the film in methyl alcohol for 3 minutes. Pour 1 ml stain with 20 ml buffer solution in Petri dish. Place a piece of thin glass rod in dish. Place the fixed slide, film side downwards in dish with one end resting on the rod. ( Sufficient stain ) Over night incubation, wash it, dry and examine.

30. ACRIDINE ORANGE STAINING Acridine orange R has a marked affinity for nucleic acids When cells stained with it are viewed by UV rays, the RNA components fluoresce orange-red and DNA yellow-green Useful in studying growth of viruses in host cells and for revealing scanty bacteria in wet films.

31. ACRIDINE ORANGE STAINING (cont.) A drop of 0.01% acridine orange in centre of slide Then add one drop of specimen in the slide. Apply cover slip and focus it using UV fluorescence microscope. (40X objevctive )

32. BIPOLAR STAINING WAYSON STAINING CARBOL THIONINE STAINING Solution A (Basic fuchsin , methylene blue, ethyl alcohol) Solution B (Phenol 5%) Stain the smear after filtration using both solutions one by one. Thionine stock solution Phenol Distilled water Filter and use this stain

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