Basics of gram staining
- 1. STAININGTECHNIQUES Presenter : Dr.Ruchika JR Microbiology deptt GMC PATIALA
- 2. Why there is need of staining ?
- 3. Methods of making the film or smear preparations .To be made on glass slides/coverslips ,should be grease free otherwise uneven films will be made
- 4. • Wipe slide with clean dry cloth • Holding end with forceps. • Roast it free by passing 6-12 times through Bunsen flame ( method no.2 ) • Moisten the finger with water and rub it on the surface of fine sand soap then smear the surface of the slide. • Remove the soapy surface with clean cloth, making it grease free.
- 5. How do we know if slide is perfectly clean?
- 17. Gram's Stain is a widely used method of staining bacteria as an aid to their identification. .
- 18. Why there is need of gram staining? Gram stain interpretation gives information about the presence or absence of bacterial disease and can guide the initial antibiotic treatment. Gram stain also provides additional information about the host immune response and quality of the specimen. A well prepared sample can show the organism colour size, shape and arrangement, allowing cellular morphology to further separate bacteria into four major groups. Cocci are spherical or oval, bacilli are rod like or cylindrical, vibrios are comma shaped or curved like and spirochetes are flexible.
- 19. Certain bacteria when treated with one of the basic para-rosaniline dyes and then with iodine, ‘fix’ the stain so that subsequent treatment with a decolourization agent does not remove the colour.
- 20. Gram positive cocci Gram negative cocci Gram positive bacilli Gram negative cocci
- 21. ORIGINAL GRAM’S STAIN Heat fix the slide and allow it to cool Stain with Carbol gentian violet * 60 sec Wash with water Stain with Gram’s iodine (iodine + KI) * 30 sec Decolorize with 95% alcohol till no further violet comes away Wash thoroughly with water Counterstain with 1% aqueous safranin * 2 min Bismarck brown may also be used Wash with water, air dry & examine Gram positive Purple Gram negative Pink
- 22. MECHANISM OF GRAM STAINING The violet dye and iodine combine to form an insoluble ,dark purple compound in the bacterial protoplasm and cell wall. This compound is dissociable with the decolorizer Removal is much slower from gram positive than from gram negative
- 23. MODIFICATIONS OF GRAM STAINING The original gram stain also has certain limitations. The original gram stain has certain limitations in identification of some organisms. Sometimes gram positive organisms may appear gram negative or vice versa due to excessive heat, prolonged decolourisation etc. and one more limitation is, it cannot be applied in tissue sections. Many modifications of the original gram staining technique have been published. For eg. The test developed by Christain Gram in 1884 was modified by Hucker in 1921. The modified procedure provides greater reagent stability and better differentiation of organisms.
- 24. MODIFICATIONS OF GRAM’S STAIN MODIFI- CATION PRIMARY STAIN MORDANT DECOLORIZER COUNTER STAIN Original Gram stain Carbol Gentian Violet Lugol’s Iodine 95% Alcohol 1% Aqeous Safranin or Bismarck brown Hucker Modification Crystal Violet + Ammonium oxalate Gram’s Iodine 95% Alcohol 0.25% Safranin Kopeloff Modification Crystal Violet with NaHCO3 Iodine + NaOH Acetone-Alcohol 2% Safranin Kopeloff- Beerman Modification Gentian Violet with NaHCO3 Iodine + NaOH 100% Acetone 1:1000 watery basic fuchsin Burke’s Modification 1% Aqueous Crystal Violet with NaHCO3 Iodine 100% Acetone 2% Safranin or Neutral red
- 25. Staining methods for acid fast bacteria
- 26. STAINS FOR ACID FAST BACTERIA MODIF ICATION PRIMARY STAIN DECOLORIZING AGENT COUNTER STAIN Ehrlich’s original method Aniline gentian violet Strong HNO3 Picric acid Ziehl Neelsen method Strong Carbol fuchsin f/b heating 20% H2SO4 Loeffler’s methylene blue/ Malachite green Brucella differential stain Dilute Carbol fuchsin without heating 0.5% Acetic acid Loeffler’s methylene blue Kinyoun modification Strong carbol fuchsin without heating 3% HCl in 70% alcohol Loeffler’s methylene blue
- 27. ZIEHL- NEELSEN STAINING Modification of Ehrlich original method ( gentian violet followed by strong nitric acid . Modifications implememted after suggested by Ziehl and neelsen. Ordinary dyes unsuitable ,not able to penetrate tubercle bacilli. Therefore req. a staining solution conatianing PHENOL APPLICATION OF HEAT MADE TO PENETRATE BACILLI ABLE TO WITHSTAND POWERFUL DECOLORIZING AGENT STILL REATIN THE STAIN ,WHEN EVERYTHING ELSE HAS BEEN DECOLORIZED .
- 28. ZIEHL- NEELSEN PROCEDURE Carbolfuchsin Basic fuchsin 3g 90-95% Ethanol 10ml 5% Aqueous solution of Phenol 90ml Acid-alcohol 3ml of conc. HCl is slowly added to 97ml of 90- 95% ethanol Methylene blue counterstain 0.3g of Methylene blue chloride is dissolved in 100ml of distilled water Malachite green counterstain 5gm in 500ml of distilled water
- 29. USE OF ALCOHOL AS SECONDARY DECOLORIZATION After primary decolorization with sulphuric acid , Film treated with 95% alcohol(ethanol) as secondary decolorization. Basis :tubercle bacilli are both acid and alcohol fast • Advanatge : decolorization completed more quickly • Other acid fast bacilli which may be confused with tubercle bacilli are decolorized by alcohol • margins and underside of the slide are more completely cleaned
- 30. ZIEHL- NEELSEN PROCEDURE contd.. Cover a heat-fixed smear with a rectangular 2X3cm filter paper Add 5-7 drops of carbolfuchsin to thoroughly moisten the filter paper Heat the stain-covered slide to steaming but do not allow to dry Remove the paper with forceps, rinse slide with water & drain Decolorize with acid-alcohol until no more stain appears in the washing (2 min) Counterstain with methylene blue * 1-2 min Rinse, drain & air-dry Mycobacteria stain Red Background stains Light blue
- 31. ACID FAST STAIN
- 32. ZIEHL- NEELSEN PROCEDURE Mycobacterium tuberculosis in sputum
- 33. MODIFICATIONS OF THE ZIEHL- NEELSEN PROCEDURE DECOLORIZER ORGANISM STAINED 20% H2SO4 M. Tuberculosis 5% H2SO4 M. leprae 1% H2SO4 Actinomycetes & Nocardia, Cryptosporidium 0.5%-1% H2SO4 Nocardia Spp. (asteroides, brasiliensis, caviae) 0.25% -0.5% H2SO4 Staining of spores (keratin)
- 34. MODIFICATIONS OF THE ZIEHL- NEELSEN PROCEDURE Oocyst of Cryptosporidium Oocyst of Isospora
- 35. ALTERNATE METHODS FOR ACID FAST STAINING • COOPER MODIFICATION : cooper carbol fuchsin + 5% nitric acid in 95% alcohol is the decolourizer + Coope brilliant green counterstain • GABBETT MODIFICATION : Carbolfuchsin + Single stain “Gabbett methylene blue” acts as both decolourizer and counterstain • MULLER-CHERMOCK CARBOLFUCHSIN-TERGITOL COLD STAIN METHOD : carbolfuchsin with 1 drop of tergitol per slide. No heat. Methylene blue counterstain. • Exposure of smears to carbolfuchsin for 15-60min at temp ranging from 37-80°C has also been reported
- 36. AURAMINE FLUOROCHROME PROCEDURE contd.. Cover a heat-fixed smear with Carbol auramine and allow to stain for 15min. Do not heat or cover with filter paper. Rinse & drain. Decolorize with acid-alcohol * 2 min. Rinse with water & drain Flood the smear with potassium permanganate for at least 2 min but not more than 4 min. Rinse with tap water & drain ON EXAMINATION Mycobacteria stain Yellow- orange Background remains dark
- 37. AURAMINE FLUOROCHROME PROCEDURE (TB )
- 38. STAINING FOR METACHROMATIC GRANULES Organisms containing metachromatic granules : 1. Corynebacterium diphtheriae 2. Neisseria 3. Bordetella 4. Yersinia 5. Pasteurella 6. Francisella 7. B. pseudomallei 8. H. influenzae 9. Spirillum 10. Gradnerella vaginalis 11. Chromobacterium violaceum The diphtheria bacillus gives its characteristic volutin-staining reaction best in young cultures (18-24hr) on a blood or serum medium
- 39. METACHROMATIC GRANULES ON ALBERT STAIN
- 40. STAINS TO DEMONSTRATE BACTERIAL ULTRASTRUCTURE
- 41. STAINING OF CAPSULES STAIN CAPSULE DEMONSTRATION Gram’s stain Unstained b/w bacterium & background India Ink (Wet & Dry methods) Unstained against black background. Also useful to demonstrate bacterial slime Eosin relief staining Unstained b/w red(eosin) cytoplasm & red background Modified Gin’s technique Unstained b/w violet cell (crystal violet) & india ink background Hiss Mix material with serum. Heat fix. Wash with 20% CuSO4 soln. Dry. Capsule appears colourless or pale lavender b/w purple cell body & purple background MacNeal Colourless or pale with Macneal tetrachrome stain Lawson Wright’s stain + Glycerine + phosphomolybdic acid mordant. Capsule is colourless Capsules are highly ordered polymers of sugars and proteins that surround some bacterial cells, and can be easily dislodged by heat or water. Accordingly, capsule stains are not heat-fixed and water is never used to rinse
- 42. STAINING OF CAPSULES Gram stain (Klebsiella) Eosin relief stain
- 43. STAINING OF CAPSULES India ink Fluoresceine FITC
- 45. Other methods • The Capsule Stain (Welch method
- 47. The Flagella Stain Flagella are too fine (12–30 nm in diameter) to be visible in the light microscope. However, their presence and arrangement can be demonstrated by treating the cells with an unstable colloidal suspension of tannic acid salts, causing a heavy precipitate to form on the cell walls and flagella In this manner, the apparent diameter of the flagella is increased to such an extent that subsequent staining with basic fuchsin makes the flagella visible in the light microscope. microscopy.
- 48. STAINING OF FLAGELLA Bacterial flagella are fine, threadlike organelles of locomotion & can be seen directly using only the electron microscope (metal-shadow films & phosphotungstic acid). The thickness of the flagella needs to be increased 10 times for light microscopy by coating them with mordants such as tannic acid and potassium alum. Then they need to be stained with basic stains METHOD STAIN USED Gray method Fuchsin Leifson’s method Pararosaniline Modified Leifson Basic fuchsin with tannic acid West method Silver nitrate Difco’s method Crystal violet Leifson- Hugh modification Dibasic potassium phosphate Silver plating stain Silver nitrate + tannic acid + FeCl3 Modified Dieterle’s silver stain for spirochaetes Specially used for Legionella pneumophila . Alcoholic uranyl nitrate + AgNO3
- 49. STAINING OF FLAGELLA Gray’s Method Modified Leifson’s Method
- 50. SATINS FOR DEMONSTRATING FUNGI
- 51. LACTOPHENOL COTTON BLUE STAIN (LPCB) aka Paurier Blue Fluid mounts can be prepared from cultures with this stain for microscopic observation of fungi Phenol 20g Lactic acid 20g Glycerine 40g Cotton blue 0.05g Distilled water 20ml A small drop of Lactophenol cotton blue is placed on a glass slide Fungal growth from culture is teased apart into the stain Apply a coverslip. Then heat the preparation gently The slide can be preserved by sealing the edges of the coverslip with clear fingernail polish or asphalt tar varnish diluted with toluidine blue(preserves the specimen longer)
- 52. LACTOPHENOL COTTON BLUE STAIN (LPCB) contd.. Lactic acid preserves the fungal structure Cotton blue stain is absorbed by the hyaline fungal structures to make them more distinct Phenol kills the fungus Glycerol is a preservative & prevents drying up of the preparation as well
- 54. CALCOFLUOR WHITE STAIN Calcofluor white 1g Distilled water 100ml This colourless dye (originally used as an optical whitener) specifically binds to cellulose and chitin & depending upon the filter system applied it fluoresces brilliant apple green or blue- white colour under UV light. Clinical material (Eg : centrifuged deposit of CSF) is taken on a slide Mix one drop of the working solution of calcofluor white with an equal volume of 10% KOH and the material View under fluorescence microscope with a UV light source (wavelengh longer than 410-450nm)
- 56. NEGATIVE STAINING • Burri’s India Ink method • Fleming’s Nigrosin method (10% nigrosin + 0.5% formalin) Nigrosin , India ink & congo red are acidic dyes. Such negatively charged dyes are repelled by the negative charge of cellular cytoplasm. That is why they cannot stain bacterial cells. Instead, they gather around cells, leaving them clear & unstained against a dark background. The method can be used to demonstrate spirochetes from syphilitic chancres.
- 57. INDIA-INK OR NIGROSIN WET MOUNT 1. India ink (Sandford or Pelikan) preserved in 0.5% phenol 2. Nigrosin (Granular) 10g Formalin (10%) 100ml The nigrosin solution is placed in a boiling water bath for 30 min. 10% formalin is added & then lost by evaporation. The reagent is filtered twice. Take a loopful of specimen in a drop of water on a clean glass slide Add a drop of Nigrosin or India ink Cover with thin coverslip and examine under low, then high magnification
- 58. India ink wet mounts demonstrating the capsule of Cryptococcus neoformans Encapsulated organisms like Cryptococcus fail to absorb the ink and the colourless capsule is clearly outlined against the black background of the ink particles on the outside and the centrally located cell on the inside.
- 59. PERIODIC ACID SCHIFF (PAS) Cryptococcus in tissue Fungal hyphae in sputum
- 60. GIEMSA STAIN Histiocyte containing numerous yeast cells of Histoplasma capsulatum
- 61. MAYER’S MUCICARMINE STAIN (for Cryptococcus and Rhinosporidium) Cryptococcus in sputum of HIV + patient
- 62. POTASSIUM HYDROXIDE (KOH mounting fluid) 10- 20% KOH assisted with a little heat digests protein debris and dissolves the cement which holds the keratinized cells together. It provides an advantageous refractive index to reveal fungal hyphae. Dimethyl sulphoxide (DMSO) or Glycerine are added to prevent rapid drying of the fluid so that the slide can be observed for upto 48 hours. DMSO additionally acts as an excellent cleansing agent. Fungi in scrapings can be made more prominent by the addition of 1 part Parker Superchrome blue- black to 9 parts KOH before heating. Pus & sputum samples can be maintained longer for examination by combining 1:1 KOH (5%) in 28% Glycerine.