GRAM STAINING ,DIFFERENT TYPE OF 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

46. FLAGELLAR STAINING

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

53. Aspergillus Chlamydospore
Mucor
Macroconidia

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)

55. CALCOFLUOR WHITE STAIN
BLASTOMYCOSIS

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.