VARIOUS STAINING METHODS IN MICROBIOLOGY

1. DR.MD .SAVARKAR

2. STAIN / DYE
 A stain is a substance that adheres to a
cell, giving the cell color.
 The presence of color gives the cells
significant contrast so are much more
visible.
 Different stains have different affinities
for different organisms, or different parts
of organisms
 They are used to differentiate different
types of organisms or to view specific
parts of organisms

3. Dyes
 Organic salts with positive and
negative charges
 One ion is colored –chromophore
 Basic dye: positive ion is colored
◦ MeBlue+ Cl-
 Acidic dye: negative ion is chromphore

4. Basic Dye
 Works best in neutral or alkaline pH
 Cell wall has slight negative charge at
pH 7
 Basic dye (positive) attracted to cell
wall ( negative)
 Crystal violet, methylene blue,
safranin

5. Acidic Dye
 Chromophore repelled by negative cell
wall
 Background is stained, bacteria are
colorless
 Negative stain-look at size, shape
 Eosin, India ink

6. Staining
 Increase contrast of microorganisms
 Classified into types of stains
◦ Simple stain:
◦ Differential stain:
◦ Structural or special stains

7. Simple Stains
 One dye, one step
 Direct stain using basic dye
 Negative stain using acidic dye

8. Differential Stains
 More than one dye
 Gram stain, acid fast
 Primary dye
 Decolorizing step
 Counter stain

9. Special/ Structural Stains
 Identify structures within or on cells
 Different parts of cell are stained
different colors

10. Why we should Stain
Bacteria??

11. Staining helps in observation of
Bacteria

12. Staining techniques
 Direct staining - The organism is
stained and background is left
unstained
 Negative staining - The background is
stained and the organism is left
unaltered

13. Simple Staining
 The staining process involves immersing the
sample (before or after fixation and mounting)
in dye solution, followed by rinsing and
observation.
 Many dyes, however, require the use of a
mordant, a chemical compound that reacts
withthe stain to form an insoluble, coloured
precipitate.
 When excess dye solution is washed away, the
mordanted stain remains.
 Simple staining is one step method using only
one dye.
 Basic dyes are used in direct stain and acidic
dye is used in negative stain.

15. Simple staining techniques is used to
study the morphology better, to show
the nature of the cellular contents of
the exudates and also to study
the intracellular location of the
bacteria

16. Bacterial arrangement
- Clusters
(group).
- Chains.
- Pairs (diploids).
- No special
arrangement.

17.  Commonly used simple stains are
 Methylene blue
 Dilute carbol fuchsin
 Polychrome methylene blue

18. Loeffler’s Methylene Blue
 Methylene Blue, Loeffler’s
 Dissolve 0.3 g of methylene blue
 In 30mL of 95% ethyl alcohol;
 add 0.01 g of potassium hydroxide
and
 100 mL of DI water;
 stir, and filter. (bacterial stain)

19. Loeffler’s Methylene Blue
Method of Staining
Flood the smear with methylene blue,
allow for 2 minutes, pour off the stain and
allow the air to dry by keeping in a
slanting position and by this the organism
will retain the methylene blue stain
Use
Methylene blue staining is used to make
out clearly the morphology of the
organisms eg. H.influenzae in CSF,
Gonococci in urethral pus

20. Polychrome Methylene Blue
 Preparation
 Allow Loeffler’s Methylene blue to ‘ripen’
slowly.
 Methylene blue stain is kept in half filled
bottles, aerate the content by shaking at
intervals.
 Slow oxidation of methylene blue forms a
violet compound and Stain gets
polychrome property.
 The ripening nearly takes 12 months and
this is hastened by addition of 1%
potassium carbonate

21.  Use
 Mc Fadyean reaction
B.anthracis blue bacilli Is surrounded
by purple capsular material

22.  Carbol Fuchsin (Ziehl-Nielson)
 Dissolve 1 g of basic fuchsin
 In 10 mL of 100% ethyl alcohol
(absolute); set aside.
 Dissolve 5 g of phenol in 100 mL of DI
water.
 Add the two solutions together
and stir. (bacterial stain, bacterial spores,
and various cytoplasmic inclusions)

23. Dilute Carbol Fuchsin
 Preparation
Prepare carbol fuchsin and dilute it to 1/15
using distilled water
 Method of staining
Flood the smear and let stand for 30
seconds, wash with tap water and blot gently
to dry
 Use
 To stain throat swab from patients of
suspected Vincent’s angina(Borrelia are
better stained).
 IT is used as a counter stain in Gram
staining.
 To demonstrate the morphology of Vibrio
cholerae (comma shaped)

24. Simple Staining Easier to
Perform
But has Limitations

25. Differential Stains
 use two or more stains and allow the
cells to be categorized into various
groups or types.
Both techniques allow the observation
of cell morphology, or shape, but
differential staining usually provides
more information about the
characteristics of the cell wall
(Thickness).

26. GRAM STAINING
ACID-FAST STAINING

27. Gram staining
 Named after Hans
Christian Gram,
differentiates
between Gram-
positive purple and
Gram-negative pink
stains and is used to
identify certain
pathogens.

28. Gram staining - Principles
 Used to determine gram status to classify
bacteria broadly. It is based on the composition
of their cell wall.
 Gram staining uses crystal violet to stain cell
walls, iodine as a mordant, and a fuchsin or
safranin counterstain to mark all bacteria.
 Gram status is important in medicine; the
presence or absence of a cell wall will change
the bacterium's susceptibility to some
antibiotics.
 Gram-positive bacteria stain dark blue or violet.
Their cell wall is typically rich with
peptidoglycan and lacks the secondary
membrane and lipopolysaccharide layer found

31. Cell structure differentiates Gram
positive from Gram Negative

32. 1. Crystal violet acts as the primary stain. Crystal violet
may also be used as a simple stain because it dyes
the cell wall of any bacteria.
2. Gram’s iodine acts as a mordant (Helps to fix the
primary dye to the cell wall).
3. Decolorizer is used next to remove the primary
stain (crystal violet) from Gram Negative bacteria
(those with LPS imbedded in their cell walls).
Decolorizer is composed of an organic solvent, such
as, acetone or ethanol or a combination of both.)
4. Finally, a counter stain (Safranin), is applied to
stain those cells (Gram Negative) that have lost the
primary stain as a result of decolorization
Gram Staining Steps

33. Gram Staining technique

34. Gram Staining ProcedureGram Staining Procedure
Gram Staining Procedure

35. Color of
Gram + cells
Color of
Gram – cells
Primary stain:
Crystal violet
Purple Purple
Mordant:
Iodine
Purple Purple
Decolorizing agent:
Alcohol-acetone
Purple Colorless
Counterstain:
Safranin
Purple Red
Differential Stains: Gram
Stain

36. Structure and Reactivity to
Gram Staining.

37. Gm+ve cocci & Gm-ve bacilli

38. Gram Reaction
 GRAM POSITIVE
 GRAM NEGATIVE

39. ACID FAST
STAINING

40. ACID-FAST STAINING
 The Ziehl – Neelsen stain, also known
as the acid-fast stain, widely used
differential staining procedure.
 The Ziehl – Neelsen stain was first
described by two German doctors;
 Franz Ziehl (1859 to 1926), a
bacteriologist and Friedrich Neelsen
(1854 to 1894) a pathologist.

41.  In this type some bacteria resist
decolourization by both acid and alcohol
and hence they are referred as acid fast
organisms.
 This staining technique divides bacteria
into two groups namely acid-fast and
non acid-fast.
 This procedure is extensively used in
the diagnosis of tuberculosis and
leprosy.

42. Principle
 Mycobacterial cell walls contain a
waxy substance composed of mycolic
acids.
 These are β-hydroxy carboxylic acids
with chain lengths of up to 90 carbon
atoms.
 The property of acid fastness is
related to the carbon chain length of
the mycolic acid found in any
particular species

43. Ziehl- Neelsen Procedure
 1.Make a smear. Air Dry. Heat Fix.
 2. Flood smear with Carbol Fuchsin
stain
◦ Carbol Fuchsin is a lipid soluble, phenolic
compound, which is able to penetrate the
cell wall
 3. Cover flooded smear with filter
paper
 4. Steam for 10 minutes. Add more
Carbol Fuchsin stain as needed
 5. Cool slide
 6. Rinse with DI water

44.  7. Flood slide with acid alcohol (leave 15
seconds). The acid alcohol contains 3%
HCl and 95% ethanol, or you can
declorase with 20% H2SO4
 8. Tilt slide 45 degrees over the sink and
add acid alcohol drop wise (drop by drop)
until the red color stops streaming from
the smear
 9. Rinse with DI water

45.  10. Add Loeffler’s Methylene Blue stain
(counter stain). This stain adds blue
color to non-acid fast cells. Leave
Loeffler’s Blue stain on smear for
1minute
 11. Rinse slide. Blot dry.
 12. Use oil immersion objective to view.

47. Ziehi-Neelsen acid fast staining
procedure

48. 1 2 3
4 5
6 7

49. How the Acid fast bacteria
appear

50. Staining of M.lepra
 M.lepra are less
acid fast than
M.tuberculosis
group of
organisms,
 The
concentration of
H2So4 is
reduced to 5 %
for decolorising

51. SPECIAL STAINS
 Stains for Metachromatic granules
 Stain for spores
 Stain for capsules
 Stain for flagella

52. Albert’s Staining for
C. diphtheriae
 In all cases of suspected cases of
Diphtheria, stain one of the smears
with Gram stain
 If Gram stained smear shows
morphology suggestive of C.diptheria,
proceed to do Albert staining which
demonstrates the presence or
absence of metachromatic granules.

53.  C.diptheria are thin Gram positive bacilli,
straight or slightly curved and often
enlarged (clubbing) at one or both ends
and are arranged at acute angles giving
shapes of Chinese letters or V shape
which is characteristic of these
organisms .
 Present in the body of the bacillus are
numerous metachromatic granules
which give the bacillus beaded or barred
appearance.
 These granules are best demonstrated
by Albert’s stain.
Appearance of C.diptheria

54.  Albert stain I
 Toluidine blue
0.15gm
 Malachite green 0.20
gm
 Glacial acetic acid 1.0
ml
 Alcohol(95%) 2.0 ml
 Albert stain II
 Iodine 2.0 gm
 Potassium iodide 3.0
gm
 Distilled water 300 ml
Albert staining

55. Albert staining Procedure
 Cover the heat-fixed smear with Albert stain I.
Let it stand for two minutes.
 Wash with water.
 Cover the smear with Albert stain II. Let it stand
for two minutes.
 Wash with water, blot dry and examine.
 To demonstrate metachromatic granules in
C.diphtheriae. These granules appear bluish
black whereas the body of bacilli appear green
or bluish green.

56. How the C.diptheria
appear
 To demonstrate
metachromatic
granules in
C.diphtheriae.
These granules
appear bluish
black whereas
the body of bacilli
appear green or
bluish green.

57. Capsule staining
 Purpose - reveal the presence of the
bacterial capsule.
 The water-soluble capsule of some
bacterial cells is often difficult to see by
standard simple staining procedures or
after the Gram stain.
 The capsule staining methods were
developed to visualize capsules and yield
consistent and reliable results
 Capsule may appear as clear halo when a
fresh sample is stained by Grams or
Leishman stain.

58. INDIA INK PREPARATION
 Principle:
◦ Specimen placed in a drop of India ink
becomes darkly colored because of the
carbon particle in the ink. Hyaline
structures such as capsules and cell
walls will be highlighted against a dark
background of inked colored specimen
creating an illusion of darkfield
microscopy.
 Reagent: 1:1 dilution of the ink

59.  Procedure:
◦ Place a drop of the specimen (body fluid or
from culture) on a clean glass
◦ Put a drop of India Ink, mix and overlay a
cover slip
◦ Examine under low power and high power
with a bright field microscope
 Result:
◦ India ink creates a dark background against
which hyaline fungal cell wall and capsules
can be seen
◦ Limitation: wbc may be confused as fungi

60.  Uses
 India ink is used to
demonstrate capsule
which is seen as
unstained halo
around the
organisms
distributed in a black
background eg.
Cryptococcus

61. Nigrosin
 Saturated: Dissolve 3 g of nigrosin
 (water soluble) in 100 mL of DI water.
 Stir and filter if necessary. (biological
stain for protozoa)

62. Nigrosin used for Negative
Staining
 The negative stain is
particularly useful for
determining cell size
and arrangement. It can
also be used to stain
cells that are too
delicate to be heat-
fixed.

63. Endospore Staining
 Bacterial endospores are
metabolically inactive, highly
resistant structures produced by some
bacteria as a defensive strategy against
unfavorable environmental conditions.

64. PROCEDURE
 Primary stain - is malachite green, which
stains both vegetative cells and
endospores and heat is applied to help
the primary stain penetrate the endospore.
 Decolorized with water, which removes
the malachite green from the vegetative
cell but not the endospore.
 Safranin – counterstain any cells which
have been decolorized.
 At the end of the staining process,
vegetative cells will be pink, and
endospores will be dark green

65. Flagella stain
 Flagella are fragile appendages
 Cannot be seen under ordinary
microscope
 Hence the surface is coated with a
precipitate to form a colloidal substance
 This precipitate serves as a layer of
stainable material

66. Components
 1% Osmic acid
 Mordant
10% Tannic acid
Sat.potassium alum
10% Ferric chloride
 Fontana’s silver solution

67. USE
 This is used to
demonstrate the flagella
and the organisms stain
black and flagella appear
light brown

68. FUNGAL STAINING

69. Taxonomical classification:
 Phylum class
 Zygomycota zygomycetes
 Ascomycota ascomycetes
 Basidiomycota basidiomycetes
 Deuteromycota deuteromycetes
(fungi imperfecti)
CLASSIFICATION OF FUNGI

70. KOH Wet Mounts
 Principle:
◦ KOH softens most tissues, dissolves fat
droplets, bleaches many pigments and
dissolves the “cement” that holds keratinized
cells together; glycerine clears tissue debris,
thus making it easier to demonstrate presence
of fungal elements.
 Reagents:
◦ 10 – 20 % KOH:
 KOH pellets 10 – 20 grams
 Glycerine (optional) 10 ml
 Distilled water 90 ml

71.  Procedure:
◦ Place a small amount of specimen on a
clean glass slide
◦ place 1-2 drops of KOH on the specimen
and overlay a cover slip
◦ Allow the preparation to stand for 10-30
minutes in a wet chamber.
 You can gently heat preparation to hasten
the action of KOH
 Do not over heat for it may crystallize the
KOH
◦ Examine preparation under low then high
magnification. Take note for the presence
of fungal elements (hyphae and/or spores)

74. Fungal staining methods
Stains used:
 Lactophenol Cotton Blue - very popular
for quick evaluation of fungal structures;
stains chitin in cell walls of fungi.
 Periodic Acid - Schiff Stain (PAS) - stains
polysaccharide in the cell wall of fungi.
Fungi stain pink-red with blue nuclei.
 Gomori Methenamine Silver Stain - silver
nitrate outlines fungi in black due to the
silver precipitating on the fungi cell wall.

75. Figure 1. Hematoxylin and Eosin
staining of Aspergillus. GMS staining of
Aspergillus.

76. Alcian Blue staining of
Cryptococcus
PAS staining of Cryptococcus.

77. Lactophenol Cotton Blue
 Principle:
◦ The morphology of fungal elements are
preserved and stained better.
 Reagents:
◦ Lactic acid & Phenol
 Kills the organism
◦ Glycerin
 Prevents easy dehydration
◦ Cotton blue
 Dye or stain

80.  Gridley Stain - Hyphae and yeast stain dark blue or
rose. Tissues stain deep blue and background is
yellow.
 Mayer Mucicarmine Stain - will stain capsules of
Cryptococcus neoformans deep rose.
 Fluorescent Antibody Stain - extremely specific
method of detecting fungi in tissues or fluids.
 Papanicolaou Stain - good for initial differentiation
of dimorphic fungi. Works well on sputum smears.
 Gram Stain - generally fungi are gram positive; but
Actinomyces and Nocardia are gram variable.

81.  Acid-Fast Stain - used to differentiate
the acid-fast Nocardia from other
aerobic Actinomyces.
 Giemsa Stain - used for blood and
bone marrow specimens.
 India Ink - demonstrates the capsule of
Cryptococcus neoformans in CSF
specimens.

82. EYE SCRAPINGS & ASPIRATE for
KERATOMYCOSIS
 KOH & LPCB, look for
◦ Septate hyaline hyphae
 Aspergillus species
 Fusarium species
◦ Coenocytic hyaline
hyphae
 Mucor species
◦ Pseudohyphae and
yeasts
 Candida species

83.  A fluorescent stain for rapid detection
of yeasts, fungi and parasitic
organisms. Calcofluor White is a non-
specific
fluorochrom that binds to cellucose
and chitin in cell walls
Calcofluor White Stain

84. Calcofluor White Stain
 Composition:
 Calcofluor White M2R 1g/l
 Evans blue 0.5g/l
 Storage:
 Store at room temperature and
protected from light.

85. Calcofluor White Stain
 Directions:
 1. Put the sample to be examined onto a
clean glass slide.
 2. Add one drop of Calcofluor White
Stain and one drop of 10% Potassium
Hydroxide
 3. Place a coverslip over the specimen
and let stand for 1 minute.
 4. Examine the slide under UV light at
x100 to x400 magnification

86.  Principle/ Interpretation:
 Calcofluor White Stain is a non-specific
fluorochrome that binds with cellulose
and chitin contained in the cell walls of
fungi and other organisms.
 The staining procedure with Calcofluor
White Stain is a rapid method for the
detection of many yeasts , pathogenic
fungi and Microsporidium,
Acanthamoeba, Pneumocystis,
Naegleria, and Balamuthia species..

87.  Evans blue present in the stain act as a
counterstain and diminishes background
fluorescence of tissues and cells when
using blue light excitation (not UV).
 A range of of 300 to 440 nm (Emmax
433nm; 0.1 M phosphate pH 7.0;
cellulose) can be taken for emission wave
length and the excitation occurs around
355nm.
 Fungal or parasitic organisms appear
fluorescent bright green to blue, while

88.  Attention cotton fibers will fluoresce
strongly and must therefore be
differentiated from fungal hyphae.
 As well amebic cysts are fluorescent but
trophozites will not stain or fluoresce,
Background fluorescence can be
diminished by examining under blue light
or by using different filter combinations
(emission and excitation filters).
 One drop of 10% potassium hydroxide
solution can be added for better
visualization of fungal elements

89. Calcoflour stain
 Calcoflour mounts for
systemic mycoses ,
look for (flourescence)
◦ Pseudohyphae and
yeasts (blood)
 Candida species
◦ Septate, hyaline at
right degrees angle
(bronchial lavage)
 Aspergillus species

90. THANK
YOU