Stains and staining techniques

1. Aswartha Harinathreddy
Assistant Professor

2. Staining of Specimens:
 The living microorganisms can be directly examined with the
light microscope, they often must be fixed and stained to increase
visibility, and study morphological features, and preserve them
for future study.
Smearing: is a process in which bacterial culture is spread
uniformly or in the form of thin layer on glass slide.
Fixation:
 Fixation is the process by which the internal and external
structures of cells and microorganisms are fixed in position.
 It inactivates enzymes, proteins and DNA so that they do not
change during staining and observation.

3. Fixation : is the immobilization of bacterial cells and
cell components.
Fixation is carryout to kill bacterial cells (pathogenic).
It increases the permeability of cell membrane to stain.
It prevents autolysis of cells.
Fixation prevents cell swelling.

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5.  There are two different types of fixations.
(1) Heat fixation: Bacteriologists heat-fix bacterial smears by
gently flame heating. This heat-fix preserves overall
morphology of cells.
(2) Chemical fixation: Chemical fixatives penetrate cells and
react with cellular components, usually proteins and lipids and
inactivate them.
Common fixative mixtures contain such components as
ethanol, acetic acid, mercuric chloride, formaldehyde, and
glutaraldehyde.

6. Dyes and Simple Staining
 The many types of dyes used to stain microorganisms have
two common features.
 (1) They have chromophore groups (Chromophore is a
coloured chemical substance that has ability to provide a
color to dye)
 (2) They can bind with cells by ionic, covalent, or
hydrophobic bonding.
 For example, a positively charged dye binds to negatively
charged structures on the cell.

7. Dyes divided into two general classes based on the nature of their
charged group.
1. Basic dyes:
 Methylene blue, crystal violet, safranin, malachite green have
positively charged groups.
 Basic dyes bind to negatively charged molecules like nucleic
acids and many proteins. Because the surfaces of bacterial cells
also are negatively charged, basic dyes are most often used in
bacteriology.
2. Acid dyes:
 Nigrosine, Picric acid, Eosin and India ink possess negatively
charged groups (COOH,OH).
 Acid dyes, because of their negative charge, bind to positively
charged cell structures.

8.  The first staining techniques developed by Robert Koch.
 First time used dye Aniline by Robert Koch.
Two types Staining's:
Simple staining and Differential staining

9. Simple staining :
 The simple stain can be used as a quick and easy way to
determine cell shape, size and arrangements of bacteria.
 The simple stain is a very simple staining procedure involving
single solution of stain. Any basic dye such as methylene blue,
safranin, or crystal violet can be used to color the bacterial cells.
 Since the surface of most bacterial cells and cytoplasm is
negatively charged, these positively charged stains adhere
readily to the cell surface.
 After staining, bacterial cell morphology (shape and
arrangements) can be appreciated.

10. Preparation of a smear and heat fixing:
 Using a sterilized inoculating loop, transfer loopful of liquid
suspension containing bacteria on to a slide.
 Spread the bacterial culture uniformly or in the form of thin layer
on glass slide.
 Allow the smear to dry thoroughly (or) Heat-fix the smear
cautiously by passing the underside of the slide through the burner
flame two or three times.
 It fixes the cell on the slide. Do not overheat the slide as it will
lysis the bacterial cells.

11. Staining of smear:
 Cover the smear with methylene blue and allow the dye to
remain in the smear for approximately one minute.
 (Staining time is not critical here; somewhere between 30
seconds to 2 minutes should give you an acceptable stain, the
longer you leave the dye in it, the darker will be the stain).
 Using distilled water wash bottle, gently wash off the excess
methylene blue from the slide by directing a gentle stream of
water over the surface of the slide.
 Wipe the back of the slide and blot the stained surface with
blotting paper.

12.  Place the stained smear on the microscope stage smear
side up and focus the smear using the 10X objective.
 Choose an area of the smear in which the cells are well
spread in a monolayer.
 Centre the area to be studied, apply immersion oil directly
to the smear, and focus the smear under oil with the 100X
objective.

13. Result:
 The bacterial cells usually stain uniformly and the color of
the cell depends on the type of dye used.
 If methyene blue is used, some granules in the interior
of the cells of some bacteria may appear more deeply
stained than the rest of the cell, which is due to presence
of different chemical substances.

14. Differential staining
 Differential Staining is a staining process which uses more than
one chemical stain. Using multiple stains can better differentiate
between different microorganisms or structures/cellular
components of a single organism.
 One commonly recognizable use of differential staining is the
Gram stain.
 Gram staining uses two dyes: Crystal violet and Safranin (the
counterstain) to differentiate between Gram-positive bacteria
(large Peptidoglycan layer on outer surface of cell) and Gram-
negative bacteria.

15.  Gram-Staining
 Acid fast staining
 Endospore staining
 Flagella Staining
 Capsule staining

16. Gram staining:
 Gram staining method, the most important procedure in
Microbiology, was developed Hans Christian Gram in 1884.
 Gram staining is still the cornerstone of bacterial
identification and taxonomic division.
 This differential staining procedure separates most bacteria
into two groups on the basis of cell wall composition:
 Gram positive bacteria (thick layer of peptidoglycan-90% of
cell wall)- stains purple Gram negative bacteria (thin layer
of peptidoglycan-10% of cell wall and high lipid content) –
stains red/pink

17. Classic Gram staining techniques involves following steps:
 Fixation of bacterial cells to the surface of the microscope slide either by
heating or by using methanol.
 Application of the primary stain (crystal violet). Crystal violet stains all cells
blue/purple.
 Application of mordant: The iodine solution (mordant) is added to form a
crystal violet iodine (CV-I) complex; all cells continue to appear blue.
 Decolorization step: The decolorization step distinguishes gram-positive from
gram-negative cells. The organic solvent such as acetone or ethanol (95%),
removes the blue dye complex from the lipid-rich, thin walled gram negative
bacteria to a greater degree than from the lipid poor, thick walled, gram-
positive bacteria. The gram negative bacteria appear colorless and gram
positive bacteria remain blue.
 Application of counter stain (safranin): The red dye safranin stains the
decolorized gram-negative cells red/pink; the gram-positive bacteria remain
blue.

18. Principle of Gram Stain:
 The crystal violet CV+ ions that penetrate through the wall and
membrane of both Gram-positive and Gram-negative cells. The CV+
interacts with negatively charged components of bacterial cells, staining
the cells purple.
 When iodine added, iodine (I-) interacts with CV+ to form large crystal
violet iodine (CV-I) complexes within the cytoplasm and outer layers of
the cell.
 The decolorizing agent, interacts with the lipids of the membranes of both
gram-positive and gram negative bacteria. The outer membrane of the
Gram-negative cell (lipopolysaccharide layer) is lost from the cell,
leaving the peptidoglycan layer exposed.
 Gram-negative cells have thin layers of peptidoglycan, one to three layers
deep with a slightly different structure than the peptidoglycan of gram-
positive cells.

19.  With ethanol treatment, gram-negative cell walls become leaky
and allow the large CV-I complexes to be washed from the cell.
 The highly cross-linked and multi-layered peptidoglycan of the
gram-positive cell is dehydrated by the addition of ethanol.
 The multi-layered nature of the peptidoglycan along with the
dehydration from the ethanol treatment traps the large CV-I
complexes within the cell.
 After decolorization, the gram-positive cell remains purple in
color, whereas the gram-negative cell loses the purple color and
is only revealed when the counterstain, the positively charged
dye safranin, is added.

21. Acid-fast stain:
 The acid-fast stain also known as the Ziehl–Neelsen stain, was
first described by two German doctors: the bacteriologist Franz
Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854–
1898).
 It is a special bacteriological stain used to identify acid-fast
organisms, mainly Mycobacteria.
 Mycobacterium tuberculosis is the most important of this group
because it is responsible for tuberculosis (TB).
 Other important Mycobacterium species involved in human
disease are Mycobacterium leprae, Mycobacterium bovis,
Mycobacterium africanum.

22.  Acid-fast organisms like Mycobacterium contain large
amounts of lipid substances within their cell walls called
mycolic acids.
 These acids resist staining by ordinary methods such as
a Gram stain.
 The reagents used are Ziehl–Neelsen carbol fuchsin,
acid alcohol, and methylene blue.
 Acid-fast bacilli will be bright red/pink after staining.
 Non acid fast blue in color.

23. Classic steps involves following:
Fixation of bacterial cells to the surface of the
microscope slide either by heating or by using
methanol.
Application of the primary stain:
 When the smear is stained with carbol fuchsin, it
solubilizes the lipid material present in the Mycobacterial
cell wall but by the application of heat, carbol fuchsin
further penetrates through lipoidal wall and enters into
cytoplasm.
 Then after all cell appears red. Then the smear is
decolorized with decolorizing agent i.e ACID
ALCOHOL(3% HCL in 95% alcohol)

24.  But the acid fast cells are resistant due to the presence of
large amount of lipoidal material in their cell wall which
prevents the penetration of decolorizing solution.
 The non-acid fast organism lack the lipoidal material in
their cell wall due to which they are easily decolorized,
leaving the cells colorless.
 Then the smear is stained with counterstain, methylene
blue.
 Only decolorized cells absorb the counter stain and take
its color and appears blue while acid-fast cells retain the
red color.

25. Application of Reagent
Cell colour
Acid fast
Non-acid
fast
Primary dye Carbol fuchsin Red Red
Decolorizer Acid alcohol Red Colorless
Counter stain Methylene blue Red Blue

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27. Endospore staining:
 When vegetative cells of certain bacteria such as Bacillus spp and
Clostridium spp are subjected to environmental stresses such as
nutrient deprivation, they produce metabolically inactive or
dormant form-endospore.
 C. botulinum and C. tetani are the causative agents of botulism and
tetanus, respectively.
 Bacillus anthracis cause anthrax, and Bacillus clausii is the
probiotic.
 These structures are extraordinarily resistant to environmental
stresses such as heat, ultraviolet radiation, gamma radiation,
chemical disinfectants, and desiccation.

28.  The spore often is surrounded by a thin, delicate covering called the
exosporium.
 A spore coat lies beneath the exosporium, is composed of several
protein layers, and may be fairly thick. It is impermeable and
responsible for the spore’s resistance to chemicals.
 The cortex, which may occupy as much as half the spore volume,
rests beneath the spore coat. It is made of a peptidoglycan that is less
cross-linked than that in vegetative cells.
 The spore cell wall (or core wall) is inside the cortex and surrounds
the protoplast or core. The core has the normal cell structures such as
ribosomes and a nucleoid, but is metabolically inactive.

30. Principle of Spore Staining:
 A differential staining technique (the Schaeffer-Fulton
method) is used to distinguish between the vegetative cells
and the endospores.
 A primary stain (malachite green) is used to stain the
endospores. Which is strong stain that can penetrate the spore
coat of an endospore.
 Endospores resist to staining, the malachite green will
be forced into the endospores by heating. In this technique
heating acts as a mordant.

31.  There is no need of using any decolorizer in this spore
staining as the primary dye malachite green bind relatively
weakly to the cell wall but penetrate into the spore wall.
 If washed with water the dye come out of cell wall however
not from spore wall. Water is used to decolorize the
vegetative cells.
 Malachite green dye is water-soluble and does not adhere
to the cell wall vegetative cells have been disrupted by heat,
because of these reasons, the malachite green rinses easily
from the vegetative cells.

32.  As the endospores are resistant to staining, the endospores are
equally resistant to de-staining and will retain the primary dye
while the vegetative cells will lose the stain.
 The addition of a counterstain or secondary stain
(safranin) is used to stain the decolorized vegetative cells.
 The endospores appear green in colour.
 The vegetative cells appear in pink/red colour.

33. Procedure:
 Fixation of bacterial cells to the surface of the microscope slide
either by heating or by using methanol.
 Application of the primary stain: Smear covered with the solution
of malachite green which is strong stain that penetrate the spore
coat of endospore.
 The slide is kept on a suitable stand and heated with steam for 5
min. (Mordant).
 The slide washed under tap water.(Decolourising agent).
 The slide is counter stained with safrain for about 30 sec.
 Then the slide washed with distilled water, dried and observe
under microscope.

34. Capsule staining:
 Some bacteria have a layer of material lying outside the cell
wall. When the layer is well organized and not easily
washed off, it is called a capsule or glycocalyx.
 A glycocalyx is a network of polysaccharides extending
from the surface of bacteria.
 For example, Bacillus anthracis has a capsule of poly- D-
glutamic acid.
 Capsules are clearly visible in the light microscope when
negative stains or special capsule stains are employed.

35.  They help bacteria resist phagocytosis by host
phagocytic cells.
 The glycocalyx also aids bacterial attachment to
surfaces of solid objects in aquatic environments or to
tissue surfaces in plant.
 Example: Streptococcus pneumoniae, Klebsiella
pneumoniae Haemophilus influenzae and
Pseudomonas aeruginosa.

36. Principle:
 Bacterial capsules are non-ionic, so neither acidic nor basic stains
will adhere to their surfaces.
 Therefore, the best way to visualize them is to stain the background
using an acidic stain (e.g., Nigrosine, congo red) and to stain the
cell itself using a basic stain (e.g.,crystal violet, safranin, basic
fuchsin and methylene blue).
 There are two methods:
A. India ink method
B. Anthony’s stain method

37. India ink method:
 In this method two dyes, crystal violet and india ink are used.
 The capsule is seen as a clear halo around the microorganism
against the black background.
 The background will be dark (color of india ink).
 The bacterial cells will be stained purple (bacterial cells takes
crystal violet-basic dyes as they are negatively charged).
 The capsule (if present) will appear clear against the dark
background (capsule does not take any stain).

38. Anthony’s stain method:
 In this type of capsule staining procedure, the primary
stain is crystal violet, and all parts of the cell take up the
purple crystal violet stain.
 There is no mordant in the capsule staining procedure.
 A 20% copper sulfate solution serves a dual role as both
the decolorizing agent and counter stain.
 It decolorizes the capsule by washing out the crystal
violet, but will not decolorize the cell.
 As the copper sulfate decolorizes the capsule, it also
counter stains the capsule.
 Thus, the capsule appears as a blue halo around a purple
cell.

40. Flagella staining:
 Most motile bacteria move by use of flagella (
flagellum), threadlike appendages extending from the
plasma membrane and cell wall.
 They are about 15 or 20 μm long. Flagella are so thin
they cannot be observed directly with a bright-field
microscope, but must be stained with special
techniques .

41.  Bacterial species often differ distinctively in
their patterns of flagella distribution.
 Monotrichous bacteria: (trichous means
hair) have one flagella; if it is located at an end,
it is said to be a polar flagellum. Ex: Vibrio
cholera.
 Lophotrichous bacteria have a cluster of
flagella at one or both ends. Ex: Psedomonas.
 Amphitrichous bacteria: (amphi means “on
both sides”) have a single flagella at each pole.
Ex: Spirillum.

42. Flagellar Ultrastructure
 Transmission electron microscope studies have
shown that the bacterial flagellum is composed of
three parts.
1.Filament, 2.Basal body, 3. The hook.
 (1) The filament: is a hollow and cylinder
constructed of a single protein called flagellin. it is
20-nanometer-thick hollow tube.
 The longest portion is the filament which extends
from the cell surface to the tip.
 The filament ends with a capping protein.

43.  (2) A basal body is embedded in the cell and the most
complex part of a flagellum.
 (3) The Hook a short, curved segment, it links the
filament to its basal body and acts as a flexible coupling.
The hook is made of different protein subunits.
 The hook and basal body are quite different from the
filament.
 In E. coli and most gram-negative bacteria, the basal body
has four rings. The outer L and P rings associate with the
lipopolysaccharide and peptidoglycan layers, respectively.
The inner S and M ring contacts the plasma membrane.
 Gram positive bacteria have only two basal body rings, an
inner ring connected to the plasma membrane and an
outer one probably attached to the peptidoglycan.

45. Flagella stain by using Leifson’s method:
 The Leifson’s stain is made up of tannic acid ,basic fuschin
stain and alcohol.
 When we treat Leifson’s stain with cell the tannic acid get
attach to the flagella and alcohol get evaporated.
 After evaporation of alcohol the thickness of flagella is
increased due to deposition of tannic acid.
 Where as Basic fuschin stain the Flagella.
 After this give a gentle stream of water wash treatment to a
slide.
 Now treat the slide with 1 % methylene blue treatment for 1
minute.

46.  After observation under microscope we can observe
that flagella appear red in colour and bacterial cell
appear blue in colour.

47. THANK YOU…….