BACTERIAL STAINING:

PREPARATION OF SPECIMENS
FOR LIGHT MICROSCOPY
PURPOSE OF STAINING
1. Observe and appreciate the
appearance of microorganism
2. Differentiate one microorganism or group
of microorganism from another
3. Identification of microorganisms and their
special structures
Basic Dyes
→cationic dyes with positively charged (pentavalent
nitrogen) that adhere to the negatively charged
molecules
Example: Crystal Violet, Methylene Blue
Malachite Green and Safranin
Acidic dyes
→anionic dyes with negatively charged groups
(carboxyl and phenolic) that bind to positively
charged cell structures
Example: Eosin, Acid Fuchsin and Nigrosin
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Note!!!
Bacteria are slightly negatively charged at pH 7
FIXING
→kills the microorganisms and fixes them to the slide
→preserves various parts of microbes in their natural
state with only minimal distortion
Example:
a. Heat-fixed
b. Methanol Fixation
→ 95% Methanol for I minute
→preserves morphology of host
cells, bacteria
→especially useful for examining
bloody specimen material
STAINING
✓Three kinds of staining techniques:
1. SIMPLE
2. DIFFERENTIAL
3. SPECIAL
1. SIMPLE STAINS
→single stain is used
→highlight the entire microorganism so that
cellular shapes and basic structures are visible
→stain is applied to the fixed smear for a certain
length of time and then washed off, dried and
examined
Example: Methylene Blue, Carbolfuchsin,
Crystal Violet, Safranin
2. DIFFERENTIAL STAINS
→react differently with different kinds of
bacteria and thus can be used to
distinguish them
Example:
✓Gram Stain
✓Acid-Fast Stain
A. GRAM STAIN
→classifies bacteria into two large groups:
a. Gram-positive
b. Gram-negative
Counterstains
→stains that have a contrasting color to the primary
stain
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Note!!!
Gram-positive cells→ retain the dye and remain
purple
Gram-negative cells → do not retain the dye; they are
colorless until counterstained with a red dye
MORDANT
→ chemically bond the alkaline dye to the
bacterial cell wall
→ chemical added to the solution to intensify the
stain
→increase the affinity of a stain for a biological
specimen
→coat a structure to make it thicker and easier to
see after it is stained with a dye
Example: GRAM’S IODINE
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Note!!!
✓Valuable information for the treatment of disease
Gram-positive bacteria→ tend to be killed easily by
PENICILLINS and CEPHALOSPORINS
Gram-negative bacteria → generally more resistant
because the antibiotics cannot penetrate
the lipopolysaccharide layer
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PRINCIPLE
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***Based on the differential structure of the cellular
membranes and cell walls
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Gram-Positive Organisms
→contain a highly cross-linked layer of peptidoglycan
that retains the primary dye-Crystal Violet-following the
application of the mordant-iodine (I)
→ iodine and crystal violet form a complex within the
peptidoglycan
→when decolorized---CV-I complex remains within the
cell
Appearance: Dark Purple to Deep Blue
Gram-Negative Organisms
→do not contain a thick cross-linked layer of
peptidoglycan
→CV-I complexes are not trapped within
the peptidoglycan
→decolorizer dehydrates the outer cellular
membrane, leaving holes in the membrane
and effectively washing or removing the
CV-I complex from the cells
→Secondary stain: Safranin
Appearance: Pink to Deep Magenta
Indirect Smear
✓Report the Gram stain organism’s cellular
shape, morphology, and Gram reaction
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Note!!!
QUALITY CONTROL
Gram-positive: Staphylococcus aureus
Gram-negative: Escherichia coli
General Rules of Gram Staining!!!
1. All COCCI are GRAM-POSITIVE except for
Neisseria, Veilonella and Branhamella
(Moraxella)
2. All BACILLI are GRAM-NEGATIVE except for
Arcanobacterium, Actinomyctes, Bacillus,
Clostridium, Corynebacterium, Erysipelothrix,
Eubactrium, Gordonia, Kuthria, Lactobacilli,
Listeria, Mycobacteria, Nocardia,
Propionibacterium and Tsukamurella
3. All spirochetes are GRAM-NEGATIVE
Reasons Why Gram-Positive Bacteria Becomes
Gram-Negative
1. Removal of MgRNA by precipitation with bile salts
2. Autolysis, aging and temperature of incubation result
to loss of gram-positivity
***Antibiotic-treated bacterial cell have atypical
staining reaction
3. Acidic solution of Gram’s Iodine
4. Technical error→ Overdecolorization
Reasons Why Gram-Negative Bacteria
Becomes Gram-Positive
1. Incomplete decolorization
2. Thick smear
B. ACID-FAST STAIN
→binds strongly only to bacteria that have a
waxy material in their cell walls
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Principle
***Acid-fast organism contain MYCOLIC ACID in
their outer membrane, making the cells waxy and
resistant to staining with aqueous based stains
such as the Gram stain
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Example: Mycobacteria spp, Nocardia
Carbolfuchsin
→ primary stain
Heat or Tergitol
→ allow the stain to penetrate into the waxy
surface of acid-fast microorganisms
3% Acid Alcohol
→Ethanol and Hydrochloric Acid
→ removed excess stain
Methylene Blue or Malachite Green
→ secondary stain
Expected Results
Acid-Fast Organisms: PINK
Nonacid-Fast Oganisms: DARK
BLUE
***Background material
should stain BLUE to BLUE-
GREEN
Ways to Facilitate Acid-Fast Staining
1. Use of heating or steaming process for 5-7
minutes to temporarily remove the mycolic acid,
while the smear is flooded with stain
2. Increasing the concentration of dye and phenol
in the staining reagent
3. Prolonged contact of the specimen with the
primary stain
4. Addition of a wetting agent like TERGITOL
METHODS OF ACID-FAST STAINING
A. Ziehl-Neelsen
→Hot Staining Method
B. Kinyoun’s Method
→Cold Staining Method
C. Pappenheim Method
→Differentiate M. smegmatis from M.
tuberculosis
M. smegmatis: decolorized by the mixture
of rosolic acid and alcohol
coloring it BLUE
M. tuberculosis: not decolorized and
remains RED
D. Baumgarten Method
→Differentiate M. tuberculosis from M.
leprae
M. tuberculosis: does not readily take
up the stain and
appears BLUE
M. leprae: easily stained by dilute
alcoholic fuchsin
coloring it RED
E. Auramine-Rhodamine Method
→ selective for the cell wall of AFB
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Note!!!
✓Mycolic acid renders the bacterial cell resistant to
decolorize-ACID-FAST
✓Acid-Fastness is affected by colonial age, medium for
growth and UV light
✓Ziehl-Neelsen method→ ideal for concentrated smears
partially acid-fast bacilli---Nocardia spp
✓Acid-alcohol is composed of Hydrochloric Acid and
Ethanol
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3. SPECIAL STAINS
→used to color and isolate specific
parts of microorganisms
→endospores and flagella, and reveal
the presence of capsules
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A. CELL WALL STAIN

Dyar Method
C. INDIRECT/NEGATIVE STAINING
→colorless bacteria against a colored
background
→excellent technique for studying bacterial
vacuoles and viral morphology
NEGATIVE STAINING FOR CAPSULES
✓Demonstrating the presence of a capsule means of
determining the organism's virulence
Appearance:
***Bacteria as light colored bodies against a
dark background
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Cell surface repels acidic stain as a result of
bacterial cells being negatively charged
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Example: INDIA INK OR NIGROSIN DYE
D. CAPSULAR STAINING
a. Hiss’s Copper Sulfate Method: capsules appear faint blue
halos around dark blue to purple cells
b. Gin’s Method: capsules unstained with margin delineated by
ink; bacteria will be stained
c. Anthony’s Method: capsule is unstained against a purple
background; cells are deeply stained
d. Welch’s Method: capsules stains a pale violet
e. Muir’s Method: cells are stained red and the capsule blue
f. Tyler’s Method
g. Wadsworth’s Method
h. MacNeal
i. Lawson
E. ENDOSPORE (SPORE) STAINING
→ cannot be stained by ordinary methods
because the dyes do not penetrate the wall of
the endospore
Schaeffer-Fulton Endospore Stain
→most commonly used endospore stain
MALACHITE GREEN
→primary stain
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Note!!! Heat
→steam for about 5 minutes
→helps the stain penetrate the endospore wall
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SAFRANIN
→counterstain
→stain portions of the cell other than endospores
Appearance: Endospores appear GREEN
within Red or Pink cells
SPORE STAIN
a. Dorner’s Method: spores stain red; bacterial cells almost
colorless against a dark gray background
b. Wirtz and Conklin: spores are green; bodies of bacteria
are red
c. Acetic Acid Method
F. FLAGELLAR STAINING
→tedious and delicate staining procedure
→uses a mordant and the stain CARBOLFUCHSIN
to build up the diameters of the flagella until
they become visible under the light microscope
✓Treating the cells with an unstable colloidal suspension of
TANNIC ACID SALTS cause a heavy precipitate to form on
the cell walls and flagella
✓Diameter of flagella is increased to such an extent that
subsequent staining with BASIC FUCHSIN makes the
flagella visible in the light microscope
FLAGELLAR STAIN
a. Leifson Method: bacterial bodies blue; flagella red
b. Gray’s Method
c. Fischer and Conn
d. Casares-Gil’s
e. Loefflers
f. Van Ermengen’s
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Note!!!
Mordant→ TANNIC ACID : swells, coats and forms
precipitate with the flagella