Staining Flashcards
PREPARATION OF SPECIMENS
FOR LIGHT MICROSCOPY
BACTERIAL STAINING
PURPOSE OF STAINING
- Observe and appreciate the
appearance of microorganism - Differentiate one microorganism or group
of microorganism from another - Identification of microorganisms and their
special structures
cationic dyes with positively charged (pentavalent nitrogen) that adhere to the negatively charged molecules
Basic Dyes
Example of BASIC DYES
Crystal Violet, Methylene Blue
Malachite Green and Safranin
anionic dyes with negatively charged groups
(carboxyl and phenolic) that bind to positively
charged cell structures
Acidic dyes
Example of ACIDIC DYES
Eosin, Acid Fuchsin and Nigrosin
(TRUE OR FALSE)
Bacteria are slightly negatively charged at pH 7
TRUE
kills the microorganisms and fixes them to the slide
FIXING
preserves various parts of microbes in their natural
state with only minimal distortion
FIXING
Example of FIXING
a. Heat-fixed
b. Methanol Fixation
95% Methanol for I minute
Methanol Fixation
preserves morphology of host
cells, bacteria
Methanol Fixation
especially useful for examining
bloody specimen material
Methanol Fixation
Three kinds of staining techniques:
- SIMPLE
- DIFFERENTIAL
- SPECIAL
single stain is used
SIMPLE STAINS
highlight the entire microorganism so that
cellular shapes and basic structures are visible
SIMPLE STAINS
stain is applied to the fixed smear for a certain
length of time and then washed off, dried and
examined
SIMPLE STAINS
Example of SIMPLE STAINS
Methylene Blue
Carbolfuchsin
Crystal Violet
Safranin
react differently with different kinds of
bacteria and thus can be used to
distinguish them
DIFFERENTIAL STAINS
Example of DIFFERENTIAL STAINS
Gram Stain
Acid-Fast Stain
classifies bacteria into two large group
Gram-positive
Gram-negative
retain the dye and remain
purple
Gram-positive cells
do not retain the dye; they are
colorless until counterstained with a red dye
Gram-negative cells
chemically bond the alkaline dye to the
bacterial cell wall
MORDANT
chemical added to the solution to intensify the
stain
MORDANT
increase the affinity of a stain for a biological
specimen
MORDANT
coat a structure to make it thicker and easier to
see after it is stained with a dye
MORDANT
Example of MORDANT
GRAM’S IODINE
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
tend to be killed easily by
PENICILLINS and CEPHALOSPORINS
Gram-positive bacteria
generally more resistant
because the antibiotics cannot penetrate
the lipopolysaccharide layer
Gram-negative bacteria
contain a highly cross-linked layer of peptidoglycan
that retains the primary dye-Crystal Violet-following the
application of the mordant-iodine (I)
Gram-Positive Organisms
iodine and crystal violet form a complex within the peptidoglycan
Gram-Positive Organisms
when decolorized—CV-I complex remains within the cell
Gram-Positive Organisms
Appearance of GRAM-POSITIVE ORGANISMS
Dark Purple to Deep Blue
do not contain a thick cross-linked layer of
peptidoglycan
Gram-Negative Organisms
CV-I complexes are not trapped within
the peptidoglycan
Gram-Negative Organisms
decolorizer dehydrates the outer cellular
membrane, leaving holes in the membrane
and effectively washing or removing the
CV-I complex from the cells
Gram-Negative Organisms
Secondary stain in GRAM-NEGATIVE ORGANISMS
Safranin
Appearance of GRAM-NAGATIVE ORGANISMS
Pink to Deep Magenta
Report the Gram stain organism’s cellular
shape, morphology, and Gram reaction
Indirect Smear
QUALITY CONTROL
Gram-positive:
Gram-negative:
Gram-positive: Staphylococcus aureus
Gram-negative: Escherichia coli
General Rules of Gram Staining!!!
- All COCCI are GRAM-POSITIVE except for
Neisseria, Veilonella and Branhamella
(Moraxella) - All BACILLI are GRAM-NEGATIVE except for
Arcanobacterium, Actinomyctes, Bacillus,
Clostridium, Corynebacterium, Erysipelothrix,
Eubactrium, Gordonia, Kuthria, Lactobacilli,
Listeria, Mycobacteria, Nocardia,
Propionibacterium and Tsukamurella - All spirochetes are GRAM-NEGATIVE
All COCCI are GRAM-POSITIVE except for
_________________
Neisseria, Veilonella and Branhamella
(Moraxella)
All BACILLI are GRAM-NEGATIVE except for
________
Arcanobacterium, Actinomyctes, Bacillus,
Clostridium, Corynebacterium, Erysipelothrix,
Eubactrium, Gordonia, Kuthria, Lactobacilli,
Listeria, Mycobacteria, Nocardia,
Propionibacterium and Tsukamurella
(TRUE OR FALSE)
All spirochetes are GRAM-POSITIVE
FALSE
All spirochetes are GRAM-NEGATIVE
Reasons Why Gram-Positive Bacteria Becomes
Gram-Negative
- Removal of MgRNA by precipitation with bile salts
- Autolysis, aging and temperature of incubation result
to loss of gram-positivity
***Antibiotic-treated bacterial cell have atypical
staining reaction - Acidic solution of Gram’s Iodine
- Technical error Overdecolorization
Reasons Why Gram-Negative Bacteria
Becomes Gram-Positive
- Incomplete decolorization
- Thick smear
binds strongly only to bacteria that have a
waxy material in their cell walls
ACID-FAST STAIN
Principle of ACID-FAST STAIN
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
Acid-fast organism contain in ______
their outer membrane, making the cells waxy and
resistant to staining with aqueous based stains
such as the Gram stain
MYCOLIC ACID
Example of ACID-FAST ORGANISM
Mycobacteria spp, Nocardia
primary stain in ACID-FAST STAIN
Carbolfuchsin
allow the stain to penetrate into the waxy
surface of acid-fast microorganisms
Heat or Tergitol
-Ethanol and Hydrochloric Acid
-removed excess stain
3% Acid Alcohol
secondary stain in ACID-FAST STAIN
Methylene Blue or Malachite Green
Expected Results
Acid-Fast Organisms:
Nonacid-Fast Oganisms:
***Background material
should stain _____
Acid-Fast Organisms: PINK
Nonacid-Fast Oganisms: DARK BLUE
***Background material
should stain BLUE to BLUE-GREEN
Ways to Facilitate Acid-Fast Staining
- Use of heating or steaming process for 5-7
minutes to temporarily remove the mycolic acid,
while the smear is flooded with stain - Increasing the concentration of dye and phenol
in the staining reagent - Prolonged contact of the specimen with the
primary stain - Addition of a wetting agent like TERGITOL
METHODS OF ACID-FAST STAINING
Ziehl-Neelsen
Kinyoun’s Method
Pappenheim Method
Baumgarten Method
Auramine-Rhodamine Method
Hot Staining Method
Ziehl-Neelsen
Cold Staining Method
Kinyoun’s Method
Differentiate M. smegmatis from M.
tuberculosis
Pappenheim Method
(Pappenheim Method)
decolorized by the mixture
of rosolic acid and alcohol
coloring it BLUE
M. smegmatis
(Pappenheim Method)
not decolorized and
remains RED
M. tuberculosis
Differentiate M. tuberculosis from M.
leprae
Baumgarten Method
(Baumgarten Method)
does not readily take
up the stain and
appears BLUE
M. tuberculosis
(Baumgarten Method)
easily stained by dilute
alcoholic fuchsin
coloring it RED
M. leprae
selective for the cell wall of AFB
Auramine-Rhodamine Method
Mycolic acid renders the bacterial cell resistant to
decolorize-_______
ACID-FAST
is affected by colonial age, medium for
growth and UV light
Acid-Fastness
ideal for concentrated smears
partially acid-fast bacilli—Nocardia spp
Ziehl-Neelsen method
Acid-alcohol is composed of __________
Hydrochloric Acid and Ethanol
used to color and isolate specific
parts of microorganisms
SPECIAL STAINS
endospores and flagella, and reveal
the presence of capsules
SPECIAL STAINS
SPECIAL STAINS
CELL WALL STAIN
INDIRECT/NEGATIVE STAINING
NEGATIVE STAINING FOR CAPSULES
CAPSULAR STAINING
ENDOSPORE (SPORE) STAINING
FLAGELLAR STAINING
Dyar Method
CELL WALL STAIN
colorless bacteria against a colored
background
INDIRECT/NEGATIVE STAINING
excellent technique for studying bacterial
vacuoles and viral morphology
INDIRECT/NEGATIVE STAINING
Demonstrating the presence of a capsule means of determining the organism’s virulence
NEGATIVE STAINING FOR CAPSULES
NEGATIVE STAINING FOR CAPSULES APPEARANCE
Bacteria as light colored bodies against a
dark background
(TRUE OR FALSE)
Cell surface repels acidic stain as a result of
bacterial cells being negatively charged
TRUE
Example of INDIRECT/NEGATIVE STAINING
INDIA INK OR NIGROSIN DYE
CAPSULAR STAINING
Hiss’s Copper Sulfate Method
Gin’s Method
Anthony’s Method
Welch’s Method
Muir’s Method
Tyler’s Method
Wadsworth’s Method
MacNeal
Lawson
capsules appear faint blue
halos around dark blue to purple cells
Hiss’s Copper Sulfate Method
capsules unstained with margin delineated by
ink; bacteria will be stained
Gin’s Method
capsule is unstained against a purple
background; cells are deeply stained
Anthony’s Method
capsules stains a pale violet
Welch’s Method
cells are stained red and the capsule blue
Muir’s Method
cannot be stained by ordinary methods
because the dyes do not penetrate the wall of
the endospore
ENDOSPORE (SPORE) STAINING
most commonly used endospore stain
Schaeffer-Fulton Endospore Stain
primary stain in ENDOSPORE STAINING
MALACHITE GREE
helps the stain penetrate the endospore wall
Heat steam for about 5 minutes
-counterstain
-portions of the cell other than endospores
SAFRANIN
Appearance in SAFRANIN
Endospores appear GREEN
within Red or Pink cells
SPORE STAIN METHODS
Dorner’s Method
Wirtz and Conklin
Acetic Acid Method
spores stain red; bacterial cells almost
colorless against a dark gray background
Dorner’s Method
spores are green; bodies of bacteria
are red
Wirtz and Conklin
tedious and delicate staining procedure
FLAGELLAR STAINING
uses a mordant and the stain CARBOLFUCHSIN
to build up the diameters of the flagella until
they become visible under the light microscope
FLAGELLAR STAINING
FLAGELLAR STAINING uses a mordant and the stain _____
to build up the diameters of the flagella until
they become visible under the light microscope
CARBOLFUCHSIN
(FLAGELLAR STAINING)
Treating the cells with an unstable colloidal suspension of _____ cause a heavy precipitate to form on the cell walls and flagella
TANNIC ACID SALTS
(FLAGELLAR STAINING)
Diameter of flagella is increased to such an extent that subsequent staining with ______ makes the flagella visible in the light microscope
BASIC FUCHSIN
FLAGELLAR STAIN
Leifson Method
Gray’s Method
Fischer and Conn
Casares-Gil’s
Loefflers
Van Ermengen’s
bacterial bodies blue; flagella red
Leifson Method
Mordant -> ______ : swells, coats and forms
precipitate with the flagella
TANNIC ACID
