Escolar Documentos
Profissional Documentos
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Discussion
Examination of Microorganisms
and Staining Techniques
1. Wet Mount
drop of medium with microorganisms is
placed on a slide and covered with
coverslip
shape, movement of microorganisms
Advantage: live organisms are observed
Disadvantage: dries out quickly
2.Hanging drop
drop of liquid containing microorganism is
placed on a cover slip and inverted and
suspended on a slide with a
concavity/depression
Shape, movement of microorganisms
(organisms with flagella)
more complex technique
Advantage: allows for longer-term observation
and more reliable observation of motility
Purpose of Fixation:
Preservation of internal and external
features of cells
Cellular enzymes are inactivated
Cell structures are hardened
Organism dies AND adheres strongly to
the glass slide
2 Types of Fixation:
1.Heat fixation
flame heating bacterial smear
preservation of morphology but NOT internal
structures
2.Chemical fixation
chemical fixatives penetrate cells and preserves
intracellular components
Acetone
Ethanol
Acetic acid
Mercuric chloride
Formaldehyde
Glutaraldehyde
Common Dyes
Basic Dyes
Safranin
Carbol fuchsin
Crystal violet
Methylene blue
Malachite green
- commonly used
Acidic Dyes
Eosin
Acid fuchsin
Congo red
Staining Techniques
1. Simple Stain
basic dye
Purpose:
cell shape and basic structures become
visible
Highlight microorganisms shapes and
arrangements
Morphology
Shape
cocci
bacilli
Curved and spiral
Arrangement
cocci in pairs, in clusters,
in chains, tetrads and
sarcinae
bacilli in singles, in pairs
and in chains
spirillum in singles
Cocci in chainlike
patterns are called
streptococci
Cocci in groups of
four are called tetrads
Cocci attached in
groups of 8 are called
sarcinae
Bacilli in singles, in
pairs or in chains
Diplobacilli
streptobacilli
2. Differential Stain
differentiates between types of organisms
uses 2 or more dyes to stain organism
a. Grams Stain
- Hans Christian Gram
- Gram positive and Gram negative
b. Acid fast stain
- Acid fast organisms
- Non-acid fast organisms
Gram Positive
Gram Negative
purple
red
Peptidoglycan in c. wall
Thick layer
Thin layer
Lipopolysaccharide in cell
walls
Absent
Present
Gram positive
CV-I complex is difficult to
remove during
decolorization
Purple in color
Gram negative
decolorizer dissolves lipids
in cell wall of Gram
negative bacteria
CV-I Complex is removed
Colorless
retain the color of
counterstain
red
Gram Positive
Gram negative
Erroneous Result
1.
2.
3.
4.
Salmonella sp.
Shigella sp.
Neisseria meningitidis
Haemophilus influenzae
Escherichia coli
Klebsiella pneumoniae
Proteus sp.
Pseudomonas aeruginosa
Acid-Fast Staining
Ziehl-Neelsens Method
Acid fast organism
retain red color because
carbolfuchsin is soluble
in cell wall lipids (red)
3. Special Stains
Used to color and isolate specific parts of
microorganisms
Endospores, flagella, capsule
Endospore
Most commonly used endospore stain
(Schaeffer-Fulton endospore stain)
Malachite green (primary stain)
safranin (counterstain)
Spores ( green) , bacterial cells (red)
MOTILITY TEST
CATALASE TEST
Test to detect whether the bacteria
produce enzyme catalase
Differentiation between Staphylococcus
and Streptococcus genera
Catalase is an enzyme used by bacteria to
induce the reaction of reduction of
hydrogen peroxide into water and oxygen
CATALASE TEST
COAGULASE TEST
Pathogenicity test
Tests to detect if bacteria produce
coagulase enzyme
Coagulase is an enzyme produced by
microorganism that enables the
conversion of fibrinogen to fibrin
Two methods:
1. slide method
2. tube method
+ Result-