CHAPTER 4
Microscopy,
Staining,
and
Classification
Microscopy and Staining Overview
MICROSCOPY
The use of light or electrons
to magnify objects
Microscopy
METRIC UNITS
Millimeter mm measuring small organisms (tick)
Micrometer μm measuring cells (5-25 μm)
Nanometer nm measuring viruses (25 nm)
Microscopy
General Principles of Microscopy
Wavelength of radiation
Magnification of image
Resolution
Contrast
Figure 4.1
Electromagnetic
Spectrum
Microscopy
General Principles of Microscopy
Wavelength of radiation
Magnification of image
Resolution
Contrast
Figure 4.2 – Magnification of an
image results when light refracts
(bends) as it passes through a lens
Microscopy
General Principles of Microscopy
Resolution
Ability to distinguish two points that are close together
The better the resolution, the better two nearby objects are
distinguished from one another (clarity)
Contrast
Differences in intensity between two objects or between an
object and its background
Staining increases contrast
Microscopy
Figure 4.3
Microscopy
Figure 4.6 Four kinds of light microscopy
Microscopy
Figure 4.7 Fluorescence microscopy – a dye is used:
Mycobacterium tuberculosis stained with auramine O
viewed under UV light to fluoresce
Microscopy
Figure 4.8 Immunofluorescence – dye is linked to an Ig:
Yersinia pestis (cause of bubonic plague)
Microscopy
Figure 4.11 Scanning electron microscope (SEM) images
Microscopy
Figure 4.12 Probe microscopy
Microscopy
Microscopy
Microscopy
Identifying Microorganisms
How are MO identified?
Stained:
* Simple stains
* Differential stains
* Special stains
Classified:
* Physical characteristics
* Biochemical tests
* Serological tests https://bit.ly/2Cm9y55
* Phage typing
* Nucleic acids (G + C content)
Staining
Preparing Specimens for Staining
Smear:
Fixing does three things:
1. kills organism
2. preserves their morphology
3. anchors specimens to the slide Heat Fixation:
Figure 4.13
Staining
Principles of Staining
Dyes are usually salts
Chromophore is the colored portion of the dye
Acidic dyes
Basic dyes
https://bit.ly/2LQey14
Staining
Principles of Staining
Acidic dyes, work best at low pH
Contains anionic chromophores (-vely charged)
Bind to +vely charged molecules
Basic dyes, work best at high pH
Contains cationic chromophores (+vely charged)
Binds to -vely charged molecules
These dyes are more common b/c most cells are negatively
charged
Staining
Simple Stains
Composed of single basic dye
Crystal violet, safranin, or methylene blue
Used to determine
Size of cells
Shape of cells
Arrangement of cells
Figure 4.14 Simple stains – E.coli and
Staphylococcus aureus
Simple Stains
Differential Stains
Uses more than one dye
Distinguish between different cells or structures
Common differential stains include:
Gram stain (developed in 1884, Hans Christian Gram)
Acid-fast stain
Endospore, capsule, flagellar staining
Histological stains (GMS and HE)
Differential Staining
Differential Stains: Gram Stain
The Gram stain technique is as follows:
Crystal violet (primary stain) is added to a heat-fixed
bacterial smear
Iodine (mordant) is added forming an insoluble crystal violet-
iodine complex (CV-I complex)
Acetone-alcohol (decolorizing step) is used to rinse the
sample
Safranin (counterstain) is added to the sample
Differential Staining
Figure 4.15 The Gram staining procedure:
Differential Staining
Differential Stains: Gram Stain
Results of the acetone-alcohol treatment on…
Gram negative bacteria:
Dissolves the outer membrane
Damages the thin peptidoglycan layer
CV-I washes out
Gram positive bacteria:
Slightly damages the thick peptidoglycan
Dehydration makes it less permeable
CV-I is retained
Differential Staining
Differential Stains: Gram Stain
Gram-positive Gram-negative Gram-positive Gram-negative
Heat-fixed smear
Crystal violet
Iodine
(acts as a mordant)
Acetone-alcohol
solution (decolorizer)
Counterstain
with safranin
Differential Staining
Differential Stains: Acid–Fast
Acid–fast bacteria
Contain waxy cell walls rich in mycolic acid
Retain red-colored primary dye after exposure to an acid
wash
Non–acid–fast cells
Do not have mycolic acid
Red primary stain is washed away after exposure to an acid
wash
Important diagnostic tool for detecting:
Mycobacterium and Nocardia species
Differential Staining
Differential Stains: Acid–Fast
Ziehl-Neelsen method
Carbol-fuchsin (primary dye) is
added to a heat-fixed smear
Sample is steamed for several
minutes to drive the red dye into
the bacteria
Acid-alcohol (decolorizing agent)
is used to rinse the sample
Methylene blue (counterstain) is
added to the sample
Differential Staining
Structural Stains
Simple stains that are used to identify specific
microbial structures include:
* Flagellar stains – determine # and location
* Negative stains – capsule stains
* Endospore stains – endospore detection
Structural Stains
Histological Stains
Two common stains used for tissue specimens:
Gomori methenamine silver (GMS) stain
* Screens for the presence of fungi
Hematoxylin and eosin (HE) stain
* Uses a basic dye (H) and acidic dye (E) Aspergillus sp.
Control Histology Slides
* Outline tissue features
* Biofilm detection in chronic infections
histology.med.yale.edu
Differential Staining
Staining
Taxonomic and Identifying Characteristics
1. Physical characteristics
2. Biochemical tests
3. Serological tests
4. Phage typing
5. Analysis of nucleic acids
Classification/Identification
Physical Characteristics
Used to identify some
microorganisms:
Protozoa, fungi, algae, and
parasitic worms can often be
identified based only on their
morphology (shape)
Some bacterial colonies have
distinct appearance used for
identification
Classification/Identification
Biochemical Tests
Distinguish among prokaryotes by their ability to utilize or
produce certain chemicals
Biochemical tests to identify pathogens, if they can be grown in
a lab
Figure 4.21 - CHO utilization test:
* Use phenol red, CHO, and durham tube
(this is the control)
* Colour change-yellow, acid produced
* Colour change-yellow, with acid and gas
Classification/Identification
Figure 4.22 One tool for the rapid identification of
bacteria, the automated MicroScan system
Classification/Identification
Serological Tests
The study of antigen-antibody reactions in laboratory settings
Many microorganisms trigger an immune response that results
in antibody production, allowing their identification
Figure 4.23
An agglutination test, one
type of serological test
Classification/Identification
Serological Tests
This type of test can ID pathogenic strains of bacteria
E.coli O157:H7
O157 is the antigen of the cell wall
H7 is the antigen of the flagella
This strain of bacteria produces ‘Shiga toxin’
Causes severe foodborne disease
The toxin is the expression of the lambda prophage genes in
this E.coli’s genome
A virus infected a bacterium; that bacterium infects you
Classification/Identification
Phage Typing
Bacteriophages (phages) are viruses that infect bacterial cells
Phages are specific for the hosts they infect
Figure 4.24 Phage typing for
Salmonella enterica serotype
Typhi
Classification/Identification
Analysis of Nucleic Acids
Nucleic acid sequence (DNA or RNA) can be used to
classify and identify microbes
Prokaryotic taxonomy now includes the G + C content
(percentage) of an organism's DNA
Ranges from 20-80% in prokaryotes
G+C x 100
A+T+G+C
Classification/Identification
