11/21/2025
MICROSCOPY
ROLE OF MICROSCOPY
• The basic flow of procedures involved in the laboratory diagnosis of
infectious diseases:
• Direct examination of patient specimens for the presence of etiologic agents
• Growth and cultivation of the agents from the specimens
• Analysis and identification of the cultivated organisms
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PRINCIPLES OFBRIGHT-FIELD (LIGHT) MICROSCOPY
• Visible light is passed through the
specimen and then through a series of
lenses that bend the light in a manner that
results in magnification of the organisms
• The total magnification achieved is the
product of the magnification power of the
objective and ocular lenses :
• The objective lenses, the closest to the
specimen-10X, 20X, 40X, and 100X
• The ocular lens, is the lens we look through-
10X
• Magnification of 1000X allows for the
visualization of fungi, most parasites, and
most bacteria
• Not for observing viruses
ROLE OF MICROSCOPY
• Resolution:
• A description of microscope’s ability to distinguish detail
• Resolving power:
• The closest distance between two objects that when magnified still allows the two objects to
be distinguished from each other
• The greater the resolving power, the smaller the minimum distance between two objects that
can still be distinguished
• The resolving power of most light microscopes allows bacterial cells to be
distinguished from one another
• Does not allow bacterial structures, internal or external, to be detected
• To achieve the best level of resolution with 1000X magnification, oil immersion
must be used
• 100X is called the oil immersion lens
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TERMS IN MICROSCOPY
• Resolution:
• A description of microscope’s ability to distinguish detail
• Resolving power:
• The closest distance between two objects that when magnified still allows the two objects to
be distinguished from each other
• The greater the resolving power, the smaller the minimum distance between two objects that
can still be distinguished
• The resolving power of most light microscopes allows bacterial cells to be
distinguished from one another
• Does not allow bacterial structures, internal or external, to be detected
• To achieve the best level of resolution with 1000X magnification, oil immersion
must be used
• 100X is called the oil immersion lens
ROLE OF OIL IMMERSION
• Oil immersion increases resolution and clarity by
reducing light refraction
• Without oil:
• When light passes from the glass slide into the
air gap and then into the lens, it bends (refracts)
due to the difference in refractive index
between air and glass
• With immersion oil:
• The oil has a similar refractive index to glass, so
when placed between the slide and the lens,
light passes through with minimal bending
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STAINING TECHNIQUES
• Gram Stain
• One of the most important bacteriologic techniques within the
microbiology laboratory
• Provides a mechanism for the rapid presumptive identification of
pathogens
• All clinically important bacteria can be detected using this method
STAINING TECHNIQUES
• Gram Stain
• Procedure
• The application of the primary stain, crystal violet (CV) for 30 sec
• The application of a mordant for 30 sec
• Forms a CV-I complex and then causes cross-linking of this complex in the bacterial
cell wall
• The decolorization step distinguishes gram-positive from gram-negative cells
• Decolorizer is applied for few seconds
• After decolorization, organisms that stain gram-positive retain the crystal violet and
those that are gram-negative lose crystal violet
• Addition of the secondary stain or counterstain safranin for 30 sec
• Stains the colorless gram-negative bacteria pink or red
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STAINING TECHNIQUES
STAINING TECHNIQUES
• Acid-Fast Stains
• The acid-fast stain is specifically designed for bacteria whose cell walls
contain long-chain fatty (mycolic) acids
• The classic acid-fast staining method called Ziehl-Neelsen
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CELL WALL STRUCTURE: ACID FAST VS NON ACID-FAST BACTERIA
STAINING TECHNIQUES
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STAINING TECHNIQUES
• Acid-Fast Stains
PHASE-CONTRAST MICROSCOPY
• Phase-contrast microscopy does not use a fixed smear preparation
• A wet preparation or wet mount is used instead
• It uses beams of light passing through the specimen that are partially deflected by
the different densities or thicknesses (refractive indices) of the microbial cells or
cell structures
• The greater the refractive index of an object, the more the beam of light is slowed,
which results in decreased light intensity
• These differences in light intensity translate into differences that provide contrast
• The greater the refractive index of an object the darker the object
• No staining is used for this microscope
• This offers the advantage of allowing observation of viable microorganisms
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PHASE-CONTRAST MICROSCOPY
FLUORESCENT MICROSCOPY
• Principle of Fluorescent Microscopy
• Certain dyes, called fluorophores or fluorochromes, can be raised to a higher
energy level after absorbing ultraviolet (excitation) light
• When the dye molecules return to their normal, lower energy state, they
release excess energy in the form of visible (fluorescent) light
• Fluorescent stains used for staining objects to enhance their visibility
• When the excitation light is emitted from above it is called (epifluorescence)
• One of the disadvantages of fluorescent microscopy is photobleaching
• The dye or a fluorophore molecule will permanently loose ability to fluoresce
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FLUORESCENT MICROSCOPY
FLUORESCENT STAINING TECHNIQUES (FLUORESCENT MICROSCOPY)
• Fluorescent staining techniques can be divided into two main categories
based on the composition of the staining reagents::
• 1-Fluorochroming
• A fluorescent dye or fluorophore is used alone
• The most common fluorochroming methods used in diagnostic microbiology include:
• Acridine orange, auramine-rhodamine, and calcofluor white
• 2-Immunofluorescence
• Fluorescent dyes have been linked (conjugated) to specific antibodies
• Combines the amplified contrast provided by fluorescence with the specificity of
antibody-antigen binding
• Used to directly examine patient specimens for bacteria that are difficult or slow to
grow (Legionella spp., Bordetella pertussis)
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FLUORESCENT STAINING TECHNIQUES (FLUORESCENT MICROSCOPY)
THE MOST COMMON FLUOROCHROMING METHODS
• Acridine Orange
• It binds to nucleic acids primarily DNA and RNA
• Auramine-Rhodamine
• The waxy mycolic acids in the cell walls of mycobacteria have an affinity for the
fluorochromes auramine and rhodamine
• Calcofluor White
• The stain calcofluor white can bind to chitin of cell walls of fungi
• This enhances fungal visibility in tissue and other specimens
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ACRIDINE ORANGE FLUOROCHROMING VS GRAM STAIN
FLUOROCHROMING METHODS
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IMMUNOFLUORESCENCE METHODS
DARK-FIELD MICROSCOPY
• Resemble phase-contrast microscopy in that there is no use of dyes or stains
to achieve contrast
• The condenser does not allow light to pass directly through the specimen
but directs the light to hit the specimen at an oblique angle
• Only light that hits objects, such as microorganisms in the specimen, will be
deflected upward into the objective lens for visualization
• All other light that passes through the specimen will miss the objective lens, thus
making the background a dark field
• Detects certain bacteria with thin dimensions in patient specimens
• Treponema pallidum
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DARK-FIELD MICROSCOPY
A treponema pallidum seen in
tissue using immunohistochemical
techniques and specific antibody
ELECTRON MICROSCOPY
• The electron microscope uses electrons instead of light to visualize small objects
• Instead of lenses, the electrons are focused by electromagnetic fields and form an
image on a fluorescent screen
• Because of its substantial resolution this technology allows 2,000,000X
magnification
• Electron microscopes are of two general types:
• The transmission electron microscope (TEM)
• Passes the electron beam through objects and allows visualization of internal structures
• The scanning electron microscope (SEM)
• Uses electron beams to scan the surface of objects and provides three-dimensional views of
surface structures
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ELECTRON MICROSCOPY
DIGITAL AUTOMATED MICROSCOPY
• Digital microscopes are microscopes without eyepieces
• A digital camera acts as a detector
• Images are displayed on a screen or monitor
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