MICROSCOPY

REYMEL B. MAGORA, RMT, MPH

MICROSCOPY
→use of a microscope to magnify objects too small
to be visualized with the naked eye so that their
characteristics are readily observable
Applications:
1. Rapid preliminary organism identification
2. Rapid final identification of certain organisms
3. Detection of different organisms present in the
same specimen
4. Detection of organisms not easily cultivated in the
laboratory
5. Evaluation of patient specimens for the presence
of cells indicative of inflammation or
contamination
6. Determination of an organism’s clinical significance
7. Provide pre-culture information
8. Determine which tests and methods should be used
for identification and characterization of
cultivated organisms
9. Provide a method for investigating unusual or
unexpected laboratory test results
BRIGHT-FIELD (LIGHT) MICROSCOPY
PRINCIPLES OF 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 present in the
specimen
A. MAGNIFICATION
B. RESOLUTION
→extent to which detail in the magnified object is
maintained
→ability of the lenses to distinguish fine detail and structure
→determined by numerical aperture and wavelength of light
Resolving power
→closest distance between two objects that when
magnified still allows the two objects to be distinguished
from each other
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Note!!!
Shorter the wavelength of light used in the instrument, the
greater the resolution
Immersion Oil
→ specific optical and viscosity characteristics designed
for use in microscopy
→used to fill the space between the objective lens and
the glass slide onto which the specimen has been
affixed
→enhances resolution by preventing light rays from
dispersing and changing wavelength after passing
through the specimen
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Note!!!
Refractive Index→ measure of the light-bending ability of a
medium
1000× magnification→ required for optimal detection and
characterization of bacteria
C. CONTRAST
→needed to make objects stand out from the
background
→achieved by staining techniques that highlight
organisms and allow them to be differentiated from
one another and from background material and debris
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Kohler Illumination
→ designed to provide maximum illumination and
resolution when observing images using a microscope
PHASE-CONTRAST MICROSCOPY
→detailed examination of internal structures in living
microorganisms
→not necessary to fix or stain the specimen
Principle:
→based on the wave nature of light rays
→ light rays can be in phase (their peaks and valleys
match) or out of phase
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Reinforcement (relative brightness)→ wave peak of light rays
from one source coincides with the wave peak of
light rays from another source
Interference (relative darkness)→ wave peak from one light
source coincides with the wave trough from another
light source
Set of light rays:
a. direct from light source
b. reflected or diffracted from a particular
structure in the specimen
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Diffraction→ scattering of light rays as they touch a
specimen's edge
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Note!!!
Two sets of light rays are brought together, form an
image of the specimen on the ocular lens,
containing areas that are relatively light and
through shades of gray, to black
FLUORESCENT MICROSCOPY
PRINCIPLE OF FLUORESCENT MICROSCOPY
Fluors or Fluorochromes
→raised to a higher energy level after
absorbing ultraviolet (excitation) light
→return to their normal, lower energy
state, they release excess energy in the
form of visible (fluorescent) light
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Fluorescing objects appear brightly
against a dark background
Excitation filter
→passes light of the desired wavelength to
excite the fluorochrome
Barrier filter
→prevents the excitation wavelengths from
damaging the eyes of the observer
Fluorescent Dyes
✓Acridine Orange, Auramine, Fluorescein
Isothiocyanate (FITC)
→requires BLUE excitation light
Exciter filter: 450-490 wavelength
Barrier filter: 515 wavelength
✓Calcofluor White
→requires a VIOLET excitation light
Exciter filter: 355-425 wavelength
Barrier filter: 460 wavelength
 A. FLUOROCHROMING
→ direct chemical interaction between the fluorescent
dye and a component of the bacterial cell
Advantage:
➢enhances contrast and amplifies the observer’s ability
to detect stained cells tenfold greater than light
microscopy
Examples:
✓Acridine orange stain
✓Auramine-rhodamine stain
✓Calcofluor white stain
1. ACRIDINE ORANGE
→binds to nucleic acid
→used to confirm the presence of bacteria in
blood cultures
→stains all nucleic acids---nonspecific
→bright orange fluorescence
→does not discriminate between gram-negative
and gram-positive bacteria
→used for detection of cell wall–deficient
bacteria grown in culture
2. AURAMINE-RHODAMINE
→have affinity to waxy mycolic acids in the cell walls of
mycobacteria
→non-specifically bind to nearly all mycobacteria
→appear bright yellow or orange against a greenish
background
→used to enhance detection of mycobacteria directly
in patient specimens
→initial characterization of cells grown in culture
3. CALCOFLUOR WHITE
→bind in the cell walls of fungi
→directly detect fungi in clinical material
→observe subtle characteristics of fungi grown in
culture
→visualize some parasites such as microsporidia
 B. IMMUNOFLUORESCENCE
→antibodies are conjugated to a fluorescent dye
→dye-antibody conjugate detect, or “tag,” specific
microbial agents
→microorganisms become readily detectable by
fluorescent microscopy
→combines the amplified contrast provided by
fluorescence with the specificity of antibody-
antigen binding
→Legionella spp., Bordetella pertussis, and
Chlamydia trachomatis

Example:
FITC→ intense, APPLE GREEN
fluorescence
→most commonly used
DARK-FIELD MICROSCOPY
→involves the alteration of microscopic technique rather
than the use of dyes or stains to achieve contrast
→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 will be deflected upward into
the objective lens for visualization
→other light that passes through the specimen will miss
the objective, making the background a dark field
→used to detect SPIROCHETES
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Appear extremely bright against a black field
ELECTRON MICROSCOPY
→uses electrons instead of light to visualize small objects
→ electrons are focused by electromagnetic fields and
form an image on a fluorescent screen
→magnifications in excess of 100,000×
Two General Types
1. TRANSMISSION ELECTRON MICROSCOPE (TEM)
→ passes the electron beam through objects and allows
visualization of internal structures
2. SCANNING ELECTRON MICROSCOPE (SEM)
→ uses electron beams to scan the surface of objects
and provides three-dimensional views of surface
structures