MICROSCOPY
Dr Ekta Chourasia
Department of Microbiology
MICROSCOPY
• Microscope invented by Antony Van Leeuwenhoek in
17th century.
• Required for the morphological study of microorganisms.
• USES - to magnify the image.
- to achieve maximum resolution.
- to provide sufficient contrast for observation
Resolution : the extent to which details in the
magnified object are maintained.
Resolving Power (RP) : the smallest distance by
which 2 points can be separated and still be observed
as 2 distinct / different points.
RP (eye) – 200 µm
RP (visible light) – 300 nm
RP (electron microscope) – 0.1 nm
Types of Microscopes
• Optical or light microscope
- Simple
- Compound
• Phase contrast microscope
• Dark field (dark ground) microscope
• Fluorescent microscope
• Electron microscope
Optical (Light) Microscope
Optical (Light) Microscope
• Principle : when visible
light passes through the
specimen
&
then
through a series of
lenses, the light gets
reflected in such a
manner that it results in
magnification of the
organisms present in the
specimen.
• Magnification = objective x eyepiece
• To achieve maximum resolution with 1000x
magnification, oil immersion must be used
• Oil prevents dispersion of light after light passes
through the specimen.
• Images produced have very little contrast,
therefore dyes are used to stain the specimen.
Phase Contrast Microscope
• Improves the contrast
• Makes the structures within the cells evident that
differ in their thickness or refractive index.
• Also, differences in the refractive index of cell &
the surrounding medium make them clearly
visible.
Phase Contrast Microscope-Principle
• When beams of light
pass
through
the
specimen,
it
is
partially scattered by
the microbial cells or
cell
structures.
Scattering depends on
the
thickness/
refractive indices of
various structures
• High refractive index - more
scattering of light. A scattered light
also loses its velocity when travelling
through the object and is not in phase
with the unaltered light. Therefore
appears as dark spot whereas the
unobstructed light appears as bright
spot. These differences in the
intensity provide light & dark
contrast to the image.
Advantage : since staining is not
involved, live organisms can be
observed.
Dark Field (Ground) Microscope
• Specimens appear as bright images against a nearly black
background.
• Dark field condenser with a central circular stop – does not
allow light to directly fall on the specimen.
• Light passes only around the edges of the condenser.
• Light rays which hit the object in the specimen are
deflected upwards into the objective for visualization, rest
will not enter and give a dark background.
Spirochetes under dark ground
illumination
Uses of Dark Field microscopy
• Very useful in finding extremely small,
unstained and / or moving objects.
• Organelles like cilia, flagella, vacuoles and
cell nuclei can be clearly seen.
Fluorescent Microscope
• Microorganisms or tissue cells are stained with
dyes or compounds called fluorochromes.
• Examined under microscope with ultra – violet
radiation instead of visible light.
• They convert light of shorter (UV) wavelength
into visible light and so become luminous –
Fluoresce.
• Wavelengths absorbed & emitted are specific for
specific fluorochromes.
Fluorochromes
•
•
•
•
Acridine orange : Orange
Auramine-Rhodamine : Yellow
Calcofluor white : White
Fluorescein Isothiocyanate (FITC) : Green
Modification of Fluorescent
Microscope
• Immunofluorescence : Antibodies labeled
with fluorochrome used to specifically stain
a particular bacterial species.
• Uses of IF : viruses, direct examination of
C.trachomatis, B.pertussis
Electron Microscope
• Invented by Knoll & Ruska in 1936.
• Uses electrons in place of light.
• Electrons are focussed by electromagnetic
field.
• Image is formed on a fluorescent screen or
is taken on a photographic material.
• Resolving power is 100,000 times more
than light microscope.
Types of EM
• 2 types : Transmission EM ( TEM )
Scanning EM ( SEM )
SEM allows the study of cell surfaces with greater
contrast & higher resolution than TEM.
• Disadvantages : Only dead & dried objects can be
examined, since the medium is vacuum.
Cell morphology is distorted.
• Uses : for viruses, microbes less than 0.1 to 0.2.