Medical Microbiology and Parasitology

Optical Techniques (Microscopy)

Name:
Year and Section:
Microscope Type
A. Light Microscope
Bright-field microscope

Distinguishing Features
Specimens are rendered visible because of
the differences in contrast between them &
the surrounding medium
Consists of two series of lenses which function
together to resolve the image(usually 100x
objective lens with 10x ocular lens=1000x)
Needs dyes for the bacterial cells or their
organelles to be more easily seen

Typical Image of Microbe

Principal Uses
Most commonly used for microbiology
courses

Phase contrast microscope

Light waves passing through transparent

objects emerge in different phases depending
on the properties of materials through which
they pass
The effect is amplified by a special ring in the
objective lens-forming dark image on light
background

Magnification of microorganism
without staining

Dark-field micros

Has special condenser that both blocks direct

light rays & deflects light off a mirror on the
side of the condenser at an oblique anglecreating a dark field
Has quite high resolution

Used for observing Treponema

pallidum, a spirochete smaller than 0.2
m

Fluorescence mi

Visualizes specimens that fluoresce; those

which do not is stained with fluorochromes

For diagnostic technique Flurorescentantibody (FA) technique/

Immunofluorescence
Ex. Antibodies to Legionella
pneumophila are chemically labeled
with fluorescein isothiocyanate(FITC)

Differential Interference
contrast microscope

Employs polarizer to produced polarized light,

Because of slight differences in refractive
index of the substance each beam pass
through, the combined beams are not totally
in phase creating an interference effect which
intensifies subtle differences in cell structure.
Spores, vacuoles, granules appear 3D
Uses beam of electrons projected from an
electron gun and directed or focused by an
electromagnetic condenser lens onto a thin
specimen
The image is visualized by allowing it to
impinge on a screen that fluoresces when
struck with the electrons.

Used for observing unstained cells

because of its ability to generate images
that reveal internal cell structures that
are less apparent by bright-field
techniques

Scanning electron microscope

Has lower resolving power than Tem, but

provides #D images of the surface of
microscopic objects and shadowing
Electrons are focused by means of lenses into
a very fine point
Interaction of electrons releases radiation

Confocal Scanning Laser

Microscope

Couples a laser light source to a light

microscope By precisely illuminating only a
single plane of the specimen, illumination
intensity drops off rapidly above and below
the plane of focus, stray light from other
planes of focus are minimized
Equipped with computer software

Used for shadowing, freeze-drying

specimens and negative staining with
and electron-dense material such as
phosphotungstic acid or uranyl
salts(without these there would not be
enough contrast to detect details of
specimen)
Widely used in numerous biological
science of disciplines, from cell biology
and genetics to microbiology. It is also
used in quantum optics and nanocrystal imaging and spectroscopy

Scanning Probe Microscopes

Measure surface features by moving a sharp

probe over the objects surface
Gives 2D image of atoms and gives better
resolution

Atomic

Gives 3d image profile of nano-objects, nonconducting objects like DNA & protein

Transmission electron
microscope

Magnification of viruses with diameters

of 0.01-0.2 m

For imaging surface at atomic level,

conductance of single molecule

For imaging, measuring, manipulating

matter at the Nano-scale. Ex.
Interaction between proteins of E.coli