Light is refracted (bent) when passing from one medium to another
Lenses bend light and focus the image at a specific place known as
the focal point; the distance between the center of the lens and the focal
point is the focal length
The Light Microscope
The bright-field microscope produces a dark image against a brighter
background
Resolution
Microscope resolution refers to the ability of a lens to separate
or distinguish small objects that are close together; magnification (total)
is the product of the magnification of the objective lens and the magnification
of the ocular (eyepiece) lens
The major factor determining resolution is the wavelength of light
used
The dark-field microscope produces a bright image of the object against
a dark background and is used to observe living, unstained preparations
The phase-contrast microscope enhances the contrast between intracellular
structures that have slight differences in refractive index and is an excellent
way to observe living cells
The differential interference contrast microscope is similar to the
phase-contrast microscope except that two beams of light are used to form
brightly colored, three-dimensional images of living, unstained specimens
The fluorescence microscope exposes a specimen to ultraviolet, violet,
or blue light and shows a bright image of the object resulting from the
fluorescent light emitted by the specimen
Preparation and Staining of Specimens
Fixation refers to the process by which internal and external structures
are preserved and fixed in position and by which the organism is killed
and firmly attached to the microscope slide
Heat fixing is normally used for bacteria; this preserves overall
morphology but not internal structures
Chemical fixing is used to protect fine cellular substructure and
the morphology of larger, more delicate microorganisms
Dyes and simple staining are used to make internal and external structures
of the cell more visible by increasing the contrast with the background
Differential staining is used to divide bacteria into separate groups
based on their different reactions to an identical staining procedure
Gram staining is the most widely used differential staining procedure
because it divides bacterial species into two roughly equal groups-gram
positive and gram negative
The smear is first stained with crystal violet, which stains all
cells purple
Iodine is used as a mordant to increase the interaction between
the cells and the dye
Ethanol or acetone is used to decolorize; this is the differential
step because gram-positive bacteria retain the crystal violet whereas
gram-negative bacteria lose the crystal violet and become colorless
Safranin is then added as a counterstain to turn the gram-negative
bacteria pink while leaving the gram-positive bacteria purple
Acid-fast staining is a differential staining procedure that can
be used to identify two medically important species of bacteria-Mycobacterium
tuberculosis, the causative agent of tuberculosis, and Mycobacterium leprae,
the causative agent of leprosy
Staining specific structures
Negative staining is widely used to visualize diffuse capsules surrounding
the bacteria; those capsules are unstained by the procedure and appear
colorless against a stained background
Spore staining is a double staining technique by which bacterial
endospores are left one color and the vegetative cell a different color
Flagella staining is a procedure in which mordants are applied to
increase the thickness of flagella to make them easier to see after staining
Electron Microscopy
The electron microscope focuses beams of electrons to produce an image
In transmission electron microscopy (TEM), electrons scatter when they
pass through thin sections of a specimen; the transmitted electrons (those
that do not scatter) are used to produce an image of the internal structures
of the organism; TEM has a resolution about 1,000 times better than that
of the light microscope (0.5 nm versus 0.2 mm)
Specimen preparation for the electron microscope involves procedures
for cutting thin sections, chemical fixation, and staining with electron-dense
materials (analogous to the procedures used for the preparation of specimens
for light microscopy); other preparation methods include shadowing or freeze-etching
The scanning electron microscope (SEM) uses electrons reflected from
the surface of a specimen to produce a three-dimensional image of its surface
features; many SEM have a resolution of 7 nm or less
Newer Techniques in Microscopy
The confocal microscope is often used to examine fluorescently stained
specimens
It uses a focused laser beam to illuminate just one point on the
specimen
A detector measures the amount of illumination from each point, creating
a digitized signal
After examining many points (optical sections), a computer combines
all the digitized signals to form a three-dimensional image with excellent
contrast and resolution
Scanning Probe Microscopy
The scanning tunneling electron microscope uses a sharp probe to
create an accurate three-dimensional image of the surface atoms of a specimen;
the resolution is such that individual atoms can be observed
The atomic force microscope is similar to the scanning tunneling
microscope in that it uses a scanning probe; however, in this microscope
the probe maintains a constant distance from the specimen and is useful
for surfaces that do not conduct electricity well
