Microbiology, 5/e

Lansing M Prescott, Augustana College
Donald A Klein, Colorado State University
John P Harley, Eastern Kentucky University

The Study of Microbial Structure: Microscopy and Specimen Preparation

Study Outline

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

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

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

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

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

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

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

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