Chapter 3

Units of Measurement
 Microorganisms are measured in micrometers, μm (10-6 m) and in
nanometers, nm (10-9m).
Microscopy: The Instruments
 Diagram the path of light through a compound microscope.
Define total magnification and resolution.
 Identify a use for darkfield, phase-contrast, differential interference
contrast, fluorescence, and scanning acoustic microscopy, and
compare each with brightfield illumination.
 Explain how electron microscopy differs from light microscopy.
 Identify uses for the transmission electron microscope (TEM),
scanning electron microscope (SEM), and scanned-probe
microscopes.
 A simple microscope consists of one lens; a compound microscope has
multiple lenses.
Light Microscopy
 The most common microscope used in microbiology is the compound
light microscope (LM).
 The total magnification of an object is calculated by multiplying the
magnification of the objective lens by the magnification of the ocular
lens.
 The compound light microscope uses visible light.
 The maximum resolution, or resolving power (the ability to distinguish
two points) of a compound light microscope is 0.2 μm; maximum
magnification is 1500×.
 Specimens are stained to increase the difference between the
refractive indexes of the specimen and the medium .
 Immersion oil is used with the oil immersion lens to reduce light loss
between the slide and the lens.
 Brightfield illumination is used for stained smears.
 Unstained cells are more productively observed using darkfield, phase-
contrast, or DIC microscopy.

 The darkfield microscope shows a light silhouette of an organism
against a dark background. It is most useful for detecting the presence
of extremely small organisms.
Scanning Acoustic Microscopy
 Scanning acoustic microscopy (SAM) is based on the interpretation of
sound waves through a specimen.
 It is used to study living cells attached to surfaces such as biofilms.
Electron Microscopy
 Instead of light, a beam of electrons is used with an electron
microscope.
 Instead of glass lenses, electromagnets control focus, illumination, and
magnification.
 Thin sections of organisms can be seen in an electron micrograph
produced using a transmission electron microscope (TEM).
Magnification: 10,000–10,000,000×. Resolving power: 10 pm .
 Three-dimensional views of the surfaces of whole microorganisms
can be obtained with a scanning electron microscope (SEM).
Magnification: 1000–500,000×. Resolution: 10 nm .
Preparation of Specimens for Light Microscopy
 Explain the purpose of simple staining.
 List Gram stain steps, and describe the appearance of gram-
positive and gram-negative cells after each step.
 Compare and contrast the Gram stain and the acid-fast stain.
 Explain why each of the following is used: capsule stain, endospore
stain, flagella stain.
Preparing Smears for Staining
 Staining means coloring a microorganism with a dye to make some
structures more visible.
 Fixing uses heat or alcohol to kill and attach microorganisms to a slide.
 A smear is a thin film of material used for microscopic examination.
 Bacteria are negatively charged, and the colored positive ion of a basic
dye will stain bacterial cells.
 The colored negative ion of an acidic dye will stain the background of a
bacterial smear; a negative stain is produced.

Simple Stains
 A simple stain is an aqueous or alcohol solution of a single basic dye.
 A mordant may be used to improve bonding between the stain and the
specimen .
Differential Stains
 Differential stains, such as the Gram stain and acid-fast stain,
differentiate bacteria according to their reactions to the stains.
 The Gram stain procedure uses a purple stain, iodine as a mordant, an
alcohol decolorizer, and a red counterstain.
 Gram-positive bacteria remain purple after decolorization; gram-
negative bacteria do not, and appear pink from the counterstain.
 Acid-fast microbes, such as members of the
genera Mycobacterium and Nocardia, retain carbolfuchsin after acid-
alcohol decolorization and appear red; non–acid-fast microbes take up
the methylene blue counterstain and appear blue.
Special Stains
 Negative staining is used to make microbial capsules visible.
 The endospore stain and flagella stain are special stains that are used
to visualize specific structures in bacterial cells.