MICROBIOLOGY (BIOL& 260)
CHAPTER 3
MICROSCOPY AND MICROORGANISMS
HOW WE MEASURE MICROORGANISMS
1. metric system is used
2. based on 10’s
3. common units
a. millimeter=1/1000 meter
b. micrometer=1/1,000,000 meter
c. nanometer=1/1,000,000,000 meter
COMPOUND LIGHT MICROSCOPE COMPONENTS
1. illuminator
2. condenser
3. objective lens
4. ocular lens (eyepiece)
TYPES OF LIGHT MICROSCOPES
1. Bright-field Microscopy
a. usual setting of the typical microscope
b. field of vision is brightly illuminated
2. Darkfield Microscopy
a. organism is light to a dark field
b. used to view unstained organisms
3. Phase-Contrast Microscopy
a. light rays are refracted out of phase with one another
b. used to visualize internal organelles of living cells
4. Differential Interference Contrast Microscopy (DIC)
a. similar to phase-contrast microscopy
b. uses two beams of light
c. produces brightly-colored image with a “three-dimensional” look
5. Fluorescence Microscopy
a.some organisms naturally absorb flourochromes
b. immunofluorescence--use of antibodies with a fluorescent tag against a specific
antigen (microbe)
6. Confocal Microscopy
a. flurochromes are used
b. laser is used to "slice" the organism giving very clear two-dimensional images
c. "slices" can be "put together" by a computer to generate a three-dimensional image
ELECTRON MICROSCOPY
1. Transmission electron microscopy
a. thin slice of specimen has beams of electrons pass through it, denser areas prevent
passage of electrons
b. heavy metal stains are used to provide a greater contrast
2. Scanning electron microscopy
a. provides a three-dimensional view of the specimen
b. electrons strike the surface and are deflected in different directions (picked up by
electron detectors)
SCANNED-PROBE MICROSCOPY
1. Scanning Tunneling Microscopy
a. a tungston probe is used to scan a specimen in order to view the bumps and
depressions of atoms on the surface
b. greater resolving capabilities when compared to the electron microscope (can
resolve up to 1/100 the size of an atom)
2. Atomic Force Microscopy
a. metal and diamond probe is forced down into the specimen
b. a three-dimensional image is produced
STAINING PROCEDURES FOR THE LIGHT MICROSCOPE
1. Simple stain--purpose is to provide visibility and shape of the organism
2. Differential stain--allow for difference in color based on the type of organism
a. gram stain
b. acid-fast stain
3. Special stain
a. negative stain--usually used to examine bacteria for capsules (gelatinous covering
around the bacteria)
b. flagellar stain--stains are applied to “build-up” the flagella large enough to see
(similar to applying mascara to eyelashes)
c. endospore stain--heat is used to drive the primary stain into the spore; the rest of the
cell is stained with a counterstain
GRAM STAIN
1. Crystal violet (primary stain) is added
2. Iodine is added--iodine binds to the crystal violet to create a large complex which
cannot leave the thick peptidoglycan layer of gram + cells
3. Alcohol is added to wash the crystal violet from gram - cells (they don’t retain the
crystal violet-iodine complex
4. Safranin (red) gives color to gram - cells
ACID-FAST STAIN
1. Used to detect pathogens of tuberculosis and leprosy
2. Carbolfuchsin (primary stain) driven in with heat
3. Acid-alcohol removes the carbolfuchsin from non acid-fast cells
4. Methylene blue (counterstain) gives color to non acid-fast cells
LEARNING OBJECTIVES FOR CHAPTER 3
Following successful study of this chapter students should be able to:
1. Define or describe each of the following terms: millimeter, micrometer, nanometer,
compound light microscope, illuminator, condenser, objective lens, ocular lens,
resolution, smear, mordant, primary stain, counterstain, capsule, virulence, endospore,
flagella
2. List and describe the distinctive characteristics for each of the following microscopy
techniques: brightfield microscopy, darkfield microscopy, phase-contrast microscopy,
differential interference contrast microscopy, fluorescence microscopy, confocl
microscopy, transmission electron microscopy, scanning electron microscopy, scanningtunneling microscopy, atomic force microscopy
3. Contrast acidic dyes with basic dyes
4. Describe the purposes behind simple staining, differential staining (gram stain and
acid-fast stain), and special stains (negative stain, endospore stain, flagella stain)