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Chapter 3—Observing Microorganisms Through a Microscope.
I.
II.
Units of Measurement. [Table 1—students should be familiar with all except “US Equivalents”].
a. 1 m = 1000 mm
b. 1 mm = 10-3 m = 1000 µm
c. 1 µm = 0.000001 m = 10-6 m = 10-3 mm
d. 1 nm = 0.000000001 m = 10-9 m = 10-6 mm
e. 1000 nm = 1 µm
f. 0.001 µm = 1 nm
g. Typical eukaryotic cells = 10 µm to 500 µm.
h. Typical prokaryotic cells = 1 µm to 10 µm.
i. Typical virus = 20 nm to 1000 nm (1 µm).
Microscopy: The Instruments.
a. A simple microscope has only one lens.
b. Compound Light Microscopy. Fig. 1.
i. Note: learn to name the parts of the microscope based on the figure shown in Lab
Module 3, Microscope Basics.
ii. Total magnification = magnification of objective lens  magnification of ocular lens.
Total Magnification Setups for a Typical Light Microscope.
Ocular Lens Magnification
Objective Lens Magnification
10 X
4X
10 X
10 X
10 X
40 X
10 X
100 X (oil immersion)
Total Magnification
40 X
100 X
400 X
1000 X
c. Resolution is the ability of the lenses to distinguish between two points. Fig. 2.
i. A microscope with a resolving power of 0.4 nm can distinguish between two points if
they are at least 0.4 nm apart.
ii. Shorter wavelengths of light provide greater resolution.
iii. The wavelength of white light cannot resolve structures smaller than about 0.2 µm;
therefore, the best compound light microscope can not magnify something more than
about 2000 X.
d. Refractive index is the light-bending ability of a medium.
i. The light may bend in air so much that it misses the small diameter of the highmagnification lens. Fig. 3.
1. Immersion oil is used to keep light from bending.
e. Brightfield Illumination.
i. Dark objects are visible against a bright background.
ii. Light reflected off the specimen does not enter the objective lens.
f. Darkfield Illumination. (Do Not Cover)
i. Light objects are visible against a dark background.
ii. Light reflected off the specimen enters the objective lens.
g. Phase-Contrast Microscopy. (Do Not Cover)
i. Accentuates diffraction of the light that passes through a specimen.
h. Differential Interference Contrast Microscopy. (Do Not Cover)
i. Accentuates diffraction of the light that passes through a specimen; uses two beams of
light.
i. Fluorescence Microscopy. (Do Not Cover)
i. Uses UV light.
ii. Fluorescent substances absorb UV light and emit visible light.
III.
iii. Cells may be stained with fluorescent dyes (fluorochromes).
j. Confocal Microscopy. (Do Not Cover)
i. Uses fluorochromes and a laser light.
ii. The laser illuminates each plane in a specimen to produce a 3-D image.
k. Electron Microscopy.
i. Uses electrons instead of light.
ii. The shorter wavelength of electrons gives greater resolution.
l. Transmission Electron Microscopy (TEM). (Do Not Cover)
i. Ultra thin sections of specimens.
ii. Light passes through specimen, then an electromagnetic lens, to a screen or film.
iii. Specimens may be stained with heavy metal salts.
iv. 10,000X-100,000X; resolution 2.5 nm.
m. Scanning Electron Microscopy (SEM). (Do Not Cover)
i. An electron gun produces a beam of electrons that scans the surface of a whole specimen.
ii. Secondary electrons emitted from the specimen produce the image.
iii. 1000X-10,000X; resolution 20 nm.
n. Scanned-Probe Microscopy. (Do Not Cover)
i. Scanning tunneling microscopy uses a metal probe to scan a specimen.
1. Resolution 1/100 of an atom.
ii. Atomic force microscopy uses a metal and diamond probe inserted into the specimen.
1. Produces 3-D images.
Preparation of Specimens for Light Microscopy.
a. Live or unstained cells have little contrast with the surrounding medium. However, researchers
do make discoveries about cell behavior by looking at live specimens.
b. A thin film of a solution of microbes on a slide is called a smear.
c. A smear is usually “fixed” to attach the microbes to the slide and to kill the microbes.
d. Preparing Smears for Staining.
i. Stains are salts that consist of a positive and a negative ion.
1. In a basic dye, the color is due to a cation (+).
2. In an acidic dye, the color is due to an anion (-).
ii. Bacteria have a slight negative charge at pH 7, so basic dyes are attracted to and stain
bacteria.
1. Crystal violet, methylene blue, malachite green, and safranin are all basic dyes.
iii. Acidic dyes typically stain the background, not the bacterium itself.
1. Eosin, acid fuchsin, and nigrosin are all acidic dyes.
iv. Staining the background instead of the cell is called negative staining.
e. Simple Stains.
i. Use of a single basic dye is called a simple stain.
1. This stains the entire microorganism to show basic shape and structure.
ii. A mordant may be used to intensify the stain, increase the affinity of the stain for the
specimen, or to coat the specimen to enlarge it.
f. Differential Stains.
i. These stains react differently with different kinds of bacteria, and allow us to tell the
difference between them.
ii. Gram Stain.
1. The Gram stain classifies bacteria into two large groups: gram-positive and gramnegative.
2. Steps in making a Gram stain: Fig. 12.
a. A bacterial smear is made and heat fixed.
b. The smear is flooded with crystal violet for 60 seconds and then rinsed
with distilled water.
i. Crystal violet sticks to peptidoglycan in the cell wall.
1. It is a primary stain since it colors all cells.
c. The smear is then flooded with iodine for 60 seconds then rinsed with
distilled water.
i. Iodine is a mordant, increasing the affinity of the crystal violet for
the specimen.
d. The smear is then flooded with an acetone/ethanol wash (decolorizing
agent) for 5 seconds then rinsed with distilled water.
i. The acetone/ethanol washes the crystal violet out of Gram-negative
cell walls. The Gram-positive cell walls retain the crystal violet as
long as the acetone/ethanol wash does not last too long.
ii. This step is the “differential step” in the Gram staining process.
Gram-positive cells look purple after this step; Gram-negative cells
look clear.
e. The smear is then flooded with safranin stain for 1.5-2.0 minutes, then
rinsed with distilled water.
i. Safranin is pink in color and stains all bacteria. However, Grampositive cells have a purple cell wall due to the crystal violet, and
the pink cytoplasm doesn’t show through. Gram-negative cells,
which have no stain because they were cleared in the differential
step show up with pink cytoplasm.
1. Safranin is a counterstain, since it has a different color than
the primary stain (crystal violet).
f. The slide is then dried in bibulous paper. Once dry, the slide is ready to be
viewed.
g. Gram staining gives consistent results when done on young, growing
bacteria.
3. Gram-positive bacteria.
a. They have a thick peptidoglycan layer in their cell walls (disaccharides
and amino acids) which is the part that stains purple due to the crystal
violet and iodine washes during Gram staining.
b. They tend to be killed easily by penicillin, cephalosporins, and detergents.
4. Gram-negative bacteria.
a. They have a thin layer of peptidoglycan in their cell walls, and aren’t able
to hang on to the little crystal violet that they pick up once they are
flooded with the acetone/ethanol wash during Gram staining.
b. They contain a layer of lipopolysaccharide (endotoxin) as part of their cell
walls.
c. They are more resistant to antibiotics, which can’t penetrate the
lipopolysaccharide layer very well.
d. They tend to be easily killed by streptomycin, chloramphenicol, and
tetracycline.
g. Differential Stains: Acid-Fast Stain. (Do Not Cover)
i. Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast.
ii. Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually
counterstained (with a different color basic stain) to see them.
h. Special Stains. (Do Not Cover)
i. Negative staining is useful for capsules.
ii. Heat is required to drive a stain into endospores to make an endospore stain.
iii. Flagella staining requires a mordant to make the flagella wide enough to see.