Chapter 3 – Observing Microorganisms through a Microscope
Units of Measure
-
Use metric system
Most microorganisms measured in micrometer (µm = 10-6) or nanometers (nm = 10-9)
Microscopy
Simple microscope = 1 lens; similar to magnifying glass
Compound microscopes = multiple lenses
o
Light Microscope – uses visible light to look at specimens.
 Compound light microscope: series of lenses and uses visible light as its source
of illumination.
 Light passes from light sources (illuminator), through the condenser,
through the specimen, into the objective lends, through the ocular lens
(eye piece).
o Total magnification = objective lens x ocular lens
o Resolution = the ability of the lens to distinguish two points a
specified distance apart
 White light has a resolving power of 0.2 µm
o Refractive index = measure of the light-bending ability of the
medium (air or oil).
 The higher the magnification, the poorer the resolution.
Immersion oil is useful because it helps focus the light
better, thus giving better resolution at higher
magnification.
 Under normal conditions, the field of vision in a compound light microscope is
brightly illuminated. By focusing the light, the condenser produces a brightfield
illumination.
Types of Microscopy
1.
Darkfield microscopy – used to examine live microorganisms that are invisible in the ordinary
light microscope, cannot be stained by conventional methods, or are so distorted by staining
that their characteristics then cannot be identified.
a. Uses a darkfield condenser instead of the normal condenser.
i. The disk blocks light that would enter the object lends directly. Only light that is
reflected off the specimen enters the objective lens.
1. Specimen will appear light against a black background.
2. Phase-contrast microscopy – permits detailed examination of internal structures in living
microbes. The specimens don’t have to be fixed to the slide or stained.
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a. Basically works because as light passes through, some of the light will hit the specimen
directly and some of the light will not. This “phase” difference can be observed in the
ocular lens.
i. Very useful for seeing internal structures.
Differential Interference Contrast (DIC) microscopy
a. Similar to phase contrast, but uses two beams of light instead of one, as well as prisms,
which split the light beams, adding color to the specimen. Better resolution than phase
contrast, and the image appears almost 3D.
Fluorescence Microscopy
a. Uses fluorochromes (fluorescent dyes) to stain specimens. These stained specimens are
then subjected to UV light, thus appearing to luminescence (Bright object against a black
background).
i. Fluorescent-antibody/immunofluorescence staining.
Confocal Microscopy – used to reconstruct 3D images.
a. Specimens are stained with fluorochromes, but only one plane of a small region of the
specimen is illuminated with short wavelength (blue) light. Each plane corresponds to
an image of a fine slice that has been physically cut from the specimen. Each plane is
scanned until the entire specimen is done.
i. Most confocal images are recreated on a computer to help construct the 3D
image.
1. Images can be rotated on the computer screen to see the whole thing.
2. Particularly useful for looking at the entirety of the cell and its
components, as well as physiological pathways.
Two-photon microscopy – similar to confocal microscopy except it uses long (red) wavelengths,
causes less damage to cells due to creation of oxygen radicals, can be used on thicker
specimens, and can give physiological results in real-time.
Scanning Acoustic Microscopy – consists of interpreting sound waves sent through the
specimen. Used to study living cells, attached to another surfaces, such as a cancer cell, artery
plaque, and bacterial biofilms.
Electron Microscopy – uses a beam of electrons instead of light. Used to examine structures that
are too small to be resolved with a light microscope. Images are always black and white but can
be artificially colored to accentuate certain details. Uses electromagnetic lenses to focus the
beam of electrons onto the specimen.
a. TEM – transmission electron microscope – a finely focused beam of electrons from an
electron gun passes through a specially prepared, ultrathin section of the specimen.
i. Made clearer by using positive-negative staining.
ii. Limitations: electrons have limited penetrating power, so the specimen must be
ultrathin = no 3D aspect. Specimens must be fixed, dehydrated, and viewed
under a vacuum to prevent the electrons from scattering. The treatments kill
the specimen and cause distortion, as well as possibly leaving artifacts.
b. SEM – scanning electron microscopy – provides a 3D image of the specimen. Basically
works by scanning the surface of a specimen with a beam of electrons, so it’s useful for
studying the surface of intact cells and viruses.
9. Scanned-Probe Microscopy – use various kinds of probes to examine the surface of a specimen
at close range, without modifying the specimen or exposing it to damaging radiation.
a. STM – scanning tunneling microscope – uses a tungsten probe to scan a specimen,
revealing the bumps and depressions of the atoms on the surface of the specimen.
Gives really good pictures of DNA.
b. ATM – atomic force microscopy – a metal-and-diamond probe moves along the surface
of the specimen, and its movements are recorded. Very useful for scanning biological
substances and detailing molecular processes, such as the assembly of fibrin.
Preparation of Specimens for Light Microscopy
A. Staining – coloring an organism with a dye that emphasizes certain structures.
a. Stains are salts composed of a positive and negative ion, one of which is colored and is
known as the chromophore.
i. Basic dyes – positive ion
ii. Acidic dyes – negative ion
1. Basic dyes are used more frequently, because bacteria are slightly
negatively charged, so a positive ion is attracted to them.
b. Types of staining:
i. Simple staining – used to highlight a microorganism’s overall shape and
arrangement. Sometimes a mordant is added to intensify the stain.
ii. Differential – used to distinguish between different types of bacteria
1. Gram stain – differentiates between gram-positive and gram-negative
bacteria
2. Acid-fast stain – useful for identifying Mycobacteria and Nocardia
species
iii. Special – used to color and isolate various structures, such as capsules,
endospores, and flagella; sometimes used as a diagnostic aid.
1. Negative – used to demonstrate the presence of capsules because they
do not accept most stains.
2. Endospore stain – malachite green, when applied to the specimen, and
heated, stains the spore.
3. Flagella stain – used to indicate the presence of flagella. A mordant is
used to build up the diameters of flagella until the become visible in the
microscope when stained with carbolfuschin.