Microscopy
Compound Microscope
• The Compound Microscope is the type of
microscope we use in the lab. It uses visible light
as it’s light source for illumination.
• The average compound microscope has resolving
power of .2m (1m = .000001m)
• The resolving power is the ability of the
microscope to distinguish between 2 pts, or to
show detail.
• Staining is used to make the specimen contrast
sharply with the medium so that it is easier to see.
Fluorescence Microscopy
• This particular microscope uses fluorochromes to attach to
organisms and allow them to fluoresce. Fluorochromes are
small molecules that only fluoresce once they have
attached to their target.
• Immunofluorescence is a particular type of marker used in
fluorescence microscopy. The fluorochromes are first
attached to a specific antibody (Ab). The antibody is
usually specific for a particular type of cell, virus, or other
type of specimen.
• This type of microscopy is useful for detecting viruses and
other org. within cells, tissue, or other clinic specimen that
normally we wouldn’t be able to see or detect otherwise.
• For example, let’s say that we suspect
someone has Hepatitis. Hepatitis is caused
by a virus. Viruses are so small that they
can’t be seen under a compound
microscope. In addition, they also spend
much of their replication time within host
cells and you can’t see into a cell with a
compound microscope.
– So we take a sample of blood from the patient.
– Then we get some antibodies that are specific
for the hepatitis virus.
– Next we put the fluorochrome on the antibody.
–.
– Then we put the fluorochrome, antibody
combination together on the specimen.
– If the virus is present, then the antibody will
bind to the virus or virus infected cell. Upon
binding, it will “turn on the switch” for the
fluorochrome. The fluorochrome will now
fluoresce indicating the specimen is positive for
that virus.
– If the virus is not present, then the antibody
won’t be able to bind and the fluorochrome
won’t fluoresce.
Electron Microscopes
• Electron Microscopes are used to examine objects
smaller than .2m. There are 2 types that are
used.
• SEM: Scanning Electron Microscope
– This type of electron microscope creates and image of
the surface structures of the organism.
– It bounces a beam of electrons (e-s) off the organism to
create the image. As the electrons come into contact
with the specimen the e-s bounce off in different
directions. A computer then records the direction it
went and extrapolates the shape of the surface of the
specimen by the direction that each e-s bounces off.
• TEM: Transmission Electron Microscopy
– The Transmission Electron Microscipe uses ultra thin
slice of the specimen to create images of inside cellular
structures.
– In this case the image is created by sending the e-s
through the specimen and recording the trajectory of
the e-s as they exit out the other side.
• These are both great ways for us to learn more
about a particular organism. Unfortunately a live
specimen can never be viewed this way because
the preparations are toxic to the cells.
Biological Stains Used in the Lab
• To prepare specimens to be viewed under the
compound microscope there are two types of
stains that are used.
– 1. Basic Stain: The basic stain is composed of a
positively (+) charged ion that attaches to the negatively
(–) charged cell membrane.
• Crystal violet and safranin are examples of basic stains.
– 2. Acidic Stain: An acidic stain is composed of a
negatively (-) charged ion that is repelled by the
negatively (–) charged membrane.
• Eosin and Acid fuschin are examples of acidic stains.
• There are two different ways that we can use basic
and acidic stains to better view and identify
microbes.
– 1. Simple Stain: A simple stain is when we use one
stain, basic or acidic and color the organism to view
under the microscope. It helps us to determine the
shape of the organism.
– 2. Differential Stain: A differential stain is when we
combine 2 or more stains to gather more information.
• For example, a gram stain uses 2 stains. One stain indicates
the organism is G+ and the other indicates the organism is G-.
• Other types of differential stains are endospore stains, capsule
stains, acid-fast stains, and flagella stains.
The Basic Gram Stain Procedure
• Let’s say that you have a culture that has two different
kinds of organisms growing in it. One organism is a cocci
and the other is a rod. You want to determine what the
gram reaction is for each.
• The first step is to heat fix the specimen. (You smear the
specimen on the slide and wave it through the bunsen
burner to slightly bake it onto the slide. You don’t want it
to wash off during the staining procedure.!)
• The second step is to apply the Primary stain. The
primary stain is always the first stain applied in a
differential staining procedure. In this case the primary
stain is crystal violet. It is a basic stain so it attaches to all
the cells present because they all have a (-) charged cell
membrane.
• The next step is to apply iodine to the specimen.
The iodine acts as a sealant to tightly bind the
primary stain to the G+ cell wall. This is called a
mordant.
• Then the specimen is washed with alcohol. The
alcohol dissolves lipids. Remember that the Gcell wall is made up of an outer membrane that
contains lipids. The alcohol wash removes the cell
wall from the G– organism.
• Next the secondary stain is applied. In a
differential stain, the secondary stain is the second
type of stain used in the process. In this case,
safranin is the secondary stain that is used. It is a
basic stain too. It attaches only to the unstained
cells, ie the G- organisms on the slide.
• The final result is that the G+ cells are
stained purple and the G- cells are stained
magenta or pink.
• The gram stain works simply because there
is a difference in the cell walls between G+
and G- organisms.
• See “Insight 4.2” on page 98 of your
textbook for an excellent explanation of the
gram stain procedure.
Endospore stain
• An endospore is a special structure made by some
bacteria to store genetic material for a new cell.
• Because endospores are so resistant to many
environmental pressures they can’t be stained with
ordinary dyes because don’t penetrate the wall.
• The Primary stain for an endospore stain is
malichite green. Heat is used with this stain to
force it into endospore.
• The Secondary stain is safranin. Safranin stains
the cell wall of the microbe giving a contrast
between the endospore and the organism.
Capsule Stain
• Remember that a capsule often indicates
virulence.
• A Negative stain (India Ink) is used to show the
outline of the capsule. It will not bind to capsule
but it will stain around the capsule.
• The the Secondary stain, methyl-blue is applied to
stain the organism inside the capsule.
• The clearing around the stained orgaism is the
capsule.
Acid-fast Stain
• Some microbes have cell walls that are constructed a little
differently that how we have discussed. The organism,
Mycobacterium has a waxy substance in its cell wall called
mycholic acid. The mycholic acid makes it difficult to
stain this organism using a basic dye. So a different type
of stain is used for it.
• The acid-fast stain is used to distinguish mycobacterium
from other types of rods.
• Carbol fuschin is the primary stain for this technique. Like
the endospore stain, heat is used to force the stain on the
organism.
• A Secondary stain is added after to bind to any organism
that did not bind to the primary stain.
Flagella Stain
• A flagella stain is a very tedious and delicate
staining procedure.
• It uses a mordant and carbol fuschin to build up
the diameter of the flagella until they become
visible.
• This type of staining procedure is valuable
because the arrangement of the flagella can be
used as a diagnostic aid.
• No homework for this chapter.