CHAPTER 3 OBSERVING MICROORGANISMS THROUGH
A MICROSCOPE
Magnification is necessary to observe and study microbes. Also, we know all microbes are small,
but we must still be able to measure their size. Units of the metric system are
used. The meter is the standard
unit, but since this is roughly equivalent to one yard, we must use much
smaller units for microbes.
Centimeter (cm) = 0.01 meter (2.54 cm = 1 inch)
Millimeter (mm) = 0.001 meter (25.4 mm = 1 inch)
Micrometer
(mm) = 0.001 mm
(25,400 mm = 1 inch)
Nanometer (nm) = 0.001 mm
(25,400,000 nm = 1 inch)
Van Leeuwenhoek’s best microscopes had a single
magnifying lens, which he ground himself, and provided approximately 300X
magnification. About 1830, an
effective compound (2 lens) microscope was developed. Improvements in this led to the modern
compound light microscope. Using
light rays to illuminate the specimen, magnifying lenses in the ocular and the objectives
can provide a total magnification of up to 1800 - 2000X, although most of these
go up only to 1000X.
Modern microscopes are compound light microscopes. This means
that there are 2 lenses that magnify, the ocular lens
and the objective lens. Light rays provide the illumination.
Brightfield microscopy---this is the most common use of the light microscope. Specimens
are usually stained to make them more easily visible. Light rays pass though the condenser
lens and are directed straight up into the objective lens, passing through the
specimen on the way. The field of
view, seen through the ocular, is brightly illuminated, with the specimen
appearing slightly darker if unstained, or brightly colored due to dyes.
The total magnification provided can be determined by
multiplying the ocular magnification times the objective magnification. The
very best compound light microscope might have a top magnification near 2000X.
Magnification alone
is not the only concern--- a clear picture in which fine detail can be seen is
also required. This is known as
resolution (resolving power)--- ability of the lenses
to distinguish between 2 points a specific distance apart. For light microscopes, this is 0.2 mm. This is the limiting factor in useful
magnification by light microscope. Resolving power is a function of the
wavelength of light from the light source and the numerical aperture of the
condenser and objective lens. The shorter the wavelength, the
greater the resolution. Blue light has the shortest wavelength, so that
is the reason for the blue filters.
Unstained specimens may be difficult to locate and observe with
bright-field microscopy, but in some situations stains are undesirable.
·
All stains kill the
microbes, so living microbes must be observed unstained.
·
Some microbes do not stain
well.
·
Staining procedures may
distort the microbe or slightly alter the size.
If stains are not appropriate, one of the following methods
would work better than bright-field:
1. Darkfield Microscopy---this type of
microscope uses a modified condenser. This condenser contains an opaque disc
that blocks direct light. Instead of directing all light rays straight through
the specimen into the objective, only those rays which are reflected off the
specimen reach the objective. The
result of this is that the objects on the slide glow against a dark background.
2. Phase-Contrast Microscopy---this
technique permits better viewing of internal structures of cells without use of
stains, so living cells can be viewed. Areas and structures of different
densities appear as various shades of gray. This also requires a special condenser.
3. Differential Interference Contrast (DIC)
Microscopy---similar to phase-contrast but gives higher resolutions and
brighter color.
·
Fluorescence Microscopy-Special
dyes (fluorochromes) can absorb short-wave length light and give off light at a
longer wave length. Observed
through a fluorescence microscope, which uses an UV light source, objects
stained with these dyes glow with neon colors against a dark background. Some bacteria can be directly stained with these special dyes to show their
presence in a sample, but the more common use is the fluorescent-antibody
technique (immunofluorescence).
Antibodies against certain organism are stained with a fluorescent
dye. These antibodies are added to
a slide containing the specimen.
The slide is gently washed.
If the antibodies did not match the bacteria or viruses in the specimen,
the antibodies are washed away. If
the antibody matches, it remains on the slide and can be viewed as it
fluoresces. This technique is a
rapid, reliable diagnostic test.
·
Confocal Microscopy-Used in
conjunction with a computer to construct 3D images of the specimen, sort of a
CT scan through a microscope.
Objects smaller than 0.2 mm
cannot be clearly viewed with a light microscope. They require the
use of an electron microscope.
Viruses and tiny internal cell structures would be examples. These microscopes use a beam of
electrons, with its much shorter wavlength, for illumination instead of light
rays, so the resolution is much greater (2.5 nm compared to 0.2mm
for light microscopes). Images produced appear on a screen and are
photographed. Types of electron microscopes:
·
Transmission Electron
Microscopy (TEM)--- the beam of electrons is passed through a very thin-sliced
specimen. Electromagnetic lenses
are used. Objects are most often
magnified 10,000- 100,000X or even more.
Since the beam of electrons has much less penetrating power than light
rays, the specimen must be sliced very thin. Even most bacteria would have to be
sliced. Specimens are “stained” with thin coats of metal salts to
make them more clearly visible. The
image is focused on a fluorescent screen or photographic plate. Disadvantages:
1) Specimens are
dead
2)Preparation
may shrink and distort the specimen
·
Scanning Electron
Microscopy (SEM)---this technique “scans” the outside of the specimen
and gives a 3-D view. Objects are
usually magnified 1000-10,000X.
Entire cells, bacteria and viruses can be viewed.
·
Scanning Tunneling and
Atomic Force Microscopy---both produce 3D images
of the surface of a molecule.
It is not impossible to view unstained microbes with
bright-field microscopy. This is
usually done by one of the following methods:
·
Temporary wet mount---a drop of liquid containing the microbes is placed on a
slide. A cover slip is placed over
the drop and the slide is viewed.
Lowering the condenser and dimming the light a little may help. Dabbing vaseline
around the edges of the cover slip will keep the specimen wet longer.
·
hanging drop---a
special slide with a hollowed out “well” in the center is used. A
drop of liquid containing the microbes is placed on a cover slip which has dabs
of Vaseline on the corners. The special slide is lowered over it, concave side
down, until it touches the Vaseline. The slide is then flipped over and viewed.
Most of the time, microorganisms are stained by applying
colored dyes to make them more easily viewed. This usually begins by spreading a thin
film of the specimen over a slide.
This is called a smear. It
is allowed to air dry, and then is passed through the flame of a Bunsen burner
to heat fix the smear. This is
mainly to stick the organisms to the slide, but the heat and drying also kill
most of them. The slide is now
ready for the desired staining procedure.
Some dyes are attracted to the outside of bacterial cells,
which are usually slightly negative in charge. These dyes are called basic
dyes, and the colored portion of their molecules is positively charged. These
include:
·
Crystal violet
·
Methylene blue
·
Safranin
·
Carbolfuchsin
·
Malachite green
Other dyes are not attracted to the bacteria and color the
background, leaving the bacteria unstained. These are called acidic dyes. The
colored portion is negatively charged, and is repelled by the outside of
bacterial cells. These include
·
Nigrosin
·
India ink
·
Eosin
·
Simple stains---one stain is applied to a heat-fixed smear and rinsed
off. All microbes would take on the
color of the dye.
·
Differential stains---stains will react differently with different types of
bacteria
This is the most important staining technique in
microbiology. It was developed in
1884 by Dr. Christian Gram. It is a
differential stain used to sort bacteria into 2 groups, gram-positive and
gram-negative, which is the first big step in identification. The gram reaction may also aid in
determining the proper treatment of the disease.
Steps in the gram stain:
·
Crystal violet is applied to a heat-fixed smear for 30 seconds and gently
rinsed off. This is the primary
stain.
·
The smear is thoroughly
covered with Gram’s iodine, which is left for 1 minute and then
rinsed. The iodine acts as a
mordant, fixing the crystal violet into the cell wall of gram-positive
bacteria.
·
95% ethanol (alcohol) is then applied with the slide at an angle, washing
over the stained area. This is the
decolorizing step. The purple dye
will wash off of the Gram-negative bacteria, but remain in the cell wall of
Gram-positive bacteria.
·
Safranin is applied for 30 seconds. This is the counter-stain. Then rinse, blot dry and examine.
Gram-positive organisms will be purple (blue), Gram-negative
will be red (pink). This difference
is due to differences in the structure of the cell wall between the 2 groups.
This stain identifies bacteria that incorporate a waxy substance
called mycolic acid into their cell wall. The main acid-fast bacteria are
members of the genus Mycobacterium,
which includes the bacteria that cause tuberculosis and leprosy, and the genus Nocardia, which are mostly soil
organisms but occasionally show up as pathogens in lung and skin infections..
Steps:
·
Carbolfuchsin is steamed
into a heat-fixed smear for 5 minutes. (Primary stain)
·
Slide is cooled and gently
but thoroughly rinsed.
·
Acid-alcohol is applied
(decolorization) to remove fuchsia color from all bacteria except acid-fast.
·
Methylene blue is applied
as the counterstain. Rinse, blot dry and examine
·
Acid-fast organisms will be
fuchsia (hot pink); non-acid-fast will be blue.
1. capsular stain----some types of bacteria are surrounded by a slimy outer
covering called a capsule. The capsule does not accept colored dyes, so the
procedure involves a negative staining technique and can be done in a number of
different ways. In lab, we will use the method known as Gin’s method.
Steps:
·
A small drop of India ink
is applied to one end of a clean slide
·
The drop is diluted with a
drop of saline
·
Bacteria are mixed in
·
The mixture is spread to
form a thin film over the slide, which is allowed to throughly air dry (no
heat-fixing)
·
Methylene blue is added to
generously cover the area of the smear and left for 3 minutes
·
Very gently rinse. Some of the smear will wash away even if you are
careful but some should be left.
·
Do
not blot, but excess water can be removed by just barely touching the
slide with an absorbent paper towel.
·
The capsules should appear
as clear areas against a dark background.
Inside each capsule, a purple bacterium should be seen.
2. endospore stain-----Endospores
are special dormant forms of bacteria that are highly resistant to adverse
conditions such as heat, drying, toxic chemicals, lack of nutrients, etc. Since only a few genera of bacteria are able
to form endospores, this can be a good identification feature.
Steps
in Schaeffer-Fulton Endospore Stain:
·
Malachite green is steamed
in to a heat-fixed smear for 5 minutes.
·
The slide is gently but
thoroughly rinsed.
·
Safranin is applied for 30
seconds. The slide is washed, blotted dry and examined. Endospores retain the green dye and appear
as tiny round to oval bodies.
Regular vegetative cells will stain pink from the safranin.
3. flagella staining---this is a
tedious, difficult procedure which we won’t attempt. It involves a special procedure to
deposit a thickening material on the flagella, which are too fine to be seen by light microscope otherwise. (Electron microscopy could show them easily).
SUMMARY OF STAINS TABLE
3.3 P. 72