1.1 Setting up and using the microscope
Introduction
Microorganisms
or microbes are microscopic living organisms, which mean they are less likely
to be visible to the naked eye. In order to be seen, microorganisms need to be
magnified. Thus, microscope is used to observe and study microorganisms.
Although microscopes come with various type of models and applications (such as
electron microscope), the most basic microscope with simple way of handling
will be the light microscope. Despite their disadvantages comparing to other
high-end microscope, they are still capable of viewing microorganism. This instrument
is commonly used by beginners such as student.
The
part of light microscope
The following description is generalized to cover a typical microscope (see figure 1.1)
The
light microscope uses light to illuminate specimens and glass lenses to magnify
image. Hence, the illuminating process requires a strong source of white light
which is generated by the light bulb installed in the base of the microscope.
The on-off switch turns on the current to the bulb; the voltage control dial
controls the brightness of the bulb. When the light bulb is turned on, it
produces white light which will travel through the condenser before hitting the
specimen. The position of the condenser stage can be adjusted using the
condenser focusing knob to ensure that the light is well-condensed and highly
focused before it exits the condenser to give the best illumination of the
specimen. Condenser is used to collect and focus the light from the illuminator
on to the specimen.
Diaphragm
controls the amount of light reaching the specimen. It is located above the
condenser and below the stage. The diaphragm and condenser together control
both the focus and quantity of light applied to the specimen.
A
flat platform that supports the slide being analysed is called stage. The stage
can be adjusted forwards and backwards and from left to right by using the
coaxial knobs which able the viewer to centre the slide preparation in the
light path. Stage clips are used when there is no mechanical stage. The viewer
is required to move the slide manually to view different sections of the
specimen.
Coarse
adjustment knob is a knob present on the arm of a microscope. The main function
of this knob is to move the specimen back or forth to adjust the slide
containing specimen in order to bring it to focus and show the best image
possible. The coarse adjustment should be carefully moved and adjusted to
attain desired results. Fine adjustment knob is a sub part of the coarse
adjustment knob. It is used to bring the specimen into sharp focus.
The
objective lens is the part of microscope responsible for magnifying the image
of specimen. There are four objective lenses with magnification of 4X, 10X, 40X
and 100X. Nosepiece houses the objectives. By rotating the revolving nosepiece,
viewer should be able to change the objectives to the one with desired
magnification power into the light path to increase or decrease the
magnification.
Lastly,
the eyepiece tube receives the light that passes through the objective lens
then redirects it to the eyepiece lens. The eyepiece has a magnifying power of
10x by its own, thus further magnifying the image from the objectives,
producing the final image will be seen by the viewer of the microscope.
Magnification
and resolution
Magnification
is simply the number of times an image's size is enlarged where size is
measured in the degrees of an angle formed by lines running from either end of
the image to the vertex at the viewer's eyes. By multiplying the power of the
objective lens with the power of the eyepiece lens, the total magnification of
the image seen can be calculated. Hence, there are four different magnifications:
4x
objective X 10x eyepiece = 40x magnification
10x
objective X 10x eyepiece = 100x magnification
40x
objective X 10x eyepiece = 400x magnification
100x
objective X 10x eyepiece = 1000x magnification
The
resolving power of a microscope determines the degree of detail that is
visible. Resolution is expressed as the minimum distance that can be resolved.
In other words, resolution is the ability to distinguish two separate points as
being separate and distinct. One of the factors that affect resolution is by
adjusting the condenser diaphragm. By closing the diaphragm, contrast of image
is increased but the resolution is decreased whereas by opening the diaphragm,
contrast is decreased but resolution is increased.
Therefore, both magnification and resolution
are equally essential. The image can be magnified through magnification.
However, a blurred image will be seen unless the resolution is excellent. On
the contrary, an excellent resolution can be obtained, but no detail will be
seen through the microscope without having good magnification.
Objective
1)
To learn to use a simple bright-field microscope correctly.
Material and reagents
Microscope
slide and cover-slip
Procedure
A) Preparation:
1. Sat on the
stool with knees under the bench and the microscope is moved so that both
eyepieces were looked through without straining to ensure own comfortability.
2. After the power
lead of the microscope is plugged in and the power is turned on, the microscope
light is turned on using the main on-off switch.
3. The light
intensity is adjusted using the brightness control. The normal adequate
position is 5.
4. The revolving
nosepiece is rotated to bring the 4x objective lens into the light path.
5. A clean slide
is being taken and a line is marked on the slide using a marker pen. The slide
is placed on the stage and it is secured by using the spring clip. Then, the
slide is moved into the light path using the coaxial stage control knobs.
6. Both eyepieces
were being looked through and were adjusted until a single circle of light is
seen. A note is made in the class manual of the setting on the
interpupillary distance scale for future reference.
7. The tube length
adjustment (diopter) ring on the right eyepiece is rotated to match the interpupillary distance setting obtained in 1.6.
8. The marker pen
mark is focused by adjusting the coarse and fine adjustment knobs. This is done
by using one's right eye only.
9. The left
eyepiece is focused using the tube lens adjustment (diopter) ring. This time is
done using left eye only. A note is again made in class manual of the diopter
ring setting for future reference. A perfect binocular vision should be
achieved by now.
B) Low power (10x)
objective viewing:
1. A marker pen
marked slide is replaced with a specimen slide.
2. A view of the
specimen is obtained by focusing using fine adjustment knob and by moving the
stage. The power is changed to 10x objective by watching from the side of the
microscope.
3. By placing an
object, such as an inoculating loop or pencil tip in the centre of the glass
above the light source, the condenser is focused. Then, the condenser light is
adjusted so that the object is in focus.
4. In order to
throw the object out of focus, the condenser is being lowered just
sufficiently.
5. To optimize
image definition and contrast, one eyepiece is removed, looked down the empty
tube and the condenser diaphragm is adjusted so that its edge can just be seen
inside the circle of light. A better image may be obtained by reducing the
aperture further for specimen of poor contrast. The eyepiece is replaced and
re-focused with fine adjustment.
C) High power
(40x) objective viewing:
1. The specimen is
focused with the 10x objective. The power is changed to 40x objective by
watching from the side of my microscope.
2. The condenser
is raised to within 1 cm of the slide.
3. The specimen is
focused using fine focus and light intensity is increased using the brightness
control if necessary.
4. The condenser
diaphragm is adjusted for optimum contrast as above.
D) Oil immersion
(100x) objective viewing:
1. The specimen is
focused with the 40x objective. The 100x objective is carefully selected by
watching from the side of your microscope. The objective is not allowed to
touch the slide.
2. The objective
is carefully turned to one side of the light path then one or two drops of oil
is placed onto the slide. The objective is rotated so that I is again in the
light path.
3. The condenser
is raised as close as possible to the slide.
4. The specimen is
focused using fine control and the light intensity is increased using the
brightness control if necessary.
5. The condenser
diaphragm is adjusted for optimum contrast as above.
E) After use:
1. The specimen slide
is removed and discarded into the appropriate discard container.
2. The light
brightness control is reset to its lowest setting.
3. The lowest
power objective is reset to the working position.
4. The oil from
the 100x objective is cleaned using lens tissue.
5. The microscope light
at the on-off switch and the power at the power point are turned off. The cord
is disconnected and it is wrapped carefully around the base of the microscope.
6. The cover is
replaced.
F) Care of the
microscope:
1. A microscope
must be carried carefully, holding it firmly by arm and supporting it at the
base. The instrument is kept upright.
2. The microscope
should never be placed close to the edge of the bench.
3. Do not tamper
with or remove any parts. Ask for help if the microscope does not seem to be
functioning properly.
4. The lenses are
not to be handled with our fingers. Specified lens tissues are used only for
cleaning lenses.
5. Liquids,
particularly acid and alcohol, are not allowed to come into contact with any
part of the microscope.
6. A cover slip is
always used when examining objects or organisms mounted in water or other
fluids.
7. The stage is
always being lowered before placing or removing a slide.
8. Before putting the microscope away, the
lowest power objective is always being put in working position and the cover is
replaced.
Results
Specimen observed:
Shigella dysenteriae
100x
magnification
400x
magnification
1000x magnification
Discussion
1. Microscope is positioned so
that we can observe specimen through the eyepieces
comfortably.
2. Specimen is observed start from
lowest power objective (10 x), followed by high
power (40 x) and oil immersion objective
(100 x) to obtain a clearer and
magnified image.
3. When we observe the specimen:
a. the light intensity is controlled
using illumination intensity knob to allow correct
amount of light enter
microscope.
b. The iris diaphragm is adjusted
to obtain higher resolution of the image.
c. The coarse-focus adjustment
knob is used, followed by fine-focus adjustment knob to
ensure image clear and more
focused.
4. The specimen that we observed
is Shigella dysenteriae.
a. Shape: rod
b. Size: tiny
c. Surface: shiny and smooth
d. Color: Red-stained
Conclusion
Light microscope is an instrument to observe microorganism which we
cannot see with our naked eyes. Each part of microscope has its own functions
and they can be adjusted to obtain best view of image onto specimen. The higher
the magnification, the clearer and the bigger the image.
1.2
Examination of cells
Introduction
In order to
examine the extremely minute microorganisms’ cells more precisely, we need to
study the cells using oil immersion objective after staining. The staining of
cells can distort of even destroy the cells.
There is another
method called wet mount method which enables us to examine the microorganisms’
cells in their natural state in a quick and easy way without using special
equipment. Throughout this method, we can observe the shapes and sizes of
living microorganisms and even determine the motility of the cells.
Objective
-
To provide an experience in the use of microscope.
- To learn the
proper technique of using wet mount method.
- To illustrate
the diversity of cells and microorganisms.
Materials
and reagents
Culture
Immersion oil
Lens tissue
A microscope slide
containing stained microorganisms
Inoculating loop
Bunsen burner
Slide and
coverslip
Procedure
A) Stained cells:
1. Microscope is
set up as described above and the slide is examined under the oil immersion
lens.
2. The shape and
size of the organisms and any structure that are visible are observed. The
observation is drawn out.
B) The wet mount:
1. A sterile
Pasteur pipette is used to aseptically transfer one drop of culture to the
centre of a glass slide.
2. A marker pen is
used to mark a coverslip so that it can help to focus on the microorganisms.
3. The coverslip
is turned downwards so that the marker pen mark faced down. Then one edge of
the coverslip is placed onto the slide and it is lowered gently so that the
drop of culture is being covered. The culture will spread between the coverslip
and the slide.
4. The slide is
placed on the microscope stage and 4x objective is used to focus on the
culture. Two or three groups of highly motile Escherichia coli.
5. The cell is
observed using the 10x and the 40x objectives. Some smaller moving objects may
be detected by a closed observation. They will be some of the larger types of
bacteria. Whatever is being seen is drawn out.
6. The cells are
observed using oil immersion lens. The condenser and diaphragm are adjusted.
Drawings are made and comment is given on anything of interest observed.
7. This procedure is
repeated with other culture.
Results
Bacteria observed:
Cocci, Bacilli, Spirilli
1000x
magnification
Specimen Observed: Escherichia coli
1000x
magnification
Discussion
1. Light will refract in different
ways under 100 x objective lens, causing the image to
look unfocused. However, oil
have similar refractive index as glass, so most of the
light can be refracted through
the oil into objective lens. This increases the resolution
of the image due to greater
light-gathering ability and reduces light scattering as light
passes from the specimen to the
objective lens.
2. Two microorganism are observed using oil immersion technique:
a. Cocci, bacilli, spirilli:
Shape: spherical (cocci), rod (bacilli), spiral (spirilli)
Size: tiny
Surface: smooth
Color: Red-stained
b. Escherichia coli
Shape: rod
Size: tiny
Surface: smooth
Color: red
Conclusion
By using oil immersion technique which has magnification of 1000 x, we
can clearly view and observe the morphology of living microorganisms while
still preserving good resolution.
References
http://botit.botany.wisc.edu/botany_130/microscope/Resolution.html
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