Introduction
Microorganisms
are organisms in microscopic size, which means they are less likely to be visible to the naked eye unless aided by a
microscope.Therefore, in order to study these amazing life forms, we need to
first understand and master the way to operate a microscope.The development of microscope started back in 14th
century and until today, there are various type of microscopes available for
researchers all around the world, ranging from a simple optical microscope to a
super-resolved fluorescence microscope. Despite the major development of
microscope, light microscope is still the best choice of microscope for viewing
microorganisms.
The parts
of light microscope
The figure below showed several parts of a typical light
microscope.
(Figure 1.1 Major Parts of a Light Microscope)
The light microscope uses white light to illuminate
the specimen. Thus, the illuminating process requires a strong source of white
light which is generated by the light bulb installed in the base of the
microscope. 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.
The diaphragm or also known as condenser diaphragm is
used to adjust the amount of light entering the condenser. You should be able
to find a lever on the side of the condenser and by turning it, you should be
able to adjust the size of the opening at the center of the diaphragm until
only the right amount of light enters the objective lenses. This is important
as objective lenses with high magnification (e.g. 100x objective) will need
intensive light unlike other objective lenses with lower magnifying power.
Insufficient light will cause the image to be dimmed and blurred.
Next will be the stage, where our slides of specimen
will be settled down. On the stage, there is a small size metal clip, known as
stage clips used to hold the slides in place. After setting the slide properly,
you may adjust the stage left and right so that the spot where the specimen is
located and be shifted into the path of light by using the mechanical stage
knob.
The course and fine focus knobs will be the ones
controlling the distance of the stage from the objective lenses. Initially, it
will be more convenient to rotate the course focus knob as this allows the
stage to be raised faster. You should adjust the stage until you can see the
figure of the specimen, then only enhance the quality of image by adjusting the
fine focus knob. While you are looking through the microscope, you should
always use the fine focusing knob only for adjustment purposes as adjusting by
using the course focus knob will most likely make you lose sight of the whole
image.
In order to produce a magnified primary image, the objective
lens focuses the light which passed through the specimen. A typical microscope
usually has four objectives with different magnifications (4x, 10x, 40x and
100x) attached to the revolving nosepiece. By rotating the revolving nosepiece,
you should be able to change the objectives to the one with desired
magnification power into the light path to increase or decrease the magnification.
Last but not least, the eyepiece is where the light
ray exits the microscope, you will be able to see the magnified image from the
eyepiece provided all the adjustment are accurate to produce a sharp image. The
eyepiece will collect the light from the objectives and refocus it again. A
typical microscope’s eyepiece has a magnification power of 10x by its own, thus
further magnifying the image from the objectives, producing the final image
will be seen by the users of the microscope.
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 observer's eye. 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. There are four magnifications in the
microscope used:
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 resolution
or resolving power of a microscope is defined as the the smallest
distance between two points on a specimen that can still be distinguished as
two separate entities. It is the most
important determinant of how well a microscope will perform and is determined
by the numerical aperture and light wavelength. 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 while by opening the diaphragm, contrast is decreased but resolution
is increased.
Both magnification and resolution
are equally important. The image can be magnified through magnification.
However, a blurred image will be seen unless the resolution is excellent. On
the other hand, an excellent resolution can be obtained, but no detail will be
seen through the microscope without having good magnification.
Objective
1.) To acquire and improve the
skills on using a simple bright-field microscope in proper way
2.) To understand the basic
concept of magnification and resolution of a microscope and its importance
3.) To gain the knowledge
about the way to take care a microscope correctly after use
Material and reagents
Microscope slide and
cover-slip
Procedure
Setting up:
1.) Sit on stool with both
knee under the bench and the microscope is moved so that both eyepieces can be
looked through without straining. Make sure the posture of sitting is
comfortable enough to carry on the experiment.
2.) The microscope light is
being turned on by using the main on-off switch after plugging in the power
lead of the microscope and turning on the power.
3.) The light intensity is
adjusted using the brightness control. Position 5 is normally adequate.
4.) The revolving nosepiece is
rotated in order to bring the 4x objective lens into the light path.
5.) A clean slide is taken. A
line is being marked on the slide by using a marker pen. The slide is then
placed on the stage by using the spring clip to secure it. The slide is moved
into the light path using the coaxial stage and control knobs.
6.) Both eyepieces is looked
through and they are being adjusted until a single circle of light can be seen.
A note in class manual of the setting on the interpupillary distance scale is
made 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 using the right eye
only.
9.) The left eyepiece is
focused by the tube lens adjustment (diopter) ring using the left eye only.
Again for future, a note in class manual of the diopter ring setting is made. A
perfect binocular vision would be obtained now.
Low power
(10x) objective viewing:
1.) A marker pen
marked slide is replaced with a specimen slide.
2.) The specimen
is focused using fine adjustment knob and the stage is moved in order to obtain
a view of the specimen. Watching from the side of the microscope, it changes to
10x objective.
3.) The condenser
is focused by placing an object, such as an inoculating loop or pencil tip, in
the centre of the glass above the light source. The condenser light is adjusted
so that the object is in focus.
4.) The condenser
is then lowered just sufficiently to throw the object out of the focus.
5.) To optimize
image definition and contrast, one eyepiece is removed, the empty tube is
looked down and the condenser diaphragm is adjusted so that its edge can just
be seen inside the circle of light. For specimen of poor contrast, a better
image may be obtained by reducing the aperture further. The eyepiece is replaced and is
re-focused using fine adjustment.
High
power (40x) objective viewing:
1.) The specimen is been focused on with
the 10x objective. The power of objective is changed to 40x by watching the
microscope from the side.
2.) The condenser is raised to within 1
cm of the slide.
3.) The specimen is been focused using
fine focus knob.
4.) The condenser diaphragm is adjusted
for optimum contrast as above.
Oil
immersion (100x) objective viewing:
1.) The specimen is focused on with the
40x objective. The objective with 100x power (oil immersion) is
selected while
watching the microscope from the side. The objective is prevented from touching
the slide.
2.) One or two drops of oil are placed onto the slide after the objective is
turned to one side of the light
path. The objective is rotated to be in the light
path.
3.) The condenser is raised as close as
to the slide as possible.
4.) The fine focus knob is adjusted to
focus on the specimen.
5.) The condenser diaphragm is adjusted
for optimum contrast as above.
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 100x objective is
cleaned using lens tissue.
5.) The light source of the microscope is
turned off at the on-off switch and the power is turned off at the power point.
The cord is disconnected and wrapped around the base of the microscope.
6.) The cover is replaced.
Care
of the microscope:
Microscopes in general are precision
pieces of equipment and should be well maintained, and cared for right from the
beginning. It is a delicate and expensive instrument. Proper care and
maintenance of microscope will ensure this equipment will provide years of
usage as well as pinpoint and accurate information.
1.) Hold the microscope firmly by the
base and the metal support arm when carrying the microscope. Avoid picking the
microscope up by the stage, as this can cause misalignment. Always keep the
instrument upright.
2.) The microscope should never be placed
near to the edge of the table. It should always be placed 6-inch away from the
edge of the table.
3.) If the microscope does not seem to be
functioning properly, do not try to tamper with, remove any parts or fix it.
Seek help from the lab assistant.
4.) Do not handle the lenses with your
fingers as microscope lenses can easily be scratched and should be treated with
great care. Use only specified lens tissues moistened with distilled water or
lens cleaning solution and rubbed gently using a circular motion for cleaning
lenses.
5.) Do not allow liquids, particularly
acid and alcohol, to come into contact with any part of the microscope.
7.) Always put the lowest power objective
to the working position and replace the cover before putting the microscope
away to avoid dust and debris accumulation.
1.2
Examination of cells
Introduction
Bacteria
are very minute organisms under the Kingdom Prokaryote. Since their sizes are
very minute and invisible to naked eye, so they can only be observed through a
very high power lens in a microscope. Bacteria can be pathogenic and therefore
aseptic technique has to be practiced to ensure the safety in workplace.
Bacteria
can be motile or immotile and can exist in any shape or sizes. However,
standard preparation method such as staining can distort or even destroy the
cells. Therefore, in order to study the bacteria in its natural state, we have
to use wet mount technique.
Objectives
1.) To
learn the proper technique in preparing wet mount
2.) To practise the accurate aseptic
technique
Materials
and Reagents
Lactobacillus
sp. culture
Immersion oil
Lens tissue
Inoculating loop
Bunsen burner
Slide and coverslip
Procedure
1.) The laboratory table is sterilized
with lysol and alcohol solution.
2.) The Bunsen burner is turned on.
3.) The inoculating loop is then
sterilized by placing it at the hottest part of the flame until it is glowing
red.
4.) The cap of bottle which contains the cell
culture is sterilized by swirling it three times back and forth at the flame.
5.) The cap of bottle is opened with one
hand near the flame.
6.) The mouth of the bottle is then
sterilized by swirling it three times back and forth at the flame again.
7.)
By
using the sterilized inoculating loop, the cell culture is picked up and
transferred onto a microscope slide.
8.) The inoculating loop and the mouth
of the bottle are then sterilized again.
9.) The one edge of the coverslip is
placed onto the slide and lowered gently so that the drop of culture is covered. The culture is spread between the coverslip and the slide.
10) The cells are observed using oil
immersion lens. The condenser and diaphragm are adjusted. Anything interesting is drawn.
Result
The sample specimen of bacteria types: Lactobacillus sp.
Colour: Transparent white
Shape:
Rod-shaped
Lactobacillus
sp. under microscope at 100x objective
viewing with oil immersion
Discussion
Lactobacillus
sp. is a genus of Gram positive rod-shaped
bacteria (bacillus). In this 100x objective viewing micrograph, the bacteria
appeared to be translucent because it was not stained. Lactobacillus sp. is well-known because of its major use in food
and beverage industry. These bacteria are able to metabolize glucose and
excrete lactic acid as its by-product. This genus comprises more than 180
species and some of the most common species are Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus brevis. In human, this
bacterium can be found in mucosal membrane such as gastrointestinal tract.
There it serves as a beneficial flora that inhibits the growth of harmful
bacteria by creating an acidic condition in the membrane. In food industry, Lactobacillus sp. is often used not only
in the production of fermented dairy product such as yoghurt and cheese, they
are also used in non-dairy product such as sauerkraut. In some traditional food
like kumiss, Lactobacillus has known
to suppress the pathogenic bacteria in the foodstuff. Besides in food industry,
Lactobacillus sp. is also used in
therapeutic purposes such as treating diarrhea in children. Some strains are
even shown to have anti-tumor and anti-cancer in experiment.
Conclusion
With a very high power objective lens
(100x) under oil immersion, the minute bacteria can be seen clearly. With wet
mount observer can see the bacteria in its natural state (unstained and
untreated). Proper aseptic technique is important because some of the
pathogenic agents can cause serious illness or death if they are not being
handled properly.
Reference:
2) Obtain from http://ibg102.wordpress.com/2013/04/05/lab-1-principles-and-use-of-microscope/
on 1 November 2014
3) Obtain from http://botit.botany.wisc.edu/botany_130/microscope/Resolution.html
4.11 on 4 November 2014
4) Obtain from http://zeiss-campus.magnet.fsu.edu/articles/basics/resolution.html on 4 November 2014
5) Obtain from http://www.microscopyu.com/articles/formulas/formulasresolution.html on 4 November 2014
6) Obtain from http://www.ehow.com/about_6611572_relationship-between-magnification-resolution.html on 4 November 2014
7) Obtain from http://www.geog.ucl.ac.uk/about-the-department/support-services/laboratory/light-microscopy/basic-rules-for-caring-for-microscopes on 5 November 2014
8) Obtain from http://www2.hendrix.edu/biology/CellWeb/Techniques/usingscopes.html on 5 November 2014
9) Obtain from http://www.microscope-shop.com.au/microscope_maintenance.html on 5 November 2014
10) Obtain from http://www.en.m.wikipedia.org/wiki/Lactobacillus on 2 November 2014
11) Obtain from http://www.nlm.nih.gov/medlineplus/druginfo/natural/790.html
12) Obtain from https://microbewiki.kenyon.edu/index.php/Lactobacillus on 2 November 2014
This is amazing. Thank you so much for freely sharing all your hard work! We are going to definitely use a lot of this!
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