Basic Microbiology Microscopy Discussion Questions I need someone to answer these questions. I will attach the files. If you have any questions let me know

Basic Microbiology Microscopy Discussion Questions I need someone to answer these questions. I will attach the files. If you have any questions let me know. University of Bridgeport
Katsuhiro Kita, Instructor of Record
Patricia Clark, Laboratory Instructor
Exercise 2: Basic Microbiology Microscopy
Introduction
To examine microorganisms the use of specific equipment such as compound microscopes are
used to bring the specimens into view. As we cover the significant historical keystones of microbiology
perhaps the most important invention was the microscope. Robert Hooke’s illustrations of
microorganisms using optical (light) microscopy1 had a significant impact on the scientific community.
Antonie van Leeuwenhoek, referred as the first person to observe “single” cells, further increased the
magnification of microscopes during the 1670s. The invention of the microscope led to a golden age of
microscopic discovery and helped disprove the theory of spontaneous generation2, as detailed in Chapter
1 of the lecture textbook.
Traditionally, light microscopy in microbiology is used for viewing microbes (bacteria, fungi,
viruses). Learning proper usage of a compound microscope is crucial for viewing specimens. However,
most microbes such as bacteria and viruses possess morphologies too tiny even for most modern
compound microscopes to view. Therefore, learning different staining techniques: Gram staining, spore
staining, acid/fast stain is another integral step for distinguishing different groups of bacteria. Electron
microscopy is necessary for detailing the cellular structures (such as pili).
Compound Microscope
All optical microscopes consist of several common parts as shown in Figure 2.1. Take note of each
component’s function as they are essential to the microscope.
Lenses
A light microscope has objective lenses (4x, 10x, 40x and sometimes 100x) attached to the nosepiece (or
turret) which is attached to the body tube (long tube) which is attached to the ocular (or eyepiece) lens
(small lens on top of body tube). Be careful the lens does not come into contact with your hand or
specimen. One scratch can permanently disable the lens or permanently damage a preserved specimen
slide.
Focus Knobs
The focus knobs can be found on either the body tube, or side of the body under the stage (inverted).
Typically, there are two knobs: 1) coarse adjustment (outer/larger) and 2) fine-adjustment
(inner/smaller). If for some reason the knobs appear loose, ask the instructor or TA for assistance.
Stage
A rectangular metal platform with the hole in the center to allow light to pass from the light source to lens
above (up-right microscopes) the stage. The simplest stage has two clips to hold a slide glass. There is
Robert Hooke’s “Micrographia…” was published in 1665 and is widely regarded as inspiring a significant interest to the field
of microscopy.
2 Spontaneous generation: an obsolete body of thought proposing all living organisms simply came into being without any
descent from other organisms.
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also a mechanical stage that can precisely move the specimen vertically or horizontally by adjusting
small knobs on the grid.
Light Source (Illumination)
The light source is located at the base of the microscope. Usually a tungsten lamp may be found at the
base; however, less sophisticated microscopes may use a reversible mirror to correct light. If the mirror is
the case, the flat side is used for artificial light and the concave side is used to correct sunlight (or any dim
light; this side is more efficient to correct light).
Condenser
The condenser is found right underneath the
stage. Since light becomes scattered from its
original trajectory, the condenser plays an
important role to collect and concentrate light
toward the specimen. The condenser also has
another component, an iris diaphragm. This
diaphragm can narrow the diameter of the
light path – it does not only simply regulate
the light, but also helps increase the contrast
of images.
Magnification
You can find the magnification of each
objective lens on the side of lenses (4X, 10X
etc.). It is essential to learn how to calculate
the total magnification. The following
calculation can be found below:
Total magnification = magnification of
objective lens × magnification of ocular (eye
piece)
Note: Although most microscopes are
equipped with 10X ocular lenses, some could
have a 20X.
There is a distinction between magnification
and resolution. Magnification is the degree
of virtual enlargement of a sample, but
resolution is the ability to distinguish two
objects (see below).
Figure 2.1 Component names of compound microscope.
Images You See Are Upside Down
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Unlike low magnification apparatuses such as magnifying glasses, optical microscopes utilize the twolens system. Light collected through an objective lens will be focused again (image is inverted), forming
the intermediate image plane (or sometimes called as “real image”) (Figure 2.2). This means that the
object in the field of view will move to
right when you move the stage to left, and
the object will move to the bottom when
you move the stage toward top.
Resolution (Resolving Power)
Resolution (optical) is the ability to distinguish two objects. It indicates the minimum distance between
objects distinguished by an optical system to be used (Figure 2.3). Resolution of microscopes depends on
numerical aperture (diameter of objective lenses and focal length-specific value; each lens has the specific
numerical aperture) of each objective lens. Usually, numerical aperture may go up upon the
magnification, but you need to confirm it. When numerical aperture is above certain value, some media is
necessary to fill between the specimen and the objective lens (water, immersion oil etc.) to efficiently
collect light. This is because of the other specific value called as refractive index (the degree of light bent
between the interface of two different materials; for example, between water and glass), which is critical
for the angle of light collected by objective lenses.
Resolution also change upon the wavelength of illumination. As you could imagine from the
simplest equation, shorter wavelength will give you better resolution when the numerical aperture of the
lens is the same (Electron microscopy utilize X-ray, which wavelength is much shorter than light used in
optical microscopy. Therefore, electron microscopy can resolve very small structures, even large protein
complexes in a cell.).
Figure 2.3 Showing the resolution of two spots; A = resolved, B & C; not resolved (J.C.
Waters (2009) J. Cell Biol., 185, 1135)
Laboratory Exercise
Tips:
1. When you switch to high magnification objective
lenses (40x and 100x), take extra caution not to hit
the specimen with the tip of objective lenses. This
can happen during the procedure 6. If this procedure
Figure 2.2 Concept of image inversion
used in compound microscopes.
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is not carried out with care, the lens and the glass slide can crack. When you switch the
objective lens to the higher magnification lenses, you may move down the stage if necessary.
Carefully monitor the distance between the objective lens and the specimen when you turn the
turret.
2. Image brightness is inversely proportional to the magnification. Practically, the eye pieces are the
same so that your field of view gets dimmer when you select higher magnification of objective
lenses. You may adjust the light intensity (and condenser) accordingly when you change
objective lenses.
3. If you use an oil immersion objective lens (magnification will be 60X or 100X), you need to have
immersion oil between the slide and the lens. Do not use the 100x lens without oil unless
explicitly instructed to do so.
Procedure A: Setting a Microscope
1. Before setting up a microscope, make sure there is no observable defect.
2. Plug the electric cord into an electric outlet on the bench. Doublecheck the cord is not loose. Turn
on the power to check that the illumination works. If not, tell your lab instructor (the bulbs
eventually burnout and need to be replaced).
3. Make sure that the stage is set to the lowest position when you mount the slide. Place the slide on
the center of the stage and hold it with the stage clips or the mechanical stage’s holder.
4. Move the turret (nosepiece) to select a low power (magnification) of an objective lens (such as
4X or 10X). Raise the stage up all the way (You will reach the position where you cannot raise
the stage anymore.).
5. Slowly use the coarse-adjustment knob while looking the sample through the eye piece. When
you see the shadow of samples (close to the focal plane), switch to the fine-adjustment knob for
fine focusing. Developing the skill to bring your sample into view takes time. If you are having
trouble bringing your sample into view, ask
your TA or instructor to assist.
6. After finding the sample you may change the
objective lens to a higher magnification. Most
optical microscopes are parfocal, meaning that
microscopes would keep approximately the
same focal plane when one objective lens is in
focus. For example, if the sample is in focus on
the 4x when you switch to a higher
magnification there should be little need to refocus the image.
7. It is necessary to observe at least several areas
(field of view) of your specimen to ensure that
the results are accurate (methods such as gram
staining require this procedure as specific
densities of bacterial cells can cause
misinterpretation of the result).
Figure 2.4 working distance and objective lenses.
Procedure B: Observation of Fixed Specimens
1. For the first exercise of microscopic observation, we will use fixed specimens to observe samples.
2. Obtain slides of microorganisms from the lab instructor. Specimens are heat-fixed onto the slides,
a process that inactivates the bacteria.
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3. Follow the protocol in procedure B. The post-lab questions include recording (sketch) of
microorganisms.
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Post-lab Questions: Exercise 2
1. Summarize the purpose, or provide the definition, of following microscope components.
a. Coarse- and fine-adjustment knobs:
b. Condenser:
c. Magnification:
d. Resolution:
2. Why is it important to carefully watch the distance between an objective lens and the specimen?
How can the specimen get damaged by quickly changing the objective lens too quickly?
3. Provide the calculation of how to determine the total magnification.
4. List how to properly prepare your microscope for storage (i.e. is the stage left in the uppermost
level or lower most, which objective lens should be selected?).
5. Sketch at least one cocci and one rod bacteria below with low and high power (magnification).
Your sketches should state the following information: name of organism, cell shape and
magnification.

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