Professional Documents
Culture Documents
Illustrated Laboratory Activity 1 Observing Cells Using Microscope
Illustrated Laboratory Activity 1 Observing Cells Using Microscope
and other unneeded objects away from your work area. A clear work area promotes efficiency
and results in fewer accidents.
Electric Cord
Microscopes have been known to tumble off of tabletops when students have entangled a foot
in a dangling electric cord. Don’t let the light cord on your microscope dangle in such a way as
to hazard foot entanglement.
Lens Care
At the beginning of each laboratory period check the lenses to make sure they are clean. At the
end of each lab session be sure to wipe any immersion oil off the immersion lens if it has been
used.
Dust Protection
In most laboratories dustcovers are used to protect the instruments during storage. If one is
available, place it over the microscope at the end of the period. Draw and Label the parts of a
binocular microscope.
MEASURING MAGNIFICATION
A compound microscope has two sets of lenses and uses light as the source of illumination. The
light source is called an illuminator and passes light through a condenser and through the
specimen. Reflected light from the specimen is detected by the objective. The objective is
designed to redirect the light waves, resulting in the magnification of the specimen.
There are typically four objectives, each having a different magnification. These are 4×, 10×,
40×, and 100×. The number indicates by how many times the original size of a specimen is
magnified, so the 4× objective magnifies the specimen four times the specimen size. The eyepiece
of the microscope is called the ocular eyepiece and it, too, has a lens—called an ocular lens—that
has a magnification of 10×.
You determine the magnification used to observe a specimen under a microscope by multiplying
the magnification of the objective by the magnification of the ocular lens. Suppose you use the
4× objective to view a specimen. The image you see through the ocular is 40× because the
magnification of the object is multiplied by the magnification of the ocular lens, which is 10×.
Many microscopes have several objectives connected to a revolving nosepiece above the stage.
You can change the objective by rotating the nosepiece until the objective that you want to use
is in line with the body of the microscope. You’ll find the magnification marked on the objective.
Sometimes the mark is color-coded and other times the magnification is etched into the side of
the objective.
RESOLUTION
The area that you see through the ocular eyepiece is called the field of view. Depending on the
total magnification and the size of the specimen, sometimes the entire field of view is filled with
the image of the specimen. Other times, only a portion of the field of view contains the image of
the specimen. You probably noticed that the specimen becomes blurry as you increase
magnification. Here’s what happens. The size of the field of view decreases as magnification
increases, resulting in your seeing a smaller area of the specimen. However, the resolution of the
image remains unchanged, therefore you must adjust the fine focus knob to bring the image into
focus again. Resolution is the ability of the lens to distinguish fine detail of the specimen and is
determined by the wavelength of light from the illuminator.
CONTRAST
The image of a specimen must contrast with other objects in the field of view or with parts of the
specimen itself to be visible in different degrees of brightness. Suppose the specimen was a thin
tissue layer of epidermis. The tissue must be a different color than the field of view, otherwise
the tissue and field of view blend, making it impossible to differentiate between the two. That is,
the tissue and the field of view must contrast. The illuminator shines white light onto the
specimen. White light contains all the light waves in the visible spectrum. The specimen absorbs
some of the light waves and reflects other light waves, giving the appearance of some color other
than white. Light waves that are reflected by the specimen are measured by the refractive index.
The refractive index specifies the amount of light waves that is reflected by an object. There is a
low contrast between a specimen and the field of view if they have nearly the same refractive
index. The further these refractive indexes are from each other, the greater the contrast between
the specimen and the field of view.
Unfortunately, refractive indexes of the specimen and the field of view are fixed. However, you
can tweak the refractive index of the specimen by using a stain. The stain adheres to all or part
of the specimen, absorbing additional light waves and increasing the difference between the
refractive indexes of the specimen and the field of view. This results in an increase in the contrast
between the specimen and the field of view.
Oil Immersion
A challenge facing microbiologists is how to maintain good resolution at magnifications of 100×
and greater. In order to maintain good resolution, the lens must be small and sufficient light must
be reflected from both the specimen and the stain used on the specimen. The problem is that too
much light is lost; air between the slide and the objective prevents some light waves from passing
to the objective, causing the fuzzy appearance of the specimen in the ocular eyepiece.
The solution is to immerse the specimen in oil. The oil takes the place of air and, since oil has the
same refractive index as glass, the oil becomes part of the optics of the microscope. Light that is
usually lost because of the air is no longer lost. The result is good resolution under high
magnification.
TYPES OF LIGHT COMPOUND MICROSCOPES
Bright-Field Microscope
The bright-field microscope is the most commonly used microscope and
consists of two lenses. These are the ocular eyepiece and the objective.
Light coming from the illuminator passes through the specimen. The
specimen absorbs some light waves and passes along other light waves
into the lens of the microscope, causing a contrast between the
specimen and other objects in the field of view. Specimens that have
pigments contrast with objects in the field of view and can be seen by
using the bright-field microscope. Specimens with few or no pigments
have a low contrast and cannot be seen with the bright-field
microscope. Some bacteria have low contrast.
Phase-Contrast Microscope
The phase-contrast microscope bends light that passes through the
specimen so that it contrasts with the surrounding medium. Bending
the light is called moving the light out of phase. Since the phase-
contrast microscope compensates for the refractive properties of the
specimen, you don’t need to stain the specimen to enhance the contrast
of the specimen with the field of view. This microscope is ideal for
observing living microorganisms that are prepared in wet mounted
slides so you can study a living microorganism.
Fluorescent Microscope
Fluorescent microscopy uses ultraviolet light to illuminate
specimens. Some organism fluoresce naturally, that is, give off
light of a certain color when exposed to the light of different color.
Organisms that don’t fluoresce naturally can be stained with
fluorochrome dyes. When these organisms are placed under a
fluorescent microscope with an ultraviolet light, they appear very
bright in front of a dark background.
Observation:
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
Laboratory Guide Questions:
From the lesson above, answer the following as best as you can. Please write your answers in
your Learning portfolio.
1. In the laboratory, you experience some difficulties in using the microscope. Describe the
steps you would take to correct the following problems:
a. Inability to bring the specimen into sharp focus.
b. Insufficient light while viewing the specimen.
c. Debris in the microscopic field.
2. For what purpose would you adjust each of the following microscope components during
a microscopy exercise?
a. Iris diaphragm:
b. Coarse-adjustment knob:
c. Fine-adjustment knob:
d. Condenser:
e. Mechanical stage control: