Exercise #1: Use of the Microscope: Introduction to Cell

Lab Format: The microscopy lab consists of two major sections. In the first part,
your instructor will work with the class as a whole, to explain the operation of the
compound light microscope. It will be explained how it differs from other types of
microscopes. Each student will have his or her own microscope to use for this
portion of the lab. It is imperative that each student understands and masters the
use of the microscope in order to complete the observation of the cells and
organisms in the lab exercise and to prevent damage to the scopes. In the second
part of the lab, you put your knowledge of the microscope to work in order to
complete the remainder of the lab exercise. The lab is set up in “stations”, each of
which has a specific cell type for you to observe (animal cells, plant cells, pond
water organisms, prokaryotes and plant-like protists). There may be one or more
examples of cells or organisms to observe at each station. It is crucial that each
student complete all of the objectives as stated. Be sure to spend as much time as
Structure and Variation
PART I (Week 1)

Objectives: At the conclusion of this lab, you should be able to:

 Use the compound microscope efficiently
 Identify and define the functions of the parts of compound microscope
using proper scientific terminology
 Define key terms of microscopy: magnification, resolution, working
distance, contrast, brightness of field, diameter of field, depth of focus,
parfocal.
 Understand the limitations of the compound light microscope as
compared to the scanning and transmission electron microscopes.
 Estimate the size of an object on a microscope slide using mathematical
formulae.
 Make a wet mount slide using proper technique and learn the technique of
“wicking.”
 Distinguish between and identify via specific characteristics….
o the differences between eukaryotic and prokaryotic cells
o the differences between animal and plant cells

I. Introduction to Microscopy:
An appropriate way to begin the study of biology is to acquire a good working
knowledge of microscopy. Microscopes are among the most basic of tools used by
all biologists. They allow us to see things that would be impossible to see with the
naked eye. Why? ……….
 Because the unaided eye cannot detect anything smaller than 0.1 - 0.2
mm in diameter, many cells, tissues, and small organisms cannot be seen
without the aid of a dissecting or compound light microscope.
 Because the electron microscope which allows us to see things down to 0.1
nm provides a means of observing viruses, nucleic acids, and proteins
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which would not be visible with the compound or dissecting scopes.
 Because we need to increase contrast to visualize components of the cell, we
can fix, stain, or otherwise treat materials for observation by microscopy and
this allows us to see certain “micro-sized" features in great detail.

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Magnification vs. Resolving Power:

Magnification is defined as image enlargement. Resolution or resolving

power is the shortest distance that two objects may be to each other and still be
seen as two objects.

Microscope quality depends upon the ability to resolve, not magnify, objects.
Imagine taking a picture with a camera of a descriptive, artistic poster from
across a large room. On the photograph, you might not be able to see either the
details of the artwork or the description on the poster. You could enlarge the
photograph, but you would not increase your ability to see the details on the
poster. It would still be blurry. So, the same is true with microscopes. Without
good resolving power in a microscope, you can enlarge and enlarge but you will
never be able to see the fine detail. The image will be larger but not more
distinct. This is called empty magnification.
Resolution depends in part upon the angle formed between the objective lens
and the light rays refracted from the specimen. The larger the angle through
which light may be gathered from the specimen into the objective lens, the
better the resolution. This means a higher quality lens system permits more of
the edges of the lens to be used, providing more information and hence, better
resolution.
The nature of the medium between the specimen and the objective lens also
influences the extent light rays refracted by the specimen are collected by the
objective lens. Air occupies the space between the objective lens and the cover
slip on the slide. Rays that emerge from the cover slip refract and may not fall
upon the objective lens. However, if the air space is replaced with oil, the rays
emerging from the cover slip (glass) will not be bent. More light rays now are
collected by the objective lens. You will need to use oil and the oil immersion
objective lens in order to observe bacterial cells later in the lab.
Finally, resolution depends upon the wavelength of light illuminating the
specimen, the shorter the wavelength, the better the resolution. Blue light (400
nm) provides better resolution than red light (700 nm). The microscopes in your
lab may therefore be equipped with blue tinted glass or have a holder in which
various filters (colored glass) may be placed.

Table I: Comparison of Various Microscopes to the human eye

Resolution Approximately “X”
Unaided eye .2 mm or 200 m 1X
Dissecting scope 3-5 m 35X
Compound Light scope 0.2 m 1000X
Scanning EM 5-10 nm 150,000X
Transmission EM 0.1 nm 300,000X

Stereoscopic Dissecting Microscope

The stereoscopic dissecting microscope (Fig. 1) presents a three-dimensional
visualization of specimens which are too large or opaque to be viewed with the
compound microscope. They utilize a 1ight source positioned so that light may
reflect off the surface of the specimen into the lens system. These microscopes
also have what is termed a large working distance (distance between objective
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and the stage) to allow for the visualization of large or whole mount specimens.
The total magnification obtainable is greater than the naked eye but far less than
the compound microscope.

Figure 1. The Dissecting Microscope .

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Compound light microscope

A compound light microscope (Fig. 2) is one in which light is transmitted

through a specimen from below and then through a system of two lenses, an
objective lens and an ocular lens, thus the term compound. It requires that the
specimen to be observed be small, thin and relatively transparent in order
to allow the light to be transmitted through it and make the image visible. It can
be used to view whole and living specimens if they are small enough. Areas of
the specimen, which are clear will allow the light to pass through while areas
which are opaque prevent the light rays from passing through the object.
The contrast provided thus allows us to view the specimen. Often, various stains
or dyes may be applied to the specimen. These stains or dyes bind to certain
structures and therefore provide better contrast between organelles or structures
within the specimen. The compound light microscope provides an image that is
mainly two dimensional. It will be the compound light microscope, which you
use most often in this course.

Arm

Base

Figure 2: The Compound Microscope

Image of the CX21 Olympus microscope (http://www.olympusamerica.com)

Using the Compound Light Microscope:

You should familiarize yourself with the proper handling of the scope. Your
instructor will explain the components and operation of the compound light
microscope. Be sure that you observe the following rules each time you use the
microscope this semester.
 Carry the microscope with two hands: one under the “base” and one holding the
“arm” (Fig. 2).
 Be certain that you start and end your use of the microscope with the lowest
power objective locked in position.
 When cleaning the lenses use only “LENS PAPER” supplied at your lab bench.
 When observing a wet mount slide or using stains, be sure that you do not tilt
your microscope.
 Do not stain slides while they are on the stage of the microscope. Apply
stains while your slide is on your lab bench. Remove excess stain prior to
placing the slide on the stage of your scope.
 Be sure that you keep the stage dry and clean. Dirt and moisture can cause rust
and corrosion.

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 Never remove parts of the microscope or make adjustments to screws, etc.
 Turn off the light source prior to unplugging your microscope.
 Wrap the cord around the scope just above the base, never over the
mechanical stage or across the objective lenses.
 Replace the dust cover (if available) on your scope prior to replacing it in the
storage cabinet.
 Notify your instructor of any malfunctions.
At this time, you may obtain your microscope from the green cabinet at
the side of the lab if they are not already at your bench. Be sure to
carry it properly.
Identification of microscope components:

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 Although many of you have used a microscope before, your instructor will
lead you through the identification of the parts of the microscope and their
proper use and/or function. This is two reasons:

 To assure that you properly use the scope in order to view all of the
various cells and organisms in today’s lab.
 To assure that this expensive piece of equipment does not incur damage through
improper use.

Beginning at the top of your microscope, locate the parts shown below
as your instructor points them out and demonstrates their use. Then
label them in Figure 3 on the next page.

Table II: Parts of the Microscope and Their Function

Part Function Additional Notes
Ocular (eye -Magnifies the inverted image -Mounted on the body
piece) produced by the objective tube
lens before it reaches the eye. -Magnifying power
should be stamped on
the barrel, e.g. 10x.
-Not firmly attached
and can fall out if
microscope is
inverted.
Body Tube -By holding the oculars on one
end and the nosepiece on the
other, light rays travel
through it from the objective
lens
to the oculars.
Objective lens -By rotating the nosepiece -Held in place on a
objective lenses of various revolving nosepiece.
magnifying powers are -Generally, 3 or 4
brought into line with the objective lenses found
illuminated specimen & on the nosepiece:
ocular lens scanning objective,
-Objective lenses produce an low-power,
inverted high power and oil
& magnified image. immersion
Arm -Provides a carrying handle
and attaches the nosepiece
and ocular lenses to the
lower parts of the scope.
Mechanical -Platform upon which slide is -Slide release lever
Stage & Stage mounted must be pulled back in
Manipulator order to mount the
Knobs slide in the “L-shaped”
holder.
-Release lever to hold
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 the slide in place
within the holder
-Two knobs below the
stage: one allows
slide to move forward
and backward the
other to move slide
right and
left
Condenser -Lens mounted under the -Moved vertically by
stage which focuses and turning knob under
condenses the light entering stage.
the stage and through the
specimen

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Iris diaphragm -Controls light intensity by -Located at the bottom
controlling amount of light of the condenser
passing through the -Flat lever is used to
specimen. adjust the amount of
-controls contrast of objects light as objectives
are changed
Coarse -Moves stage or -The larger knob
adjustment objective lenses closest to the arm of
knob vertically to focus lens the microscope
system on specimen. -Should be used only
–Does this by increasing or with low power
decreasing the distance objectives
between the lens and the
cover slip of the slide.
Fine adjustment -Responsible for finer focusing -The smaller knob
knob extending outward
from the coarse
adjustment knob.
Base -Contains the light source for -May have a rheostat
the scope on the base which
controls the
intensity of the light
source.

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Figure 3: Parts of the Compound Light Microscope

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Magnification

Total magnification in the compound light microscope is

calculated by multiplying the magnification of the ocular by the
magnification of the objective you are using.

Observe the ocular lens of your microscope. What is the power of

magnification stamped on the barrel of the lens? Record this value in the table
below.

In the table below, record the magnifying power of each objective lens on your
microscope, starting with the shortest objective known as the scanning
objective. By rotating the nose-piece you can also record the magnification of the
second shortest objective referred to as the low power objective. Continue with
the next shortest objective, which is the high-power objective and the longest
of the objectives referred to as the oil immersion objective.

Complete the table below by calculating the total magnification of the

microscope when using each of the objective lenses.

Table III: Total magnification of the compound microscope when using

each objective lens

Objective Objective Ocular Total

Magnificat Magnificat Magnification
ion ion
scanning
low
high
oil immersion

Contrast

Contrast is the difference in intensity (and/or color) of light transmitted by

different parts of the specimen or by the specimen and surrounding medium.
Necessary contrast can be obtained by doing one or more of the following:
 Using stains that selectively color the specimen or specific parts of the
specimen.
 By adjusting the rheostat (if available) to control the amount of light
emitted by the light source in the base.
 By adjusting the amount of light that penetrates the specimen. Do this by
adjusting the iris diaphragm on the condenser lens. Locate the lever
controlling the iris diaphragm, and gently slide it from left to right. Look
through the opening in the stage as you do this. What happens to the light
rays entering the condenser?

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Operation of the Compound Light Microscope:

Each time you begin using your microscope the following sequence of events
should be followed in order to use the microscope effectively. You will be using a
prepared slide to learn the proper use of the scope. Your instructor will guide you
through and help you should you have any trouble along the way.

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1. Clean the ocular and objective lenses with lens cleaning solution and lens
tissue only, not Kim-wipes or a paper towel.
2. Turn the light source on and adjust the rheostat (if available) about halfway.
3. Fully open the iris diaphragm. Raise the sub-stage condenser to its topmost
position using the sub- stage condenser height adjustment knob and then
just lower it a small amount so that it is just below the top of the stage in
the opening.
4. Adjust the inter-pupillary distance of the oculars. Do this while looking into
the oculars and adjusting the distance between them until you see only one
distinct circle of light. Be sure you have both eyes open. You only need to do
this once.
5. If your scope allows, make adjustments for visual acuity by doing the
following: Using the fine adjustment knob, adjust the focus by looking through
the ocular with your right eye only. Finally, close your right eye and use the
adjustment on the left ocular lens to bring the specimen into focus for your
left eye. These steps compensate for any differences in visual acuity between
your eyes.
6. Remove the slide labeled “letter e” from your slide box. With the scanning
power objective (4X) in place, put the prepared slide of the "e" on securely in
the slide holder of the mechanical stage oriented in the same way as it would
appear in a newspaper. Make sure you have the nosepiece raised when you
are putting a slide on the stage.
7. Watch the objective lens from the side as you lower the objective using the
coarse adjustment knob. Center the specimen in the field of view by
moving the slide with the stage manipulator knobs.
8. While looking though the oculars, use the coarse adjustment knob to slowly
raise the nosepiece until the specimen comes into view. Then use the fine
adjustment knob to bring the specimen into sharp focus.

In the box below draw the letter “e” as it actually appears on the stage when
viewed with the naked eye. Now draw the image of what you see through the
microscope.

Actual View on Image Seen on Microscope (4X objective =40X total

Slide magnification)

Does the letter image appear in the same orientation as the letter when
viewed with the naked eye as mounted on the stage?
Why or Why not?

Move the slide to the left. In which direction does the letter image

appear to move? Move the slide towards you. In which direction

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 does the letter image appear to move?

Practice moving the slide, until you are comfortable with the manipulations
necessary to achieve the desired directional movements of the image.

9. Center the specimen in the field of view and switch to the low power objective
(10X). It is important to always center the specimen before you switch to a
different objective; this assures that your specimen will be in the field of view
of the higher power objective.

Does the image of the letter “e” still appear the same as when you viewed it
with the 4X objective?
If not, how does it differ?

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 Draw the image as it appears using the 10X objective in the box below.

Image using 10X

objective= 100X

Now, switch to the high-power objective (40X). Note that these lenses are
Parfocal, i.e., once
focused on low power, the microscope will remain basically focused for all
powers. Therefore, you will need only to adjust the light intensity and focus
with the fine adjustment. Adjust the focus using the fine focus adjustment
knob only! If you use the coarse adjustment knob you may hit the slide and
damage both the slide and the lens. You may need to increase the amount of
light entering the field of view by readjusting the iris diaphragm. (Remember
contrast.)

Has the image of the letter “e” changed?

If so, how?

In the box below, draw what you see in your field of view using the

40X objective.

Image using 40X

objective = 400X

How has brightness of field, (i.e. how much light you see) been affected?

Did you have to adjust the light using the iris diaphragm?
Did you have to increase or decrease the light as magnification increases?

10. When you have finished looking at a slide with the high-power objective
raise the objective away from the slide and then remove the slide. You should
never remove a slide from the microscope stage while the high-power
objective is in place. Set the slide aside for use later.

REMEMBER: ALWAYS MAKE PRELIMINARY OBSERVATIONS UNDER LOW

POWER AND SWITCH TO A HIGHER POWER IF FURTHER DETAILS ARE
TO BE DISCERNED.
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Measurement of specimen under the microscope

It is frequently necessary for a biologist to know the approximate dimensions of a

specimen under the microscope. First, we must determine how to calculate the
diameter of the field of view. Then we can use this information to estimate the
size of a particular specimen.

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 Calculating the diameter of field of view in both millimeters and
micrometers.
An important relationship exists between magnification and diameter of the field
of view. When the magnification is doubled, the field of view is cut in half. Place a
clear plastic millimeter ruler across the stage so that the edge is along the
diameter (center) of the 4X field and one of the vertical ruler markings is lined up
along the edge of the field. Be sure you are using the millimeter side of the ruler.
Take a reading.

Diameter of field using the 4X objective = mm (mm stands for

millimeters)

Generally, scientists refer to the diameter of field in terms of micrometers ( m)

rather than millimeters (mm). To convert your answer from millimeters to
micrometers you must know that there are 1000 micrometers in every 1
millimeter. To make this conversion you will need to move the decimal place for
your answer in millimeters, three places to the right. Now convert your answer
above from mm to m.

Diameter of field using the 4X objective =

m. Repeat the procedure after changing to the 10X

objective.
Diameter of field using the 10X objective = mm, = m

Now, use the 40X objective. Can you measure the diameter of the 40X field as
easily as you did in the l0X field? _______ Why or why not? ______________

__________________

To calculate the diameter of the 40X field of view, you need to use the following
equation.

Diameter of field
Magnification for for higher
lower power objective
power equals Objective
Diameter of field for lower power objective Objective
Magnification for higher power

Using the known distance measurement for the diameter of field of the 10X
objective, and known magnifications for either the 10X objective, you can
calculate the diameter of the field of the 40X objective.

Example: If your field of view for the 10X was 2 mm (2000 m) you would
perform the calculation as shown below for determining the diameter for 40X
magnification. HPD is the abbreviation for High Power Diameter, which is the
diameter with the 40X objective. The 2000 µm value represents the low power
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 diameter (LPD) in this example. You can see that the Low Power Diameter value
for the 10X objective is in the denominator on the left of the equation, and the
magnification that gave this value (10X) is in the numerator on the right side of
the equation.

Que
stion: In the space provided, use your data to solve for HPD (diameter of field
when using the 40X objective).

Calculated diameter of Field of 40X objective is = m, or _________________

millimeters.

If you like, you can now predict the diameter of the 100X (oil immersion) field
diameter. Remember, you used a ruler to measure the diameter of field for 4X
and 10X objective, but in you must use this information to calculate the 40X
and 100X field diam

Record your answers for each of the diameters in the table below.

Table IV: Diameter of field

Objective Magnification 4X 10 40X 100

X X

Total Magnification

mm= mm= mm= mm=

Diameter of Field (mm and m = m = m = m =

m)

True or False. The diameter of the field of view is inversely proportional to the
magnification. ___________

Using the Diameter of Field to Estimate the Size of the Specimen

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Knowing the diameter of the field of view allows you to estimate the size of a
specimen. Using the slide of the letter "e", estimate the size of the letter by
examining the slide on scanning power (4X). You will need to use the following
formula to estimate how many "e's" or any other specimen may fit across the
Size of specimen = diameter of field of view for a
given magnification number of
field of view. specimens estimated to fit across the

The following example may not represent your “e” but is here to demonstrate the
use of the equation.

Ex.: 5 mm (not diameter of field for all types of microscopes at 4X) = 1.67
mm = size of my "e" I estimate three "e's" can fit across the slide.

Remember, this is only an estimate, but it allows biologists to determine

relative size of a specimen.

How large is your "e"? mm when using the 4X objective?

Working Distance and Depth of Field in a Compound Microscope

You have observed your microscope slide with the scanning, low, and high-
power objectives. You noted that as magnification increases, the diameter of the
field of view decreases and the brightness of the field reduces.
Working distance is measured as the distance between the bottom of the
objective lens and the top of the cover slip on your slide. You may have also
noted that the working distance decreased as you increased the magnification.
This is the reason you should never focus on thick specimens with a high-power
objective.

Now, you need to consider depth of focus.

Depth of Field

Depth of focus refers to the ability of the microscope to focus in on different

levels or “depths” of a specimen at any given time. The compound light
microscope has limited depth of focus. That is, it can only view clearly one plane
of thickness at a time. The higher the magnification, the fewer layers you will see
or the thinner the focal plane and the smaller the working distance.

Remove the slide labeled “colored threads” and place it on your microscope
while using the 4X objective. Focus on the area where the strands intersect.
Can you see all the layers?

Now switch to 10X and then to 40X. Does the intersection of the threads differ
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in appearance?
If so, how?

Using the fine focus knob, move it slightly away from you and then toward you.
What changes do you notice in the depth of focus?

PART II: Introduction: The Cell Theory

The Cell Theory is one of the foundations of modern biology. The major principles of
this theory are:
 All living things are composed of one or more cells.
 The chemical reactions of living cells take place within cells.
 All cells originate from pre-existing cells.
 Cells contain hereditary information, passed from one generation to another.

All cells can be divided into two types: prokaryotic vs. eukaryotic. Eukaryotic
cells will contain a membrane bound nucleus and membrane bound organelles.
Those that are prokaryotic do not have a membrane bound nuclei or membrane
bound organelles. You will be observing examples of cells, which are either
Prokaryotic (such as bacteria) or Eukaryotic (such as plant and animal cells).
In addition, you will observe samples from pond water and plant-like protists.
This will expose you to living organisms observed under the microscope.
By the end of lab, you should be able to distinguish between Prokaryotic
and Eukaryotic cells and within the Eukaryotic category, the difference
between plant and animal cells. You will also be able to make a wet mount
slide using proper technique.

The lab has been set up so that each bench is a different station. Station 1
explains the difference between Prokaryotic and Eukaryotic cells and
describes the basic structure of each. The remaining stations will present
examples of both Prokaryotic cells and Eukaryotic cells or organisms for
you to observe.

Station 1: Cell Structure of Prokaryotic and Eukaryotic Cells

Station 2: Prokaryotes: Bacteria
Station 3: Eukaryotes: Animal Cells
Station 4: Eukaryotes: Plant cells
Station 5: Eukaryotes: Pond Water Organisms (including but not
limited to the Protists Euglena and Paramecium) and Plant-Like
Protists (including Spirogyra and Clamydomonas)
Station 6: Examples of Various Microscopes and the types of images they
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produce

At this point, you should place your belongings under the lab bench and take
only the lab exercise with you as you move through the stations. It is not
necessary to do the stations in any particular order, but you should make every
attempt to complete all stations.
Making a Wet Mount Slide
At several stations you will be asked to make a “wet mount” slide. The
following instructions will guide you through how to make a proper wet mount
slide. Be sure to refer to the directions each time you are required to mount a
specimen. Once you obtain a clean slide do the following:
 Obtain a clean slide.
 Add a drop of suspension (can be water, saline, or protoslo depending on the
specimen to be mounted)
 Add your dry object or specimen
 Holding a coverslip between your thumb and first finger, touch one edge of
the coverslip to the edge of the solution on the slide and hold it at a 45 °
angle as indicated in Figure 1 below. The liquid will “flow” along the edge of
the coverslip.
Cover- liqui
Slide

Figure 1: Preparation of a Wet Mount

 While keeping the one edge of the cover slip touching the liquid, release the
other side of the slip so that it “pushes” the liquid ahead of it. This will reduce
the number of air bubbles that will be trapped under the coverslip making
your specimen much easier to view. If you have too many air bubbles, repeat
the procedure.
 Observe your specimen
 When you are done, you should discard the entire slide which you made into
the Cardboard container in front of the instructor’s desk, marked “GLASS
DISPOSAL”

AS YOU MOVE FROM STATION TO STATION, PLEASE BE

SURE TO CLEAN UP AFTER YOURSELF! ALL SLIDES
WHICH YOU MAKE
YOURSELF SHOULD BE REMOVED FROM THE SCOPES AND
DISPOSED IN THE APPROPRIATE DISPOSAL CONTAINER. ALL
GLASS SLIDES WILL BE DISPOSED IN THE CARDBOARD
“GLASS” BOX WITH THE
EXCEPTION OF THE SLIDES YOU MAKE OF YOUR CHEEK
CELLS. THOSE SLIDES MUST BE DISPOSED IN THE ORANGE
BAGS IN THE BEAKERS AT THE BENCH. THESE ARE
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CONSIDERED BIO-H AZARD AND WILL BE DISPOSED OF
PROPERLY BY OUR PREP TEAM.

Station 1: Cell Structure of Prokaryotic and Eukaryotic Cells

All living organisms have one common feature and that is that they are
composed of cells. In the seventeenth century, Robert Hooke first
observed slices of cork and noted that the cork appeared as though it was
composed of small, somewhat cuboidal units. He referred to these units
as “cells”.
All cells vary in size, organization and function and yet they all share three basic
structural features. First, they all have a plasma (or cell) membrane, which
surrounds and encloses the living material within and controls the passage of
materials into and out of the cell. Second, at some stage of their life, they all
contain a region of DNA (deoxyribonucleic acid) which stores the genetic
information making the cell unique and directing the activities of the cell. Lastly, all
cells contain cytoplasm. The cytoplasm will contain all other organelles or
substances, which are outside of the DNA region but lie within the plasma
membrane.
All cells can be divided into two basic categories, based on their internal
structural organization. The first of these are the Prokaryotic cells (or
Prokaryotes). These include the cells called bacteria and archaea (or
archaebacteria). All other forms of life are composed of Eukaryotic cells
(referred to as
Eukaryotes) . The term “karyon” from the Greek, means “kernel” and refers to
the nucleus of the cell. The prefix “eu” mean true, while the prefix “pro” means
before. Thus, the term eukaryote refers to cells having a “true nucleus” while the
term prokaryote (“before nucleus”) is used to refer to cells lacking a membrane
bound nucleus.
Although Prokaryotic cells still have a nucleoid region containing DNA, the
DNA will not be separated from the rest of the cell by a nuclear membrane. The
DNA of a prokaryotic cell is actually one double- stranded DNA molecule in the
form of a ring. In essence, it is a singe chromosome. In addition to the nucleoid
region, a prokaryotic cell may also have much smaller rings of DNA called
plasmids, which consist of only a few genes. You will hear more about plasmids
later in the semester. Thus, prokaryotic cells are believed to be very similar to
those cells, which first appeared on earth nearly 3.5 billion years ago.
Prokaryotic cells also lack many of the organelles typically found in the
cytoplasm of the eukaryotic cell. One however, can find ribosomes within the
cytoplasm of a prokaryote. In an electron micrograph, the ribosomes will appear
black and give the cytoplasm a granular appearance. Virtually all prokaryotic
cells do have a tough outer cell wall, which surrounds the plasma membrane.
Some prokaryotes may also have a
flagellum, which aids in movement of the cell. The flagellum of prokaryotes is,
however, different from that of eukaryotes. They are not covered by an extension
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of the plasma membrane and they are about one tenth the width of a eukaryotic
flagellum. In addition, there are some prokaryotes, known as cyanobacteria,
which contain chlorophyll and can photosynthesize and produce their own
nutrition from the sun’s energy.
Examine the drawing at your bench showing various structures present in a
Prokaryotic cell. The bacteria cell shown here is representative of a prokaryotic
cell. This bacterial cell would be only micrometers in length. Label the Figure 2
of the prokaryotic cell to show the following structures:

Nucleoid region, plasmid, ribosome, cell membrane, cell wall,

flagellum

Figure 2: Typical Prokaryotic Cell

The eukaryotic cell is believed to have evolved from the prokaryotic cell and is
therefore much more complex. All Eukaryotic cells are bound by a plasma
membrane just as the prokaryotic cells. Some eukaryotic cells are additionally
surrounded by a cell wall. These would be the plant cells. Animal cells do not
have the additional cell wall.
The eukaryotic cell has many different membrane-bound organelles, which
serve to subdivide the cell into different functional compartments. Examples of
such organelles are the nucleus, mitochondria, endoplasmic reticulum,
and the Golgi bodies. Many of these organelles will not be visible to you using
the compound light microscope. You would need to use an electron microscope
to view them. All of the organelles of the eukaryotic cell will not actually be
viewed today. They will be discussed here in order that you might become
familiar with their functions. It will help you distinguish the eukaryotic cell from
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the prokaryotic cell. Detailed descriptions of these organelles will be covered in
lecture.
Using the information at this station, determine the function of each of the
following organelles:
Structure Functi
on
Nucleus
Mitochondria
Endoplasmic
Reticulum
Ribosomes
Golgi Bodies
Plasma Membrane
Use the available resources at this bench to help you label the missing
structures on the diagrams. Label the following structures in diagram
below.
Microtubules, nucleolus, ribosomes, smooth endoplasmic reticulum,
Golgi body, mitochondria, plasma membrane, nucleus

The typical plant cell has some additional features. Label these on the plant cell below.
Cell Wall, Chloroplast, Central Vacuole

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 Station 2: Prokaryotes –Bacteria cells and Cyanobacteria
Bacteria are generally single-celled organisms, although they may occur in
colonial forms (groups of undifferentiated cells, or in simple multi-cellular forms
with differentiated cells. Most true bacteria have one of three basic shapes:
spherical or cocci; rod-shaped or bacilli; and spiral-shaped or spirilla.

While bacteria are best known for their pathological effects, human beings could
not exist without them.
Some bacteria recycle organic material from dead organisms and waste products,
keeping the ecological cycles in balance. Other bacteria such as E. coli actually
inhabit your own gut and are necessary for your survival. Bacteria are also used
to convert milk to yogurt and produce other products like cheese and sourdough
bread. Cyanobacteria , such as Oscillatoria and Anabaena in Figure 3,
photosynthesize in the world’s oceans, providing food for other organisms, fixing
carbon dioxide and nitrogen, and producing oxygen for other organisms to
consume.

Figure 3: On the left are the prokaryotic Oscillatoria and on the right
areAnabaena
http://botit.botany.wisc.edu/images/130/Bacteria/Cyanobacteria/Oscillatoria/Oscillatoria_MC.jpg
http://faculty.baruch.cuny.edu/jwahlert/bio1003/images/anabaena.jpg

Bacteria cells are quite small, but it will be possible to view them with a
compound light microscope using higher powers if magnification. The slides at
this station may be observed at 40X but will show more detail when observed
using the 100X or oil immersion lens. Ask your instructor to help you when using
the oil immersion lens and be sure to clean off the objective when finished.

Cocci Bacillus
Spirillum

Look at the prepared slides of bacteria cells. Draw the general shape of each type
of bacteria. Can you discriminate the cocci, from the bacilli from the spirilla?
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Draw an example of each from what you view in the microscopes at this station.

Station 3: Eukaryotes- Animal Cells

Animal cells are Eukaryotic cells. Your own cells are examples of eukaryotic
animal cells used here to demonstrate characteristics of a typical animal cell.
Typical animal cells have an outer cell membrane (also referred to as the
plasma membrane) and contain a nucleus and cytoplasm with multiple
organelles.
Most of the other organelles will not be visible using the compound light
microscope.

Human epithelial cells are sheets of cells that cover the body surfaces, both
internally and externally. The cells that line the mucous membranes of the mouth,
the surface of the lungs, and the outer layers of skin are all epithelial cells.

Make a wet mount slide of your epithelial cells, which line your inner cheek.
 Use the flat side of a toothpick to gently scrape the lining of your cheek.
 Mix the scraping into a drop of saline or water on a microscope slide and
properly add the cover slip to reduce the amount of air bubbles trapped under
the cover slip.
 Place all used toothpicks in the designated disposal container on your lab bench.
 Put on your goggles and a pair of gloves and stain the slide with methylene
blue in the following manner. Place a small drop of methylene blue
along one edge of the cover slip. Then using a Kim- wipe, place the edge of the
Kimwipe at the opposite side of the cover slip from the methylene blue. This
will draw the methylene blue under the cover slip. This process is termed
"wicking" and will reduce the possibility of applying too much stain to your
specimen.
 View these cells under low power and then increase the total magnification to
400X.
 Sketch a few cells in the space below. Include the magnification and label the
nucleus, cytoplasm, and cell membrane. It is important to remember that
you really cannot see the cell membrane because it is too thin to be visible
with the light microscope, but you can assume that it surrounds the
cytoplasm. Look carefully at one of the nuclei and locate the nucleolus within
it. (You may need to look at several cells.)

What cell structures are highlighted by the stain?

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Station 4: Eukaryotes-Plant Cells
Plant cells are eukaryotic. They contain chlorophyll, have cell walls made up
of cellulose in addition to a plasma membrane and most make their own food
through photosynthesis. They are therefore, called photoautotrophs. Today,
you will look at typical plant cells.
Example 1: Elodea leaf cell - Elodea is an aquatic plant, made of two layers of
cells. You may already be familiar with it, since it is typically used in fish
aquariums. Since it is photosynthetic, the cytoplasm will contain green
chloroplasts, which contain the pigment chlorophyll. Chloroplasts circulate
within the cytoplasm because of cytoplasmic streaming. Figure 4 below shows
the elodea plant. You will be using one leaf from this plant to view the individual
cells which make up the leaf.

Figure 4: Elodea Plant. Image taken from http://ipm.ppws.vt.edu/scott/weed_id/belodea12-

28.jpg

 Remove a leaf from the tip of an Elodea sprig and place it on a microscope
slide in a drop of distilled water.
 Place a cover slip on the Elodea leaf.
 Examine the specimen under the compound microscope. View the leaf
under low power and notice that the leaf consists of many cells. When you
are looking at the Elodea leaf, focus on the uppermost layer of cells to get a
clear view.
 Switch to high power and carefully examine an individual cell. You should be
able to identify the cell wall , which encloses the cytoplasm . The plasma
membrane presses against the cell wall and is not visible with the
compound microscope. Within the cytoplasm are bright green
chloroplasts , the organelles in which photosynthesis takes place. These
organelles appear green because they contain chlorophyll, a green pigment.
 In the center of the cell is a clear vacuole, the central vacuole .
 Look to see if the chloroplasts are moving around the central vacuole.
Chloroplasts can be observed circulating within the cytoplasm because of
cytoplasmic streaming.
 Sketch one or two Elodea cells in the space below. Label the organelles listed
above in bold type.

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 Now prepare a slide using the onion, which is available at this station. To
obtain a very, very thin piece of onion, first cut a small piece of onion and then
slowly and gently snap it in half pulling the thin outer layer away as you do.
This outer layer, which is appears as a thin layer of skin is what you wish to
use for this slide. Once you have the layer placed on a slide, add a drop of
Lugol’s solution to the onion and cover it with a cover slip. View the onion
cells and note how they differ from the elodea.
 Draw what you see below. Do the onion cells have chloroplasts? _

Station 5: Eukaryotes-Pond Water Organisms (including but not

limited to Paramecium and Euglena) and Plant-like Protists
(including Stentor and Clamydomonas)
At this station you will be examining samples of pond water which contain
various living organisms.
Most of these will be single-celled Eukaryotes referred to as protists. Protists
are the most diverse of all eukaryotes . In fact, protists vary in structure
and function more than any other group of organisms. They include animal-like,
plant-like, or fungal-like characteristics. Most of the approximately 60,000
known species of protists are unicellular, but there are some colonial and
multi-cellular species. Most protists are unicellular, and justifiably considered
the simplest eukaryotic organisms.
Examples of protists include the plant-like protists such as the Euglena as
well as the animal-like protists such as the Stentor and Paramecium. These
animal-like protists are usually motile and lack a cell wall. Some engulf food into
vacuoles, where it is digested. Some animal-like protists have contractile
vacuoles that take in and pump out excess water to maintain water balance.
Animal-like protists usually move by flagella, cilia, or amoeboid movements
(extensions of the cell). There will be a variety of organisms, which you might
find in your sample of pond water. The plant-like protists possess chloroplasts
containing chlorophyll and most but not all of them have a cell wall. The plant-like
protists can therefore make their own food through photosynthesis.

Using the Pond Water Provided follow the directions carefully to

make a wet-mount slide and observe some examples of eukaryotic
protists.
 Make a wet-mount slide as described above, using a drop of pond water but
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 DO NOT STIR UP THE POND WATER. It is best to take a dropper and squeeze
the top, to empty the air from the dropper PRIOR to sticking the tip into the
pond water. Keep the rubber stopper squeezed while the tip is under water
until find where you wish to take your sample.
 Try to take your sample from areas around the grains, placed in the water as
a food source. It is most likely that you will get more organisms from that
location than if you take your sample from the surface of the dish.
 Once you have your wet mount made, view your slide and see if you can
identify any of the organisms you find. There are several diagrams at the
station, which may help you with this. Don’t be dismayed if you cannot
identify your organisms. Just enjoy! You may have to move your slide
forward, backward or side to side in order to view your living specimen.
Draw what you see below.

 Why must you continually regulate the fine adjustment to keep a moving organism
in focus in a wet mount?

Plant
Like

Protists
Chlamydomonas, a very simple representative, is thought to be the group
from which land plants arose. Chlamydomonas are single-celled and have two
anterior flagella, which provide locomotion. These organisms are commonly
found in freshwater and soil, obtaining energy from sunlight via photosynthesis.
They have only one photosynthetic organelle or chloroplast that is cup-shaped
and located at the end of the cell opposite the flagella and no cell wall.

Spirogyra is also a freshwater green alga but differs from Chlamydomonas in

that it is multi-cellular rather than single-celled. The cells arrange in long
filaments and have characteristically spiral shaped chloroplasts, which run the
length of the cell. The nucleus of each cell is suspended within a large cell
storage compartment or vacuole.
Examine the picture of Spirogyra and Chlamydomonas at the station. Make
wet mount slides of Chlamy-domonas and Spirogyra. Focus your scope by
starting with the scanning objective (4X) first and then l0X and finally 40X.
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Sketch what you see and label any structures you are able to identify as
described above.

Chlamydomonas Spirogyra

Station 6: Comparison of Photomicrographs from Various

Microscopes

Although you have not discussed all of the types of microscopes available, it
was mentioned at the beginning of the lab that organisms or structures which are
too small to visualize using the compound light microscope, could be viewed using
an electron microscope. There are two types of electron microscope available.
The first is the transmission electron microscope (TEM) which provides an
image similar to that of the compound light microscope in that you can view
structures within an organism or specimen. The second is the scanning
electron microscope (SEM) which is analogous to the dissecting scope in that
it provides an image of the surface of the specimen. Both use electromagnets to
magnify and focus a beam of electrons on a photographic plate. In each case, the
specimen viewed is specially prepared by treating it with heavy metals. This
increases the contrast of the structures observed. The specimen must be very
thinly sliced, in the case of TEM. Both of these electron microscopes have a much
greater resolving power than, either the dissecting or compound light
microscopes.

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Examine the examples of photomicrographs (pictures of organisms viewed through
a microscope) available at this station.
How do the images taken with the transmission electron microscope differ from
those of the compound light microscope?

How do the images of specimens taken with the scanning electron microscope
differ from those taken with the transmission electron microscope and the
compound light microscope?

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