Laboratory Objectives: LAB EXERCISE: Microscopy and The Cell
Laboratory Objectives: LAB EXERCISE: Microscopy and The Cell
Laboratory Objectives
After completing this lab topic, you should be able to:
1. Identify the parts of compound and stereoscopic microscopes and be proficient in their
correct use in biological studies.
2. Describe procedures used in preparing materials for light microscopy.
3. Describe features of specific cells and determine characteristics shared by all cells
studied.
4. Discuss the evolutionary significance of increasing complexity from unicellular to
multicellular organization and provide examples from the lab.
Introduction
According to cell theory, the cell is the fundamental biological unit, the smallest and
simplest biological structure possessing all the characteristics of the living condition. All
living organisms are composed of one or more cells, and every activity taking place in a
living organism is ultimately related to metabolic activities in cells. Thus, understanding
the processes of life necessitates an understanding of the structure and function of the
cell.
The earliest known cells found in fossilized sediments 3.5 billion years old (called
prokaryotic cells) lack nuclei and membrane-bound organelles. Cells with a membrane-
bound nucleus and organelles (eukaryotic cells) do not appear in the fossil record for
another 2 billion years. But the eventual evolution of the eukaryotic cell and its internal
compartmentalization led to enormous biological diversity in single cells. The evolution
of loose aggregates of cells ultimately to colonies of connected cells provided for
specialization, so that groups of cells had specific and different functions.
This early division of labor included cells had specific and different functions. This early
division of labor included cells whose primary function was locomotion or reproduction.
The evolution of multicellularity appears to have originated more than once in eukaryotes
and provided an opportunity for extensive adaptive radiation as organisms specialized
and diversified, eventually giving rise to fungi, plants, and animals.
Given the fundamental role played by cells in the organization of life, one can readily
understand why the study of cells is essential to the study of life. Cells, however, are
below the limit of resolution of the human eye. We cannot study them without using a
microscope. The microscope has probably contributed more than any other instrument to
the development of biology as a science. Two types of microscopes are named according
to the source of illumination used: light microscopes and electron microscopes. We will
be using light microscopes exclusively in our study of cells, and we will view electron
micrographs of cell structures not visible with the light microscope.
Microscopes of one kind or another are used by all biologists in numerous subdisciplines:
genetics, molecular biology, neurobiology, cell biology, evolution, and ecology. The
knowledge and skills you develop today will be used and enhanced throughout this
course and throughout your career in biology. It is important, therefore, that you take the
time to master these exercises thoroughly.
The microscope is designed to make objects visible that are too difficult or too small to
see with the unaided eye. There are many different kinds of light microscopes, including
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phase-contrast, darkfield, polarizing, and UV. These differ primarily in the source and
manner in which light is passed through the specimen to be viewed.
The microscopes in biology lab are usually compound binocular or monocular light
microscopes, some of which may have phase-contrast attachments. Compound means
that the scopes have a minimum of two magnifying lenses (the ocular and the objective
lenses). Binocular microscopes have two eyepieces, monoculars have only one
eyepiece, and light refers to the type of illumination used, that is, visible light from a
lamp.
Place the microscope on your desk with the arm of the microscope nearest you. With the
aid of the illustration, locate the various parts of the instrument. Learn to understand the
function of each part. You should also become familiar with the terms frequently used in
microscopy, some of which are described in the following.
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Inclination Joint Permits tilting of parts attached to arm; should be used only with
permission of the instructor.
Pillar Supports microscope arm and its attached parts.
Base Bottom portion that supports the microscope; provides carrying support.
Condenser Concentrates and directs light through specimen.
Iris Diaphragm Controls area of illumination through the condenser.
To determine magnification:
If the eyepiece is marked 10 X and the objective in position is marked 10 X, the specimen
is magnified 100 times (10 X 10). If the eyepiece is 10 X and the objective in position is
43 X, then the specimen is magnified 430 times (10 X 43 = 430).
Working distance:
The distance between the specimen and the objective lens is the working distance. This
distance decreases as the magnification is increased. Therefore, the greater a specimen is
magnified, the greater is the chance for the specimen and the lens of the objective coming
into contact. This requires that the student exercise care when the high power objective or
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the oil immersion objective is in position. On a commercially prepared slide this could
mean the difference between a broken slide and one that is kept in good condition.
On most microscopes the low power objective is blocked by a stop screw from coming
too close to a slide. This objective is most commonly used so the stop is standard.
However, the high power does not have a stop and is clearly a potential threat for
breakage. Turn the adjustment knobs so that the body tube is moved away from the slide
to prevent breakage.
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With your eye to the eyepiece and your hand on the coarse adjustment knob, slowly focus
upward until the specimen is in view. You should have to move the coarse adjustment
knob very little before the specimen is in view. Once the specimen is in fairly good focus,
it may be made more distinct by slight adjustments of the fine adjustment knob.
If you wish to observe the specimen under high power, decide upon the portion of the
specimen you wish to examine, and move this to the exact center of the low power field.
Rotate the high power objective in position. Do this slowly, making sure that as the
objective moves into position, it does not hit the stage or the slide.
Open and close the diaphragm several times to obtain the best lighting.
Most microscopes are parfocal, that is, once the specimen is in focus on low power, it
will remain in focus when you change to high power. A slight turn of the fine adjustment
knob will make the focus sharper.
Return the low power objective to position when you are finished.
Note that focus on the lamp will produce the ground glass or frosted glass effect. Refocus
to get rid of this problem.
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Preparation of a wet mount:
Clean the microscope slide and coverslip with water and some clean paper toweling or
paper tissues. It is wasteful to use the expensive lens paper for cleaning blank slides.
Handle the slide and coverslip by the edges so that body oils and other bodily releases do
not get onto them.
If the specimen is dry or nearly so water will have to be added with it. Place this type of
specimen on the center of the clean glass slide. Put a single drop of water on the
specimen. If the water is too quickly absorbed, add some more. Then, following the
procedures illustrated below, cover the specimen in such a manner that the coverslip is
lowered onto the water-specimen gently to keep out air bubbles.
Water should not be found on top of the coverslip because it might contaminate the
objectives of the microscope.
For cultures which are fluid, place the drop from the culture medium onto the slide and
add the coverslip. There is no need to add water in the initial set-up.
If this preparation begins to dry, add a bit of water at the edge of the coverslip so that it
can seep under the coverslip and thus keep the specimen wet.
Always keep the bottom of the microscope slides clean and dry so that the microscope
stage is not messed up or the mechanical stage cannot move the slide smoothly.
Blank slides, those without labels, may be washed and dried with paper toweling or other
inexpensive materials.
However, commercially prepared slides, those with labels, must be cleaned with lens
paper to remove finger prints and smudges. Using any other materials may ruin the slide.
Stains:
Stain are used to color so that a specimen being viewed with light coming through it will
appear more distinct. Obviously, a specimen which is naturally colored will not need any
additional staining. The most commonly used stain in this laboratory text is diluted
iodine. Iodine is a very useful stain because it is easy to prepare, stores for long periods
without deterioration, and it colors the specimens which will be used in these exercises.
However, iodine is toxic and will kill most forms of life. There is no danger to the student
in these procedures ordinarily. Some care is necessary in handling the stain because it
will not wash out of many types of clothing.
VITAL STAINS are also used to stain and color certain specimens in a natural state. The
advantage of this type of stain is that it either does not kill or kills slowly so that one may
observe the living organism. In most instances the vital stain is no more harmful than
food coloring.
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Let the preparation stand for at least a minute so that the methyl cellulose and fluid can
mix. If the preparation is too thick, tap the coverslip gently to spread out the mixture.
Protoslo is a clear material also used to slow fast-moving microscopic forms.
Neither one of theses slowing materials is colored. They will not add any color to a
specimen.
Dissecting scope:
When a specimen is large and thick, it is not possible or practical to view it under the lens
of a compound microscope, which requires that light pass through anything on view.
With the dissecting scope, it is the whole specimen which is to be viewed and which can
be viewed in three dimensions.
By using the dissecting scope, it is not necessary to have a special staining or to develop
difficult techniques of preparation so that the specimen can be observed. This type of
scope is not a substitute for the compound scope because it serves in a different capacity
as another tool in the biology laboratory.
Typically, there are two objectives and though their power of magnification varies, they
usually are 5 X or 10 X objectives. The objectives can be moved into position by rotation.
There is a metal plate that serves as a stage or platform on which the specimen is placed.
One side is white for use in any way convenient for viewing a specimen.
In most instances a specimen is lighted by some sort of lamp with the light directed onto
the specimen from above, and to light up or shade areas of a specimen.
Materials
clear ruler lens paper
coverslips dropper bottle with distilled water
prepared slides: letter and crossed thread blank slides
Kimwipes
Introduction
In this exercise, you will learn to use the microscope to examine a recognizable object, a
slide of the letter e. Recall that microscopes vary, so you may have to omit steps that refer
to features not available on your microscope. Practice adjusting your microscope to
become proficient in locating a specimen, focusing clearly, and adjusting the light for the
best contrast.
Procedure
1. Clean microscope lenses.
Each time you use the microscope, you should begin by cleaning the lenses. Using lens
paper moistened with a drop of distilled water, wipe the ocular, objective, and condenser
lenses. Wipe them again with a piece of dry lens paper.
*Use only lens paper on microscope lenses. Do not use Kimwipes, tissues, or other
papers.
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2. Adjust the focus on your microscope:
a. Plug your microscope into the outlet.
b. Turn on the light. Adjust the light intensity to mid-range if your microscope has
that feature.
c. Rotate the 4 X objective into position using the revolving nosepiece ring, not
the objective itself.
d. Take the letter slide and wipe it with a Kimwipe tissue.
Each time you study a prepared slide, you should first wipe it clean. Place the
letter slide on the stage, and center it over the stage opening.
*Slides should be placed on and removed from the stage only when the 4 X
objective is in place. Removing a slide when the higher objectives are in
position may scratch the lenses.
e. Look through the ocular and bring the letter into rough focus by slowly
focusing upward using the coarse adjustment.
f. For binocular microscopes, looking through the oculars, move the oculars until
you see only one image of the letter e. In this position, the oculars should be
aligned with your pupils. In the margin of your lab manual, make a note of the
interpupillary distance on the scale between the oculars. Each new lab day,
before you begin to use the microscope, set this distance.
g. Raise the condenser to its highest position, and fully close the iris diaphragm.
h. Looking through the ocular, slowly lower the condenser just until the graininess
disappears. Slowly open the iris diaphragm just until the entire field of view is
illuminated. This is the correct position for both the condenser and the iris
diaphragm.
i. Rotate the 10 X objective into position.
j. Look through the ocular and slowly focus upward with the coarse adjustment
knob until the image is in rough focus. Sharpen the focus using the fine
adjustment knob.
*Do not turn the fine adjustment knob more than two revolutions in either
direction. If the image does not come into focus, return the 10 X and refocus
using the coarse adjustment.
k. For binocular microscopes, cover your left eye and use the fine adjustment
knob to focus the fixed (right) ocular until the letter e is in maximum focus. Now
cover the right eye and, using the diopter ring on the left ocular, bring the image
into focus. The letter e should now be in focus for both of your eyes. Each new
lab day, as you begin to study your first slide, repeat this procedure.
l. You can increase or decrease the contrast by adjusting the iris diaphragm
opening. Note that the maximum amount of light provides little
contrast. Adjust the aperture until the image is sharp.
m. Move the slide slowly to the right. In what direction does the image in the
ocular move?
n. Is the image in the ocular inverted relative to the specimen on the stage?
o. Center the specimen in the field of view; then rotate the 40 X objective into
position while watching from the side. If it appears that the objective will hit the
slide, stop and ask for assistance.
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*Most of the microscopes have parfocal lenses, which means that little
refocusing is required when moving from one lens to another. If your scope is
not parfocal, ask your instructor for assistance.
p. After the 40 X objective is in place, focus using the fine adjustment knob.
*Never focus with the coarse adjustment knob when you are using the high
power objective.
q. The distance between the specimen and the objective lens is called the working
distance. Is this distance greater with the 40 X or the 10 X objective?
In this exercise, you will examine the features common to all eukaryotic cells that are
indicative of their common ancestry. However, you will observe that all cells are not the
same. Some organisms are unicellular (single-celled), with all living functions
(respiration, digestion, reproduction, and excretion) handled by that one cell. Others form
random, temporary aggregates, or clusters, of cells. Clusters composed of a consistent
and predictable number of cells are called colonies. Simple colonies have no
physiological colonies have physiological connections and specialization of groups of
cells. Multicellular organisms have large numbers of cells with specialized structure and
function, and no one cell can exist successfully by itself.
Unicellular Organisms
Materials
microscope slides coverslips
Amoeba culture Paramecium culture
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Procedure
1. Prepare wet mounts of both amoeba and paramecium samples.
a. Using a clean pipette (it is important not to interchange pipettes between culture
dishes), transfer a drop with several amoebas or paramecia to your microscope
slide. To do this, squeeze the pipette bulb before you place the tip under the
surface of the water. Disturbing the culture as little as possible, pipette a drop of
water with debris from the bottom of the culture dish.
b. Cover your preparation with a clean coverslip.
c. Under low power on the compound scope, scan the slide to locate an amoeba.
Center the specimen in your field of view, then switch to higher powers.
d. Identify the following structures in the amoeba:
Cell membrane is the boundary that separates the organism from its surroundings.
Ectoplasm is the thin, transparent layer of cytoplasm directly beneath the cell membrane.
Endoplasm is the granular cytoplasm containing cell organelles.
The nucleus is the grayish, football-shaped body that is somewhat granular in
appearance. This organelle, which directs the cellular activities, will often be seen
moving within the endoplasm.
Contractile vacuoles are clear, spherical vesicles of varying sizes that gradually enlarge
as they fill and then empty its contents into the surrounding environment. These vacuoles
serve an excretory function for the amoeba.
Food vacuoles are small, dark, irregularly shaped vesicles within the endoplasm. They
contain undigested food particles.
Pseudopodia (false feet) are fingerlike projections of the cytoplasm. They are used for
locomotion as well as for trapping and engulfing food in a process called phagocytosis.
d. Follow the same procedure for Paramecium and identify the following
structures:
Cilia: minuscule cilia that envelop the paramecium and are used for locomotion.
Contractile vacuole: cavity of the paramecium that is able to contract.
Food vacuole: cavity of the paramecium responsible for digestion.
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Micronucleus: one of the less important central organelles of a paramecium.
Oral groove: canal of the paramecium used to ingest nutrients.
Gullet: cavity of the pharynx.
Ectoplasm: vitreous superficial layer of a paramecium.
Endoplasm: central part of a paramecium.
Large nucleus: the most important central organelle of a paramecium.
Canals of contractive vacuole: division of the contractile cavity of a paramecium.
Trochocyst: root of a vibrative cilium of a paramecium.
Figure 4. Paramecium.
*Draw examples of what you see for both Amoeba and Paramecium cultures.
Materials
microscope slides
coverslips
Protococcus and Volvox cultures
Introduction
Unlike unicellular organisms, which live independently of each other, colonial organisms
are cells that live in groups and are to some degree dependent on one another. The
following organisms show an increasing degree of interaction among cells. Protococcus
(Figure 5) is a terrestrial green alga that that forms loose aggregates on the bark of trees
grows on the north sides of trees and is often referred to as moss. The size of the cell
groupings is random, and there are no permanent connections between cells. Each cell is
surrounded by a cell membrane and an outer cell wall. Volvox (Figure 6) is an aquatic
green alga that also is common in aquaria, ponds, and lakes. In this complex colony, the
individual cells are interconnected by cytoplasmic strands to form a sphere. Small
clusters of cells, called daughter colonies, are specialized for reproduction that the cells
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of this organism form a large complex colony. Approximately 500 to 50,000 cells
(depending on the species) are permanently united, there are cytoplasmic connections
between cells, and some cells are specialized for reproduction.
Procedure
1. Prepare wet mounts of both Protococcus and Volvox samples.
a. Identify the following structures in Volvox:
Individual cells all possess the following structures: cell wall, nucleus, vacuole,
chloroplasts, flagella (two per cell).
Cytoplasmic strands form connections between adjacent cells.
Daughter colonies are smaller spheres within the larger colony. These are produced
asexually, and when they are large enough, they will be discharged from the parent
colony into the surrounding environment.
Figure 5. Protococcus.
Figure 6. Volvox.
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*Draw examples of what you see for both Protococcus and Volvox cultures.
Multicellular Organisms
Materials
microscope slides coverslips
dropper bottles of water Elodea
toothpicks methylene blue
Introduction
Multicellular organisms are composed of groups of specialized cells, called tissues that
together perform particular functions for the organisms. Tissues, in turn, may be grouped
to form organs, and organs may be grouped into organ systems. In this lab study, you
will examine some of the cells that compose the basic tissue types of plants and animals.
Procedure
Plant Cells
1. The major characteristics of a typical plant cell are readily seen in the leaf cells of
Elodea, a common aquatic plant (Figure 7). Prepare a wet mount and examine one of the
youngest (smallest) leaves from a sprig of Elodea under the compound
microscope.
2. Identify the following structures:
The cell wall is the rigid outer framework surrounding the cell. This structure gives the
cell a definite shape and support. It is not found in animal cells.
Protoplasm is the organized contents of the cell, exclusive of the cell wall.
Cytoplasm is the protoplasm of the cell, exclusive of the cell wall.
The central vacuole is a membrane-bound sac within the cytoplasm that is filled with
water and dissolved substances. This structure serves to store metabolic wastes and gives
the cell support by means of turgor pressure. Animal cells also have vacuoles, but they
are not as large and conspicuous as those found in plants.
Chloroplasts are the green, spherical organelles often seen moving within the cytoplasm.
These organelles carry the pigment chlorophyll that is involved in photosynthesis. As the
microscope light heats up the cells, cytoplasm and chloroplasts may begin to move
around the central vacuole in a process called cytoplasmic streaming, or cyclosis.
The nucleus is the usually spherical, transparent organelle within the cytoplasm. The
structure controls cell metabolism and division.
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Figure 7. Elodea. Elodea is an aquatic plant commonly grown in freshwater aquaria. The
cell structures may be difficult to see because of the three-dimensional cell shape and the
presence of a large central vacuole.
Animal Cells
1. Animals are multicellular heterotrophic organisms that ingest organic matter. They are
composed of cells that can be categorized into four major tissue groups, epithelial,
connective, muscle, and nervous tissue. In this lab study, you will examine epithelial
cells. Similar to the epidermal cells of plants, epithelial cells occur on the outside of
animals and serve to protect the animals from water loss, mechanical injury, and foreign
invaders. In addition, epithelial cells line interior cavities and ducts in animals. Examine
the epithelial cells (Figure 8) that form the lining of your inner cheek. To obtain a
specimen, follow this procedure:
a. With a clean toothpick, gently scrape the inside of your cheek several times.
b. Roll the scraping into a drop of water on a clean microscope slide, add a small
drop of methylene blue, and cover with a coverslip.
c. Using the compound microscope, view the cells under high power.
Observe that these cells are extremely flat and so may be folded over on themselves.
Attempt to locate several cells that are not badly folded, and study their detail.
2. Identify the following structures:
The cell membrane is the boundary that separates the cell from its surroundings.
The nucleus is the large, circular organelle near the middle of the cell.
Cytoplasm is the granular contents of the cell, exclusive of the nucleus.
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Figure 8. Human epithelial cells.
*Draw examples of what you see for both plant and animal cells.
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BIO201 Laboratory Assignment: Microscopy
Exercise for today:
Follow procedures as outlined in manual and answer the following questions:
2. Is the image in the ocular inverted relative to the specimen on the stage?
3. Working distance. Is the working distance greater with the 40 X or the 10 X objective?
4. Compute the total magnification of the specimen being viewed (the letter e and crossed
threads).
a) What is the total magnification of the letter e when viewed in focus with the 40
X objective?
b) What would be the total magnification if the ocular were 20 X and the
objectives were 100 X?
c) Focus on the region where the threads cross. Are both threads in focus at the
same time?
d) Rotate the 10 X objective into position and focus on the cross. Are both threads
in focus at the same time?
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5. Measuring the diameter of the field of view. (Using a clear ruler).
b) Measure the diameters of the field of view for the 4X, 10 X and 40 X
objectives:
Draw examples of what you see for Amoeba, Paramecium, Protococcus, Volvox, Elodea
and human epithelial cells.
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