Download laboratory 1 introduction to the microscope and comparison of sizes

LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
LABORATORY 1
INTRODUCTION TO THE MICROSCOPE AND
COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
A. Bacterial Shapes and Arrangements
B. Yeasts
C. Measurement of Microorganisms
D. Focusing Using Oil Immersion Microscopy
DISCUSSION
In this lab, you will become familiar with the use of the microscope (particularly oil immersion microscopy) and will
compare the relative size and shape of various microorganisms.
A. BACTERIAL SHAPES AND ARRANGEMENTS
Bacteria are unicellular prokaryotic microorganisms that divide by binary fission, a process by which one
bacterium splits into two. For a review of prokaryotic versus eukaryotic cellular characteristics, see Unit 1, section
IB in your Lecture Guide.
There are three common shapes of bacteria: the coccus, the bacillus, and the spiral.
1. Coccus
A coccus-shaped bacterium is usually spherical, although some appear oval, elongated, or flattened on
one side. Most cocci are approximately 0.5 – 1.0 micrometer ( m) in diameter and may be seen, based
on their planes of division and tendency to remain attached after replication, in one of the following
arrangements (see Fig. 1A):
a. Division in one plane produces either a diplococcus arrangement (cocci in pairs) or a
streptococcus arrangement (cocci in chains), as shown in Figs. 1A, 1E, and 1C.
b. Division in two planes produces a tetrad arrangement (cocci forming a square of four), as
shown in Fig. 1A.
c. Division in three planes produces a sarcina arrangement (cocci forming a cube of eight), as
shown in Figs. 1A and 1D.
d. Division in random planes produces a staphylococcus arrangement (cocci in irregular, often
grape-like clusters), as shown in Figs. 1A and 1B.
As you observe these different cocci, keep in mind that the procedures used in slide preparation may
cause some arrangements to break apart or clump together. The correct form, however, should
predominate. Also remember that each coccus in an arrangement represents a complete, individual, onecelled organism.
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
Fig. 1:
Arrangement of Cocci
A= Arrangement of cocci
B= Staphylococcus arrangement
C= Streptococcus arrangement
D= Sarcina arrangement
E= Diplococcus arrangement
2. Bacillus (rod)
A bacillus or rod is a hotdog-shaped bacterium having one of the following arrangements (see Fig. 2A):
a. Bacillus: a single bacillus (see Fig. 2B).
b. Streptobacillus: bacilli in chains (see Fig. 2C).
c. Coccobacillus: oval and similar to a coccus.
A single bacillus is typically 0.5-1.0 m wide and from 1-4 m long. Small bacilli or bacilli that have just
divided by binary fission may at first glance be confused for cocci so they must be observed carefully.
You will, however, be able to see bacilli that have not divided and are definitely rod-shaped as well as
bacilli in the process of dividing.
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
Fig. 2:
Arrangements of Bacilli
A= Arrangements of bacilli
B= Single bacillus
C= Streptobacillus
3. Spiral
Spiral-shaped bacteria occur in one of three forms (see Fig. 3A):
a. Vibrio: an incomplete spiral or comma-shaped (see Fig. 3D).
b. Spirillum: a thick, rigid spiral (see Fig. 3B).
c. Spirochete: a thin, flexible spiral (see Fig. 3C).
While some spirochetes are over 100 m in length, the spirals you will observe today range from 5 m to
40 m long.
B. YEASTS
Yeasts, such as the common baker's yeast Saccharomyces cerevisiae, are unicellular fungi. They usually
appear spherical and have a diameter of 3 - 5 µm. Yeasts commonly reproduce asexually by a process called
budding (see Fig. 4). Unlike bacteria, which are prokaryotic, yeasts are eukaryotic. For a review of prokaryotic
versus eukaryotic cellular characteristics, see Unit 1, section IB in your Lecture Guide.
C. MEASUREMENT OF MICROORGANISMS
The approximate size of a microorganism can be determined using an ocular micrometer, an eyepiece that
contains a scale that will appear superimposed upon the focused specimen.
The ocular micrometers provided are calibrated so that when using 1000X oil immersion
microscopy, the distance between any two lines on the scale represents a length of approximately
one micrometer (see Fig. 5). Remember this does not hold true when using other magnifications.
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
Fig. 3:
Spiral Forms
A= Spiral forms
B= Spirillum
C= Spirochete (arrows)
D= Vibrio
Fig. 4:
A Budding Yeast
Note budding (arrows).
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
Fig. 5:
Ocular Micrometer
When using oil immersion microscopy, the distance between any two
lines is one micrometer. The streptococcus seen here is five
micrometers long.
D. FOCUSING WITH OIL IMMERSION - Olympus CH-2 Microscope (see Fig. 7)
1. Before you plug in the microscope, turn the voltage control dial on the right-hand side of the base of
the microscope to 1 (see Fig. 6). Now plug in the microscope and turn it on (see Fig. 7).
2. Place the slide in the slide holder (see Fig. 6), center the slide using the two mechanical stage control
knobs under the stage on the right-hand side of the microscope, and place a rounded drop of immersion
oil on the area to be observed.
3. Rotate the white-striped 100X oil immersion objective until it is locked into place. This will give a
total magnification of 1000X.
4. Turn the voltage control dial on the right-hand side of the base of the microscope to 9 or 10 (see Fig.
6). Make sure the iris diaphragm lever in front under the stage (see Fig. 6) is almost wide open (toward
the left-hand side of the stage), and the knob under the stage on the left-hand side of the stage controlling
the height of the condenser is turned so the condenser is all the way up (see Fig. 7).
5. Watching the slide and objective lens carefully from the front of the microscope, lower the oil immersion
objective into the oil by raising the stage until the lens just touches the slide. Do this by turning the
coarse focus (larger knob; see Fig. 6) away from you until the spring-loaded objective lens just begins
to spring upward.
6. While looking through the eyepieces, turn the fine focus (smaller knob; see Fig. 6) towards you at a
slow steady speed until the specimen comes into focus. (If the specimen does not come into focus
within a few complete turns of the fine focus control and the lens is starting to come out of the oil, you
missed the specimen when it went through focus. Simply reverse direction and start turning the fine focus
away from you.)
7. Using the iris diaphragm lever, adjust the light to obtain optimum contrast (see Fig. 6).
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
8. When finished, wipe the oil off of the oil immersion objective with lens paper, turn the voltage
control dial back to 1, turn off the microscope, unplug the power cord, and wrap the cord around the
base of the microscope.
An alternate focusing technique is to first focus on the slide with the yellow-striped 10X objective using only the
coarse focus control and then without moving the stage, add immersion oil, rotate the white-striped 100X oil
immersion objective into place, and adjust the fine focus and the light as needed. This procedure is discussed in
the Introduction portion of the lab manual on p. xii.
Fig. 6: An Olympus CH-2 Microscope
iris diaphragm lever: moving the lever to the left increases the light; moving the lever to the right
decreases the light.
stage control "A": moves the mechanical stage holding the slide forward and backward.
stage control "B": moves the mechanical stage holding the slide left and right.
coarse focus: turning the knob away from you raises the stage; turning the knob towards you
lowers the stage.
fine focus: turning the knob away from you raises the stage; turning the knob towards you lowers
the stage.
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
Fig. 7: Olympus CH-2 Microscope
SPECIMENS
Prepared slides of the following bacteria:
Staphylococcus aureus
Escherichia coli
Treponema pallidum or Borrelia burgdorferi
Spirillum species
On-line demonstration slides of the following bacteria:
Micrococcus luteus
Neisseria gonorrhoeae
Streptococcus pyogenes
Bacillus megaterium
Broth culture of Saccharomyces cerevisiae
Human hair
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
PROCEDURE
1. Using oil immersion microscopy (1000X), observe and measure the bacteria listed below.
TIPS FOR MICROSCOPIC OBSERVATIONS
Remember that in the process of making the slide, some of the coccal arrangements will clump together and
others will break apart. Move the slide around until you see an area representing the true arrangement of each
organism. Also remember that small bacilli (such as Escherichia coli) that have just divided by binary fission will
look similar to cocci. Look carefully for bacilli that are not dividing and are definitely rod-shaped as well as bacilli in
the process of dividing to confirm the true shape. Also, bacilli do not divide so as to form clusters. Any such
clusters you see are artifacts from preparing the slide. Finally, you will have to look carefully to see the
spirochetes as they are the thinnest of the bacteria. When seen microscopically, spirochetes resemble extremely
thin, wavy pencil lines.
a. Staphylococcus aureus: Staphylococcus species, as the genus name implies, have a staphylococcus
arrangement (cocci in irregular, often grape-like clusters). Measure the diameter of a single coccus.
b. Escherichia coli: Escherichia coli is a small bacillus. Measure the length and width of a typical rod.
c. Treponema pallidum: Treponema pallidum is a spirochete, a thin, flexible spiral. On this slide you are
examining a direct stain of Treponema pallidum, the bacterium that causes syphilis. Measure the length
and width of a typical spirochete.
or
Borrelia burgdorferi: Borrelia burgdorferi is a spirochete, a thin, flexible spiral. On this slide you are
examining a direct stain of Borrelia burgdorferi, the bacterium that causes Lyme disease. Measure the
length and width of a typical spirochete.
d. Spirillum species: Spirillum species appear as thick, rigid spirals. Measure the length and width of a
typical spirillum.
When finished, remove the oil from the prepared slides using paper towel and return them to their proper tray.
2. Observe the on-line demonstration slides of the following bacteria:
a. Micrococcus luteus: Micrococcus luteus can appear in tetrads, cubes of 8, or in irregular clusters. This
strain usually exhibits a tetrad or a sarcina arrangement. Note the shape and arrangement.
b. Neisseria gonorrhoeae: Neisseria species usually have a diplococcus arrangement. Note the shape
and arrangement.
c. Streptococcus pyogenes: Streptococcus species, as the genus name implies, usually have a
streptococcus arrangement (cocci in chains). Note the shape and arrangement.
d. Bacillus megaterium: Bacillus megaterium appears as large bacilli in chains (a streptobacillus). Note
the shape and arrangement.
3. Prepare a wet mount of baker's yeast (Saccharomyces cerevisiae.
a. Using a pipette, put a small drop of the yeast culture on a microscope slide and place a cover slip over
the drop.
b. Using your iris diaphragm lever, reduce the light for improved contrast by moving the lever almost all
the way to the right.
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
c. Observe using oil immersion microscopy. Measure the diameter of a typical yeast cell.
d. When finished, wash the slide and use it again for step 4. Discard the coverslip in the biowaste
disposal containers at the front of the room and under the hood.
4. Prepare a wet mount of your hair.
a. Remove a small piece of a hair from your head and place it in a small drop of water on a slide.
b. Place a cover slip over the drop and observe using oil immersion microscopy.
c.
Measure the diameter of your hair and compare this with the size of each of the bacteria and the
yeast observed in steps 1-3.
d. Discard the slide and coverslip in the biowaste disposal containers at the front of the room and under
the hood.
5. At the completion of the lab, remove the oil from the oil immersion objective using lens paper and put your
microscope away.
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
RESULTS
1. Make drawings of several of the bacteria from each of the four prepared slides and indicate their approximate
size in micrometers.
Staphylococcus aureus
Escherichia coli
Shape =
Shape =
Arrangement =
Length =
m
Width =
m
Diameter =
m
Treponema pallidum or
Borrelia burgdorferi
Spirillum species
Shape =
Shape =
Form =
Form =
Length =
Width =
m
m
Length =
m
Width =
m
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
2. Make drawings of several of the bacteria from each of the four demonstration slides and indicate their
approximate size in micrometers.
Micrococcus luteus
Neisseria gonorrhoeae
Shape =
Shape =
Arrangement =
Arrangement =
Streptococcus pyogenes
Bacillus megaterium
Shape =
Shape =
Arrangement =
Arrangement =
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
3. Make a drawing of several yeast cells and indicate their size in micrometers.
Saccharomyces cerevisiae
Diameter =
m
4. Make a drawing indicating the size of the bacteria and yeast observed above relative to the diameter of your
hair.
Hair
Diameter =
m
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
PERFORMANCE OBJECTIVES
LABORATORY 1
After completing this lab, the student will be able to perform the following objectives:
DISCUSSION
1. State three basic shapes of bacteria.
2. State and describe five different arrangements of cocci.
3. State and describe three different arrangements of bacilli.
4. State and describe three different spiral forms.
5. Describe the appearance of a typical yeast cell.
RESULTS
1. When given an oil immersion microscope, a prepared slide of a microorganism, and an ocular
micrometer, determine the size of that organism in micrometers.
2. Using a microscope, identify different bacterial shapes and arrangements.
3. Differentiate a yeast cell from a coccus-shaped bacterium by its size.
4. Compare the size of the microorganisms observed in lab with the diameter of a hair when using oil
immersion microscopy.
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LABORATORY 1:
INTRODUCTION TO THE MICROSCOPE AND COMPARISON OF SIZES AND SHAPES OF
MICROORGANISMS
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