topic: microscope lab part i: compound light...
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Lab #4 Biology 10 Gallagher Page 1 of 18
Lab #4 Biology 10 BCC
Topic: MICROSCOPE LAB
PART I: COMPOUND LIGHT MICROSCOPE
OBJECTIVES: After completing this exercise you should be able to:
Demonstrate proper care and use of a compound microscope.
Identify the parts of the compound light microscope and describe the function
of each part.
Compare magnification, resolving power, and contrast.
Demonstrate proper technique of preparing a wet mount slide.
Demonstrate inversion and depth of field.
Use the compound light microscope as an instrument of measurement.
INTRODUCTION:
The unaided human eye can detect objects as small as 0.1 mm in diameter. Most cells
are between 0.01 mm and 0.1 mm in diameter and cannot be seen without a
microscope. A microscope contains one or more lenses and is used to view detail that
cannot be seen with the unaided eye. The light microscope, by virtue of its lens
system, extends our vision a thousand times so that object as small as 0.1 micrometer
(µm) in diameter can be seen. The electron microscope further extends our viewing
capability down to 1 nanometer (nm). At this magnification it is possible to see a virus
and the outline of individual protein or nucleic acid molecules. A lens functions by
refracting (bending) light rays coming from an object and focusing them to form an
image of that object. Refraction of light is due to the angle at which it passes from one
transparent medium to another (for example, air to glass) and the difference in density
between the media. A magnifying glass is a simple light microscope. The microscope
consists of a set of lenses that focus an enlarged image of an object on the retina of
the eye. The greater the area of the retina covered by the image of a specimen, the
greater its magnification.
A: PURPOSE OF THE MICROSCOPE
The microscope is useful in making observations and collecting data in scientific
experiments. Microscopy involves three basic concepts:
Magnification: The degree to which the image of a specimen is enlarged.
Resolving power: How well specimen detail is preserved during the magnifying
process.
Contrast: The ability to see specimen detail against its background. Stains and dyes
are added to sections of biological specimens to increase contrast.
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The microscope is an expensive precision instrument. When
removing the microscope from the storage area, always
grasp it with both hands. Place one had around the arm and
the other hand firmly under the base. Hold it close to your
body for stability. Once you reach your work area, set the
microscope down gently on the table with the arm toward
you.
1. Get a microscope from the microscope cabinet and bring it
back to your desk.
B: THE COMPOUND MICROSCOPE
This section covers the parts of a compound microscope.
Make sure you can identify each of the parts listed in this section on your microscope.
1. Support Structures
Arm: Supports the body tube and the stage of the microscope
Stage: Platform where the slide is placed for viewing
Stage clip: Holds the slide firmly in place on the stage
Stage opening: The hole in the stage that allows light to pass from the lamp,
through the specimen, and into the body tube.
Base: Lowermost part of the microscope; provides a firm and steady support
Body tube: Holds the eyepiece lens and objective lens at the correct distance
for magnification.
Rotate the coarse focus knob. Does the stage or body tube move?
_____________________________________________________________
2. Lighting
Lamp: Provides the light needed to view the specimen.
Diaphragm: A disk located directly below the stage of the microscope;regulates
the amount of light passing through the stage opening.
Condenser: Focuses the beam of light; located below the stage
Does your microscope have a condenser?____________________________
Describe the diaphragm on your microscope._________________________
_____________________________________________________________ What diaphragm setting (number) allows the most light to pass through the specimen?
___ _____________________________________________________________
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What diaphragm setting (number) allows the least amount of light to pass through the
specimen?
_______________________________________________________________
3. Focus
Coarse focus knob: Larger knob used to elevate or lower the body tube or
stage a large distance with each turn.
Fine focus knob: Smaller knob used to elevate or lower the body tube or stage
a small distance with each turn; used to make fine adjustments when focusing
on a specimen.
Where are the coarse focus knobs located on your microscope?
_____________________________________________________________
Where are the fine focus knobs located on your microscope?
_____________________________________________________________
4. Optics
The compound microscope consists of a set of lenses
that gather light transmitted through a specimen and
focus this light on the retina of the eye. The diagram on
right shows the path of light as it passes from the lamp,
through the microscope, and into the eye.
The compound microscope has at least two lens
systems: an eyepiece that you look into and an
objective that magnifies the specimen.
Eyepiece lens: Located in the upper end of the body
tube and focuses light on the retina of the eye. The
power of the eyepiece is usually 10X.
How many eyepieces does your microscope have?______________________
Is it monocular or binocular? ___________________________________
Objective lenses: Attached to the revolving nosepiece. The number and magnification of the objective lenses will vary with the type of microscope.The objective lenses are housed in one end of several steel tubes that are threaded into the revolving nosepiece. The desired objective lens is placed in position by rotating the nosepiece until it clicks into place. The microscopes used in this class have either three or four objective lenses. The objectives include the scanning lens (4X), lower power lens (10X), high power lens (40X), and the oil immersion lens (100X) in some microscopes.
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The drawing below shows the distance between the objective lens and the slide. This
distance decreases with higher magnification; therefore it is important to use care
when focusing with higher magnification. Only the fine focus knob should be used.
Notice the oil immersion lens requires a drop of oil between the lens and the slide.
The Lens Table below will help you see the relationship between the unaided eye and
the magnification made possible by the light microscope. The magnification is marked
on the housing of each lens. The power of the microscope is determined by
multiplying the power of the eyepiece lens times the power of the objective lens. The
objective lenses have a color coded ring around each lens which indicates the
magnification of that lens.
5. Complete the Table below.
Lens Lens
Magnification Ring Color
Total
Magnification
Eyepiece
Scanning
Low Power
High Power
C: FOCUSING THE MICROSCOPE
6. Obtain a prepared slide from the supply area.
7. Make sure the scanning lens or the lower power lens is in place.
8. Raise the body tube or lower the stage just enough to allow you to place the
slide on the stage without hitting the objective lens.
9. Place the slide on the stage of your microscope and clip it into place. Move the
slide so the specimen is over the stage opening.
10. While looking at the microscope from the side, move the body tube all the way
down or move the stage all the way up.
11. While looking through the eyepiece, move the body tube up or move the stage
down until the specimen comes into focus.
12. Adjust the diaphragm opening until you have the best view of the specimen.
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13. With the specimen in focus and positioned in the center of the field of view,
rotate the nosepiece lens to the high power objective (40X). DO NOT move the
coarse focus. Only fine focus should be necessary to bring the specimen into
sharp focus. The ability of the microscope to remain in focus when switching
from one objective lens to the next highest power is called parfocal.
14. Adjust the diaphragm opening until you have the best view of the specimen.
12. Have your partner repeat steps 6 – 12.
13. Return the prepared slide to the supply area.
D: SPECIMEN ORIENTATION
14. Prepare a wet mount slide of an R by first cutting a capital R out of a newspaper.
(Do not use one from a headline.)
15. As shown in the illustration below, place a drop of water on the slide (Diagram
#1)
16. Add the R to the drop (Diagram #2).
17. Place one edge of a coverslip on the slide, in the water, next to the R. Use a
dissecting needle or pin to gently lower the coverslip onto the R (Diagram #3).
18. Get rid of any air bubbles by raising and lowering the coverslip until any trapped
air is released. Do not press directly down on the coverslip.
19. Place the letter R slide right side up on the stage with the low power objective
lens in place. Center the letter in the field of view.
20. Bring the R into focus under low power.
1 2
3 4
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21. Draw the R as you see it through the eyepiece with the low power lens in place.
22. Bring the R into focus under high power.
23. Draw the R as you see it through the eyepiece with the high power lens in place.
R viewed without R viewed under R viewed under
Microscope Low Power High Power
24. With the low power objective lens in place, move the slide to the right while
watching the image through the microscope.
In what direction does the image move? __________________________
25. Move the slide away from you. In what direction does the image move?
___________________________________________________________
What is the relationship between the movement of the image and the movement
of the object?
E: MICROSCOPE MEASUREMENT
Most of the objects you view under the compound microscope are smaller than two
millimeters. Obviously, measuring these microscopic objects could prove to be quite
difficult and inexact if millimeters are used as the unit of measure. To solve this
problem scientists divide the millimeter into 1000 smaller units called micrometers
(µm). Tiny objects can then be accurately measured in micrometers. In this section
you will learn how to estimate the size of the tiny organisms you view under the
compound microscope.
26. Obtain a transparent plastic ruler from
the supply area.
27. Place the plastic ruler on the stage so
that the ruler’s edge is centered in your
field of view under low power. Make sure
you use the millimeter side of the
ruler. Use the diagram below for
help.
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28. Position the ruler so one of the millimeter marks is just visible
to the left in your field of view. Use the diagram at the right for
help. Notice the distance between the mark on the left and the
next mark is one millimeter. Estimate the remaining distance
in decimal fractions of a millimeter across
the diameter of the field of view.
What is your total field of view size in millimeters under low power? ____
29. What is the low field diameter in micrometers (µm)? (1 mm = 1000 µm)
___________________________________________________________
30. Switch to high power. Look at the marks on the ruler. You will find that the high
power field of view is less than 1mm or 1000µm. For that reason, it is difficult to
estimate the diameter of the field of view using the same technique used for low
power. However, you can determine the field of view under high power by using
the formula below:
High Power Magnification Low Power Field Diameter
Low Power Magnification = High Power Field Diameter
What is the microscope’s calculated high power field diameter in µm?
31. Now that you know the diameter of your field size under both high and low
power, you can use that information to estimate the size of objects you observe
under the microscope. For example, in the diagram at the
right, 10 circular objects fit across the field of view. The
field of view is 2000µm in diameter. Since each object
takes up 1/10 of the 2000µm field diameter, the size of
each object is 200µm. You can use this
method to estimate size of objects you view under
your microscope once you know your microscope’s
field diameter.
32. Obtain the prepared slides for this section from the supply area.
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33. Focus under low or high power to view each specimen and then estimate the size
of each. Record your observations in the table below.
Specimen
Viewed under Low
or High
power
Field
Diameter
# of
specimens
that fit
across
field
Estimated
Specimen
Size
34. Return all slides to the supply area.
F: DEPTH OF FIELD
35. Obtain a microscope slide of silk fibers from the supply area.
36. Look at the slide under low power where the threads cross. Adjust the diaphragm to give the sharpest view. Are all three thread colors equally visible under low power?
__________________________________________________________
37. Look at the slide under high power where the threads cross. Adjust the
diaphragm to give the sharpest view and fine focus. Are all three thread colors
equally visible under high power?
___________________________________________________________
38. Slowly fine focus up and down to determine the order of the thread colors.
Which color is on top? _________________________________________
Which color is on the bottom? ________________________________________
How did you determine the order of the thread colors?__________________
_____________________________________________________________
39. Return the slide to the supply area.
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PART II: QUESTIONS
56. Describe how a compound microscope should be held and carried.
_____________________________________________________________
_____________________________________________________________
57. How is the total magnification of a microscope determined?
_____________________________________________________________
58. If the eyepiece on a microscope has a magnification of 10X, what is the total
magnification with a 15X objective?
___________________________________________________________
59. If the eyepiece on a microscope has a magnification of 15X, what is the total
magnification with a 45X objective?
___________________________________________________________
60. A microscope gives a total magnification of 1500X, but the image is too blurry to
be useful. What might be the problem with the microscope?
___________________________________________________________
___________________________________________________________
61. An image is located in the lower right hand corner of the field of view. How
would you move the slide to center the image?
_____________________________________________________________
_____________________________________________________________
62. Objects viewed under a compound microscope are frequently lost when
switching from low to high power. Give one reason why this happens.
_____________________________________________________________
_____________________________________________________________
63. How did the light intensity change when you switched from low power to high
power objective?
_____________________________________________________________
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64. In general, how would you have to adjust the diaphragm after switching from
low to high power?
_____________________________________________________________
65. Do you observe more or less area in your field of view when under high power
compared to low power?
_____________________________________________________________
66. If a microscope has a low power magnification of 100X and a high power
magnification of 500X, and a low power field of 1500µm, what is the high power
field in µm?
_____________________________________________________________
67. If 20 objects fit across the diameter of a low power field of view whose field
diameter is 4000µm, what would be the approximate size of each object?
_____________________________________________________________
68. Why is it more difficult to measure the diameter of the high power field of view
than the low power field of view?
_____________________________________________________________
_____________________________________________________________
69. The circle at the right represents a microscope’s field of
view with a black dot under 10X magnification. Draw
how large the dot would appear under 40X
magnification. Also, draw a circle to indicate the size of
the field of view under 40X magnification.
70. Sketch the number 4 as it appears through the lenses of
the compound microscope.
How has the lens system of the compound
microscope changed the orientation of the
numeral?
____________________________________
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71. A student focuses on a specimen at low power and carefully centers it before
changing to high power. At high power, however, he doesn’t see the part of the
specimen he was interested in. What might be the problem?
___________________________________________________________
___________________________________________________________
72. Inspired by her biology lab, a student decides to make a closer study of the food
she eats. She uses a razor blade to make a very thin section from a raw potato
and mounts it in a drop of water on a slide. To her disappointment, she can
barely make out the cells under the microscope. What might she do to improve
her results?
___________________________________________________________
___________________________________________________________
73. How is magnification different from resolving power?
Magnification Resolving Power
74. What are the advantages and limitations of studying cells using light
microscopy?
Advantages Limitations
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75. Compare and contrast transmission electron microscopy (TEM) and scanning
electron microscopy (SEM).
TEM SEM
Similarities
Differences
76. What are the advantages and limitations of studying cells using electron
microscopy?
Advantages Limitations
PART III: CELL LAB
OBJECTIVES
After completing this lab you should be able to:
1. Compare and contrast prokaryotic and eukaryotic cells,
2. Prepare wet mount slides of eukaryotic cells,
3. Identify each cell part and state its function, and
4. Distinguish between plant and animal cells.
INTRODUCTION:
In the 17th century Robert Hooke built a microscope powerful enough to see objects at greater
magnification than had previously been possible. Hooke used his microscope to examine a thin piece of
cork. While viewing this section of cork, he observed many individual units making up the cork. He
published a report in 1655 in which he called these units “cells” because they reminded him of the small
cubicles in which monks lived.
Other scientists began to use microscopes to examine many different plants and animals and these
scientists often saw structures that reminded them of the cork cells Hooke described. Over the next 150
years, scientists realized that all living things are composed of cells.
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With better microscopes, scientists observed that although cells vary in organization, size, and function,
all cells have the following structures:
• A plasma membrane defining the boundary of the living material,
• A region of DNA (deoxyribonucleic acid), which holds the genetic information, and
• A cytoplasm (everything inside the plasma membrane that is not part of the DNA region).
There are two basic types of cells: eukaryotic, those with a clearly defined nucleus and membrane-bound
organelles, and prokaryotic, those without a nucleus and membrane-bound organelles. The Greek word
karyon means kernel, referring to the nucleus. Thus, prokaryotic means “before a nucleus”, while
eukaryotic means true nucleus. The table on the next page compares the characteristics of prokaryotic
and eukaryotic cells.
Characteristics Prokaryotic Cells Eukaryotic Cells
Genetic Material
Located in nucleoid (region of
cytoplasm not bounded by membrane)
Consists of a single DNA
molecule
Located in nucleus
(membrane-bound compartment within the
cytoplasm)
Made up of DNA molecules
and protein. Organized into
chromosomes.
Cytoplasm
Small ribosomes.
Photosynthetic membranes
arising from the plasma
membrane in some species.
Large ribosomes.
Membrane-bound
organelles present.
Organelles are compartments
which perform specific cell
functions.
PART IV: PROKARYOTIC CELLS
1. Observe the microscopic structure of the 3 bacteria on demonstration. You are viewing the
bacteria with the oil immersion lens in place.
What is the total magnification?
____________________________________
2. Carefully draw that you see in the field of view.
Spirillum (cork-screw) Bacillus (Rod-shaped) Cocci (Round)
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3. Examine the drawing of the bacterium Escherichia coli to the right. The
cell has a cell wall, a structure different form the wall of plant cells but
serving the same primary function. The plasma membrane is flat against
the cell wall and may be difficult to see. Look for two components in the
cytoplasm: the small block dots called ribosomes give the cytoplasm its
granular appearance; the nucleoid, a relatively electron-transparent
region (appears light) containing fine threads of DNA.
4. Label the structures highlighted structures from #3 on the diagram below.
Part V: Eukaryotic Cells – Animal Cells
5. Obtain a clean toothpick, slide, coverslip, Barnes bottle of water, and Barnes bottle of methylene
blue from the supply area.
6. Use a clean toothpick to gently scrape the inside of your cheek.
7. Add a drop of water to the slide. Roll the toothpick with your cheek cells in the water drop. Add
a coverslip and throw the toothpick in the trash.
8. Methylene blue is a dye that will stain the cell’s nucleus darker than the cytoplasm. Stain your
sample by drawing a drop of stain under the coverslip by touching a piece of paper towel to the
opposite side of the coverslip. DO NOT remove the coverslip.
9. Locate the cheek cells using low power, the switch to high-power. Find the nucleus, a round
centrally located body within each cell.
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10. Carefully draw several cells as they appear under the microscope. Label the cytoplasm, nucleus,
and plasma membrane. Estimate the size of a typical cell. Record the cell size and
magnification used.
Magnification _____________
Cell size (µm) _____________
11. Wash and dry your slide and coverslip.
PART VI: EUKARYOTIC CELLS – PLANT CELLS
12. Use forceps to remove a young leaf from the growing tip of
an Elodea plant and prepare a wet mount slide.
13. Examine the leaf structure under low power. Then, study
the detail of several cells under high power.
14. Add a drop of safranin stain to make the cell wall more visible. Add the stain the same way you
stained your cheek cells with methylene blue. (Step #8)
15. You will notice many spherical green chloroplasts in the cytoplasm. These organelles function in
photosynthesis. The cell wall is a clear area outside the cytoplasm. The plasma membrane is
not visible because it is pressed tightly against the cell wall and because it is beyond the resolving
power of the light microscope. You may also see cytoplasmic streaming. This is evident by the
movement of chloroplasts along the cell wall. Microfilaments are responsible for this
intracellular movement. Toward the middle of the cell, you will find the large, water filled central
vacuole. This structure may take up over half of the cell interior. The nucleus, within the
cytoplasm, appears as a clear or slightly amber-colored body. It is slightly larger than the
chloroplasts.
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16. Carefully draw and label several Elodea cells in the field of view. Indicate where the plasma
membrane is located in the cells. Estimate the size of a typical cell. Label the plasma membrane,
chloroplasts, nucleus, cytoplasm, and cell wall.
Magnification ____________
Cell size (µm) _____________
17. Describe the three-dimensional shape of the Elodea cell.
___________________________________________________________________________________
___________________________________________________________________________________
18. Wash and dry your slide and coverslip.
19. Prepare a wet mount of onion epidermal cells using the technique described below.
20. Observe the wet mount with your microscope under low power then switch to high power.
21. Stain the specimen with iodine using the same technique you used in step #8. The stain will
increase the contrast and enable you to better view the nucleus, oil droplets, and cell wall.
22. The nucleus will be a large sphere within the cytoplasm. Examine the nucleus carefully and you
will spot several nucleoli inside the nucleus. Nucleoli are the areas within the nucleus were RNA
(ribonucleic acid) is produced. The rest of the nucleus is largely DNA.
23. Look for oil droplets in the form of granular material within the cytoplasm. The droplets are a
form of stored food for the cell (starch).
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24. What plant cell organelle is present in Elodea leaf cells that is absent in onion epidermal cells?
______________________________________________________________________________
______________________________________________________________________________
25. What is the observable difference between the Elodea cell and the onion epidermal cells?
______________________________________________________________________________
______________________________________________________________________________
26. Carefully draw several onion epidermal cells. Label the oil droplets, nucleus, cell wall, cell
membrane, and cytoplasm. Estimate the size of
a typical cell.
Magnification _____________
Cell size (µm) _____________
PART VII: QUESTIONS
27. Determine if each of the following characteristics is true of Prokaryotic cells, Eukaryotic cells, or
Both cell types.
______ No membrane-bound nucleus
______ Membrane-bound nucleus
______ No membrane-bound organelles
______ Contains membrane-bound
organelles
______ Ribosomes present
______ Cell membrane present
______ Chromosomes present
______ Cytoplasm present
______ Mitochondria, endoplasmic
reticulum, Golgi, and vacuoles
present
28. How does the size of the bacteria cell observed compare with the size of the human cheek
cells or Elodea cells?
________________________________________________________________________
________________________________________________________________________
29. Name 4 structures common to all cells.
__________________________ ________________________
__________________________ ________________________
30. List 3 differences between plant and animal cells.
31. Examine the cell at the right. Is the cell prokaryotic or
eukaryotic?
____________________________
How do you know?
____________________________
____________________________
____________________________