LAB MODULE I: BASIC MICROSCOPE USE
OBJECTIVES: After completing this section on basic microscope use, you should be able to
1. Establish and maintain an updated lab book.
2. Describe and demonstrate the correct handling and use of the microscope.
3. Identify the parts of the microscope and describe the function of each.
4. Describe and differentiate between resolution and magnification.
5. Calculate the resolution for an objective lens.
6. Calculate the total magnification achieved when different objective and ocular lenses are used.
7. Explain the meaning of parfocal and working distance.
8. Calibrate an ocular micrometer and calculate the size of organisms under the microscope using
correct units.
9. Properly use the microscope to observe, identify, and draw to scale a number of assigned
specimens.
10. Describe and demonstrate the proper procedure for putting the microscope back into storage.
11. Compare and contrast the images obtained by bright field, darkfield, phase and electron
microscopy.
REFERENCES:
BLMB: Unit II
ECB: Chapter 1. Introduction to Cells (pgs. 1-38)
ECB: Panel 1-1 Light and electron microscopy (pgs. 8 & 9).
ECB: Panel 1-2 Cells: the principle features of animal, plant, and bacterial cells (pg. 25).
Web site: http://www.microscopyu.com/
Sepich and Solnica-Krezel, Analysis of Cell Movements in Zebrafish Embryos, Methods in
Molecular Biology, vol 294
EXERCISES FOR BASIC MICROSCOPE USE:
1. Documentation in a lab book
2. Using the Bright Field Microscope
3. Preparing a wet mount
4. Measurements – Calibration of an ocular micrometer
5. Morphometric Measurements in zebrafish embryos
MATERIALS AVAILABLE IN LAB:
brightfield microscopes
microscope slides and coverslips
lens paper
millimeter rulers
ocular micrometer
phase contrast microscope (only one available)
prepared slides
immersion oil (Type A)
stage micrometers
living samples, including zebrafish embryos
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Exercise 1: Documentation in a Lab Book
1. All students will keep an up-to-date lab notebook.
2. Minimal items that will be required include:
a. Every page in the lab book numbered.
b. A Table of Contents at the front of the book (updated on a weekly basis).
c. Every entry must include the date and time.
3. Write your name on the edge of your lab book with a permanent marker and number the pages
within your book.
4. Reserve the first one or two pages for a Table of Contents.
5. All text in lab books must be hand-written. The insertion of all figures, tables and graphs that
are not hand-written must have prior approval from the instructor before being inserted in the
lab book.
Exercise 2: Using the Bright Field Microscope
We will start our lab by reviewing the steps for properly using the bright field microscopes. (It is
assumed that you have had some prior knowledge of microscope use.) Everybody should get a
microscope from the cabinet. After you select a microscope, carry it back to your bench space by
grasping the arm of the microscope with one hand and supporting it under the base with your other
hand. At this time, perform an inspection of the microscope as follows:
a. Carefully and completely unwind the electrical cord.
b. Check for any obvious loose or missing parts.
c. Position the stage at the lowest setting.
d. Place the lowest objective in down position.
e. Move the condenser up close to the stage.
f. Open the iris diaphragm.
g. Make sure lens paper and lens cleaner are available.
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Collect the following materials for completing the first part of today’s lab. (Item’s location, if not
available on the lab bench)
2 slides (3”x 1” x 1 mm) (A-3)
2 coverslips (22 x 22, Number 1 thickness) (A-3)
lens paper (A-4)
stage (objective) micrometer (A-2)
1 box of prepared slides/pair (A-5)
Procedure of use:
1. Plug the microscope into an electrical outlet.
2. Identify the following:
ocular – the ocular (eyepiece) is the primary
magnifying lens of a compound light microscope.
Microscopes with one ocular are called monocular
scopes while those with two are binocular scopes.
When using a monocular scope both eyes should
be kept open – don’t squint one eye.
ocular
arm
objectives
stage
focus knob
fine (inside)
course (outside)
condenser
light
Record the magnifying power (MP):
base
objectives – the objectives magnify and resolve
the specimen. They are set in a revolving
nosepiece so that the user can easily change
lenses. When changing the objective, turn the
revolving nosepiece by the grooved ring – do not
pull on the objectives directly! The objective
should “click” into a downward working position.
ocular
arm
objectives
stage
Record the magnifying power (MP) and numerical
aperture (NA) of all objectives
MP:
NA:
MP:
NA:
MP:
NA:
condenser
stage adjustment
knobs
focus knob
light
base
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light switch
Determining the resolution of a lens:
The ability of a lens to distinguish two points as separate entities is referred to as its resolving
power. Magnification only refers to an enlargement of an object. The lens in the human eye can
resolve objects ~ 0.2 mm apart (any closer and the objects are seen as one point).
The resolution of a microscope lens is limited by:
1. Quality of the lens – noted by its numerical aperture (NA)
2. Wavelength of light (400 nm-700 nm)
To calculate the resolving power of each of your objectives use the following formula:
Resolution = 0.61 (λ)
For our calculations assume λ = 450 nm (blue light)
NA
Record the resolving power for each of the microscope objectives.
10X:
40X:
100X:
stage – the stage is the platform that holds the slide specimen. Once the slide is placed properly
within the slide holder, you can easily move the slide in all directions by turning the appropriate stage
adjustment knob.
focus knobs – the focus knobs raise or lower the stage to bring the specimen into focus. The outside
knob is the course focus, the inner knob is for fine focusing.
light – the substage light serves to illuminate the specimen. The light is focused into a beam by a
condenser lens located just below the stage. The condenser can be raised and lowered. Located within
the condenser is an iris diaphragm that can be used to control the amount of light passing through the
condenser lens.
3. Make sure the stage is in its lowest position. Use a Sharpie marker to place a spot (~1 mm diameter)
on the slide close to the edge of the coverslip on a prepared slide, and then place the slide correctly
on the stage. Turn on the light switch. Using the stage adjustment knobs center the spot over the
hole in the stage.
4. Rotate the lowest power objective (10X) into its working position. (DO NOT TOUCH THE
OBJECTIVES WHEN YOU ROTATE TO A HIGHER OBJECTIVE.) Slowly turn the course focus
knob until the spot comes into view. The fine focus knob can then be used to sharpen the image. Once
you have focused on the spot move the specimen into view.
5. Once you are focused on the specimen at low power, you may change to a higher power objective.
Notice that the specimen should stay in focus as you change from one objective lens to another. This
property of a microscope to stay in focus throughout objective lens changes is referred to as
parfocal.
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Draw in your lab books what you see under the prepared slide you used using both the 10x and
40X objectives. Do not use the 100X objective!. Make sure you label your drawing, include what
you are looking at (look at the label on the slide) as well as the total magnification.
Exercise 3: Preparing a Wet Mount
Animal Cells
1. Clean and label a fresh microscope slide “Animal Cells”.
2. Place one drop of saline in the middle of the slide.
3. Take a toothpick and gently scrape the inside of your cheek.
4. Stir the cells collected from your cheek into the saline.
5. Add one drop of the stain provided.
6. Place a coverslip on top of your stained cells.
Draw in your lab books what your cheek cells look like under the 10x and 40X objectives. Do not
use the 100X objective!. Make sure you label your drawing, include what you are looking at
(cheek epithelial cells) as well as the total magnification.
Plant Cells:
1. Clean and label a fresh microscope slide “Plant Cells”.
2. Place one drop of saline in the middle of the slide.
3. Place one elodea leaf on the saline drop (no stain is needed).
6. Place a coverslip on top of the leaf, gently flatten.
Draw in your lab books what the plant cells look like under the 10x and 40X objectives. Do not
use the 100X objective! Make sure you label your drawing, include what you are looking at
(cheek epithelial cells) as well as the total magnification.
Exercise 4: Measurements – Calibration of an Ocular Micrometer
Not all the microscopes in the lab will have ocular micrometers (in fact most do not). However, we can
still determine the width of each objectives field of view and be able to estimate the size of a
specimen under the microscope.
Procedure:
1. Place a stage micrometer on the stage and find the little ruler. Each of the smallest divisions on
these stage micrometers are 0.01 mm or 10.0 µm (micrometers). Now might be a good time to review
units of measurement. (There is a table on the inside cover of you lecture textbook, that you can copy
and place in your lab book for future reference.)
2. Make note of the width and length of the pointer using each objective. (Do not use the 100X
objective!)
10X:pointer width
pointer diameter
40X:pointer width
pointer diameter
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3. If you have an ocular micrometer, you simply align the marking of both the stage and ocular
micrometers and determine the distance between each mark on the ocular micrometer. Think of the
stage micrometer as your control – the distance between the marks is always 10 µm. The distance
between the marks on the ocular micrometer changes depending on the magnification of the objective
you are using, but once you calibrate the ocular micrometer for a given objective you don’t have to do
it again.
At this time, go back to your previous drawings and make note of the size of objects you drew. How
big are cheek cells?
How long & wide are plant cells?
What are the green structures in the plant cells?
How big are they?
Exercise 5. Morphometric Measurements in Zebrafish Embryos
1. Study the figure that represents the developmental stages of a zebrafish embryo.
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