Download The Care and Feeding of the Microscope Lab 5

BIOLOGY I - LAB
Lab 4: The Care and Feeding of the Microscope
Lab 5: Cell Structure
Evelyn I. Milian
Instructor
2012
BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
OBJECTIVES OF THIS LAB
1. Learn to recognize and use the
parts of a compound light
microscope.
2. Determine magnification power.
3. Focus a microscope.
4. Learn how to make a wet mount.
5. Examine structures of plant and
animal cells.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
FIGURE 3.2
Relationships between the sizes of
various specimens and the resolution of
the human eye, light microscope, and
electron microscope.
It takes a microscope to see most cells
and lower levels of biological organization.
Cells are visible with the light microscope,
but not in much detail. An electron
microscope is necessary to see eukaryotic
cell organelles (such as the mitochondrion
and the nucleus) in detail and to observe
viruses (noncellular parasitic agents) and
molecules.
In the metric system, each higher unit is
ten times greater than the preceding unit.
Measurements: 1 centimeter (cm) = 10-2
meter (m) = 0.4 inch; 1 millimeter (mm) =
10-3 m; 1 micrometer (µm) = 10-3 mm =
10-6 m; 1 nanometer = 10-3 µm = 10-9 m.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
FIGURE 3.2 – Relative sizes of objects.
Sizes are shown on a metric scale; names in red are organisms studied in microbiology.
Chlamydia and Rickettsia are groups of bacteria that are much smaller in size than other
bacteria. The range of effective use for various instruments is also depicted.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
Compound Light Microscope (LM)
• It has multiple lenses and uses visible
light as the source of illumination.
• Visible light is passed through a
specimen and then through glass
lenses that magnify the image.
• The image from the objective lens is
remagnified by the ocular lens.
• Total magnification of a specimen =
magnification of objective lens 
magnification of ocular lens.

Example: 10X (objective lens)  10 X
(ocular lens) = 100X (total magnification)
• Maximum resolving power: 0.2 µm.
• Maximum magnification: 1000-2000X.
“X” means “times magnified”.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
COMPOUND LIGHT MICROSCOPE (LM)
• When focusing a specimen with the
microscope, the objective lenses must be
used from the lowest magnification lens
to the highest magnification lens.
• Bring the specimen into sharp focus before
trying to observe it with the next higher
magnification objective lens.
• The sequential steps for the correct use of
the objective lenses are:

4X → 10X → 40X → rotate halfway to
the next lens and add one drop of
immersion oil directly over the area of
the microscopic slide to be observed →
100X. *The immersion oil increases
the resolution and is used only with the
100X lens, or “oil immersion objective”.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
COMPOUND LIGHT MICROSCOPE
• Refractive index is the relative
velocity with which light passes
through a substance (the lightbending ability of the medium).
• When two substances have
different indexes of refraction,
light will bend as it passes from
one material into the other.
• The light may bend in air so much
that it misses the small highestmagnification lens.
• Immersion oil is used ONLY with
the oil immersion objective lens
(100X, the highest magnification)
to keep light from bending.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
Using and Focusing the Compound Light Microscope
6.
Start with the lowest power
objective lens in position, by rotating
the nosepiece. The lowest objective
is usually 4X (the shortest objective).
7.
1.
2.
3.
4.
5.
Move the stage all the way up using
the coarse focus knob.
8. Look into the ocular lens in the
eyepiece, and use the coarse focus
Switch the microscope on (make sure
knob to clarify the image.
the light works).
9. Once you have a nearly clear image,
Lower the mechanical stage as far as
use the fine focus knob to get the
it will go, using the coarse focus knob.
clearest image.
Place the microscopic slide on the
stage, securing it with a stage clamp. 10. To increase the magnification, put
the 10X objective in position.
Make sure that the slide is not upside
These parfocal microscopes will
down or backwards.
automatically be in focus or at least
Move the slide with the stage
be close to in focus.
adjustment knobs so that the area
of interest is in the center of the light
11. Use only the fine focus knob after
beam.
this point to sharpen the image,
Adjust the amount of light with the
when using 40X and 100X. * Use
diaphragm.
immersion oil with the 100X.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
LIGHT MICROSCOPY:
Dissecting Microscope (Stereo Microscope)
• It differs from the compound
microscope in that it has two
(rather than one) objective lenses
for each magnification.
• Binocular: It has two oculars.
• Objects can be observed in three
dimensions.
• The resolution and magnification
capabilities are less than in a
compound microscope.
• Magnifications usually range from
4X to 50X.
• It is often used for the microscopic
dissection of specimens.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
MICROSCOPY: Magnification, Resolution, Contrast
•
•
•
Magnification is the ratio of an object’s image size to its real size; magnification
simply makes the objects or specimens appear larger.
Resolution (resolving power) is the ability of an optical instrument to distinguish
between two points that are close together, so that they are seen as separate
units; it is the ability of the lenses to distinguish fine detail and structure (or a
measure of the clarity of the image).
 Example: A microscope with a resolving power of 0.4 nm can distinguish
between two points if they are at least 0.4 nm apart.
Contrast is the effect of a striking difference, as in color or tone, of adjacent parts
(for example, in a photograph or image). It is based on the differential absorption of
light by parts of the specimen. Contrast accentuates differences in parts of the
sample and is also an important parameter in microscopy.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
Preparation of Specimens for Light Microscopy
• Staining increases contrast—a striking difference, as in color or
tone, of adjacent parts (for example, in a photograph or image) based
on the differential absorption of light by parts of the specimen:

Microscopists improve contrast by coloring specimens with stains (dyes)
that bind to cellular structures and absorb light to provide contrast.

Live or unstained cells have little contrast with the surrounding
medium. However, researchers do make discoveries about cell
behavior looking at live specimens.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
Preparation of Specimens for Light Microscopy
• Wet Mounts

Technique in which a drop of medium
containing the organisms or cells is
placed on a microscope slide and
covered with a coverslip; it can be
used to view living microorganisms
or to stain specimens to observe them.
1) Take a small amount of the sample to be studied.
2) Place a drop on a
microscope slide.
3) Place one edge of the coverslip on
the drop and carefully pull the drop
along. When the coverslip is in
position, gently let it drop.
4) Sample is ready to observe
under the microscope.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
Preparation of Specimens for Light Microscopy:
Wet Mount with Staining
Place a drop of water on the slide.
Place your sample in the water. In
some cases you may need to spread it.
Gently lower cover slip onto liquid; start with
it upright touching one side of the liquid.
If not staining, view
under the scope
If staining, place a drop of stain on
one side of the cover slip and a piece
of absorbent paper on the other side
(to suck the stain through).
View under the microscope .
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
Bacteria
Microscopic Algae
Protozoa
Fungi: Molds
Fungi: Yeasts
Parasitic Microscopic Worms
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
Protozoa,
Algae, and Cyanobacteria
PROTOZOA: Unpigmented, eukaryotic
microoorganisms; larger than bacteria.
ALGAE: Greenish or goldenbrown eukaryotic microorganisms;
larger than bacteria.
* To observe the
cyanobacteria, you need
patience!!! Remember
that bacteria are
prokaryotic and are
smaller than eukaryotic
microorganisms.
CYANOBACTERIA:
Blue-green, prokaryotic
microorganisms; smaller
than eukaryotic
microorganisms.
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
COMPARISON OF ANIMAL CELLS AND PLANT CELLS
CHEEK CELLS: Some nuclei are visible inside the cheek cells.
ONION: Onion cells stained
with iodine (cell walls and
some nuclei are visible)
ELODEA PLANT: Leaf cells in Elodea, an
aquatic plant (weed), showing chloroplasts inside
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
Similarities and differences to pay attention to:
• Different types of cells have different features, shapes,
and sizes.

Those differences usually enable cells to function
differently from one another.

Form follows function!
• In this lab, you will be viewing different types of cells
and structures.
• Particularly, you should pay attention to similaries and
differences between:

Animal cells, plant cells, protist cells
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
Common Mistakes in Microscopy
• Common mistakes that lead to problems:
1.
Too little or too much light—check your condenser and diaphragm.
2.
Not in the correct focal plane—keep adjusting coarse and fine focus.
3.
In a bad field—not all visual fields will contain specimen. Once
focused properly, keep moving the stage side-to-side until you find
your sample.
4.
Slide upside down—when preparing your own slides, make sure to
mark the side of the slide that was inoculated with the sample (such as
bacteria or plant cells).
5.
Dirty lens will make everything look blurry—Wipe the lens with
alcohol and lens paper. * Do not use regular paper towel or drying
paper for the lens!!!
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BIOLOGY I – Labs 4 and 5: Microscope / Cell Structure
References
•
Audesirk, Teresa; Audesirk, Gerald & Byers, Bruce E. (2005). Biology: Life on Earth.
Seventh Edition. Pearson Education, Inc.-Prentice Hall. NJ, USA.
•
Campbell, Neil A.; Reece, Jane B., et al. (2011). Campbell Biology. Ninth Edition. Pearson
Education, Inc.-Pearson Benjamin Cummings. CA, USA.
•
Mader, Sylvia S. (2010). Biology. Tenth Edition. The McGraw-Hill Companies, Inc. NY, USA.
•
Presson, Joelle & Jenner, Jan. (2008). Biology, Dimensions of Life. The McGraw-Hill
Companies, Inc. NY, USA.
•
Solomon, Eldra; Berg, Linda; Martin, Diana W. (2008). Biology. Eighth Edition. Cengage
Learning. OH, USA.
•
Starr, Cecie. (2008). Biology: Concepts and Applications Volume I. Thompson
Brooks/Cole. OH, USA.
•
Tortora, Gerard J.; Derrickson, Bryan. (2006). Principles of Anatomy and Physiology.
Eleventh Edition. John Wiley & Sons, Inc. NJ, USA. www.wiley.com/college/apcentral.
•
Tortora, Gerard J.; Funke, Berdell R.; Case, Christine L. (2010). Microbiology An
Introduction. Tenth Edition. Pearson Education, Inc.-Benjamin Cummings; CA, USA.
www.microbiologyplace.com.
•
Trefil, James & Hazen, Robert M. (2011). The Sciences An Integrated Approach. Sixth
Edition. John Wiley & Sons, Inc. NJ, USA.
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