Description
During this course, we will use both bacteria and yeast as models. Based on yourobservation of the yeast slides, slides of other organisms, and EM data you haveobserved determine what type of cells yeast are. Describe below whatobservations were used in your decision. Write at least a paragraph. NOTE: you can see the procedure in the pdf below to have a brief knowledge of the things that you nee to know for this paragraph
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Lab 1
Microscope and Cells
Relevance to your life
Big Question: Why is observation a key component of all science?
Biological Relevance: Microscopes allow us to observe cells and the organelles
found within them.
Chemical Relevance: The chemistry of the components of cells and organelles
determines the methods used to view microscopic particles.
Real-World Relevance: From day one you have been using your senses to
observe and make decisions about the world around you. Learning how to
manipulate things so that you can “see” what was formerly not seen will
profoundly change your life.
Objective
– Compare and Contrast the abilities of the compound light and electron microscopes to provide
images we can perceive with our eyes.
– Learn the similarities and differences between plant and animal cells.
– Learn the types, sizes and locations of organelles in cells.
Introduction
The Cell Theory states that all living things are
composed of cells. In today’s lab you will be
observing various types of cells in order to gain an
understanding of sizes of cells and organelles that
can be found within cells. You will also be using your
power of observation; the first step in asking a
scientific question. In subsequent weeks, we will
couple your observation skills with experimental
design and data acquisition.
You will be using compound light
microscopes in today’s laboratory assignments. The
power of compound light microscopy results from its
ability to:
• view small living and non-living objects
• use colored dyes to differentiate between different
cell types, organelles or molecules that make up a
cell or organelle
• use wavelengths of light that we can see with our
eyes
• use visible light that can be transmitted through thin
sections of aqueous material (all life is water based)
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Chemistry Connection
Everything that we perceive as a
visual image, including microscopic
images, is chemistry in motion. All
light is a series of waves of differing
lengths. For us to observe anything
with a light microscope, the atoms
and molecules of the specimen must
reflect the light back to our eye.
This happens because the
wavelength of the light is reflected
by the molecules and not absorbed.
Our eye is made of light receptors
(composed of atoms and molecules)
that absorb the reflected light and
turn the received light into chemical
nerve signals that are integrated by
our brain so that we perceive the
visual image.
To gain the most benefit form this laboratory it is important that you make every effort to
learn how to use the compound light microscope properly before using it. Your TA will
demonstrate its use to you before you will be allowed to use it. Pay attention so nothing gets
broken; scientific equipment is expensive and delicate. If you don’t know how to use a piece
of equipment, just ask!
In addition to the compound light microscope, scientists use dissecting microscopes and
electron microscopes. Your TA will discuss the differences and applications for each type of
microscope. The instructor will also present electron micrographs of selected images for you
to view. Note the relative size of the cell parts and their location in the cell. While electron
microscopes are excellent for resolving very small structures like organelles, they have
several disadvantages:
• expensive to maintain and purchase
• extensive experience is needed to prepare the tissue samples
• cannot detect colors
• cannot be used to observe live organisms (water needs to be removed before
viewing in electron microscopy)
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Microscope and Cells, Lab Session 1 – Day of Class
Experiment
Part A – Instructions, 15 minutes (00:15)
Material will be presented to help you understand the context
of the experiment that you will be performing. Video Cameras
are attached to the microscopes; your TA will provide
information on how to use the camera and camera software.
There is a pull on the side of the microscope that redirects the
light to the camera. If you cannot see light coming through
the eye pieces, slide the lever.
Proper Use of the Microscope:
Your TA will demonstrate how to use the microscope properly
and will go over the parts you can and cannot touch or move. Pay attention! The
diagram below is slightly different from our microscopes. Your TA will check each
microscope and your box of slides after class to make sure you have used and cleaned
it properly and have left the microscope as it should be after use. The most important
thing is to do everything slowly and don’t force things if they do not move smoothly. Pay
attention to what you are doing and nothing will get broken.
Inform your TA if there are any problems!
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Steps for the Proper Use of the Microscope (bold words are shown on
microscope):
1. Do not move the microscope. Get out of your chair to look through it. Start on the
lowest power objective.
2. Wipe off the slide with lens paper. Place the slide on the stage and hold it in with the stage
clip. Release the stage clip slowly so you don’t break the corner of the slide.
3. Turn on the microscope, adjust the amount of light with the rheostat and open the condenser
all the way. Center the object by looking at the stage (not through the eyepieces) and
moving it with the mechanical stage controls.
4. While looking at the stage, move the stage all the way up until it stops or until you
are close to the slide. Now, adjust the eyepieces so they fit your eye width and look
through the eyepieces with both eyes open.
5. Slowly move the stage down with the coarse focus adjustment knob until the object
comes into focus. Make sure you don’t move the stage down too low. If you crank it
down too low, you will smash the bottom lens. We don’t want that to happen so
be careful!
6. Center the object of interest in your field of view. Use the fine focus to obtain the
sharpest image possible. Once you have the object in the sharpest focus, do not
move the stage up or down anymore until you are finished with that slide.
7. Now, while looking at the stage, flip the next higher power objective into place. You
will notice that the lens is now much closer to the slide (see figure below) and your
field of view is smaller. From this point on you will only use the fine focus
adjustment knob. A microscope is built to be par focal. This means that once you
are in focus on low power, you are nearly in focus and will only have to use the fine
adjustment for any higher power. So, don’t touch the coarse adjustment anymore or
you will break the slide or the lens or you will get it out of focus so much that you will
have to start over.
8. Now look through the eyepieces again and use the fine focus adjustment to get the
sharpest image. The next higher power is the 40X lens (also called the high dry lens).
Make sure there is NO OIL on the slide before you flip this one into place. Follow the
same instructions as above and remember to look at the stage while you flip the lens
into place and do not touch the coarse focus adjustment. Center the object of
interest again and use the fine focus.
To compute your total magnification, multiply the eyepiece (ocular) power
(10X) by the objective you are using. So for this lens, you will compute:
10 X 40 = 400X total magnification.
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9. Next is the oil immersion lens (100X). What is your total magnification with this lens?
The oil is used to view the object at the highest power for these microscopes. Since
light travels through the lens differently than through the air, oil is used for this power.
The oil has the same optical density, as the lens and therefore, the light will not bend
when using oil. So, move the 40X lens out of the way and place a very small spot of
immersion oil onto the center of the slide. Move the 100X lens into place and follow
the same instructions as above to focus the image. Do not use the coarse focus. If
you cannot focus the object using the fine focus, you have done something wrong
and will have to start over. Notice how close the objective lens is to the slide and
how much smaller your field of view is. When you are finished, watch the stage,
move the 100X lens out of the way and use the coarse adjustment to lower the stage
to remove the slide. Remember not to smash the bottom lens! Make sure you do not
drag the 40X lens back through the oil when you are finished. Start over with a new
slide.
10. When you are all finished with all the slides do the following:
– Remove the slide from the stage. Clean off any oil from the slide.
– Clean the stage with lens paper to remove any oil.
– Wipe the 40X lens with a new piece of lens paper to remove any oil.
– Make sure the microscope is off.
– Put the low power objective in place.
– Move the stage up to the top.
– Clean any oil off all the slides and return them to the slide box.
– Report any problems with the microscope or the slides to your TA.
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Part B – Observation, 1 hour 30 minutes (01:55)
You will need to make sketches and answer many of the questions for the lab write-up
during lab time. Please make sure you have it printed out and a copy is available for
use during class. You are going to look at many slides. Why? To gain an
understanding of a few of the many types and sizes of cells, organelles within cells and
whole organisms.
1. Use the prepared slide of letters to practice using the microscope. Follow the
instructions above so you use it properly. Put the slide on the stage so that you can
read the letters right side up by looking at the stage.
2. Look at the crossed threads and practice focusing up and down through all three
threads. Use only the 4X and 10X objectives. Do not use the 40X or 100X lens or
you will break the slide or the objectives. Always make sure the slide you are
observing is not too thick for the higher power lenses before switching.
Animal Cell
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3. Observe a typical animal cell. Using a toothpick, gently scrape your cheek to obtain
some epithelial cells. Smear the cells on a slide and add a drop of methylene blue.
Make a wet mount slide (see directions below) and observe. You may look at these
cells using any of the objective lenses. In both the animal cells and plant cells
look for the nucleus and large organelles such as mitochondria and
chloroplasts. To visualize the mitochondria use the special wet mount reagent for
mitochondria. It will precipitate a blue dye only in mitochondria.
Wet Mount Slide directions:
Place a drop of liquid (water, stain, etc.) on a clean slide. At about a 45’ angle,
bring a coverslip to the drop of liquid until it touches it. Notice how the drop of
liquid spreads out along the edge of the coverslip. Lower down the coverslip
onto the slide. There should not be any trapped air bubbles if you have done it
correctly.
Wet Mount Mitochondrial stain:
Succinic Dehydrogenase (SDH) is an enzyme found within the Krebs cycle in the
mitochondria. The reagent includes succinate (similar to citric acid found in
fruits) and a dye that turns blue and precipitates when the SDH enzyme reaction
occurs. Place a drop of the mitochondrial stain on the slide and coverslip as
above. Wait 5 min. Observe.
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4. Observe a typical plant cell. Place an Elodea leaf on a slide and make a wet mount
preparation using a drop of water. Observe the cells. Focus up and down through
the Elodea cells to gain an understanding of the three dimensional shape of these
cells. Observe the differences between the cheek cells and the Elodea cells.
Plant Cell
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5. Examine the prepared slide of a human blood smear. Sketch one field of view (find
a field of view with several types of blood cells in it) and observe the differences and
similarities in these cells as compared to the cheek cells.
5. Observe the prepared slide of hydra using only the 4X and 10X objectives. Hydra is
a member of the Kingdom Animalia (therefore multicellular).
6. You have available four different live organisms from the Kingdom Protista for observation;
Amoeba, Euglena, Volvox, Paramecium. They are unicellular organisms that have developed
an extraordinary complexity within a single cell. Carefully observe at least three of the
organisms and make sketches. Be sure to include any similarities and differences between
these three organisms. Label the organelles that you can distinguish.
7. There will also be some dissecting scopes with live planarians for you to view. These are
multicellular organisms from the Kingdom Animalia, Phylum Platyhelminthes. We will be
using these for your Regeneration Lab 8.
8. Observe the prepared slide of bacterial types. These belong to the Kingdom Monera
and are prokaryotic cells. All other cells you have observed so far are eukaryotic
cells. This slide contains Gram + and Gram – bacteria.
9. Make a smear of the live
bacteria and stain them for
mitochondria as above.
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Part C – Unknown, 35 minutes (02:30)
1. You will be provided with a few slides of unknowns (yeast and bacteria). Using your
powers of observation and what you have learned in Part B, fill out the following grid.
Prokaryote or
Eukaryote?
Plants or Animals?
Organelles? Yes or No?
Yeast
Bacteria
2. What critical observations enabled you to make this classification?
3. When everyone has completed this step, your TA will show you an electron
microscope section of a yeast cell. Were you correct?
Part D – Clean Up, 5 minutes (02:35)
Clean the slides, slide box and microscope with lens paper to remove oil and dirt.
Make sure the microscope is left properly for the next lab group. Your TA will
check to make sure you did it correctly. Clean your lab bench.
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