Lab: Microscopy CLASS SET - DO NOT REMOVE OR WRITE ON
Bielke - Biotechnology
Introduction:
During the 1st century AD (year 100), glass had been invented and the Romans were
looking through the glass and testing it. They experimented with different shapes of clear
glass and one of their samples was thick in the middle and thin on the edges. They
discovered that if you held one of these “lenses” over an object, the object would look larger.
These early lenses were called magnifiers or burning glasses. The word lens is actually
derived from the Latin word lentil, as they were named because they resembled the shape of a
lentil bean.
Around the same time, Roman philosopher Seneca (4 BC – AD65) described actual
magnification by a globe of water. “Letters, however small and indistinct, are seen enlarged
and more clearly through a globe of glass filled with water “.
Salvino D’Armate (1258-1312) from Italy is credited for making the first eye glass,
providing the wearer with an element of magnification to one eye. Roger Bacon (1214 –
1294) , an English Franciscan monk, is the first man credited for combining two lenses and
experimenting with mirrors. This led to charges of ‘witchcraft’ against him.
The development of the lens by Zechariahs Janssen (a Dutch spectacle maker) and
Robert Hooke led to the first microscope. In 1665, Hooke published a book entitled
Micrographie, introducing the world to microorganisms. Another gentleman, Anton van
Leeuwenhoek, made more advancements in lens making and opened this world even further.
He took a sample from an abscessed tooth to examine under the microscope. He called the
organisms he saw "animalcules". Your teeth still make an excellent source for environmental
isolates.
There have been considerable advances in microscopes over the past 350 years. The
most common microscope used today is called the compound bright-field microscope. A
compound microscope as its name implies, uses two or more lenses to achieve greater
magnification than a simple magnifying glass. Working together the lenses of a student
microscope can magnify an object up to 2000 times so that objects down to 1um may be
viewed. This is about as small as a mitochondrion inside a cell.
Electron microscopes:
We can see objects in the world around us because light rays (either from the Sun or from
another light source, like a desktop lamp) reflect off them and into our eyes. No-one really
knows what light is like, but scientists have settled on the idea that it has a sort of split
personality. They like to call this wave-particle duality, but the basic idea is much simpler
than it sounds. Sometimes light behaves like a train of waves—much like waves traveling
over the sea. Other times, it's more like a steady stream of particles—a bombardment of
microscopic cannonballs, if you like. You can read these words on your computer screen
because light particles are streaming out of the display into your eyes in a kind of mass,
horizontal hailstorm! We call these individual particles of light photons: each one is a tiny
packet of electromagnetic energy. Seeing with photons is fine if you want to look at things
that are bigger than photons. But if you want to see things that are any smaller, photons
turn out to be pretty clumsy and useless. Just imagine if you were a master wood carver,
renowned the world over for the finely carved furniture you made. To carve such fine details,
you'd need small, sharp, precise tools smaller than the patterns you wanted to make. If all you
had were a sledgehammer and a spade, carving intricate furniture would be impossible. The
basic rule is that the tools you use have to be smaller than the things you're using them on.
And the same goes for science. If you want to see finely detailed things that are
smaller than photons, you need to use particles that are smaller than photons to start with: in
other words, you need to use electrons. As you probably know, electrons are the minute
charged particles that occupy the outer regions of atoms. (They're also the particles that
carry electricity around circuits.) In an electron microscope, a stream of electrons takes the
place of a beam of light and allows us to see things smaller even than light itself.
Prelab Questions: MUST copy and answer these before moving on to prelab.
1. What were early “lenses” called ?
2. What is the derivative of the word “lense” ?
3. Who is credited for the first eye glass ?
4. Whose experimentation with lenses and mirrors led to charges of witchcraft ?
5. Who is credited with the first microscope ?
6. What did Anton van Lueewenhoek do ?
7. Summarize how an electron microscope works.
Objectives:
Identify the parts of the microscope
Prepare a wet mount specimen
Perform a simple staining procedure
Correctly dispose of human cells
Materials:
Microscopes
Slides and cover slips
Newsprint (the letter "e")
Toothpicks
Medicine droppers / water
Methylene blue stain
10% bleach
Procedure:
A. The Parts of the Microscope
1.
Take a microscope from the cupboard.
2.
This is a compound microscope with two sets of lenses. One set of lenses is positioned
close to the sample or object and is called the objective lens. Look at your microscope. How
many objective lenses are there ? The shortest lens has the least magnification. It is called the
low power objective. This is usually listed as 4X (X stands for "times" so this lens magnifies
four times). The 10X lens is called medium power. The high power lens magnifies 43X. This
is a frequent viewing lens for cells. Most microscopes are parfocal. This means that when
the objectives are rotated to change the magnification, very little focusing needs to be done.
The objective lenses are mounted on the round nosepiece.
3.
The second set of lenses is close to the eye and is called the ocular lens. Look at your
microscope. Do you have one ocular lens or two ? One lens is known as monocular and two,
logically enough is called binocular. The binocular lens may be viewed with both eyes and is
much easier to see. The monocular lens is viewed with one eye. The magnification of your
ocular lens is 10X.
4.
The magnification of the microscope is the magnification of the ocular lens times the
objective lens. Thus if an ocular lens is 10X and the objective lens is 40X then the object is
magnified (10X)(40X) or 400X. Draw and complete the chart below. Pay close attention to the
objectives found on the microscope YOUR GROUP is using. Objectives will vary by
microscope.
Objective Lens
4X
10X
43X or 40x
Ocular Lens
10X
10X
10X
Total Magnification
5.
The microscope slide rests on the stage. Look at your stage. Does it have 2 clips to
keep the slide in place or is there a stage manipulator ? The stage manipulator is present on
the better microscopes. It holds the slide whose location may be adjusted by using the knobs
underneath the stage. Most of our microscopes have only stage clips.
6.
On the base of the microscope is the light source. This is usually a powerful light bulb.
Locate the on-off switch and turn on your microscope. Under the stage is the iris diaphragm
which focuses the light along with the condenser which is a light focusing lens. Look through
the microscope and open and close the iris diaphragm. Do you see any change in the light
coming through the microscope?
7.
The sample is focused by using the adjustment knobs located on the side of the
microscope. The larger knob is the coarse adjustment and is used ONLY when viewing on low
power. The smaller knob is the fine adjustment. It is used when viewing on medium or high
power. Move each of the knobs. Which one makes the objective move up and down more?
NEVER focus downward when using medium or high power objectives.
NOTE: For testing and experimental purposes, this is the microscope (parts) you will
be held accountable for. This is the one that should be placed in your lab book.
8.
Sketch and label the parts of the microscope below in your lab book:
B. Using the Microscope
The following steps are used each and every time you use a microscope.
1.
Prepare a slide of the letter "e" by placing the letter from newsprint on a clean slide.
Add one drop of water and a cover slip. Orient the slide so that the "e" is right side up as you
are looking at it and then place it on the stage under the clips. Turn on the light source.
2.
Rotate the nosepiece to place the low power objective lens over the slide. Although
microscopes are supposed to be put away so that the stage is in its lowest position and the low
objective is in place, not everyone follows this rule. Be careful when you rotate the nosepiece so
that the objectives don't bump into the slide and get damaged.
3.
Rotate the coarse adjustment knob to bring the low power objective all the way down.
The coarse adjustment knob will stop . Look through the ocular. SLOWLY focus AWAY
from the "e" (toward you) until it is in focus. Use the fine focus to bring into sharp detail.
4.
Sketch the "e" in the margin as you see it. (Remember you will use a template to draw
your field of vision in the analysis and include the magnification). Is it right side up or
upside down?
5.
Manually move the slide a little to the left. (or use the moveable stage if you have one)
Which way does the "e" move while looking through the ocular ?
6.
Practice moving the "e" around. Then write a statement correlating the way you move
the slide with the direction it moves as observed through the ocular.
7.
Center the "e" in your field of vision. The field of vision is what you see as you look
through the microscope. Now, rotate the nosepiece to place the high-dry objective (43X) in
place. The microscope is par focal so it might look like the objective will crash; but as long as
you are NOT using coarse adjustment it will be fine.
8.
Look through the ocular and use the fine focus knob to get the sample in focus.
9.
What do you see under the high-dry objective?
What happened to your field of view?
10.
Return to the low power objective and wash your slide and cover slip.
DO NOT WASH THE COVERSLIP or any materials DOWN THE DRAIN
B. Yeast cells
1.
Yeast are a tiny form of fungi that exist in or on all living matter.
2.
Place one drop of the yeast mixture on a clean microscope slide. Cover with a clean
coverslip and view on low power. Change to high power and sketch and label the cell wall, cell
membrane and cell nucleus in the margin. (remember you will complete a final drawing in
the analysis) edge of the coverslip. What happened to the cell ?
C. Human Cheek Cells
1.
With a toothpick gently scrape the inside of your cheek and place the cells onto a clean
slide. Discard the toothpick in the bleach solution.
2.
Add one drop of methylene blue stain. Add a cover slip.
3.
Observe and sketch your cheek cells in the margin under low and high power.
(remember you will complete a final drawing in the analysis)
4.
ALWAYS LEAVE THE MICROSCOPE ON LOW POWER WITH THE STAGE
MOVED TO THE LOWEST POSITION and the OBJECTIVE MOVED TO THE HIGHEST
POSITION . (this prevents accidental scratching of the objectives)
5.
TURN OFF THE LIGHT WHEN FINISHED
6.
Place the slide in the place designated by your teacher.
D. Plant cells – research online after completion of the lab. Search – “Plasmolysis of elodea
cells”. Draw, label and color the plant cell in a normal isotonic solution and in a
hypertonic / saline solution. What happens to the cell membrane in the latter illustration ?
Lab: Microscopy
Analysis – The analysis MUST begin at the top of the next clean page
in your lab book.
1.
Include all required drawings below. YOU MUST USE A TEMPLATE FOR YOUR
FIELD OF VISION. (A small or large petri dish is satisfactory for this purpose).
Color and shade as needed. Label all parts (use straight lines out to the side).
Be sure to label and provide the magnification under which it was viewed.
You should have 5 illustrations: newprint (40x), yeast(430x), cheek (40x), elodea isotonic
(40x), elodea hypertonic (40x).
Questions must be copied and immediately followed by the answer.
2.
What is the total magnification on the indicated class microscope for low power?
3.
What is the total magnification on the indicated class microscope for high power?
4.
What is meant by field of vision?
5.
Why did you have to stain your cheek cells?
6.
Why was it necessary to dispose of the toothpick/cheek cell slide in bleach?
7. Why would yeast have ever been considered to be plant-like ?
8. Discuss the changes in a plant cell from an isotonic to a hypertonic solution.
http://socrates.acadiau.ca/COURSES/BIOL/REEKIE/1113/diffuse/Salt.htm
You should include the following illustrations (Using a template for field of vision, titled,
labeled correctly, colored realistically, correct magnification) following the analysis
questions.
Letter “e” – low power
Cheek cells – high power
Yeast cells – high power
Elodea – isotonic and hypertonic from website above (or other as appropriate)