Download Microscopy - Napa Valley College

Exercise 1
Microscopy
Introduction:
Today’s lab is one of the most important as we
cover both safety (see separate section) and
microscopy. The microscope is one of the most
important tools in all of biology and it has led to
major discoveries for over 400 years. Robert
Hooke was the first person to see cells when he
examined the bark of an oak tree with his
microscope in 1665. Antoni van Leeuwenhoek
developed a better microscope and was able to
see living cells, including bacteria, in 1674.
We use light microscopes in this lab as they use
light to magnify and focus the specimen.
(Electron microscopes use a beam of electrons
to perform the same functions.) Two important
features of microscopes are magnification and
resolution. Magnification is simply the
enlargement of an image—we will cover that in
more detail later in lab.
Resolution is the ability of a microscope to
reveal fine detail. Technically, it is the ability of
a microscope to distinguish the distance
between two small objects. Resolution and
magnification are not the same! If you saw
Harry Potter 2, you saw an example of
resolution. You may remember that Uncle
Dersley screwed bars over Harry’s bedroom
window, and the Wealeys flew their car to
Harry’s rescue. When Harry first sees the car, it
is so far away that the two headlights appear as
one—human eyes cannot resolve the two
headlights as separate from so far away.
However, as the car gets closer, the single light
resolves itself into the two headlights. So even
though two objects may in fact be separate, the
microscope may not be able to reveal that fact.
And if the microscope cannot resolve two
images, magnifying the image will just result in
the same blur—only bigger.
The microscopes at Solano are quite good and
are an important tool for you to master the lab
material. We will use the scopes almost every
lab, so you want to be comfortable using them.
You’ll also be introduced to the major groups of
organisms that we’ll be discussing for the rest of
the semester. These include the eubacteria,
protists, fungi, plants, and animals.
Goals/Student Learning Outcomes:
Identify the parts of the microscope and briefly
explain the function of each.
Using prepared slides or wet mounts that you’ve
made yourself, focus a specimen clearly and with
appropriate illumination.
Determine the total magnification using any of
the objective lenses.
List the kingdoms that we’ll be discussing
throughout the semester.
Activities:
The compound microscopes are located in the
tall cabinets in the middle of the tables (do not
confuse them with the dissecting scopes). There
should be one for each of you. Grab the
microscope by the arm with one hand and
down. Turn the dimmer up and light should
now appear.)
support the base with your other hand. The
power cords are in the drawers above the
cabinets; plug the cord into the microscope and
the electrical outlet, some of which are located
on top of the table while others are located just
beneath the table tops on the sides.
Obtain a prepared slide (one that has already
been made for you) with the letter “e.” Hold it
up to the light or against white paper so it’s
oriented the way you would read it. Open the
slide clip and place the “e” so that the corners
of the slide fit into the corners of the slide
holder on the stage as demonstrated in this
photograph:
See the photograph below for parts of the
microscope: on/off switch, dimmer/rheostat,
light source, iris diaphragm lever, condenser,
stage, stage adjustment knobs, slide clip,
objective lens, ocular lens, course focus
adjustment, and fine focus adjustment.
Ocular Lenses
Slide clip
Stage
Now center the “e” over the hole in the stage
by using the stage adjustment knobs; these are
located to the right and below the stage.
Objective Lenses
Stage
adjustments
Iris diaphragm
lever
Fine Focus
Dimmer
Course Focus
Light Source
On/off switch
The objective lenses (as you can see there are
four with our microscopes) are attached to a
rotating ring known as the “turret.” To change
objective lenses, please grab and turn the
turret, not the lenses themselves. Turn the
turret so that the 5X objective lens is centered
over the “e”; it should make a small “click”
when centered properly.
You now want to focus the “e.” You do so by
rotating the coarse focus knob (the larger one
on either the right or left side of the scope)
toward or away from you. Note that the stage
moves when you focus; the objective lens stays
in one place. If you don’t have the “e” in focus,
raise your hand to get help from your
instructor. At this point, you may need to better
Turn the microscope on with the switch on the
right side in the back. These scopes also come
with a dimmer (or rheostat), located above the
on/off switch. (If you turn the scope on and no
light appears, the dimmer is probably turned
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from side to side as you look at the light
emerging from the hole in the stage—you
should see it brighten and dim. You can also
control the light by adjusting the dimmer
switch; again, turn the dimmer forward and
backward while you look at the light emerging
from the hole in the stage—you should see the
light brighten and dim. After the light leaves the
specimen, it travels through the objective lens;
it then moves through a series of prisms and is
reflected by mirrors so that it emerges from the
ocular lens.
center the “e” using the stage adjustment
knobs.
Also at this point, you’ll probably want to make
two adjustments to the oculars. The first is to
adjust them to match your interpupilary
distance—the distance between your two eyes.
So looking at the “e”, move the oculars closer
together or further away so that you see just
one image. (When you’re new to microscopy,
your head may move closer to or further away
from the oculars, which complicates this step.)
You may also need to adjust the oculars if your
eyes do not have the same power. If you have
contacts or glasses, your optometrist has
compensated for the fact that your two eyes
may be different. If this is the case, you’ll want
the oculars to be in the same position so turn
both oculars so the “0” aligns with the white
dot of the housing.
What is the orientation of the “e”? Right side
up?
Readable orientation?
As you observed, the lenses of the microscope
invert the image of the “e”; why will this flipflopping NOT be a problem for us in lab?
If your two eyes are significantly different from
one another and you don’t have corrective
lenses, close your left eye and focus the “e”
with your right eye. Now close the right eye
and open the left. Rotate the left ocular until
the “e” is in good focus. You should now be
able to use both eyes to see the specimen in
good focus.
Return the “e” to its proper tray. (All the
prepared slides that you will use will come in
shallow trays, which are labeled in the front.
Please return all slides to their correct trays.)
Next, obtain a “cross thread” or “cross fiber”
slide. Three different colored threads—blue,
red, and yellow—were placed on top of one
another; however, the arrangement will vary
(they are not all the same). Again, examine the
slide against the classroom lights or white paper
so you see the intersection of the threads; open
the clip to place the slide into the corners of the
slide holder; and then center the intersection of
the threads by using the slide adjustment
knobs.
It is important to make these adjustments so
that you can complete your lab work without
eye strain.
Note that light is produced in the bulb
controlled by the dimmer; it travels through the
iris diaphragm then the condenser and then the
hole in the stage through the specimen. You
can control the amount of light that comes
through the condenser by closing down or
opening up the iris diaphragm; do so by moving
the lever from side to side. Move the lever
Using the 5X objective, focus the slide with the
coarse focus knob. Now rotate the turret to
swivel the 10X objective into place. Our
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how much of the specimen you can see. The
fields-of-view with the other objectives become
smaller with the 63X objective having a
miniscule one. WHEN USING THE
MICROSCOPE, ALWAYS START WITH THE 5X
OBJECTIVE AS THAT GIVES YOU THE BIGGEST
FIELD OF VIEW AND THE GREATEST DEPTH OF
FOCUS TO FIND THE SPECIMEN. And once you
focus a specimen with the 5X objective, always
be sure to center the specimen as you move to
the 10X objective, etc. (It is easy to lose the
specimen as you move up in magnification as
the fields-of-view become smaller and smaller.)
microscopes were constructed to be “parfocal,”
that is, once you focus a specimen with one
lens, the other lenses should be close to focus
as well—of course they will be off just a bit.
Here is where you use the fine focus (the small
focusing knob) to get a crisp image of the
threads. Starting at the top of the intersection
or at the bottom, slowly move the fine focus to
determine which thread is on top, which one is
in the middle, and which one is on the bottom.
The top thread is ____________,
the middle thread is _____________, and
the bottom thread is _____________.
For the rest of the lab period we’ll examine the
major groups—known as kingdoms in biology—
of organisms that we’ll be discussing
throughout the semester. You’ll be practicing
your microscopy techniques as you go.
Return this slide to its correct tray.
When you use the 5X objective, you are actually
magnifying the specimen a total of 50 times as
the ocular lens magnifies the specimen by 10X.
Note that total magnification is a PRODUCT of
the objective and ocular lenses, that is, you
MULTIPLY the individual magnification powers
of the two lenses to get the total.
We’ll start off with the Kingdom Eubacteria.
These small organisms, generally single-celled,
are prokaryotic because they have no
membrane organelles such as nuclei or
chloroplasts (“prokaryotic” means “before
having a nucleus” and refers to organisms that
evolved before “eukaryotic” organisms that
have them).
Our objectives magnify 5X, 10X, 40X, and 63X;
remember that the ocular always magnify 10X.
So what is the total magnification when using
the 10X objective? ______ The 40X objective?
______ The 63X objective? _____
Examine the prepared slides of two
cyanobacteria: Anabaena and Stigonema. (The
art majors in class may recognize “cyan” as a
blue-green color; and the cyanobacteria were
once called the blue-green algae.) We start
with these bacteria as they are very large for
prokaryotic organisms and should be fairly easy
to see. Both of these organisms occur in chains,
though they are still considered single-celled.
Stigonema grows in branched chains, which is
uncommon in bacteria. These organisms
respire as you do, but they also photosynthesize
and fix nitrogen—the latter two topics will be
covered in detail later in the semester. But for
The different objective lenses also have
different depths-of-focus, that is, how much of
the specimen you can see in focus from top to
bottom. The 5X has the deepest depth-of-focus
while the 63X has almost none. Consequently,
when you use any of the lenses, but especially
the 40X and 63X, you’ll need to make fine
focusing adjustments if you want to see the
entire specimen from top to bottom.
The other nice thing about the 5X objective is
that it also has the biggest field of view, that is,
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now, organisms that resembled present-day
cyanobacteria were the first organisms to
develop photosynthesis and they started adding
oxygen to the atmosphere; without
cyanobacteria (and algae and plants) there
would be no oxygen for us to breath.
Solids/liquids
of yogurt
Air bubbles
Much smaller bacteria can be found in yogurt.
Humans all over the planet have figured out
that fermenting milk is one way to preserve it.
Milk is very nutritious as it contains proteins,
lipids, and carbohydrates; but you’re probably
aware that it sours/rots very quickly, especially
if kept at room temperature (many countries
still lack refrigeration). The bacteria that
ferment milk produce acids that curdle the milk,
that is, cause the proteins to clump together
into curds as well known in the nursery line:
“Little Miss Muffet sat on her tuffet eating her
curds [the clumped proteins] and whey [the
liquid portion].” These acids also protect the
milk from different bacteria that would
decompose it. In the U.S., we eat mostly cow
yogurt, but you can also find goat yogurt. In
Tibet, they make yak yogurt, in Russia horse
yogurt, and in the Middle East camel yogurt.
refracts light as you focus up and down, which
produces something of a rainbow effect as the
colors change. However, air bubbles are not of
interest in our wet mounts and we’ll ignore
them.)
Place the yogurt slide on the stage and try to
center an area on the slide that is more
transparent as you want to avoid the
coagulated proteins—the clumps or curds—in
the slide. Be aware that bacteria are composed
mostly of water and you’ve mounted them in
water, so turn the light intensity down.
(Remember the two ways to reduce light?) As
you fine focus from the 5X to the 10X to the 40X
to the 63X, you should see very small particles
floating in the water—remember to recenter
the slide as necessary. These are the acidproducing bacteria of yogurt, which are teeny
compared to the cyanobacteria.
Using a toothpick place a very small dab of
yogurt onto a slide and add a drop of water
(remember for safety reasons there can never
be any eating or drinking in the lab). Add a
coverslip by placing one end down and then
lowering the rest of it to reduce air bubbles
that might get trapped. (When students are
first making wet mounts, it is very common to
get air bubbles, and they are huge compared to
bacteria—see the photograph in the next
column.) Coverslips flatten the specimen and
provide some protection for the objective lens.
After about two billion years of evolution from
prokaryotic ancestors, protists appeared on the
planet—this occurred about 1.5 billion years
ago. These organisms are eukaryotic as they
have membrane-bound organelles such as
nuclei, chloroplasts, and mitochondria. Many
are single-celled while others are colonial
(aggregates of cells that perform the same
functions) while others are multicellular
(composed of many cells that do different
So it is very common for students to get excited
about their “discoveries,” especially as the air
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Around 500 million years ago, fungi—our next
kingdom—evolved from protists. They come in
two main forms: filamentous molds and
unicellular yeasts. Examine the slide labeled
“Cyathus,” more commonly known as the bird’s
nest fungus. When Cyathus produces its
spores, it packages them up (the eggs) and then
protects them with the “nest.” Cyathus is a
decomposer; it is uses the energy of dead wood
to grow and reproduce.
functions, e.g., most cells photosynthesize in
algae while a smaller number of them are
specialized for reproduction).
Make a wet mount of amoeba, which are
always found at the bottom of the jar—never
on the sides or floating above. To obtain a few,
expel the air out of a dropper BEFORE you place
the tip of the dropper at the bottom of the jar.
Suck up a small amount of fluid and transfer it
to your slide; remember to gently lower a
coverslip.
Yeasts are one of the most important fungi
economically—they are used for brewing
alcohol (Budweiser is just across the highway
and the Napa Valley is just a few miles away),
baking, and genetic engineering. Place just a
small drop of the living yeast culture on a slide.
As water is the largest component of yeast cells
and water surrounds them, they are difficult to
see. Biologists overcome this problem by using
dyes that the organism or parts of the organism
absorb. To your small drop of yeast cells, add a
small drop of methylene blue; then add the
coverslip. (There should be dropper bottles of
methylene blue on the cart.)
Amoebae are single-celled organisms that move
and capture food by pseudopods—temporary
extensions of their cytoplasm. Most amoeba
are free-living as they capture and digest
bacteria, other protists, and bits of organic
matter (only a few amoebae are parasites and
you may have heard of amoebic dysentery).
Amoebae are delicate creatures and they get
beat up as they are shipped through the mail
from Sacramento; so there will be quite a few
dead ones. A little searching should reveal the
living ones.
For comparison, examine the prepared amoeba
slide labeled “Chaos carolinensis” or “Pelomyxa
carolinensis.” (“-ensis” means found in or
located in, so “carolinensis” indicated that these
amoebae were first discovered in Carolina.)
If you search a bit, you should find cells that are
reproducing asexually—a process known as
budding where a larger cell produces a small
one that begins to grow and then detaches.
A photosynthetic protist is Volvox. This is a
colonial organism in the shape of a sphere.
Examine the prepared slide of Volvox (living
Volvox is green; this prepared slide has been
stained so the organisms are unnaturally
colored red or purple). It can reproduce
asexually; when it does so, daughter colonies
develop inside the parent organism. When the
parent splits open, the daughter colonies are
released.
Also about 500 million years ago, a green alga
gave rise to plants—our fourth kingdom. Make
a wet mount of Elodea, an aquatic plant.
Remove one leaf, place on a slide, add a drop or
two of water (there are dropper bottles of
water near the sink), and then add a coverslip.
You should see chloroplasts—green organelles
that are the site of photosynthesis—moving
around the cell. (The rhythmic cycling of the
chloroplasts ensures that each one receives the
same amount of sunlight as its neighbors.)
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As the final slide for today, examine the flea.
Note the piercing/sucking mouthparts at the
front. Fleas are parasites and as they feed from
one host to another, they transmit pathogens.
For the next plant—the potato—make the
thinnest section of potato tuber that you can—
a quarter of an inch is more than enough; place
it on a slide. Now add a drop of iodine (labeled
IKI) then the coverslip. You may know that
potato tubers store starch, which turns purple
with the addition of iodine. Look for the
amethyst-like starch granules.
Historically, one of the most devastating was
the Black Plague of the 1300s (though there
were plague epidemics prior to and after this
one). Fleas transmitted the bacterial pathogen
Yersinia pestis from rats and other rodents to
humans. As the bacterium kills human cells
they turn black (hence “Black Death”) as other
bacteria decompose the dead tissue.
The animal kingdom is our last one for today.
Animals are multicellular organisms that have
no cells walls. They are also the only organisms
that have muscle and nerve tissues.
This disease is also known as “bubonic plague”
for the production of buboes—swollen lymph
nodes of the groin, arm pit, or neck. Plague
killed one-quarter to one-third of Europeans in
the 14th century.
Obtain a sterilized toothpick and GENTLY scrape
(do not pierce!) the inside of your cheek.
Transfer your cheek cells to a slide; add a drop
of methylene blue and a coverslip. You should
see clumps of flattened cells—try to find one
that is separated from the others. Your nuclei,
in the middle of the cells, are stained blue—
you’re looking at your DNA! You have 46
chromosomes in your check cells and if you
placed them end-to-end, they would extend for
over six feet!
If you’re lucky, some of the bacteria that reside
on the surface of your cheek cells will also stain
darkly blue. They are generally spherical or rodshaped and much, much smaller than the cheek
cell. (The human body is made up of 3 to 200
trillion cells, yet we have 10 times that number
of bacteria on and in our bodies! These
bacteria are generally beneficial to our health as
they synthesize vitamins and protect us from
potential pathogens.)
As humans can carry bacterial, viral, or fungal
pathogens, please discard your cheek cell slide
with coverslip and toothpick into the beaker
labeled “Cheek Cell Slides.” We will add
bleach to kill any potential pathogens before
we dispose of the slides.
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Other than magnification, how does the
microscope process the image of a specimen?
For review:
Label the parts of the microscope.
When viewing a slide for the first time, why do
you always start with the 5X objective? Give at
least two reasons.
Explain why dyes are used in lab.
List the five kingdoms that we saw in today’s
lab.
If the total magnification of an image is 400X,
what objective lens was used? __________
If it is 100X? _____
Briefly describe the two ways to add or reduce
the amount of light when using the microscope.
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