IB Biology Name _________________________
How to Use a Microscope Period ____ Date _____________
Introduction: Learning to use a microscope skillfully is one of the challenges for biology students.
Additionally, it is important for students to learn how specimens are prepared for study with a microscope.
Furthermore, students must learn how to communicate what they observe while using a microscope.
Step 1 Familiarize yourself with the parts of a microscope including the name and function of each part. Learn
how to clean this valuable instrument.
Step 2 Use the correct sequence of steps when finding & focusing a specimen.
1. Set the scope so that the low power objective is clicked into place
2. Use the coarse adjustment knob to put the stage and the objective as close together as possible.
3. Set the light so that it passes through the diaphram through the objective and into the eyepiece. If
you are looking at a very thin or small specimen, decrease the amount of light by adjusting the
diaphram; you may also need to adjust the light when you change the objectives.
4. Set the specimen onto the stage, moving it until a shadow of the specimen or movement is seen. Use
of stage clips is optional.
5. Focus with the coarse adjustment knob; fine tune the focus using the fine adjustment knob.
6. Only after doing these steps can you use higher power objectives. Center your specimen in the field
of view. Without changing the adjustment knobs, carefully turn the nosepiece to the next higher
power (yellow line). Fine focus only. Because our microscopes are parfocal, you should now see
your specimen. After focusing at this power, center the specimen, rotate the nosepiece to the next
higher power and focus with the fine adjustment knob.
Remember When you change to a higher power of magnification you can see the object in more detail,
but you cannot see as much of the field of view as you could before. The depth of field will also be
reduced as you increase the power of magnification. When hunting for something use lower powers
first.
7. Oil Immersion: This special lens is used with extremely small specimens like bacteria. To use it,
remove the scanning objective (red line) and replace it with an oil immersion lens (white line). After
finding & focusing the specimen as in steps 4,5,6, then move the objectives so that there is a space
over the slide. Put a drop of immersion oil onto the specimen. Carefully turn the oil immersion lens
into the oil. Fine focus until your specimen is clear. At this magnification (1000x) each person who
looks at your slide may need to adjust the focus using the fine adjustment only.
8. Clean the oil off of the lens immediately after using it. Immersion lenses are ruined by failure to
clean them immediately.
Step 3 Practice using the instrument. You will notice several irritating things:
--Objects seem to move the opposite way from what you are expecting.
--Live specimens often take a dive and seem to disappear before your eyes.
--You may get a specimen right on the end of the pointer, you turn the eyepiece and the pointer has
moved. Since the pointer is in the eyepiece, it moves whenever the top of the microscope is moved!
--You may get tired or you may see only "eyelashes"; Cover your eye that is not looking through the
microscope with one of your hands; OR Make a ring of your finger and thumb and put around the eye
that is looking into the eyepiece. Who knows why these things work?! They do seem to help until you
become proficient. A proficient person can keep both eyes open, look with one eye, and draw with the
other eye!! Good Luck!
Dumas 09
Step 4 Make slides for various uses. Check off each type of slide as you do it during our series of activities. Use
the skills development record
Type 1: Protozoa or Algae Place a drop of water expected to have the specimen onto a slide. Add stain if
desired. Add a coverslip. Observe.
Type 2: Simple Wet Mount Place a drop of water on a clean slide. Add a thin piece of the specimen. Add
stain if desired. Place coverslip onto the slide. Observe.
Type 3: Hanging Drop Slide Place a drop of mixture to be observed on a cover slip. Put a ring of vaseline
around the depression of adepression slide. Turn the depression slide onto the coverslip allowing
the ring of vaseline to surround the drop of mixture. Turn the whole thing right-side up. Observe.
Type 4: Fixed Bacteria Preparation Place a drop of water onto a clean slide. Using a flamed loop, transfer
bacteria from a culture to the drop of water; mix it well; spread the droplet as thin as possible.
Heat fix the bacteria to the glass by passing the slide with its water-
bacteria mixture over a flame a few times. Be careful not to boil the water, as that confuses your
view later. Some people like to wait until the water has completely dried before heating the slide.
Stain the slide using the technique of your choice. Staining and
rinsing the slide will not remove the bacteria; However, if you rub the slide to dry it, the bacteria
may be removed.
Slides prepared in this manner do not need coverslips.
STAINS: There are many types of stain used depending upon what you want to see and what kind of microscope
you are using. Stains kill the specimen. Usually the effect is a stop-action, but sometimes stains
cause distortion.
IODINE & METHYLENE BLUE are often used to make the nucleus of cells show.
GRAM'S STAIN is used to help classify bacteria since some bacteria will turn purple with this stain
because of the chemical nature of that species, but other species will remain pink in the presence of
this stain. Bacteria species fall into one or the other category. We call the purple ones Gram
Positive; the pink ones are called Gram Negative.
WRIGHT'S STAIN is used with blood to make the special granules and the nuclei of white blood
cells show up. With this stain it is possible to tell white blood cells from red blood cells.
Step 5 Drawing & Diagramming
Learn to represent what you are studying in a drawing and display it well. This means that you draw
a sample or an example of the specimen, not everything that you see. You will have to learn to see
your specimen without the garbage or artifacts that often accompany your specimen onto the slide.
Display your work so that is attractive and communicative. Review the recommendations in the
handout entitled Scientific Illustration by Russell Glenn.
Use a simple line drawing that occupies at least 14 of a page. Give your drawing a title. Label
parts. Do this using labels lettered parallel to the top edges of the page. Lines are to be drawn with
a ruler from the labels to touch the parts indicated. Label lines must not cross. Use pencil and
unlined paper.
Do not draw a circle to indicate the field of view unless you are drawing everything within the field
of view exactly as it appears. This is seldom appropriate.
A diagram is a transformation of an observation. Whereas a drawing is raw data showing what the
object looks like from full direct observation, a diagram reveals the outlines of features for the
purpose of explanation.
Step 6 Measuring
Drawings are to be drawn proportionally. This means that the relative sizes of parts are accurate.
Use a ruler to obtain accuracy when you draw.
Obtain a measuring scale. Then using the arrow with its "ruler" (ocular micrometer), estimate the
size of the organism in microns. Notice that the scale of the micrometer changes whenever you
change objectives. Next to your drawing write the actual size of your organism.
Deluxe drawings also indicate the degree of magnification of the drawing over the actual size of
the specimen. To do this, measure the dimension on your drawing that you measured of your
specimen with the micrometer. Measure your drawing in millimeters; convert millimeters to
microns by multiplying by 1000. Then divide the size of your drawing by the size of your specimen.
The answer will indicate the percent enlargement that you have drawn.
Another way of thinking about this:
actual size multiplied by what number equals drawing size
actual size times X = drawing size, where X is magnification
NOTE: The powers of magnification that you used to look at the object is determined by the lenses
that you used to do the observation. If you multiply the number on the objective times the number
on the eyepiece, you will know how much magnification the microscope provided to you eye. This
is not your drawing magnification.
Step 7 Microscope Care & Clean up:
Carry only one microscope at a time in an upright position with one hand on the arm and one hand on the base.
Set the microscope on a flat table that is orderly and has only lab equipment on it. Use only lens paper when
cleaning the lenses. Dust other parts with a soft rag. Keep the equipment dry.
Three things need to be cleaned: the Microscope, the slides & the work area
MICROSCOPE: Replace the oil immersion objective with the scanning objective
Clean all objectives and eyepiece with lens paper.
Roll the scanning objective as close to the stage as possible
Wrap the cord around the base of the 'scope
Return the 'scope to its place on the shelf or cart.
SLIDES: Slides are to be cleaned with soapy water, rinsed and polished.
WORK AREA: Return all supplies to the designated area cean, dry and polished.
SPECIMEN PREPARATION FOR MICROSCOPY
When scientists want to prepare cells or tissues for viewing under a microscope several questions must
be asked:
What do I want to see?
Can this be seen in a living cell? or not? What kind of tissue am I using? Will the state-of-being
of the tissue at the time I "fix" it influence what I am seeing. (NOTE: To "fix" tissue means to
stop it and make it stable & unchanging. Fixing may be done by some stains, by chemicals
such as alcohol, formalin and by heat.
What do I need to do to gain an accurate picture? stain? slice? fix? fracture?
Different stains make different parts show up. Some stains make one part show up while
destroying another. Most stains cause death, some fix the tissue making it stable. Some stains
are applied before cutting the tissue; others are applied afterward. This depends upon the stain
being used. Staining is called enhancement.
When something is sliced, getting it thin enough can be a problem. So, resins or paraffin are
used to infiltrate the tissue so that the tissue won't collapse as it is being sliced. But you cannot
force paraffin into a water system, so you must first dehydrate the tissue before infiltrating it.
Furthermore, when slicing a tissue, it is important to record the direction of the slice (cross-
sectional, longitudinal, or some other cut.)
Which microscope will I use?
The manner of tissue preparation will be determined by which type of microscope you will be
using. Electron Microsopes (TEM or SEM) require metal stain to deflect the electrons onto
photographic film. Without this metal enhancement, the electron would pass right through the
object. Light microscopes are able to “see” objects that are larger than the wavelength of
light. Good resolution occurs with oil immersion and light microscopes so that objects about
1 micron are clear. Electron microscopes allow scientists to see objects that are smaller than
the wavelength of light. Most of our images of organelles are from electron microscopes. The
images are all black and white as light is required to have color. Furthermore, the stains use
metals which kill the specimen. TEM examines one plane of a specimen, while SEM examines
the surface of a specimen. So the choice of microscope returns us to the question, What do I
want to see?
What errors have I introduced because of my techniques?