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Lab 2: Microscopy & Tests for Biological Molecules

Week of September 9, 2013
Name: _________________________________ Lab section: ____________

Prelab Exercises

*Complete this page as a Pre-Lab Activity*
Turn in this page when you arrive at your lab session.


1. Copy down the equation for total magnification found on page 4 of this lab.



2. Using the equation above, fill in the correct values for total magnification in the following
table (ocular magnification is 10x):
Scanning power 4x objective total magnification = _____x
Low power 10x objective total magnification = _____x
High power 40x objective total magnification = _____x

3. The large circle represents the field of view for a microscope image at 4x magnification. If the
diameter of the field of view is 4 mm, how long is the little critter were examining?
















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Lab 2: Microscopy & Tests for Biological Molecules
Week of September 9, 2013




Preparation
1. Read the entire exercise before lab and mark anything you will need to ask about. This
will prepare you to work more efficiently in lab because you might not then have to read
through all of the background information while you could be doing the activities. We
will be able to tell who is prepared and who did not read their lab material thoroughly.
2. Study the included microscope diagram so that you can locate microscope parts by name.


Reference
The attached pages on microscope parts/function, microscope care, and how to prepare a wet-
mount slide, will be a valuable resource for this lab and future labs during the semester.


Objectives

1. Know the names, functions, and correct operation of microscope parts.
2. Use good technique in preparing and studying fresh specimens.
3. Use good technique in drawing and labeling diagrams.
4. Estimate the true size of objects seen in the microscope.
5. Know the names of the basic tests for carbohydrates and proteins.
6. Be able to accurately interpret the results of the tests for biological molecules
7. Use your knowledge of the basic tests for carbohydrates and proteins to determine what is
in an unknown solution.












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Part I: Microscopy

Resolution and Magnification
Microscopes are magnifying tools used to produce enlarged images of tiny objects to bring them
into the range of resolution of the human eye.
Magnification refers to how many times bigger the image is than the object itself. It is
measured in terms of the number of times the diameter is increased10x, for example, means
that the diameter of the image is ten times the diameter of the actual object.
Resolution, or resolving power, refers to the clarity of the image and is measured as the
smallest space that can be seen between nearby objects. The human eye cannot distinguish
between objects that are closer together than about 0.1 mm; they blur together and look like a
single object. The resolving power of the eye is therefore said to be about 0.1 mm (or 100 m).
This also means that the unaided eye cannot detect an object less than 0.1 mm wide, which is
about the width of a human hair, because it blurs into the background. If the object is magnified
so that the eye looks at a larger image, it can be seenif it is magnified accurately. A microscope
(or any other magnifying system) is useful only if its resolving power is better than that of the
eye. A microscope with poor resolution but high magnification simply produces a large fuzzy
image. As photographers know, if you want to blow up a picture to a very large size and still
have sharp details, you need to start with a high-resolution image.
Resolving power depends on (1) the quality of the lenses and (2) on the wavelength of
light used. Excellent light microscopes can resolve objects that are about 0.2 m apart. This
means also that objects only 0.2 m wide can be seen with such a microscopeprovided they
are magnified enough to bring the magnified image within the resolving power of the eye. This
would require a magnification of at least 500 times, which is a little more than the microscopes
we use in lab can produce. But our microscopes do not have lenses capable of resolving such
small objects, so higher magnification would be wasted.
Electron microscopes use a beam of electrons instead of a beam of light. The wavelength
of a beam of electrons is many times smaller than the wavelength of any visible light, so an
electron microscope can resolve much smaller objects. But an image cannot be formed by
focusing a beam of electrons on the retina of the eye. Instead, the image is formed on a television
screen, and then viewed and/or photographed.
Contrast is another critical factor in microscopic visualization. Contrast refers to
differences in color or intensity between different structures. If all of the parts of an image are
the same color, or the same shade of gray, they are difficult to distinguish. At the microscopic
level, there is very little natural contrast, and so material that is prepared for the microscope must
often be stained with something that will make structural differences show up better. Stains can
do this only if they color some structures more readily than others. Different materials, therefore,
require different stains. Inevitably, though, using a stain means changing the specimen and stains
must be used cautiously and studied carefully to be sure they are not introducing meaningless
differences.
Some advanced microscopes manipulate the light coming through an object so that small
differences are enhanced, increasing contrast. On our microscopes in lab, adjusting the iris
diaphragm allows you to control the amount of light passing through the specimen on the slide,
thus affecting contrast.

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Using Our Microscopes
During the course of the semester, we will use two kinds of microscopes in lab:

Light microscope Dissecting (stereoscopic) microscope
-for slides -for large, opaque, 3D objects.















Both are compound microscopes because there are two stages at which the image is magnified on
its way to your eye. (A magnifying glass is a simple microscopethere is only one stage of
magnification.) The image is first magnified by an objective lens (that is, one close to the
object), and then the lens through which you lookthe ocular lens, which is mounted in an
eyepiece, magnifies it again. Some microscopes are monocular (one-eyepiece) and some are
binocular (two-eyepieces).

When looking through a microscope, it is important to know how much the image you see has
been magnified. Since we have compound microscopes, meaning two sets of lenses are working
to magnify the image, we must calculate total magnification using the equation below:

Total magnification = magnification of the x magnification of the
objective lens ocular lens

Magnification of the objective lenses is written on the lenses themselves. For most of our
microscopes with 3 objective lenses the magnifications are 4x, 10x, and 40x. The magnification
of the ocular lens is 10x. There may be another objective lens marked 100x which we generally
will not use as it requires a special technique, called oil immersion to produce a focused image.

For this lab and others, you will be asked to draw images of cells you are viewing under the light
microscope. Part of a properly labeled drawing includes reporting the total magnification of the
image, not simply the magnification of the objective lens.



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Monocular Compound Microscope:

Note: the fine focus knob of the Leica microscope is in a different location than shown in the
above diagram. Your instructor will show you its location.

Microscope Parts (clockwise from top)

Eyepiece contains the ocular lens (i.e., the one closest to the eye). On all our
microscopes, the ocular lens magnifies the image 10x.

Body tube contains mirrors that reflect the image toward the ocular lens

Nosepiece rotates to move different objective lenses into place above the slide

Objective lenses are the lenses closest to the object. The three lenses of our microscopes
(from shortest to longest) magnify 4x (low power, or scanning lens),
10x (medium power), and 40x (high power). The magnified image from
the objective lens passes through the body tube to the ocular lens, which
magnifies it ten times. Therefore total magnification = objective
magnification times ocular magnification.

Each lens sees only what was in the center of the field at lower power.

When the image has been focused at a lower power, the next higher lens
can be rotated into place without moving the focus knobs. Minor
adjustment of focus may be necessary at the new magnification.
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Stage supports the object being studied (normally a glass slide).

Stage clips hold the slide in place over the opening in the stage and help to stabilize it
when it is moved to view a new area.

Lamp provides light that passes through the object; absorption and refraction by
the object alter the beam of light and produce the image that is
subsequently magnified as it passes through the lenses. (Switch is also in
base.)

Condenser located under the stage and above the lamp; contains the lever for the iris
diaphragm and a lens. The lens focuses the light from the lamp on the
specimen so that it is evenly illuminated. For most purposes, the
condenser should be raised at or near its highest level.

Lever for
Iris Diaphragm operates diaphragm that opens and closes to admit more or less light from
the lamp through the object. Generally, very transparent objects and/or
high magnification require less light than intensely colored objects and/or
low magnification.


Base supports the optical parts of the microscope and must always be placed on
a flat, stable surface; one of the parts to hold when carrying the
microscope

Coarse focus knobs (on both sides of arm) move the objective lenses significantly closer to or
further away from the object. Coarse focus knobs are never used at high
power because they move the lens too fast, and at high power the lens
almost touches the slide.

Fine focus knobs are used to make delicate adjustments to focus, especially at high power.

Arm should be used to carry microscope, with the other hand supporting the
base.







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General Principles for Care and Use of Microscopes

Care of microscope
Always carry a microscope with both hands, one holding the arm and the other supporting the
base.
Clean lenses and slides with special lens tissue only.
Always use a coverslip with wet preparations.
When finished, remove slide; rotate low-power lens into position; turn coarse-focus knob all
the way away from you so the objective is close to the stage (i.e., focus down); wrap cord
around microscope and tuck plug securely in; put cover (if available) on microscope. Return
microscope to cabinet.


Use of microscope
Always secure slide with stage clips.
Begin examination of slide with scanning-power lens (shortest one), and specimen (if visible
with unaided eye) in center of opening in stage. Turn coarse-focus knob all the way forward
(i.e., focus down), then turn it gradually towards you until image comes into focus. Move slide
until desired area is in center of field. (Microscope rotates image, making it necessary to move
slide in the direction opposite to the one that seems required. Practice!)
When image is focused at scanning power, rotate the low-power lens into place without
touching focus knobs until lens is in place. Then re-focus, using either focusing knob.
Adjust light if necessary by moving iris-diaphragm lever.
If higher magnification is desired (usually necessary for study of cells), move desired area to
center of field and carefully rotate high-power lens into place. (Some slides are too thick for
this lens; dont force the lens into place.) Use ONLY the fine-focus knob to adjust focus.
Adjust the light using the iris diaphragm. (Do this frequently to see if it improves image.)



















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Skill Building Exercises:

Materials: for each pair of students
one light microscope
one slide with a printed phrase or word

Becoming confident using a microscope takes time and practice. No doubt there will be
times when you cannot find your image. Below are four common things to try in order to
bring your image into focus.

1. Slowly move the slide around on the stage. If the object you want to view is not centered on
the stage you will not be able to see it in your field of view. It might be just out of the field of
view. Remember the microscope rotates the image, so when you turn the stage knobs to
move the slide, it will be moving in the direction opposite to the direction you want the image
to move.

2. Go back to the lowest magnification where you last saw your object by rotating a different
objective lens into position. Always begin by finding your object in focus on low power then
proceed stepwise through medium and high power, making sure the image is centered and
focused before moving to the next magnification.

3. Change the focus. There are focus knobs on both sides of the microscope. The coarse-focus
knob is used only at low and medium power; the fine-focus knob is most useful at high power,
though it can be used also at other magnifications.

4. Change the amount of light coming through the object by moving the lever that opens and
closes the iris diaphragm. Generally, you will need more light as you go to a higher
magnification or if you use a slide with a lot of color. If your preparation is pale, too much
light will wash it out. Get in the habit of trying different light levels to see their effect on a
particular slide.

How to find something on the slide
1. Hold the slide so that the label is to your left. Draw the word or phrase on your slide as it
appears to your naked eye (i.e. do not put it under the microscope just yet).

2. Check to make sure the scanning power objective lens (4x) is clicked into position. You
should ALWAYS begin looking at a slide under scanning power or low power even if you
eventually plan to view the object under high power.
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3. Now, put the slide on the stage so that the label is to your left. Pinch the projecting handles
to open the jaws of the slide-holder, position the slide securely, and then carefully release the
handles. Use the knobs to move the slide so that part of the phrase is over the hole in the stage.

4. Adjust the coarse and then fine adjustment knobs as needed until one or more of the letters in
your phrase are completely in focus on low power. Draw the letter(s) as it appears through the
microscope. Label your picture with a title and total magnification.


Title: __________________

Total Magnification: ______________






5. Explain what this quick demonstration has taught you about how the lenses of the microscope
affect the orientation of your image (hint: its not just simply upside-down). If you need help
figuring out what the lenses are doing, try this: turn the stage knobs so the slide moves away
from you. Watch in the ocular lens at the same time. Is your image in your field of view also
moving away from you? Now try moving the slide to the left on your stage. Is the image in the
field of view also moving to the left?





How to keep the object in view when you change magnification
When you increase magnification, you look at a smaller part of the object, more highly
magnified. Therefore, you need to be sure that the thing you want to study is in the center of
the field before you change magnification.

6. Get the desired object in the center of the field and focused with the scanning power
objective (4x). Then without changing the focus, carefully rotate the 10x objective lens (low
power) into position and re-check the focus. Adjust it carefully, using the fine-focus knobs
so you do not overdo it.

7. Now move to the highest-power lens (40x). DO NOT TOUCH THE COARSE
ADJUSTMENT KNOB. I promise that the high power lens will fit, although it will look
like it might hit your slide. Make sure it is clicked into place. Now look through the
microscope. If you do not see anything there are 4 things to try BEFORE calling over your
instructor:
1. Adjust the FINE focus knob (DO NOT TOUCH THE COARSE KNOB).
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2. Use the knobs to move your slide around slightly since you could be looking at the
space between letters.
3. Return to low power (10x objective) and get your letter(s) in focus and centered and
then try again to switch to high power.
4. Clean the lenses of the objectives with lens paper and lens cleaner.

Estimating the actual size of an object seen with the microscope:
There are devices that can very accurately measure the size of microscopic objects. For our
purposes, however, an estimate is sufficient. A reasonable estimate can usually be made by using
the field of view (the lighted area you see through the lens) as a crude ruler.

Exercise 1: Determining the Diameter of the Field of View:

1. Using a slide with graph paper or metric ruler/grid, you will measure the diameter of the field
of view with the 4X and 10X objective only. You cannot accurately measure the field of the
highest power objective (40x), because the markings are too far apart.

a. Place the grid of the slide over the hole in the stage of the microscope right over the area
where the light shines.
b. While looking through the ocular, align the grid to measure the diameter of the circular
field of view.
c. The lines are 1mm apart. Enter the diameter of the field of view for the 4X and 10X
objectives in the table below:
d. Calculate the diameter of the field of view in microns (m) and enter it in the table
below:

Objective Lens Total Magnification
(objective x ocular)
Field of View
Diameter (mm)
Field of View
Diameter (m)
Scanning 4x
Low power 10x
High power 40x

2. Because the slide cannot be used for the highest power objective, simple math can be used
to calculate the diameter of the field of view. Look back at your drawings of the word slides.
What happened to the size of the field of view when you increased the magnification? ________
_____________________________________________________________________________

As magnification increases, the field of view decreases proportionately. Knowing this, you can
use the following formula to calculate the field of view size for the 40X objective:

Mag
4x
X FD
4x
= Mag
40x
X FD
40x
Where: Mag = magnification of that objective
FD = field of view diameter of that objective


3. Fill in the last row of the table.
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Exercise 2: Measuring an actual object

1. Obtain a slide with a word or phrase. Estimate the width of a letter e using the following
strategy:

a. Choose the best magnification at which the object (e) to be measured fits entirely
within the field.

b. Move the slide so that one edge of the object (e) is at the edge of the field.

c. Estimate how many objects of this kind could fit end-to-end (or side by side) across the
center of the field of view.

d. Determine the approximate diameter (or length, or width) of the object using the
following equation:

Estimated diameter (mm) = Field of View Diameter # times your object
(from table) could fit across the f.o.v.

Show your work below. You should include a drawing of your e at the edge of the field of
view (to scale; label total magnification) and write out the formula used.

Find the answer in mm and micrometers (m). Ask your instructor to check your work
before continuing.



Formula used: __________________
Total magnification: _____________
Diameter in mm: ___________
Diameter in m: ____________




Little swimmy critters!

Pond Water
Using a drop or two from the jar of pond water, prepare a wet mount according to the directions
below. Make two separate drawings of two different organisms you find in your sample of pond
water. Record the magnification at which you view the organisms, and estimate the length (i.e.,
the longer dimension) of the organisms in micrometers (m) using the estimation technique you
learned.



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Preparing a wet mount
Have slide, coverslip, and pencil (with eraser) ready.
Place specimen (see previous section) in middle of slide and
cover with a drop of water or stain. If the specimen is in water,
like your pond water, adding water is not necessary.
Put one edge of coverslip against slide at one edge of water
drop, and lower coverslip carefully onto drop.
Press lightly against coverslip with pencil eraser to squeeze out
air bubbles.




Pond Water Drawings:


Total Magnification: Total Magnification:
Estimated length: ! _____mm _____m Estimated length: _____mm _____m

Using Stain to Visualize Cell Structures:
Parenchyma cells of potato
Parenchyma cells are large cells commonly found in many different parts of a plant. Some types
of parenchyma cells may contain chloroplasts and carry out photosynthesis. Other parenchyma
cells may be adapted for storage of substances, like starch, which youll remember as being a
type of storage polysaccharide. Lets look at how starch is stored in the parenchyma cells of a
potato:
1. Using a wet, single-edged razor blade, cut an extremely thin slice from the freshly cut
surface of a potato.
2. Specimen Preparation: A good specimen will occupy no more than about one-fourth
of the area under the coverslip, and it must be somewhat transparent in order to be
seen. For plant material, tear off a small piece or use a razor blade to shave a thin
slice from a larger specimen. It sometimes works to cut a wedge-shaped piece first and
put it on the slide, then cut and save only the thin edge of the wedge.
3 . Prepare a wet mount, in water, of this ultrathin slice of potato. If you sliced it thinly
enough, the cover slip will lie flat and even.
4. Examine the parenchyma cells under low power, and then increase the magnification.
Look for typical plant cell components.
5. Draw what you observe in the space provided.
6. Without removing the coverslip, stain the potato slice with iodine according to the
following procedure.
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Staining a specimen that has already been prepared in water:
Its generally a good idea to examine a specimen in water before staining it, in order to
understand the effects of staining. You can use low or medium power without a coverslip, and
simply add a drop of stain and a coverslip after the initial examination. However, if you need to
use high power before staining, you must put a coverslip on the preparation. The water can be
exchanged for stain (or for any other solution) without removing the coverslip, and even without
removing the slide from the microscope stage:













Potato in water only Potato stained with iodine
400X (high power) 400X (high power)

Clean up:

BEFORE PUTTING AWAY YOUR MICROSCOPE, HAVE YOUR
INSTRUCTOR CHECK TO MAKE SURE YOU HAVE PREPARED IT
PROPERLY (lenses clean, low power objective down, no slides on the stage,
cord wrapped).
Wash and dry the microscope slides. Return the slides to your table.
Put a drop of the new solution at one edge of the
coverslip.
At the other edge, hold a strip of paper towel. As the
paper towel draws water from under the coverslip, the
solution from the other edge will flow in.
Repeat with additional drops as necessary.

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Part 2: Tests for Biological Molecules

Benedicts test for Sugars

Benedicts solution is composed of sodium carbonate, sodium citrate, and copper sulfate.
When Benedicts solution is heated in the presence of a reducing substance, the blue cupric ions
(Cu++) are reduced to red, insoluble cuprous ions (Cu+).
The color varies from greenish to orange to red to brown, depending on the amount of
reducing substances present. Thus, Benedicts solution allows us to test quantitatively for the
presence of reducing sugars.

Procedure:
1. Half fill the beaker with water and heat to a gentle boil.
2. Using a Sharpie, number the test tubes 1-7.
3. Add 1 ml (10 drops) of the correct test solution to each tube, as indicated in the table.
4. Add 2 ml (20 drops) of Benedicts solution to each of the 7 test tubes.
5. Place the test tubes in a boiling water bath for approximately 3 minutes.
6. Allow the tubes to cool to room temperature and record your results below:

General conclusions: Compare the Benedicts test results obtained on the different solutions.
How can you explain these results? ________________________________________________

_____________________________________________________________________________


Tube
#
Test
Solution
Color/appearance
before adding
Benedicts
Color
right after
adding
Benedicts
Resulting
Color
after
heating
Conclusion
(Does the substance in the
test tube contain a
reducing sugar?)
1

Water
2

Glucose
3 Sucrose
4 Fructose
5 Lactose
6 Starch
solution

7 Egg
Albumin

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Iodine Test for Starch

Iodine potassium iodide solution enters the amylase coils of the starch molecule. When
this happens, the color of the iodine potassium iodide solution changes from a yellowish orange
to a bluish black. This was seen previously when the starch in the parenchyma cells of the potato
was stained with iodine.

Procedure:
1. Obtain 4 clean test tubes and number them 1-4.
2. Add 1 mol (10 drops) of the correct test solution to each tube as indicated in the table below.
3. Add (5 drops) of Iodine Solution to each of the 4 tubes. Mix by gently shaking the tube.
*DO NOT HEAT * tubes containing Iodine solution.
4. Record any color change for each tube in the table below.


General conclusions: Compare the Iodine test results obtained on the different solutions.
How can you explain these results? _______________________________________________

____________________________________________________________________________


Biuret Test for Protein

The Biuret Test is one method of detecting the presence of proteins. The name biuret is
derived from the colored chemical complex formed between copper and proteins under alkaline
conditions. Whole proteins will give a violet color and if polypeptides are present rather than
whole proteins, a pink color results.
A number of complex substances besides proteins and polypeptides can respond with a
violet color to this test. Thus, a positive biuret test does not in itself conclusively prove the
presence of protein but since all proteins give a positive test even in high dilution, a negative
biuret test can be concluded as the near absence of protein.

Procedure:
1. Obtain 4 clean test tubes and number them 1-4.
2. Add 1 mL (10 drops) of the correct test solution to each tube as indicated in the table below.
3. Add 1 mL (10 drops) of Biuret Solution to each of the 4 tubes.
4. Wait 5 minutes. Record any color change for each tube in the following table.
Tube
#
Test Solution Color/appearance
before adding
Iodine
Color
after
adding
Iodine
Conclusion
(Does the substance in the test
tube contain starch?)
1 Egg albumin
2 Starch
3 Glucose
4 Water
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General Conclusions: Compare the Biuret test results obtained on the different solutions.
How can you explain these results? ________________________________________________



Determining the components of an unknown solution:

Obtain a sample of unknown solution. This sample contains a mixture of biological molecules
you have just learned to test. Using what you have learned, determine the types of biological
molecules present in the solution (reducing sugar, starch, protein or mixture of 2 or more
molecules). Record the process of your investigation, results, and conclusions. When you have
finished, check with your TA to find out if youre right. Then go celebrate your success (or try
to figure out what might have gone wrong)!





Clean up:

Washing test tubes:
1. Carefully pour the contents of the test tubes down the sink drain.

2. Fill the test tubes with soapy water. Then using a test tube brush, scrub the inside of the test
tube. Be sure to remove all of the red colored residue (from the sugar tests). Also, remove
your markings on the outside of the tubes.

3. Rinse well with tap water and return the tubes to your bench.

4. Invert the tubes so that they will drain.

Make sure the hot plate is turned off.
Tube
#
Test
Solution
Color
before
adding
Biuret
Color right
after
adding
Biuret
Resulting
Color after
5 minutes
Conclusion (Does the
substance in the test tube
contain protein?)
1 Egg albumin
2 Starch
3 Glucose
4 Water
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Discussion Questions

1. List the 4 basic types of biological macromolecules:



2. What are the building blocks of each of these types of macromolecules?



3. List the functions of each of the 4 types of biological macromolecules




4. With regard to biological molecules, what does it mean to be hydrophilic? What does it mean
to be hydrophobic? Can a molecule be both at the same time?






Look at the food labels:

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5. What is a polyunsaturated fat? What is a monounsaturated fat?




6. Judging by the numbers on these labels, how many food calories are there in a gram of fat?
Protein? Carbohydrate?











7. Which type of biological macromolecules would be best to use for energy storage?