TOPIC 2

MICROSCOPES AND CELLS

Objectives:

1. Identify the components of the compound light microscope and

know the functions of each. Be able to use the microscope to find and
to maintain the image of an object.
2. Understand the following terms: inversion, resolving
power,magnification and field diameter.
3. Be able to prepare wet mounts of plant and animal cells. Be able to
identify or explain the differences between them. Apply the concept
"structure is related to function" to different types of cells.
4. Understand the function of biological stains in microscopy, and be
able to apply stains properly to enhance an image.
5. Understand what causes "plasmolysis".
6. Prepare a stomatal peel, and recognize the structure and function
of stomates.
7. Understand the function of biological stains in microscopy, and be
able to apply stains properly to enhance an image.
8. Prepare a stomatal peel, and recognize the structure and function
of stomates.
9. Be able to create a bar graph using Prism.

Pre-lab Questions:

1. Convert 27.5 micrometers to centimeters. Show work below.

Give your answer in scientific notation!
Note: the quiz may give you a different value to convert.

2. Which of the following lengths is the largest? Explain your answer.

a. 12.5 cm c. 0.125 m
b. 125 mm d. 125,000 µm

3. Of the following objective magnifications, which would you use to get the
largest field diameter and why? Which would give you the smallest?
a. 4X c. 40X
b. 10X d. 100X

4. What is the function of a “stain” in microscopy?

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Web Resources:
One website provides a theoretical overview of how metric system
conversions are performed. The other site provides multiple choice questions
and answers for conversions using length, mass, and volume.

Introduction:
There are two general types of microscopes used by scientists in the
modern biology laboratory: light and electron. The major difference between
the two is based on the kind of energy used to illuminate the object. Light
microscopes use visible light passing through lenses which produces a
magnified image seen by the eye. Electron microscopes send a stream of
electrons to bombard the object. Magnetic lenses are able to focus the pattern
of reflected and transmitted electrons onto a photographic plate or television
screen.
Compound Light Microscope:
This is called "compound" because it contains at least two glass lenses.
The lens nearest your eye is called the ocular or eyepiece, and usually has a
magnification power of 10X. The lenses closest to the object are called
objectives and have varying magnifying abilities. The amount of
magnification is printed on the lens itself. Total magnification is the product
of the ocular and objective magnifications. Figure 1 shows the major parts
of a compound microscope.

Figure 1: Generalized compound light microscope.

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Magnification and Resolving Power:

Microscopes have two purposes: to magnify and to resolve images.

Magnification alone only makes small objects appear large. Resolving
power (or resolution) measures how clearly you can see details of the image.
It is usually defined as the ability to view closely adjacent objects or
structures as distinct images instead of a fuzzy blur.

The formula for determining resolution is:  = wavelength of radiation

n = refractive index of medium
between objective and object
RP= 0.6
 = half the acceptance angle of
n sin 
the objective lens.

Most of these details are unnecessary for you to know because the
numbers they represent are constant for any type of compound microscope.
Notice that resolution is directly related to , the wavelength (or color) of
light used to illuminate the object. The human eye can only see light from
450 nm- 700 nm (see Figure 2). Therefore the resolution of the compound
microscope is limited by the human eye, and is approximately 0.23 m.
Electron microscopes are powerful tools in biology because they use
electrons for visualization of the object. Electrons can have a wavelength of
up to 0.0037 nm, and therefore have a resolution of 0.2 nm. Remember, this
means that electron microscopes may be able to distinguish between two
objects that are only 0.2 nm apart.

Purple- Blue Green- Yellow Orange- Red- Far red

Figure 2: The electromagnetic spectrum showing visible wavelengths.

Visible spectrum shown in nanometers (nm).

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Focusing:
 On the microscope, click the lowest power objective into place. Raise the
objectives as high as they will go using the coarse adjustment knob.

 Place the slide on the stage, and clip it into place. The E should be
oriented so that it is right-side up to you. Using the control knobs,
move the E so that it is in the center of the light coming up from below.

 Viewing the microscope from the side, lower the 4X objective by the coarse
adjustment until it is just above the slide.

 Look through the lens and slowly turn the fine focus knob until the image
comes into clear and distinct view. Does the image appear normal or
upside-down? Sketch the image as it appears.

 Move the slide to the right. Which way does the image appear to move?

Inversion refers to the fact that the image is not only flipped, but also
reversed.

 Make sure the E is centered in the field. Move to the 10X objective. Do
not adjust the coarse adjustment first.

 The E should be visible. Ask your instructor if you can’t find it. If any
adjustment is needed, use the fine focus knob only. Can you still see all of
the E with the 10x objective?

 Change to the 40X objective. Is all of the E still visible? Did you have to
make any dramatic adjustments to the focus?

Compound light microscopes are parfocal. This means that once the object
is in focus under low power, it should remain mostly in focus as the
objectives are changed.

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Field Diameter

Field diameter describes the area you see when looking into the microscope.
Field diameter will change as the magnification is changed.

 Place a transparent ruler on the stage of the compound microscope.

Measure the field diameter at 40x and 100x magnification. Record your
data in cm and then convert this value to mm and m (micrometers). If
you have problems with the conversions, there is a practice sheet at the
end of Topic 2, or use the online resources on the lab webpage.

Field diameter at 40x in cm: in mm: in m:

Field diameter at 100x in cm: in mm: in m:

What is the relationship between magnification and field diameter?

Cell Observations:

Often it is necessary to prepare a specimen for observation. In such

cases, the object should always be placed in water, forming a wet mount. A
wet mount is prepared by placing a drop of water on the clean slide, and
placing the specimen in it. Cover the water and specimen with a coverslip.

Living Animal Cells (Human Epidermis):

 Gently scrape the inside of your cheek with a toothpick and place the
scrapings in a drop of water. Cover with a coverslip.

 Scan for the cells under 40x total magnification. When you find them,
raise the power to 100x and 430x. Adjust the iris diaphragm to get the
optimum amount of light.

 Can you clearly see the nucleus? Staining is required to add contrast to
the thin and tranparent epithelial cells. Prepare a new slide as before,
but this time add a tiny amount of methylene blue to the water and cell
mixture. Observe as before. Sketch the cells, and label the nucleus.

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Living Plant Cells:
 Elodea is a common plant of freshwater aquaria. It is an ideal material to
use for viewing plant cells because the leaves are only a few cells thick.

 Place a single leaf in a drop of water on a clean slide, and cover with a
coverslip. Do not use stain. Scan at 40x total magnification, and focus
on cells near the edge of the leaf. Raise the magnification up to 400x to
view details of the cell. Sketch some Elodea cells.

 Label the chloroplasts and the cell wall.

The chloroplasts are where light is harvested to make carbohydrates

for the plant. It appears as though the chloroplasts are attached to the cell
walls. This is not true. In fact, the interior of each plant cell is taken up by a
large sac, called a vacuole. This is so large that it pushes all other parts of
the cell up against the cell wall. You may see some chloroplasts moving
around the edge of their cell. They are moving between the vacuole
membrane and the cell wall/membrane.

 Add several drops of concentrated salt solution to the Elodea leaf.

Observe the leaf under 400X total magnification.

 Sketch the new appearance of the cells after the salt solution

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 Observing and quantifying Stomata:
Stomata are the tiny openings in the epidermis of terrestrial (land)
plants (Fig 3). Gases, primarily oxygen and carbon dioxide, can pass through
these openings to allow for photosynthesis to occur. Likewise, water will
evaporate out of the stomata, leading to potential dehydration.
Stomata typically remain open during the day and close at night,
when photosynthesis doesn’t occur, allowing for water conservation.
Stomata can also close during especially hot days, during droughts, or as a
response to plant growth regulators. Desert plants have a special form of
photosynthesis which allows them to open their stomates at night, to
perform gas exchange, and close them during the day to conserve water.
Today we will observe and count the stomates on two different types of
plants: a monocot and a fern. Monocots are a type of flowering plant which
appeared approximately 150 million years ago. Ferns are not flowering
plants at all, they actually appeared on Earth about 200 million years before
the first flowers. Nevertheless, ferns do have stomates which perform
essentially the same functions as in other plant groups.

Stomate

Epidermal cell

Fig 3: Stomatal openings in the underside of Polypodium. Seven stomates are

visible in this section.

The number, shape and size of stomata will vary from species to
species and leaf to leaf! Today we will use a simple technique to observe and
quantify the number of stomata found on leaves of different species.

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Procedure:
 You should obtain leaves from a monocot, a dicot and a fern.
 Using a small amount of clear nail polish, coat the underside of each
leaf with a THIN layer. Set the leaves aside for 30+ minutes to dry.
Coat the upper side of three additional leaves in the same way.
 Gently peel away the nail polish, and it will contain a perfect imprint
of the cells.

 Do not use water with these samples!

 Find the leaf cell imprints at 100X and then move the objective to
400X for counting.

 For each species, identify the epidermal cells and the stomata. Fix
the field of your microscope in place and count the number of stomata
visible. Stomates on the edge of the field count! Move the field of view
to a new position on the leaf and repeat the procedure. Then do a third
count in a new position.
 Repeat the procedure for each species and each surface, and record
your data in Table 1.

Table 1: Number of Stomates observed by surface and species.

Species Surface Number of Stomates

View 1 View 2 View 3
_______________________________________________________________________
upper
lower
upper
lower
upper
lower

This lab was written and revised by:

Tony Botyrius M.S.
References:

Electromagnetic spectrum modified from: http://www.yorku.ca/eye/spectrum.gif

Microscope Image. http://www.towson.edu/~cberkowe/medmicro/315lab1.html.

Stomate Image.
http://acces.ens-lyon.fr/acces/equipes/dyna/travaux/evolbio/phylovegetal/ressources-
iconographiques-pour-les-collections-phylogene/photos-
lejamble/Polypode%20stomates%20vue%20gle.jpg/view

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 Metric System and Conversions

In order to easily convert one metric measurement into another, it is

essential to understand the metric system divisions and how they relate to
the basic unit: the meter, the liter, and the gram. The table below gives this
information, and the most often used values are written in bold.

Prefix Division of Metric Unit Scientific Notation

giga (G) 1,000,000,000 109
mega (M) 1,000,000 106
kilo (k) 1,000 103
hecto (h) 100 102
deka (da) 10 101
deci (d) 0.1 10-1
centi (c) 0.01 10-2

milli (m) 0.001 10-3

micro () 0.000001 10-6

nano (n) 0.000000001 10-9

pico (p) 0.000000000001 10-12

The most common units used in Biology 1 are the milli-, centi-, and
micrometers. They relate to each other in the following way.

Meter
Gram
Liter Centi Milli Micro Nano
1 100 1000 1,000,000 1 x 109

0.1 10 100 100,000 100,000,000

0.01 1 10 10,000 10,000,000

0.001 0.1 1 1000 1,000,000

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To convert from one unit to another, you will have to use conversion factors
(how many of one unit goes into the other unit). For example, in the
conversion of 472 grams into kilograms, it should be clear that you are
changing into a larger unit, and therefore the numerical value should be
smaller, since you can’t have a larger number of kilograms than grams.

So, to convert set up an equation as below.

Value to Convert Multiplied by Conversion Factor

472 grams x 1 kilogram = 0.472 kg

1000 grams

(Note that grams cancel out of this equation, leaving kilograms)

Of course, it is also possible to go from a larger unit to a smaller one. In this

case, your numerical answer should be larger than what you begin with.

Convert 5.5 liters to milliliters.

5.5 L x 1000 ml = 5500 ml
1L

Convert 15 micrograms to milligrams

15g x 1mg = 0.015mg
1000 g

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