Exercise 2: Measurements II and the Microscope
______________________________________________________________________________
OBJECTIVES:
1. Understand measurements and conversions of the metric system.
2. Learn how to properly use both compound and dissecting microscopes.
______________________________________________________________________________
INTRODUCTION:
Numbers and measurements impact every part of our lives, and are tools that scientists,
engineers, astronauts, chefs and doctors use to analyze data, build bridges, fly orbiters into space,
adjust recipes, and prescribe medication. Collecting and analyzing numerical data allows us to
understand patterns in the natural world that are not easily observed with the naked eye, and the
natural variation that is inherent to all organisms is the major reason we need measurements. In
today’s lab you will learn about basic measurements and common instruments used by scientists
on a daily basis. Your ability to learn and use these concepts will be tested and reinforced
throughout the semester.
____________________________________________________________________________
Task 1 - MEASUREMENTS IN SCIENCE: Familiarize yourself with the metric system.
Recall from last week that a key component of the scientific method is experimentation.
This step is necessary for the collection of data that will either lend support to, or lead to the
rejection of, the hypothesis being tested. In general, data can be qualitative or quantitative.
Qualitative data describe variables based on quality (e.g. smell, appearance, texture, etc) and are
usually gathered through interviews, pictures, field notes and/or surveys. Quantitative data define
the quantity of a variable through measurements (e.g. length, area, cost, height, age, etc.). The
main disadvantage of qualitative data is that they are often too subjective (what smells good to
one individual might not smell equally well to another). Therefore, quantitative data, which can
be statistically manipulated and analyzed, are the preferred choice of most scientists because they
provide objective, less biased measures. Statistical analyses of quantitative data are used to
describe biological processes and examine how organisms respond to, adjust to, and modify their
world. We will examine both types of data in greater detail throughout the semester.
The metric system is used as the international standard to make measurements
worldwide. It is based on units of ten (see Table 1 for a list of common prefixes, their
abbreviations and the divisions/multiples of each). In contrast, the Imperial Units of
Measurement is based on historical precedent, e.g., a foot was first measured as the length of a
man’s foot. Because the metric system is widely employed throughout the scientific arena, it will
be covered in this lab. Listed in Table 2 are the commonly used metric units in biology.
1
Table 1:
Prefix
Abbreviation
Division or Multiple of Metric Unit
Deci
d
0.1
Centi
c
0.01
Milli
m
0.001
Micro
µ
0.000001
Nano
n
0.000000001
Pico
p
0.000000000001
Deka
da
10
Hector
h
100
Kilo
k
1000
Mega
M
1000000
Giga
G
1000000000
Table 2:
Unit (abbreviation)
Measures
Meter (M)
Length
Liter (L)
Volume
Kilogram (Kg)
Mass
Degree Celsius (C)
Temperature
In this task you will practice measuring length, temperature, volume and mass using the
metric system. When taking measurements we often need to convert between units. In order to
do this, we must first have information about the size of the unit we are interested in converting
to or converting from. Table 1 provides a partial list of these measures. For example, suppose we
know that the length of a table is 1.5 meters, but we want to know how many centimeters this
corresponds to. Based on Table 1, we know that the centi prefix means 0.01. Therefore, a
centimeter (cm) equals 0.01 (one hundredth) of a meter (m), or there are 100cm in 1m. To
calculate the number of cm in 1.5m, we can either:
(1) Divide 1.5 by 0.01  1.5m x (1cm/ 0.01m) = 150cm or 1.5m / 0.01 = 150cm
(2) Multiply 1.5 by 100  1.5m x (100cm/1m) = 150cm
In both examples, the meters cancel out, leaving the answer in centimeters.
2
I. CONVERSIONS
Convert the following measures:
2 meters = _______centimeters =_______millimeters
87 millimeters = _______meters = _____centimeters
II. MEASURING LENGTH
Using the rulers and meter sticks provided at your station, make measurements of all items listed
below. Be sure to include the proper units:
Length of this page: ______
Width of this page: _______
Area of this page (Area = width x length): _______
Your height: _______
Question:
What are some potential sources of error when making measurements?
III. MEASURING VOLUME
Volume is the space occupied by an object. Units of volume are usually cubed units of length,
but can also be expressed as divisions/multiples of a liter, i.e., 1L = 1000cm3 = 1000mL.
In scientific laboratories, volume is measured using pipettes, beakers and graduated cylinders. In
general, pipettes are used to measure small volumes (≤ 25mL), while larger volumes (≥ 25mL)
are measured with graduated cylinders. Remember from last week’s lab that volume readings
should be taken from the bottom of the meniscus.
1. Acquire a graduated cylinder and note of the total volume that can be measured with it:
Total volume that can be measured = _________ mL
.
2. Use the graduated cylinder to determine how many mL it takes to fill one of the available
beakers.
Total volume of beaker = _________ mL
3
IV. MEASURE VOLUME WITH WATER DISPLACEMENT
Water displacement can be used to measure the volume of a solid object. The following exercise
will demonstrate this process.
1. Weigh the rock at your station and record its mass (For instructions on measuring
mass please see Part VI of this exercise).
Mass of rock = ________ g
2. Fill the graduated cylinder at your station with 50mL of water.
3. Add the rock to the graduated cylinder with water. Notice that the volume of water
rises with the addition of the rock. Calculate the volume of the rock by subtracting the
initial volume (50mL) from the new volume.
Volume of rock = ________ mL
4. Repeat for a pencil. This data will be used later on in part VI.
Volume of pencil = ________ mL
5. Each group should record their results on the board and then record the class data in
the Table 3. Use this dataset to calculate the mean, median, range, variance and
standard deviation for each variable. For a review on these statistical calculations, see
Week 1 Task sheet.
Table 3:
Group #
Rock mass (g)
1
2
3
4
5
Mean
Median
Range
Variance
Standard Deviation
4
Volume displaced (mL)
Using the graph paper provided, plot the class data as rock mass (horizontal, X-axis)
vs. volume displaced (vertical, Y-axis) below. Label both axes accordingly (including
the units of measurement) and give your graph a title.
Question:
Based on the class data, explain the relationship between the mass of an object and its
volume.
V. USING PIPETTES
There are three general types of pipettes: 1) plastic suction pipettes, 2) glass pipettes and 3) airdisplacement pipettes (Fig. 1).
5
A
B
C
D
Figure 1. A) plastic pipette, B) pipette pump used to uptake liquid into glass pipettes (C),
and D) air-displacement pipette
Plastic pipettes are the easiest to use, however, they are the least accurate. Liquid is taken
up into the pipette by pressing the plastic bulb, prior to placing the pipette into the liquid. Once
in the liquid, the bulb is released and the liquid is taken into the pipette. To release the liquid, the
bulb is again pressed.
6
Glass pipettes are more accurate than plastic pipettes and differ in both size and the
volume (1mL, 5mL, 10mL and 25mL) that they can uptake. Graduated markings present across
the outside (see Fig. 2) indicate the total volume of liquid that the pipette can accurately measure
and also allow monitoring of the content volume being taken up into and expelled from the
pipette. In addition, on the upper end, the smallest and largest volumes that the can be measured
are also noted. For example, in Figure 2, ‘10mL in 1/10’ (C) specifies that this pipette can
accurately measure a minimum of 0.10mL (1/10) and a maximum of 10mL [measured from the
tapered tip (A) up to the 0 marking (B)]. Unlike the other two types, glass pipettes require an
external suction device, e.g. a bulb or pipette pump (Fig 1B), to uptake and expel liquids.
B
A
C
Figure 2. 10mL glass pipette with gradations
http://bioweb.wku.edu/courses/Biol121/Carbo/pipets.png
The last category of pipettes, air-displacement pipettes, are most often used by molecular
biologists. These pipettes are more commonly referred to as micropipettes because of the small
volumes that they deliver, which range from 0.2 up to 1000 microliters (µL). In general, the
volume of liquid that needs to be measured will determine which pipette model (P2, P10, P20,
P100, P200 or P1000) should be used. If we were to select a P2 for example, the 2 that follows
the P would refer to the maximum volume (2µL) that this particular pipette can uptake at a time.
Provided below are general instructions on how to use an air-displacement pipette (Fig. 4):
Pipette In:
1. Set the pipette to the desired volume.
2. Before immersing the pipette tip in a
tube of liquid, press the plunger to the
first of its two stops.
3. Slowly release the plunger to draw the
liquid into the tip.
4. Remove the pipette tip out of the tube.
Pipette Out:
1. Place the pipette tip in a tube.
2. Press the plunger to the second stop to
begin dispensing the liquid.
3. Slowly release the plunger to release the
liquid into the tube.
4. Remove the pipette tip out of the tube.
Figure 3. Air-displacement pipette instructions
7
Practice using pipettes:
Read the instructions on how to take up and release liquid with air-displacement pipettes (Fig. 4).
Your TA will demonstrate how to set a specific volume on the different pipette models. A
demonstration on the use of glass pipette was provided by your TA during Exercise 1.
A. Materials required:







Balance
2 weighing boats of the same size
1mL glass pipette
Pipette pump
P1000 pipette
Tips for the pipettes
Beaker with water
B. Procedure:
Before beginning the procedure, each person in the group should practice setting volumes as
well as taking and expelling water using the P1000 pipette.
1. Tare one weighing boat.
2. Take up 1000µL of water using a lmL glass pipette and release the water into the
tarred weighing boat.
3. Record this weight in Table 4.
4. Empty the weighing boat and dry it.
5. Repeat steps 1- 4 three times, making sure to dry and tare the weighing boat before
each trial.
6. Repeat steps 1-5 using a P1000 pipette.
7. Calculate the mean, variance, and standard deviation of the collected data
Table 4:
Trial #
1 mL
1
2
3
Mean
Variance
Standard deviation
Expected weight
(provided by TA)
8
P1000
Questions:
1. What can account for the variance observed between the 3 trials for each pipette type?
a. Was the variance the same for both pipettes?
2. Based on your data, which pipette was more accurate in measuring 1000µL of water?
Explain your rationale.
3. Which pipette was more precise? Explain.
4. If you needed to measure 50µL, which pipette would you use: P20, P100, P200, or
P1000? Explain your rationale.
a. What about 250µL?
b. What about 1329µL?
VI. MEASURING MASS
A balance/scale is used to measure the mass of an object. Using the scale at your station,
measure the mass of the items listed in Table 5.
Table 5:
Object
Mass (g)
Coin
Paper clip
Pencil
9
Rock
Empty beaker
Beaker with 100mL water
Using the data that you have already gathered calculate the density (mass/volume) of the
following items:
Density of water = ________________g/mL
Density of the pencil = _____________g/mL
Density of the rock = ______________ g/mL
VII. MEASURING TEMPERATURE
Temperature is the amount of heat present in a particular substance, and it is recorded in degrees
Celsius (oC). The Celsius scale is based on water freezing at 0 oC and boiling at 100 oC. To
convert between Fahrenheit and Celsius the following equations are used:
F = C (1.8) + 32
or
C = (F – 32) /1.8
With the thermometer at your station, measure the temperature of the items in Table 6 in Celsius
and then convert them to Fahrenheit.
Table 6:
Object
Temperature (°C)
Temperature (°F)
Room
Cold tap water
Inside refrigerator
Questions:
a. What are some advantages and disadvantages of using the metric system?
b. Why is it important for all scientists to use a standard system of measurements?
10
______________________________________________________________________________
Task 2 - USING THE MICROSCOPE
Microscopes are tools used to examine specimens too small to be observed with the naked eye.
There are two types of microscopes that you will use in this lab, compound light and dissecting
microscopes. In general, a compound light microscope is used to visualize very small items (e.g.
blood cells) while a dissecting microscope is used for observing much larger items (e.g.
mouthparts of a grasshopper).
A. Familiarize yourself with the use of the light microscope
1. Obtain TWO compound microscopes per group.
2. Identify each part labeled on the compound microscope in Figure 4 and note its function in
Table 7:
Oculars
Body Tube
Nosepiece
Arm
Objective
Slide Holder
Stage Clip
Coarse Focus
Adjustment
Stage
Condenser iris
diaphragm
Fine Focus
Adjustment
Substage
Lamp
Field Iris
Diaphragm
Base
Figure 4. Major parts of a compound light microscope
11
Table 7:
Part
Function
Objective
Stage Clip
Stage
Condenser Iris Diaphragm
Substage Lamp
Oculars
Arm
Slide Holder
Coarse Focus Adjustment
Fine Focus Adjustment
3. Plug in your microscope and turn the light source on. Rotate the objective lens to the 4X
power. It should click into place. Note: You should always start at the lowest power
available on a microscope.
Question:
Why do you think it is best to always start at the low power objective?
4. Locate the coarse adjustment. Turn it while watching the stage. See how fast or slow it
allows you to move the stage compared with the fine adjustment.
5. Adjust the ocular lenses so that they fit the width between your eyes.
6. Obtain the letter e slide from your slide box and place it on the stage (make sure it is held
by the clip). Move the stage so that the e is directly beneath the objective lens.
7. Use the coarse adjustment to move the slide to about 1cm from the objective lens. Looking
through the oculars, move the coarse adjustment until you can see the e through the lens.
12
8. Use the fine adjustment to get the e into sharp focus.
9. Move the e left and right. And then forward and backwards.
10. Change the objective lens to 10X.
Questions:
a. As you view the letter e, how is it oriented? Upside down or right side up? What
does that tell you about how the microscope processes the image?
b. How does the image move when the slide is moved to the left or right?
c. What happens to the brightness of the view when you switch from the 4X to the
10X objective?
B. Magnification
1. Examine your microscope and determine the magnification of each objective and for the
oculars. Record this information in Table 8:
Table 8:
Objective
Magnification
Ocular
Magnification
Total
Magnification
FOV Diameter
(mm)
FOV Area
(mm2)
2. Calculate the total magnification (objective magnification x ocular magnification) for
each objective (4x-40x) and record in the table above.
13
Questions:
a. How many times is the image of the e magnified when it is viewed through the highest
power objective lens?
b. If you didn’t know what you had on your slide (an e) and you began examining it at the
highest power, how could you determine it was an e?
C. Field of View
The field of view is the area you can see when you look through the lens of a microscope
(Fig. 5). Understanding the size of this field under different magnifications is important because
it allows you to estimate the size of objects in your view. The following procedure demonstrates
the determination of field of view (FOV) under various magnifications.
Figure 5. Field of view under various magnifications
14
Procedure:
1. Place a ruler (mm) on the stage of the microscope.
2. Begin with the lowest power objective.
3. Using the coarse adjustment, try to get the ruler into focus. Only use the fine adjustment
to sharpen the image. Measure the diameter of the field of view and record this in Table
8.
4. FOV can easily be determined for the low power. At higher powers you will not be able
to use the ruler because the field of view is too small (see Fig. 5). Instead, you can use the
following formulas:
FOVlow x Maglow = FOVmedium x Magmedium
Or
FOVlow x Maglow = FOVhigh x Maghigh
Use these formulas above to calculate the FOV at medium power (10X objective) and at
high power (40X objective). Record your results in the Table 8.
5. Area of a circle = π x radius2. Use this formula to calculate the area of the FOV for each
magnification and record your results in Table 8.
Questions:
a. Discuss the advantage and limitation of viewing specimens under highest magnification.
b. What about the low-power objective?
c. Which magnification provides the largest FOV? Which provides the smallest?
D. DEPTH OF FIELD
The depth of field is the thickness of the object being viewed with a microscope. The
following procedure will demonstrate how to use the microscope to determine the depth of the
field of view.
1. Place the colored thread slide on the stage of your microscope.
15
2. Start by using the lowest power objective lens. Use the coarse adjustment to get the
threads into focus. Sharpen the image with the fine adjustment knob. Try to determine
how many threads are present by focusing up and down. Once the number of threads is
known, determine what order they are in (i.e., which is on the bottom, the middle and
then top).
3. Repeat this process using the high power objective lens.
Questions:
a. How does depth of field affect viewing biological phenomena that are thick?
b. Are all three threads visible under the low power? Can they all be seen at the same time
under higher power?
c. Which objective provides the greatest depth of field?
E. Preparing Wet Mounts of Biological Specimens
1. Place a drop of “pond water” on a clean slide. Position the edge of a coverslip against the
water drop at a 45o angle and slowly lower the coverslip onto the slide. This is called a
wet mount.
2. View the slide with your microscope. Locate any organisms on your slide and draw these
in the space provided. Take note of the tips below as you look for organisms. For each
task requiring the use of blank slides and cover slips for the remainder of the semester,
your group will be responsible for cleaning, drying, and putting away all slides and cover
slips so that they can be used later in the lab or by the students in the next lab section.
16
MICROSCOPE TIPS:
This is an air bubble, NOT your specimen
These are cotton fibers, not your specimen
F. Dissecting Microscope
1. Obtain one dissecting microscope for your table.
2. Familiarize yourself with all the parts of the microscope labeled in Figure 6.
Ocular
Lens
Zoom
Magnification
Adjustment
Arm
Focus
Adjustment
Transmitted
Light Source
Stage
Base
Figure 6. Major parts of a dissecting microscope
3. Plug in the microscope and turn on the two light sources. One source provides light from
below and the other from above.
17
4. Add a small amount of “pond water” to a weigh boat or a petri dish and examine it under
the dissecting microscope.
5. Use a ruler to measure the FOV diameter at the lowest and the highest magnification.
FOV diameter low power = _____________
FOV diameter high power = _____________
6. Now calculate the FOV area for both magnifications.
FOV area low power = _____________
FOV area high power = _____________
7. Looking through the lens, move the petri dish containing the “pond water” backwards and
forwards, then left and right. Is the direction noted through the lens the same as when
observed with the naked eye?
8. Place the letter e slide on the stage. How is it oriented when you look through the lens
compared to when you examine it with the naked eye?
G. Comparison of Compound and Dissecting Microscopes
Compare the two types of microscopes we examined today in Table 9.
Table 9:
Characteristic
Dissecting Microscope
Magnification
Resolution
Size of field of view
Depth of field
18
Light Microscope