LAB 1:
“Measurement Madness!”
& Diffusion/Osmosis/Tonicity
I. Objectives:
Today’s laboratory will introduce students to several of the basic and most commonly used
laboratory techniques in cellular/molecular biology. Students will learn how to accurately
measure small volumes, from milliliters (1 mL = 1/1000th of a liter) to microliters (1 uL =
1/1,000,000th of a liter), accurately make up solutions of known molarity, and perform
calculations that involve unit conversions. The lab will also introduce students to the concepts
of diffusion, osmosis, and tonicity and demonstrate how these natural phenomena can be
visualized and studied in a laboratory setting. By the end of today’s lab, students should:

Be able to perform calculations and unit conversions involving weight, moles,
molecular weight, concentration, and volume

Be able to accurately measure and dispense small volumes of liquid

Be able to accurately prepare solutions of known concentrations starting with
solid or liquid reagents

Be able to prepare a solution for sterilization in an autoclave

Be able to accurately articulate the difference between grams and moles of a
substance

Be able to define the terms "diffusion," "osmosis," and "tonicity" and explain
why they are important for the survival of cells

Know how the rate of diffusion of a substance is affected by temperature,
concentration gradient, and molecular size and weight

Be able to define hypertonic, hypotonic, and isotonic solutions and describe
the effects of placing living cells in each type of solution.
II. Safety Considerations

This lab has minimal safety concerns. Tie back long hair and be careful not to poke
yourself or others with the serological pipettes.
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
Do not touch the agar plates containing the potassium permangenate and crystal
violet solutions because these are strong dyes that can permanently adhere to (stain)
clothing.

All of the reagents used in this laboratory (NaCl, colored water, tryptone, yeast, etc.)
are non-toxic and can be disposed of down the sink or in the trash. See specific lab
clean-up instructions at the end of this chapter.
III. Introduction:
A:
Measurement Madness!
Your success in the laboratory portion of this course, in future lab courses that utilize
molecular and cellular biology techniques, and your future career (e.g. health care, forensics,
biotechnology - should you decide on a career in science) will depend on your ability to make
accurate calculations and measurements. In today’s lab, we will develop your hands-on
measurement skills by introducing you to the tools that are commonly used to measure
volumes and weights. Since these quantities are usually very small in cell/molecular work, we
will focus specifically on the special tools that have been created to accurately measure very
small volumes and weights in the lab.
Although performing calculations and making these kinds of measurements are basic skills,
they are not necessarily easy to master, and even if you have handled some of this equipment
previously, do not assume that you are an expert, or even necessarily proficient. Be ready to
be careful and to learn and improve your skills even if you’ve done some of these things
before! If you are unsure or just not confident at any point of this lab, it is your responsibility
to ask the lab instructor or a TA for assistance.
1. MEASURING VOLUMES OF 1 ML OR LESS: CARE AND USE OF MICROPIPETTORS
Micropipettors are indispensable to molecular and cellular biologists because they allow the
accurate measurement of volumes of 1 mL or less. If you have not done so already, it is very
important that you commit to memory the following simple units of the metric system of
measurement:
1 L (liter) = 1,000 mL (milliliters) = 1,000,000 uL (microliters)
For example, 650 mL = 0.65 L and 200 uL = 0.2 mL. You will need to practice making these
kinds of conversions over and over again until they become second nature (and easy to do in
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your head). You will need to be able to do them quickly all the time in a cell/molecular biology
laboratory setting, and we will start practicing today.
The type of micropipettor we will use is shown below. Each instrument is calibrated for a
narrow range; e.g. 1-20 µL, 20-200 µL, and 200-1000 µL. We refer to each micropipettor by
the highest volume it is able to administer: P20, P200 and P1000, according to the ranges
above. Each micropipettor is labeled accordingly on the top of the plunger.
The volume is adjusted by turning the black wheel in the top-center of the handle. As the
wheel turns, the reading in the front window changes. Be careful not to extend the wheel
beyond the volume of the micropipettor (e.g., a P20 should not be adjusted to above
20 µl) since this can strip the bearings and severely damage the instrument. The
volume is read in the window in the front of the instrument. Unfortunately, each of the three
pipettors uses a different scale. The table on the next page should help you determine the
volumes for each micropipettor. Refer to this table often as you are learning how to use the
instruments, and after a while you won't need it anymore.
A micropipettor tip should always placed on the end of the shaft prior to use. There are two
sizes of tips: one for the P1000 and one that fits both the P200 and P20. To place a tip on the
shaft, open a box of tips and gently insert the narrow end of the shaft into a plastic tip. Then,
always secure the tip by gently screwing it on with your fingers. Do not pound the shaft into
the plastic tip as this may bend the shaft of the micropipettor.
plunger
tip ejector
button
volume
adjustment
wheel
window
tip ejector
shaft
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To suck liquid into the tip: Gently press down on the plunger to the first stop position.
(This is the position where you will first feel resistance to your pressing.) Then place the tip
into the liquid to be measured (below the level of the liquid’s surface) and gently and slowly
release the pressure on the plunger. Then remove the pipette from the liquid and transfer the
contents in the tip to the container you wish to fill. (The liquid will remain in the tip as you do
this because it is being held in by the vacuum you created inside the micropipettor when you
released the pressure on the plunger.)
To expel the liquid from the tip: Gently push the plunger down to the first stop position
and the continue until you reach the second (final) stop position. You may use either your
thumb or the first finger to depress the plunger, whatever is most comfortable. If you are
transferring the contents of the tip into a tube that already contains liquid, express the
contents into the liquid, below the level of its surface. If you are transferring the contents of
the tip into an empty tube, place the tip on one of the inside surfaces of the tube while
expelling. (This will enable capillary action to help you release all the contents of the tip into
the tube; if you do not do this, some liquid may remain in the tip.) As you expel, keep the
pressure on the plunger. Do not release the pressure until you have removed the tip
from the tube. (Otherwise you’ll just suck the liquid right back up into the tip!)
The best technique is always to watch the liquid to be absolutely sure that you got some in the
pipette tip and that all of it was expelled. As you become used to using the micropipettors, you
will get a good sense of how different volumes appear in a tip. This will help you avoid
pipetting errors, particularly those that occur when you accidentally pick up the wrong
pipettor.
Some pipettors have an ejector on one side. After use, the tip can be popped off by pushing
down on the ejector button at the top of the pipettor. If your pipettor does not have an
ejector, you’ll have to remove the tip with your fingers.
READING THE WINDOWS ON THE MICROPIPETTORS
PIPETTOR
P1000
P200
P20
TOP
VALUE
1000 uL
200 uL
20 uL
READING IN WINDOW
AT TOP VALUE
1-0-0
2-0-0
2-0-0
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SAMPLE
READING
0-7-7
0-7-7
0-7-7
ACTUAL VOLUME OF
SAMPLE READING
770 µL
77 µL
7.7 µL
Lab 1, Page 4
SOME DO'S AND DON'TS WITH MICROPIPETTORS
Micropipettors are expensive instruments and should be handled with extreme care.
Please, never 'mess around' or 'play' with these instruments. Each one costs about $350 and
can be easily damaged by carelessness and inattention.
Always use the appropriate pipettor for the volume you desire.
The P20 is used for volumes of 1 µL to 20 µL.
The P200 is used for volumes of 20 µL to 200 µL.
The P1000 is used for 200 µL to 1000 µL.
Remember never to turn the wheel past the maximal volume for the pipettor. This
breaks the instrument and it must then be sent in for repair (which costs about
$50).
Always hold the instrument upright; DO NOT hold the micropipettors horizontally or upside
down.
Remember to depress and release the plunger slowly so that the liquid in the pipette tip does
not splash into the shaft of the pipettor. If the shaft becomes contaminated, all downstream
samples can become contaminated.
If fluid enters the shaft, please call this to the attention of your instructor or a TA so that the
instrument may be cleaned without damage. Please do not attempt to remove the
contaminating fluid yourself.
When placing a new tip on the end of the shaft, do so with care and attention.
2. MEASURING VOLUMES OF 1 ML OR GREATER: USING SEROLOGICAL PIPETTES
To accurately measure volumes larger than 1 ml, glass or plastic serological pipettes are used.
These are available in a variety of volumes; we will be using 5 ml and 10 mL pipettes in this
class. (The pipette size refers to the largest volume that it can deliver.) A pipette pump is
used to get liquid into the pipette. You will be learning in the "To Do" section how the pumps
are used.
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3. MEASURING WEIGHTS OF 10 G OR LESS
To accurately measure the weight of a dry substance, particularly when the amount is small,
plastic weigh boats or weigh paper are generally used. A small spatula is used to carefully
transfer the powdered reagent to the weigh boat or paper and the substance is then weighed
on a scale. Since the weight of the boat or paper needs to be subtracted from the total weight
in order to get an accurate measurement, the weigh boat or paper is first placed on the scale
and the scale is "zeroed" or "tared" (set to 0.00) before the substance is added for weighing.
When making solutions in the lab, it is critical that you understand the difference between
weight (or mass), moles, molecular weight, and molarity. These terms are defined
below. Make sure you commit them to memory.
Weight is a measurement of the gravitational force acting on an object. Near the surface of
the Earth, the acceleration due to gravity is approximately constant; this means that an
object's weight is roughly proportional to its mass. Therefore, because all our
measurements will be used on planet Earth (there will be no field trips to Venus or
Mars this semester!) we will use the terms weight and mass interchangeably in this
class. Please remember, however, that weight and mass are not equivalent terms in physics.
A mole is defined as 6.02 ×1023 atoms or molecules of a substance. Note that if the molecule
has a large molecular weight, one mole of that substance will weigh more than one mole of a
molecule that has a lower molecular weight.
Molecular weight (also called formula weight) is defined as the weight of one mole of an
atom or molecule. The most common unit of expression for molecular weight is g/mol. NaCl is
a fairly small molecule, so 6.02 ×1023 molecules (one mole) of NaCl doesn’t weigh much
(58.44 grams or about 2 ounces). On the other hand, one mole of human chromosome 1 (a
molecule containing 247 million base-pairs of DNA) would weigh 160 billion grams, which is
over 170,000 tons!
Molarity (M)is defined as the number of moles per liter of a solution of a substance.
Obviously, the molarity of a solution can be manipulated by a researcher since it depends on
how much of the substance is added to how much solvent (usually water). For example, 1 liter
of 1 M solution of NaCl would contain 58.44 g of NaCl.
You will have an opportunity to practice making calculations involving these entities in the "To
Do" section of today's lab, and will learn to make solutions of a given volume and molarity.
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3. WORKING WITH SOLUTIONS
The concentrations of solutions in the lab are usually given in molarity (moles/L). However,
they may also be expressed in grams/L or as a percent. By definition, 1 g/mL = 100%, so
conversions can easily be made between these three methods of expressing solution
concentration.
For example, NaCl has a FW of 58.44. You have a 5 M solution of NaCl. What is its
concentration in g/L? What percent solution is it?
(5 moles/L)(58.44 g/mole) = 294.65 g/L
(294.65 g/L)(100%/(1 g/mL)(1 L/1000 mL) = 29.465%
You will need to learn to do these kinds of conversions quickly and easily.
In addition, you will find the following formula very helpful when working with solutions in the
lab:
(Vi)(Ci) = (Vfi)(Cfi)
where Vi = initial volume,
Ci = initial concentration
Vf = final volume
Cf= final concentration
Suppose, for example, that your stock solution of EDTA is at 2 M. You wish to make 50 mL of
a 0.8 M solution of this reagent. How would you do it? What you really need to know is how
much volume of the original solution you need to add to how much water to achieve the more
dilute solution you are after.
Vi = (Vfi)(Cfi) = (50 mL)(0.8 M)
Ci
= 20 mL
2M
So you would need to add 20 mL of 2M EDTA to 30 mL of water to make 50 mL of 0.8 M EDTA
solution.
B: Diffusion/Osmosis/Tonicity
Diffusion, osmosis, and tonicity are fundamental phenomena that govern cellular and
organismal function.
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1. DIFFUSION
Diffusion is the “random walk of particles” (paraphrased from Einstein, who first proved the
molecular dynamics of the phenomenon) due to the fact that no substance can ever be
brought to a temperature of absolute zero. Particles in solution (or in gas/air) are in constant
motion and the direction of a particle at any given time is random. The net effect of diffusion
is that over time, particles will tend to move from areas of high concentration (order) to areas
of lower concentration (less order). This is a practical example of entropy, the Second Law of
Thermodynamics. Summarized differently, things go down their concentration gradient (from
high to lower concentration)…or, said another way, left to their own accord, molecules tend to
spontaneously spread out and become less ordered over time.
For example: Imagine that you are in a room with no cross-drafts and someone at the other
end of the room strikes a match. Eventually you will smell the match, because the molecules
that are perceived (by receptors in your nose) as having "odor" have diffused throughout the
room.
When it comes to diffusion across membranes – such as into and out of membrane-bound
cells – atoms or molecules such as ions and gasses tend to move from the side where they are
highly concentrated to the side where they are less concentrated - IF they can get across the
membrane. In general, gasses and water are able to diffuse across cell membranes but
charged atoms or molecules and large molecules (like proteins) do not. They must enter and
leave the cell through various processes that will be discussed at length in lecture, including
endocytosis, exocytosis, and transport proteins embedded in the membrane. There are many
factors that can influence the rate of diffusion, including temperature, the difference in
concentration across the area through which the substance can freely diffuse, molecular size,
and solubility.
Single-celled organisms exchange nutrients and wastes with the environment via diffusion. In
multi-celled organisms, gas exchange in the lungs and between the blood and individual cells
works via diffusion.
2. OSMOSIS
Osmosis is a special case of diffusion. Osmosis refers specifically to the movement of water
across a membrane due to a concentration gradient. The net movement of water
spontaneously tends to diminish the solute concentration differences across the membrane.
Osmosis is easily observed when a wilted plant is provided water. When the plant is wilted, the
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cells have lost water and become limp. When water is added, it moves through the plant
xylem to the cells and enters them, making them turgid. As a result, the plant "perks up"
(assuming you haven't already killed it due to lack of water!).
3. TONICITY
Tonicity refers to the relative concentrations of non-penetrating (non-permeable) ions across
a membrane. When compared with blood (which has a lot of dissolved salts and other
membrane-impermeable substances), plain water is hypotonic (it has fewer non-permeable
particles in it than blood). As a result, if you were to place blood cells into plain water, there
would be a considerable concentration gradient, with a high concentration of dissolved
substance inside the cells, and essentially none outside the cell. As a result of this
concentration gradient, the tendency would be for the solute particles to cross the membrane.
However, if the particles cannot cross the membrane (i.e. they are non-permeable), the only
thing that can move to decrease the order (the concentration difference across the cell) is
water [what is the diffusion of water referred to? ________________]. Indeed, water moves
into blood cells when the cells are placed in a hypotonic solution - so much water, in fact, that
the cells lyse (burst open)!
A hypertonic solution is one that has a higher concentration of non-permeable solutes than
the solution on the other side of the membrane. If we were to place blood cells into a
hypertonic solution, the cells would shrink due to water leaving the cells in order to decrease
the concentration difference of solutes across the cell membrane.
An isotonic solution has the same concentration of dissolved solutes as that of the solution on
the other side of the membrane. In an isotonic solution, blood cells will neither lyse nor shrink.
There are many interesting applications of osmosis and tonicity. For example, to check a
human fetus for chromosomal abnormalities (such as Down Syndrome), a small amount of
amniotic fluid is withdrawn from the sac that surrounds the embryo. This sac contains cells
from the fetus that have sloughed off into the surrounding medium. The cells are then placed
in a mildly hypotonic solution, which causes water to move into the cells by osmosis. Since
animal cells lack cell walls, this causes the cells to swell and gently burst open, releasing the
chromosomes into the surrounding solution. The chromosomes are then stained, ordered, and
photographed to look for abnormalities, a process called karyotyping.
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IV. Things to Do:
NOTE: Before proceeding with these activities, make sure you can define the following terms:
solute, solvent, solution.
PART A. MEASUREMENT MADNESS!
1.
Practice using the micropipettors. Although you are working in groups of 3, each person
should practice with these delicate instruments and make sure they feel comfortable with
them and can operate them properly.
Practice pipetting the following volumes. Use the colored liquid at your bench and pipette
into a weigh boat so that you can see the size of the drop expelled. Pipette each of the
volumes below three times (into the same weigh boat) and compare the sizes of the drops
side-by-side.
a.
Set a P1000 to measure 0.5mL (500 µl) of colored water. Place the appropriate tip
on the P1000. Depress the plunger to the first stop. If you aren’t sure where the first
stop is then continue to push the plunger in further until you cannot push it in any
more. At this point you are past the 1st stop and are into the “ejection” region of the
pipettor. Past the first stop is into the un-measured region – don’t go here unless you
are ejecting your sample!
To withdraw the volume you set on the dial, you need to stop at the first stop! Read
the previous sentence again! If you ever go past the first stop and then take up
liquid, you will be beyond your desired volume and may have difficulty ejecting all that
you have taken up. Once you and your lab partners are confident that you know
where the first stop is on the plunger, depress the plunger to the first stop, submerge
the tip (only) into the liquid and then slowly release the plunger to take up the liquid.
Did the pipette tip bubble when you depressed the plunger? If so, you depressed the
plunger after the tip was submerged. This would be a mistake if you are trying to not
disturb your sample. You should always depress to the first stop PRIOR to submerging
the tip. Do not remove the tip from the liquid until the plunger is completely released.
It is advisable to watch the liquid go into the tip.
Once you have taken up the 0.5 ml of liquid, look at the tip. Are there any bubbles in
the liquid? If so, you released the plunger too rapidly so some of the sample flew up
high into the tip (and possibly contaminated/ruined the pipettor). Alternatively,
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bubbles will result in the tip if the tip comes above the surface of the sample before
the plunger is fully released. So, don’t release the plunger too fast, and keep the tip
below the surface the whole time the sample is being taken up! As soon as you have a
good 0.5 ml sample, pipette it into the weigh boat. Repeat and place the second
(and 3rd and 4th) samples near to the first sample. Do all of the samples appear to be
the same size?
b.
Repeat the above procedure with the P200, the appropriate tip, and pipetting 90 µL
of the colored solution. Each person should repeat this 3 – 4 times. You can use the
same weigh boat that you used the first time; just be sure to rinse out the weigh boat
first.
c.
Rinse out your weigh boat and dry it carefully. Using the scale, tare a weigh boat.
Pipette 1 mL DI water onto the weight boat. (The DI water comes from the lab tap
with the white cap. If you can’t find it, ask your instructor or a TA.) How much does it
weigh? Record this value in your lab notebook. Tare the weigh boat again and pipette
500 µl onto the weight boat. How much does this weigh? Now tare again and pipette
200 µl. How much does it weigh? How much does 100 µl weigh? How much does 7 µl
weigh? Be sure to record all of these values in your lab notebook.
d.
Now, PLOT the relationship between water volume and mass on graph paper
and tape the graph into your lab notebook. Is the result a straight line (i.e. is the
relationship linear)? If not, what do you see instead?
3.
Put a 10 mL pipette into the appropriate pump—do this gently but firmly. Insert the tip
of the pipette into a liquid. Slowly roll the wheel on the shaft of the pump and allow
the liquid to come to the '2' line. This will draw liquid into the pipette. Be very careful
not to allow the liquid to get into the pump!! When reading the volume, you always
read the bottom of the meniscus. Now, transfer the pipette to an empty test tube.
Press the lever-like arm on the shaft to expel the liquid from the pipette. How many
mL did you pipette? Record the volume in your lab notebook.
A small amount of liquid will remain in the tip of the pipette. This is the design of this
particular pipette. You do not need to get the last little bit out. Notice that the top of
the pipette, near the brand name and volume, is a 'TD'. This stands for 'to deliver'
and indicates that the volume delivered is accurate when a small amount remains in
the tip. All of the pipettes we will use in this class are TD pipettes.
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In contrast, some pipettes have a 'TC' designation. This means ' to carry' and in the
case of these pipettes, that last little bit of liquid does need to be expelled from the
tip.
5.
Also examine the 5 mL pipettes. What volume is the subdivision on each of
these?__________ What is the number at the bottom of each pipette?_______.
Now using the appropriate pipette type for each volume, pipette the following volumes
into the empty test tubes at your lab table. Compare the height of liquid in your test
tube with those of your lab partners (be sure the diameter of your test tubes are the
same). Are they the same?
1.8 mL
6.
3.2 mL
4.7 mL
6.7 mL
9.9 mL
Examine the reagent bottle of NaCl. Note that the MW (sometimes also referred to as
formula weight, FW) of NaCl is 58.44 g/mol. (This information should be clearly visible
on the outside of the bottle.) Note also that the bottle is marked with the name of the
manufacturer and a lot number. Record this information in your lab notebook.
7.
You will now make 100 mL of a 1 M solution of NaCl. To do this, you will first need to
calculate how much NaCl you will need to measure out in your weigh boat:
(100 mL) (58.44 g/mol) (1 mol/L) (1 L/103 mL) = 5.844 g
Take a careful look at this calculation and make sure you understand it. Cross out the
units that cancel. Note that when you do this, only grams (g) remain, and they remain
in the numerator, not the denominator.
Now place a weigh boat on the scale and tare the scale. Then, using your spatula, add
5.844 g of powdered NaCl to the weigh boat.
Fill up your graduated cylinder with about 90 mL of water. (DO NOT fill it all the way
to 100 mL because the NaCl will increase the volume a little bit when added, and you
want the FINAL volume of the solution to be exactly 100 mL.)
Add the water to your beaker and then add the powdered NaCl that you measured out.
Then add a stir bar to the beaker and place the beaker onto one of the electronic
mixers. Turn the mixer on so that the stir bar begins to move (it is metal and
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responds to a magnet in the mixer). It should not take long for the solution to become
clear as all the NaCl dissolves into the water.
Pour the entire solution back into the graduated cylinder and add water to 100 mL.
Then pour the solution back into the beaker. You now have 100 mL of a solution
of NaCl that is exactly at 1 M (mol/L). Congratulations!
By the way, did you notice how quickly the NaCl went into solution? What chemical
properties of NaCl make it so soluble in water?
8.
Rinse out the beaker and the graduated cylinder and repeat the above steps to make
50 mL of a 0.5 M solution of NaCl. Write your calculation out in your lab
notebook BEFORE coming to class so that you do not waste time in class
making the calculation. Check with your lab partners to make sure that your
calculation matches theirs before proceeding.
9.
You will now make 100 mL of LB broth. This is a nutrient-rich broth used to grow
bacteria, and you will be using the broth you make today later in the semester. Once
you have made up your broth, you will pour it into the autoclave bottle (orange cap)
for sterilization.
To make up your broth:
Add the following to an empty 250-mL glass beaker containing a stir bar:
1g
tryptone
0.5 g
yeast extract
0.5 g
NaCl
Using a graduated cylinder, collect 80 mL of tap water and add it to the beaker. Stir
the powdered ingredients into the water until you have a clear solution. (It will be
yellow in color.) Then pour the broth back into the graduated cylinder and add
additional water to exactly the 100 mL mark.
Pour the broth from the graduated cylinder into the orange-capped autoclave bottle.
Place the lid on the bottle and tighten it slightly. Do not tighten the lid all the way
because steam needs to be able to escape from the bottle during the sterilization
process. If too much steam builds up in the bottle, it may explode – or at least crack
open.
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Place a small (approx. 1 inch long) piece of autoclave tape of the side and lid of the
bottle. This tape looks white prior to autoclaving but will become striped during the
autoclaving process. The tape therefore provides a visual mark indicating that the
solution is sterile.
Finally, label the bottle using the colored labeling tape and your Sharpie. The label
should include the name of the substance (LB broth), it’s concentration (1X in this
case), the date the solution was made (today’s date), and your initials. “1X” is a
relative term for concentration and indicates that the solution should be used “as is” in
an experiment – i.e. it does not need to be diluted prior to use. 1X solutions are also
often referred to as “Working Solutions.” Sometimes, “Stock Solutions are made up at
higher concentrations – e.g. 5X, 10X, 100X, etc. These solutions usually need to be
diluted to a 1X concentration for use in the lab.
An autoclave is a type of oven that sterilizes liquids and instruments by raising them
to high temperature and pressure for a period of time. When solutions are autoclaved,
they should be placed in a special autoclave tray so that if the glass breaks, the
solution does not get all over the autoclave. An autoclave tray is located on the front
bench of the lab. Place your bottle in that tray when it is labeled and ready. Check
again to make sure that the lid on the bottle is loose, not tightened down.
When all the bottles are ready, your instructor will take the entire class on a "field trip"
to the autoclave room (located in Humboldt Hall) and demonstrate how to use the
instrument.
PART B. DIFFUSION/OSMOSIS/TONICITY
1.
DEMONSTRATION 1: OSMOSIS AND TONICITY
1.
Before class, the lab technician placed a thin slice (~1/8" inch thick) of potato in a
Petri dish containing a strong salt (10% NaCl) solution. Enough salt solution was
added to cover the potato slice. Another slice was also prepared and placed in a Petri
dish with distilled water.
2.
Using your fingers or the tweezers provided, pick up the potato slices and compare
how they feel. What is the difference? Record the results in your lab notebook.
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3.
Now interpret the results. Does this experiment demonstrate osmosis, diffusion,
tonicity or a combination of these? Explain.
2.
DEMONSTRATION 2: DIFFUSION
1.
Before class, the lab technician prepared six Petri dishes of 1% agar. (You’ll learn
more about agar in Laboratory 2.) The agar was added to water, boiled, and then
cooled, allowing it to solidify. After cooling, a few crystals of potassium permanganate
(KMnO6; MW - 158 g/mol) were added to the center of three of the agar plates and a
few crystals of crystal violet (C25H30CIN3; MW = 408 g/mol) were added to the center
of the other two.
2.
One plate with potassium permanganate and one plate with crystal violet were then
incubated at 37 degrees C (body temperature) overnight, one set of plates was
incubated at room temperature overnight, and one set of plates was incubated in the
lab refrigerator overnight. All the plates were incubated for exactly the same length
of time and placed on the lab bench immediately before the lab period began.
3.
Examine the plates. How does the MW of a substance influence its rate of diffusion
through the agar? How does temperature influence its rate of diffusion? Explain.
3.
DEMONSTRATION 3: MOLES, WEIGHT, AND MOLECULAR WEIGHT
1.
Both baggies (A and B) weigh exactly the same amount.
2.
Assuming that the contents of these baggies represent molecules, which baggie (A or
B) contains the most moles of the substance? Which substance (A or B) has the
greater molecular weight (g/mol)?
3.
Working with your lab partners, try to figure out a way to determine how many moles
of rice grains are contained in the baggie of rice.

What information do you need in order to make your calculation?

How can you go about getting that information, using the equipment and materials
available to you in the laboratory?

This is a solvable problem – see if you can figure it out!
BIO 2 Lab Manual, Fall 2008 Version 8/27/2008
Lab 1, Page 15
V.
Lab Clean-Up
Before leaving the lab today:

Place all used serological pipettes in the large plastic containers provided.

Rinse your test tubes, spatula, stir bar, beaker and graduated cylinder, making
sure they are free of colored water, salt solution, or powdered reagents.

Wipe out weigh boats with a kim wipe and stack them the way you found them.
(They can be reused by the next lab section.)

Inform the instructor or TA if you are low or out of micropipettor tips or serological
pipettes.

Empty the contents of your solid waste beaker into the trash and rinse it out.

Empty the contents of your liquid waste beaker into the sink and then rinse it out.

Arrange all items on your bench the way you found them and wipe down the work
area of your bench with the disinfectant.

Wash your hands.
BIO 2 Lab Manual, Fall 2008 Version 8/27/2008
Lab 1, Page 16