LAB 3:
“Measurement Madness!”
Diffusion/Osmosis/Tonicity
and
Diversity of Bacteria (Continued)
I. Objectives:
Today’s laboratory will have three distinct sets of exercises. The first exercise will introduce you to several of
the basic and most commonly used laboratory techniques in cellular and molecular biology. The second set of
exercises will introduce you to the concepts of diffusion, osmosis, and tonicity and demonstrate how these
natural phenomena can be visualized and studied in a laboratory setting. In the third set of exercises we will
carry on the experiments that we began last week examining the diversity of bacteria in the environment. By
the end of today’s lab, you should be able to:

Accurately measure and dispense small volumes of liquid ranging from 1 µl to 5000 µl

Perform calculations and unit conversions involving weight, moles, molecular weight,
concentration, and volume

Accurately prepare solutions of known concentrations starting with solid or liquid reagents

Accurately articulate the difference between grams and moles of a substance

Define the terms "diffusion," "osmosis," and "tonicity" and explain why they are important
for the survival of cells

Explain how the rate of diffusion of a substance is affected by temperature, concentration
gradient, and molecular size and weight

Define hypertonic, hypotonic, and isotonic solutions and describe the effects of placing
living cells in each type of solution.

Plate bacteria

Describe how selective media and indicator media can be used to begin to isolate and
identify bacteria.
II. Safety Considerations

Do not touch the agar plates used to observe diffusion. These plates contain either potassium
permanganate or crystal violet which are strong dyes that can permanently adhere to (stain) clothing.

All of the chemical 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.
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
The bacteria (and fungi) that we have sampled from the environment could be harmful so wear gloves
when handling bacteria and performing serial dilutions

Do not touch the colonies on any of the plates

Leave the lids on the plates at all times
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
know the following simple units of the metric system of measurement:
1 L or l (liter) = 1,000 mL (milliliters) = 1,000,000 µL (microliters)
For example, 650 mL = 0.65 L and 200 µL = 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 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 force the wheel beyond the volume of
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the micropipettor (e.g., a P20 should not be adjusted to above 20 µl) since this can strip the gears
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 be placed on the end of the shaft prior to use. There are two sizes
of tips for the pipettors that we will be using: 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, after removing the tip from the box, you may also gently twist it on further with your fingers to ensure
a snug fit. 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
To measure a liquid: 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
(just 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 then
slightly past this position. It is possible to continue until you reach the second (final) stop position—but try
to completely dispense the liquid by just passing the first stop. 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
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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
SAMPLE
READING
0-7-7
0-7-7
0-7-7
ACTUAL VOLUME OF
SAMPLE READING
770 µL
77 µL
7.7 µL
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 $285 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-$150).

Always hold the instrument upright;

NEVER hold the micropipettors with the tip above horizontal.

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.
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
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.
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.
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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.
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
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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) cannot. They must enter and leave the cell through various processes that will be discussed at
length in lecture, including endocytosis, exocytosis, and transport through 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.
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 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
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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.
PART C. MICROBIAL BIODIVERSITY (CONTINUED)
1. BACTERIAL DIVERSITY AND NUMBERS
Today we will also examine the plates you prepared last week from your environmental samples. Each
colony on an agar plate like those your prepared represents a clonal population of bacterial cells. That is, all
the cells in the colony arose from the division of one original cell that adhered to that spot on the agar prior to
incubation of the plate. Depending on the type of bacteria, the colony will take on a characteristic morphology
or appearance that can be described and recorded to get an idea of the diversity of bacteria in the
environmental source that was tested.
The diagram below shows the different types of colony morphologies that you might see on your plate and
the terminology that microbiologists use to describe them. Note that the morphology includes vertical as well
as horizontal characteristics of growth. In addition, the color of the colony should be recorded as this can
provide important information.
8
2. COLIFORMS
One major group of bacteria that you are likely to find on your environmental plates is the coliforms.
Coliforms are Gram-negative, rod-shaped bacteria that are capable of fermenting lactose. There are over 27
genera of coliforms, including Citrobacter, Enterobacter, Escherichia, and Klebsiella. Their common ability to
ferment lactose makes them unusual and allows scientists to isolate and study them apart from other
bacteria. On BHI agar, coliforms (as well as some other types of bacteria) form cream-colored colonies with
smooth edges. Therefore, when you examine your plates, colonies with this kind of morphology may be a
coliform and it may be E. coli. There is no way to tell without further analysis.
One unusual species in the coliform family is Escherichia coli (E. coli). E. coli is unique among
coliforms in that it grows exclusively in the intestinal tracts of several mammalian species including humans,
cows, and pigs. Due to the fact that it lives in the intestines, E. coli can be found in large numbers in
mammalian feces, since the bacteria adhere to the feces as they are deposited from the body. Once released
to the external environment, however, E. coli does not usually survive more than about 8 hours.
Since E. coli does not grow outside the mammalian intestines, the presence of live E. coli in a water
sample always indicates recent fecal contamination of the water. Therefore, while coliforms as a group are a
normal and innocuous resident in fresh-water, the presence of E. coli in a water sample is considered a
positive test for fecal contamination. In fact, if a water sample has as little as one E. coli cell/mL, the water is
considered unsafe to drink.
E. coli can be distinguished from other coliforms by its growth and color reaction on eosin methylene blue
(EMB) agar. EMB is a selective and differential medium in that it promotes the growth of coliforms (selective) and
differentiates between E. coli and other coliforms (differential). Coliforms metabolize lactose in the medium (no
other sugar is available) and release acid as a waste product. EMB agar can distinguish E. coli and other
coliforms based on the amount of acidic waste product that is released. E. coli generates a large amount of
acid while all other coliforms produce a small amount of acid. The change in pH caused by the release of
acidic waste products causes the precipitation of the eosin (a red dye) and (sometimes) methylene blue (a blue
dye) from the medium. On EMB, the large amount of acid released by E. coli will cause the precipitation of both
the eosin and methylene blue dyes, causing the E. coli colony to become metallic green in color. All other
coliform colonies will be pink (or pink with a dark center) since the small amount of acid they release will only
precipitate the eosin (red) dye. Occasionally, non-coliform bacteria will also grow on EMB agar, but they usually
grow slowly and remain dark in color.
In today’s lab, you will examine your environmental plates and choose one colony (per lab group) that
appears to be a coliform. You will then take part of the colony and plate it onto EMB agar. Your plate will be
incubated at 37
o
C to determine whether or not the colony you chose is a coliform, and if it is, if it is E. coli.
In our lab next week you will isolate the DNA from the bacteria you have cultured and begin to test it for
the presence of genes specific to coliforms and to E. coli.
9
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 precision 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, 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?
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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
deionized (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?
2.
For volumes of 1-10 ml serological pipets are often used. P5000 pipets are available, but in many
instances are less convenient than serological pipets.
3.
Examine the 5 and 10 ml pipets that are available in the lab. What volume does each division
represent?
4.
Serological pipets require a device of some sort to draw up and dispense liquids (we do not allow
mouth pipetting in any instance). We’ll have several of these available for you. Try out the pipet
pump and pipet bulb with both 5 ml and 10 ml pipets. Before doing this, note whether the pipets you
are using are marked TD or TC. TD pipets are designed “To Deliver”. These pipets are designed to
leave a bit of liquid in the tip once the sample is dispensed. Therefore, do not dispense the last little
bit that remains. TC pipets are “To Contain” and are designed to deliver the correct amount if all of
the contents are “blown out” of the tip (using the device—not your mouth).
5.
Practice drawing up and dispensing using the colored liquid supplied or just use water.
6.
Next, pipet 2.5 ml into a weigh boat, weigh it and record its weight. How much should it weigh. How
close were you to that weight? Repeat this for 5, 7.5, 8.2 and 10 ml.
7.
How could you measure 12 ml with one of the 10 ml pipets supplied?
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PART B. DIFFUSION/OSMOSIS/TONICITY
DEMONSTRATION 1: OSMOSIS AND TONICITY
1. Before class, a thin slice (~1/8" inch thick) of potato was placed 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.
3. Now interpret the results. Does this experiment demonstrate osmosis, diffusion, tonicity or a
combination of these? Explain.
DEMONSTRATION 2: DIFFUSION
4. Before class, six petri dishes of 1% agarose were prepared. The agarose 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 agarose plates and a few crystals
of crystal violet (C25H30CIN3; MW = 408 g/mol) were added to the center of the other two.
5. One plate with potassium permanganate and one plate with crystal violet were then incubated at 37
o
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.
6. Examine the plates. How does the MW of a substance influence its rate of diffusion through the
agarose? How does temperature influence its rate of diffusion? Explain.
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?
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
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!
4. Once you have figured out how to calculate the number of moles of rice grains in the baggie, calculate
how much ONE mole of rice would weigh.
PART C. BACTERIAL DIVERSITY
RECORDING BACTERIA SPECIES DIVERSITY
1. Put on a pair of gloves.
2. Retrieve your two BHI agar plates from last time. Using the morphology chart provided in the introductory
section to this lab, record the number of different types of colonies you observe and record your results in
your lab notebook, using the following table format. (You should have this table copied into your lab
notebook before coming to class as part of your lab protocol write-up.)
Environmental Colony
Source
Shape
Colony
Color
Colony
Elevation
# of this Type
of Colony
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3.
Use the books at the front of the room to try to identify what type of bacteria are on your plate. Note
that many bacteria have similar morphologies on BHI agar and it may not be possible to know for
certain what you are seeing. Just do the best you can.
4.
Now, consider the hypothesis you developed last week and the samples you have collected. Record
any data that you can obtain from your plates that will allow you to address your hypothesis.
a.
Can you represent your data in a way that helps you understand your results?
b.
Do you need to conduct additional experiments to address your original hypothesis? Briefly
describe at least one additional experiment or modification of our first plating experiment that
could be done to better address your hypothesis.
PART B. SELECT A COLONY FOR PLATING ON EMB AGAR
1.
We are going to use DNA analysis in upcoming labs to help us identify the specific types of bacteria on
your plates. Since it is challenging for this big group to look at many different types of bacteria by
DNA analysis, we will all look for one type of bacteria, E. coli, in order to illustrate how this can be
done in general. As described in the introduction, we can begin to look for E. coli using the indicator
medium EMB.
2.
Put on a fresh pair of gloves.
3.
At least one of the plates in each lab group should have a cream-colored colony with smooth edges.
Compare your plates with those of your lab partners and find one colony with this type of morphology.
Try to choose a colony that is isolated (not touching or overgrown by other colonies.)
4.
Once you have identified a colony, use a sterile cotton swab to remove a small bit of the colony. Then
swipe the cotton swab across a plate of EMB agar, spreading the colony cells as evenly as you can.
5.
Place the lid on the plate and wrap a piece of tape around the plate to hold the lid in place. Then write
the date, your lab section number, and your initials (for all members of your lab group) on the tape
using your Sharpie pen.
6.
Place your plate in the 37
o
C incubator oven, noting its location so that you can retrieve it next lab
period.
7.
Your original plates can be discarded in the biohazard container at the front of the lab.
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 kimwipe and stack them the way you found them. (They can be
reused by the next lab section.)
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
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.

As noted above, your plates from last time should be discarded in the biohazard bag

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.
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