LAB 3:
Bacteria Colony Morphology & Mouthwash Experiment
I. Objectives
The purpose of today’s lab is to help students distinguish between different
bacterial species based on colony morphology and to enumerate the types of
environmental species. This lab will also introduce the concept of serial
dilutions and Kirby-Bauer tests. By the end of today’s lab, students should:

Be able to identify different aspects of bacterial colony morphology on agar
plates

Understand that the different colony morphologies (phenotypes) reflect
genetic differences among bacterial species

Be able to perform a Kirby-Bauer test and understand how it works
II. Safety Considerations for this Lab

WEAR GLOVES WHEN DEALING WITH THE ENVIRONMENTAL BACTERIA
PLATES AND WHEN TOUCHING THE KIRBY-BAUER DISCS (Why?)

Do not touch the bacterial colonies on the environmental plates.

Dispose of the environmental plates as biohazardous waste. (Why?)
III. Introduction
A. BACTERIA DIVERSITY AND NUMBERS
Each colony on an agar plate 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
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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.
B.
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.
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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 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 degrees C to determine whether or not the colony you chose is
a coliform, and if it is, if it is E. coli.
You will also take part of the colony and use it to “seed” the LB broth you made last time. The LB
broth will then be incubated overnight, shaking, at 37 degrees C to promote the growth of the
bacteria. After the overnight incubation, there will be about 109 bacteria per mL, and you will
eventually isolate the DNA from the bacteria in the culture and test it for the presence of genes
specific to coliforms and to E. coli.
C.
EFFECT OF MOUTHWASH ON GROWTH OF ORAL BACTERIA
In today’s lab, we are also going to test oral bacteria for their susceptibility or resistance to
possible anti-microbial agents found in Listerine and Scope mouthwashes. The companies that
make Scope and Listerine claim that their products kill or inhibit oral bacteria, but is this really
true?
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A simple way of determining the susceptibility of a microorganism to an antimicrobial agent is
to spread the test bacteria (in this case, from the mouth) over the entire surface of an agar
plate. Filter paper disks saturated with the test solution (for this experiment Scope and
Listerine) are added to the bacteria-seeded agar. The test solution then diffuses into the agar
medium. As the solution diffuses from the filter paper into the agar, the concentration of the
solution decreases as a function of the square of the distance of diffusion. At some particular
distance from each disk, the antimicrobial agent is diluted to a point that it no longer inhibits
microbial growth. The effectiveness of a particular antimicrobial agent results in the production
of growth-inhibition zones that appear as clear areas surrounding the disk from which the
agent diffused (see figure below). The diameter of the zones can be measured with a ruler and
the results of such an experiment constitute an anti-biogram. This procedure is known as the
Kirby-Bauer test and provides a rapid assessment of anti-bacterial effect for any water
soluble compound. The controls for this experiment are water (negative control), which should
not kill the bacteria, and ciprofloxacin (positive control), an antibiotic that should kill all oral
bacterial species.
Kirby-Bauer test.
Observe growth-inhibition
zones
IV. THINGS TO DO
PART A. 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
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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
3.
Colony
Color
Colony
Elevation
# this Type
of Colony
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 think: Did this plating experiment have a hypothesis? If so, what was it?
PART B. SELECT A COLONY FOR PLATING ON EMB AGAR
1.
Put on a fresh pair of gloves.
2.
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.)
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3.
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.
4.
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.
5.
Place your plate in the 37 degree incubator oven, noting its location so that you can
retrieve it next lab period.
PART C. “SEED” LB BROTH FOR OVERNIGHT GROWTH
1.
Retrieve your LB broth from the front or side bench of the lab. Note that the autoclave
tape is now striped, indicating that the LB solution is now sterile.
2.
Using a sterile Q-tip, once again remove a small part of the same colony you used for
Part B.
3.
Remove the lid from the bottle of LB broth and dip the Q-tip into the broth. Make
sure you are wearing gloves so that you don’t introduce any other bacteria
into the broth!
4.
Immediately replace the lid. Tighten it down all the way and then untighten it about
one half of a turn. This will ensure that oxygen can get into the bottle while the
bacteria are growing. Coliforms require the presence of oxygen, so your culture will
not grow if the lid is tightened down all the way.
5.
Replace the label on the bottle. The new label should have the name of the culture
(“Possible coliform” is a good title to use), the date, and the initials of all of your lab
partners.
6.
Bring your bottle to the front of the room. The lab technician will help you load it into
the shaking incubator. When all the bottles are in place, the incubator will be set to 37
degrees and started. After the overnight incubation, the culture will be removed and
placed in the refrigerator. The cells in the culture will eventually die (why?), but their
DNA will still be intact and available for isolation and study.
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PART D. TEST SCOPE AND LISTERINE IN A KIRBY-BAUER ASSAY
1.
Working in your lab group, obtain three filter paper disks and three small pieces of
aluminum foil. Place one filter disk onto each piece of foil. DO NOT mark the filter
disks. Using a P200 micropipettor, add 50 uL of Scope mouthwash to the first filter
disk, 50 uL of Listerine mouthwash to the second filter disk, and 50 uL of sterile water
to the third filter disk. Allow the disk to sit for 5 minutes to fully absorb the liquid
before adding to the agar plate.
2.
Obtain a BHI agar plate from the front bench. Using a wax pencil, draw four
quadrants on the agar side of the plate (not the lid) and label the quadrants 1-4. Using
a sterile cotton swab, have one person in the group thoroughly swab his/her mouth,
concentrating on the tongue and cheeks, so that the swab is saturated. Then roll the
swab back and forth across the surface of the agar plate as follows:
a.
left to right
b.
top to bottom
c.
side to side at a diagonal angle
It is very important to make certain that the entire surface of the agar plate
has been inoculated with the swab. Otherwise the assay will not work
properly. When you are done, dispose of the swab in the trash.
3.
Using a pair of STERILE forceps, add the filter disks to the agar plate as follows:
a.
Scope filter disk to quadrant 1
b.
Listerine filter disk to quadrant 2
c.
Water filter disk to quadrant 3 (negative control)
d.
Ciprofloxacin antibiotic filter disk to quadrant 4 (positive control); Available at the
front bench
Gently tap the disks once they are on the agar with the STERILE forceps to ensure
disk adherence to the agar surface.
4.
Secure the lid to the plate with tape and label the lid with the initials of your group
members and your lab section number. Take it to the 37 degree incubator and place it
lid-side down, noting its location in the incubator.
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V. Lab Clean-Up

All tubes used in the serial dilution experiment should be emptied into the sink and
returned to your tube rack for clean-up by the lab technician.

The leftover plates from the environmental bacteria/colony morphology experiment
should be discarded in the large biohazard bag at the front of the class.

Gloves can be discarded in the regular trash.

Wash your hands before leaving lab.
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