Vi-An Nguyen

BIO 210
Lab report #1
Microbial Growth
Background information:
In lab, there were four bacteria that were introduced and learned about in class. The first
was Staphylococcus epidermidis. This is a non-motile and non-spore cocci shaped bacterium. It
showed up purple on a gram stain, and forms grape-like clusters. This bacterium has an optimal
temperature range of 26-37C. S. epidermidis is found in the human body more on the outside
skin like wounds. With that, this bacterium can cause urinary tract infections
(http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.ht
ml).
On the other hand, the bacterium Escherichia coli is rod-shaped, Gram-negative, and
forms in small clusters or short chains. The bacterium Proteus mirabilis was a unique bacterium
because it swarmed, and has an optimal temperature of 37C
(http://www.who.int/mediacentre/factsheets/fs125/en/). This type of bacterium is found and
thrives in the gut of humans and overall does not cause any harm to the human body. However,
some strands can cause food poisoning (http://www.medicalnewstoday.com/articles/68511.php).
It is also possible that some E. coli strands can cause urinary tract infections (RW 743).
Proteus mirabilis is a bacterium with a gram-negative reaction and is rod shaped. P.
mirabilis is different because it swarms so it is motile. The bacterium has an optimal growth
temperature at 37C (http://www.tgw1916.net/Enterobacteria/Proteus.html). P. mirabilis can
replicate and survive in moist reservoirs so can be caused by drinking contaminated water
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122780/).

Pseudomonas fluorescens is a gram-negative rod bacterium. This type of bacterium has

an optimal growth temperature of 37 or above for a response
(http://www.gutpathogens.com/content/2/1/16). P. fluorescens can be found in food, especially in
the form of biofilms. It thrives in spoiled food which can cause sickness in humans when
digested (http://www.biomedcentral.com/1472-6750/8/790).

In this lab, both selective and differential media were used to inoculate and observe the
bacteria. A selective medium specifically is used only to allow certain types of organisms to
grow and prevents any other organisms for growth. For a selective medium to be used in an
experiment what is used on the medium or the incubation temperature could be altered.
A differential medium differs from selective medium because differential is designed to show
how differently the bacteria look when they grow. Differential compares two colonies of bacteria
on a growth plate to see their characteristics and growth patterns. This also used oxidase and
catalase tests for oxygen utilization. The oxidase test is useful to identify the type of metabolism
used by the bacteria because an oxidase tests able to detect if the bacteria contained the oxidized
form of cytochrome c and seeing if the colonies of the bacteria form a dark violet color. The
oxidase test can also differentiate between Pseudomonas and Aeromonas. The catalase test is
beneficial to identify if the bacteria are aerobic which would mean that there would be a
formation of bubbles, otherwise they would be facultative anaerobes.

Methods:
In the experiment, there were five different types of agar that needed to be inoculated
with bacteria to see if there would be growth. The five different type of agar used in the lab were
blood agar, MacConkey agar, Mannitol-agar, Hektoen entric agar, and Trypticase soy agar. The

agar is already in the petri dishes and labeled with the type of agar being used. The petri dishes
should then be divided in half with a marker on the bottom side of the plate to then label each
side with a bacterium to tell them apart. After, there should be dishes containing each bacterium
for the experiment needed to be put into the agar plates. With a sterilized inoculation loop, a
colony of the bacterium needed for the agar should be taken lightly. Only a small colony should
be taken for a sample. Then transfer the bacterium to the appropriate agar plate to match up the
bacterium. Then the inoculation loop should streak the plate with a straight line towards the
middle of the line, then strokes should be drown coming out of the straight line creating an E
shape. After, all five plates should be streaked the same way and the petri dishes should be
closed right after streaking. The plates should be turned upside down with the label facing up and
then be placed in the incubator at 37C for 24 hours and then should observed for growth.
In the oxidase test, the microorganisms E. coli, P. flourescens, and S. epidermidis were
being compared to see if they were positive or not. First, a colony of the samples should be
transferred to a glass slide from a sterilized inoculating loop. Then, drop one or two drops of the
oxidase reagent onto the bacteria. Wait for a one minute or so and examine if there are any color
changes. Colonies should start turning into a light color then dark. With that, the results with
color changes would be positive.
In the catalase test, two slides will be needed because water and S. epidermidis will be
used to compare the two. A drop of water is need on one glass slide while a colony of S.
epidermidis is needed to be transferred from a sterilized inoculating loop to the glass slide. Then,
each slide will need about two to three drops of hydrogen peroxide since that is the reagent. After
waiting about a minute the slides should be checked to see if there is bubble formation. If there
were bubbles, then the reaction would be positive.

Results:
Table 1: Growth of bacteria on selective and differential media
Bacterium
MacConkey agar
Mannitol-Salt agar
Uninoculated
Dark violet
Red
S. epidermidis
No growth
No color change with
red zones with no
hydrogen sulfide
production
E. coli
Pink colonies
Yellow color
surrounded with pink surrounding colonies
zones with no
with no hydrogen
hydrogen sulfide
sulfide production
production
P. mirabilis
Colorless colonies
No color change in
with no hydrogen
colonies with no
sulfide production
hydrogen sulfide
production

Hektoen enteric agar

Green
No growth

Salmon-pink colonies
with no hydrogen
sulfide production

Black colonies with

hydrogen sulfide
produced

Table 2: Hemolytic reactions of bacteria grown on agar

Bacterium
Type of hemolysis observed
S. epidermidis
Gamma hemolysis
E. coli
Gamma hemolysis
P. mirabilis
Gamma hemolysis

For the oxidase test, the bacteria E. coli, S. epidermidis, and P. flourescens were tested to see if
they were negative or positive. In this case, E. coli demonstrated a negative result with no
reaction. On the other hand, both P. flourescens and S. epidermidis bacteria tested positive for
the oxidase test.

For the catalase test, water and S. epidermidis were compared to observe if the enzyme catalase
were present. When water was tested, the results came out negative. On the other hand, S.
epidermidis came out positive because there was a formation of bubbles after adding the reagent.

Discussion:
1. The mediums that were used in the lab were found in the BIO 210 lab manual 1st quarter,
2013, DePaul University, and (October 13, 2012). The first medium that was used in the
lab was blood agar. It is a differential medium because it has differential properties due to
hemolysis and can be identified with the kind of color zones around the colonies
(http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/selectivedifferen
tial.htm). Another medium used in the lab was macConkey agar. Since this type of agar
uses crystal violet, it is preventing the growth of any gram-positive bacteria. On the other
hand, macConkey agar compares the bacteria of their fermentation. In this case, the
medium show if the gram-negative bacteria are ferment lactose or lactose-nonfermenting
which will differ in colony colors. The use of the red indicator will show that the
bacterium is ferment lactose. Mannitol-salt agar is a selective medium because of the
sodium chloride that prevents the growth of majority of the other bacteria. It is
differential because it is comparing to see if the bacteria are mannitol fermenter or
mannitol nonfermenter by the indication of red colonies. Hektoen enteric agar has a
selective agent of bile salts that is trying to inhibit the growth of other gram positive
organisms. Also, hektoen agar can identify lactose fermenters with the appearance of
yellow color from the dyes.
2. If S. epidermidis and E. coli were growing together in a tube on nutrient broth, what can
be compared to identify which bacteria is which, the media can help with the color. For
example, knowing what the characteristics of E. coli, then that can assist in trying to
identify when comparing that bacterium to S. epidermidis. The two organisms would be
able to show whether one grows more rapidly than the other because it is possible for one

organism to grow over the other organism. It is essential to use the blood agar medium to
first separate and compare between the two bacteria. The blood agar was used as an allpurpose medium because it differentiated the organisms with the reactions on the medium
(BIO 210 lab manual, 2013).
3. P. mirabilis is one of the few organisms that are motile that was tested during the lab.
Also, it was the only organism to have had colonies with black centers on the Hektoen
enteric agar. From observing P. mirabilis, it was a lactose fermenter from the agars which
help will help identify what kind of gram reaction it would be because gram-negatives are
supposed to grow on the different mediums used in the lab. Since P. mirabilis was a
gram-negative bacterium, it was expected to grow on the agars.
4. Since blood agar was used as the all-purpose medium in the lab, it would be beneficial
for Staphylococcus aureus and S. epidermidis on blood agar because it will show the
color of its colonies to tell them apart because the bacteria are different, so the blood agar
would show the their hemolysis, thus the actions on the medium. S. aureus on blood agar
should form cream colored colonies, while S. epidermidis should form white colonies
(http://www.healthhype.com/lab-tests-for-staph.html). Tryptic soy agar is used as the
base for agar plates, so having TSA on an all-purpose agar will help identify between the
two organisms.
5. For the oxidase test, only P. flourescens and S. epidermidis had positive reactions which
can be concluded that the two organisms must use oxygen to transport their foods during
oxidative metabolism
(http://www.vumicro.com/vumie/help/VUMICRO/Oxidase_Test.htm). For the catalase
test, the organism S. epidermidis tested positive meaning that there is a catalase present

which is an enzyme that breaks down toxic products used in oxygen so the organism is
able to tolerate oxygen (http://www.ncbi.nlm.nih.gov/pubmed/1141195).
6. To identify P. mirabilis, lactose would be essential in determining the organism because
P. mirabilis was tested as a lactose fermenter and also that P. mirabilis is motile so that
differs from E. coli which is another characteristic that could differentiate them apart.
Knowing the characteristics of P. mirabilis that it is a gram-negative rod bacterium and
that it was the only organism to grow colonies with black centers in the Hektoen agar so
it was the only bacterium to have produced hydrogen sulfide.

References:
Bauman, RW. (2012). Microbiology with diseases by body system, third edition. pp 743
http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.ht
ml
Date accessed: October 12, 2013. Date last updated: September 27, 2004
http://www.scienceprofonline.com/microbiology/differential-selective-bacterial-growthmedia.html
Date accessed: October 12, 2013. Date last updated: April 2013
http://www.who.int/mediacentre/factsheets/fs125/en/
Date accessed: October 12, 2013. Date last updated: December 2011
http://www.who.int/topics/escherichia_coli_infections/en/
Date accessed: October 12, 2013. Date last updated: N/A
http://www.gutpathogens.com/content/2/1/16
Date accessed: October 13, 2013. Date last updated: November 27, 2010
http://www.gutpathogens.com/content/2/1/16
Date accessed: October 13, 2013. Date last updated: June 2011

http://www.medicalnewstoday.com/articles/68511.php
Date accessed: October 14, 2013. Date last updated: May 21, 2013
http://www.biomedcentral.com/1472-6750/8/79
Date accessed: October 14, 2013. Date last updated: October 28, 2008
http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/selectivedifferential.htm
Date accessed: October 14, 2013. Date last updated: N/A
http://www.healthhype.com/lab-tests-for-staph.html
Date accessed: October 14, 2013. Date last updated: June 10, 2013
http://www.vumicro.com/vumie/help/VUMICRO/Oxidase_Test.htm
Date accessed: October 14, 2013. Date last updated: N/A
http://www.ncbi.nlm.nih.gov/pubmed/1141195
Date accessed: October 14, 2013. Date last updated: July 1975