Kimsey Cooper

Microbiology 210 Lab

Final Unknown Report
Section#006/ Radha

Report on Identification of Mixed Culture Unknown

Lab Exercise 33

Introduction

The purpose of this experiment is to apply the knowledge gained from the entire
semester in the Microbiology Lab and apply it to be able to identify bacteria. Each
student was given a tube that had a mixture of two different types of bacteria inside. The
tube used in this experiment was tube number fourteen. Inside the tube was one gram
negative and one gram positive organism. The bacteria of which we learned about and of
which were possibilities to be inside the test tubes include: Corynebacterium xerosis,
Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermis, Clostridium
perfringens, Enterobacter aerogenes, Pseudomonas aeruginosa, Neisseria flavescens,
Proteus vulgaris, and Moraxella catarrhalis. The organisms in the test tubes must be
streaked for isolation for oxygen requirements and followed with additional tests based
on results.

Materials and Methods

The first steps taken in lab were to conduct a gram stain from the organisms in the
mixed culture using the techniques and steps in exercise seven. Using aseptic techniques
from exercise 5 in the lab manual, I then inoculated the mixed culture onto two Tryptic
Soy Agar, TSA, plates and incubated them for 24 hours in 37C in both aerobic and
anaerobic conditions. The next week, I aseptically streaked the mixed culture from the
tube again with an inoculating loop onto two different types of agar. One agar used was
Columbia CNA agar with 5% sheep’s blood for gram positive organisms and the other
agar was MacConkey agar, MAC. I inoculated both plates for 24 hours in 37C. After that,
I aseptically conducted another gram stain test using lab exercise seven to be sure of the
morphology of my organisms. On the CNA agar I checked for hemolysis and on the
MAC agar I checked for a difference in colony color. The next route I took was for gram
positive organisms.
For the gram positive organisms I first conducted a Carbohydrate Fermentation
and Hydrogen Sulfide production test. I used Triple Sugar Iron, TSI, agar slants. I
aseptically streaked the top of the agar slant and stab inoculated the agar ¾ of the way
down. I then incubated it for 24 hours at 37C. The next test I conducted was the Catalase
test. For this test I needed two clean glass slides and a bottle of 3% H2O2. I aseptically
mixed a bacterial colony onto a clean glass slide with a drop of water. Then I put one or
two drops of % H2O2 directly on top of the organism and watched for bubbling. Another
test that I conducted was on the Mannitol Salt Agar, MSA. For this test I aseptically
streaked for isolation and incubated the agar for 24 hours at 37C. After that I conducted a
Coagulase test. This test used citrated rabbit plasma as the medium. I aseptically
inoculated a loop full of the bacteria into the tube and inoculated it for 24 hours in 37C.
Finally, the last test I conducted for the gram positive organisms was the DNase test. This
test was for my organism because it was Catalase positive. For this test I aseptically
heavily spot-inoculated my organism onto two methyl green agar plates and incubated for
24 hours in 37C.
For my gram negative organisms, I conducted totally different tests. The first test
I conducted was the Oxidase test. For this test I aseptically transferred an individual
bacterial colony onto a glass slide and added a drop of water, and mixed them together. I
then transferred with my loop a small amount onto the oxidase test strip and the results
were instantaneous. The next tests I conducted were more complicated and known as the
Methyl Red Vogues Proskauer, MRVP, tests. The first step for this test was to aseptically
transfer part of my bacterial colony into a broth tube and incubate for 48 hours at 37C.
After that, I transferred 2.5 mL of the MRVP results to a clean test tube and added 0.6mL
of α-naphthol and 0.2 mL of 40% Potassium Hydroxide to the solution. I vortexed the
solution and added 5 drops of Methyl Red indicator and waited for a color change. After
that test I conducted the IMVic Series of tests. This series also included the MRVP test.
First came the Indole test. For this test I used the Sulfide-Indole-Motility deep agar, SIM,
I aseptically stab inoculated the medium ¾ of the way down and incubated for 24 hours
at 37C. After that I added 5-10 drops of Kovac’s Reagent and observed for a color
change. The next test out of that series I conducted was the Simmons Citrate test. For this
test I used the Simmon’s Citrate agar and I aseptically streaked the top of the slant and
incubated for 24 hours at 37C. After this test I conducted the Urease test. For this test I
used the urea agar slants. I aseptically streaked the agar on the top of the surface and
incubated for 24 hours at 37C. The last test I conducted for my gram negative organism
was the Nirtrate Reductase test. For this test I aseptically inoculated the nitrate broth with
my organism and incubated the tubes for 48 hours at 37C. After that I added 5-10 drops
of sulfanilic acid and α-naphthylamine to the tube for a color change. I did not have to
add the zinc dust.

Results

The results for my gram positive bacteria were first of all, gram positive cocci.
The results from the CNA agar showed that I had γ hemolysis and I saw no zones of
clearance. The results that I obtained from the TSI slant were KA--. This means that after
incubation, the slant of the agar turned pink (alkaline), the butt of the agar turned yellow
(acidic), and the butt of the agar did not have any cracking or any blackening in the
medium. The results for the Catalase test were a bubbling over the bacteria on the glass
slide which means a positive reaction for catalase. The results for the MSA were negative
for mannitol fermentation. The colonies remained translucent. The results for the
Coagulase test negative and no clotting was found in the citrated rabbit plasma. Finally,
the results for the DNase test were negative as well. There was no zone of clearance
around the spot inoculated bacteria.
The results for my gram negative bacteria were first of all, gram negative rods.
The results from the MAC agar were negative and I the colonies remained translucent.
The results I obtained from the oxidase test were positive. The end of the test strip turned
to a blueish/ purple color. The results for the Methyl Red test were positive and the
medium turned a red color. The results for the Vogues Proskauer test were negative and
the medium did not impart a rose color. The results for the Indole test were positive and
the medium turned a cherry red color. The results for the Simmon’s Citrate test were
positive for citrate permease and the medium changed from green to blue after
incubation. The results for the Urease test were positive as well and the agar changed
from a salmon-orange to a fuchsia color. The final test I conducted was the Nitrate
Reductase test. The results for this test were positive as well. The liquid broth turned red
and created a red precipitate.

Discussion

I will now discuss the purpose of inoculating all of the tests chosen for my gram
positive organism. The first test conducted was on the CNA agar. This tested for the
hemolysis pattern of my organism. The agar plate showed no zone of clearance and thus
resulted in γ hemolysis. The purpose of conducting this test is because it enabled me to
differentiate between the degrees of which the hemolysins lysed red blood cells. The
incubation times for all of the organisms for 24 hours and the temperature at 37C is
important because this is the optimal temperature to maintain agar state and also the
perfect temperature that provides the right conditions for most microbial growth. In these
tests, most of the incubation times and temperatures were the same for basically the same
reasons. The second test conducted was on the TSI agar. This agar differentiates between
the ability of organisms to ferment glucose, sucrose, or lactose and to liberate hydrogen
sulfide gas from sodium thiosulfate. My gram positive organism was KA- - which meant
that the slant was alkaline (pink), the butt was acidic (yellow), and it did not liberate the
gas (were no cracks in medium). These results showed that my organism ferments
glucose only. The third test conducted was the Catalase test. This test was to check for
the presence of the catalyst catalase which liberates molecular oxygen. This catalyst is
necessary for organisms to be able to survive in aerobic conditions. After the experiment,
it was seen that my gram positive organism did in fact bubble, and utilize catalase. The
fourth test conducted was on the MSA agar. This test was conducted for the purpose of
selective and differential halophiles. It is based on mannitol fermentation. The phenol-red
indicator helps to identify the bacteria. My colonies were shown not to ferment mannitol
and they remained translucent. The fifth test conducted was the Coagulase test. The
coagulase test is important for distinguishing between species of Staphylococci that are
catalase positive, which is why I conducted it. Coagulase is an extracellular protein
involved in blood clotting which is why the rabbit plasma was provided as the medium.
The plasma is a source of fibrinogen, citrate and EDTA which are needed to form clots.
My organism did not form a clot and therefore was negative. The last test conducted for
my gram positive bacteria was the DNase test. This test is also conducted for catalase
positive organisms. DNases aid in the spread of bacteria throughout tissues. The soy and
peptones in the media provide nutrients and the sodium chloride provides the osmotic
equilibrium for this test. The 0.2% polymerized DNA and methyl green gives the
medium a 7.5 pH. The DNase positive organisms produce a zone of clearing around the
organism. My organism did not produce that and therefore does not contain DNase.
Based on all of the results from these tests, my conclusion is that my gram positive cocci
organism is, Staphylococcus Epidermis.
I will now discuss the purpose of inoculating all of the tests chosen for my gram
negative organism. The first test conducted was on the MAC agar. This tested for the
organism’s ability to ferment lactose. The neutral red indicator serves to contrast the
lactose-fermenting from the non lactose-fermenting organisms. I observed that my
bacterial colonies remained translucent and the medium stayed a redish/ purple color.
This result means that my organism does not ferment lactose. The incubation times for all
of the organisms for 24 hours and the temperature at 37C is important because this is the
optimal temperature to maintain agar state and also the perfect temperature that provides
the right conditions for most microbial growth. The second test conducted was the
Oxidase test. This test was looking for the presence of cytochrome c oxidase which is
important in the determination of aerobic conditions. Without this enzyme, an organism
cannot survive around oxygen. If the organism is positive for this enzyme, the enzyme
oxidizes and produces indophenol blue which causes the test strip to turn blueish/ purple.
I observed this reaction so my organism utilizes oxidase and is aerobic. The second test I
performed was the MRVP test. This test differentiates between organisms by detecting
fermentative end-products following growth in a buffered peptone-glucose broth. In the
Methyl Red test, the low pH of 4 is indicated by a red medium. In the Vogues Proskauer
test, acetonin is shown by imparting a rose color to the medium after addition of 40%
KOH. My organism tested positive for the mixed-acid fermentation with the red color,
but tested negative for the 2,3-butanediol fermentation. This test needed to be incubated
for 48 hours instead of 24 hours to allow for extra time for growth of the bacteria and
because it is a broth medium. However, it was incubated at the same temperature. The
third test conducted was the Indole test. This test was performed to detect the presence of
tryptophanase which hydrolyzed tryptophan into indole, pyruvic acid, and ammonia. The
medium contains peptones rich in tryptophan and the Kovac’s Reagent allows for
detection of production of indole as the waste product. A positive reaction, which is what
I obtained, results in the medium turning a cherry red color. A second result from the
SIM deep agar is used to test for cysteine desulfurase or thiosulfate reductase which
produces hydrogen sulfide gas and a blackening color. A third result is testing for motile
bacteria and produces cracks in the agar. My organism did not produce the gas and was
not motile. The fourth test conducted was the Simmon’s Citrate test which determines if
the organism can use citrate as the sole source of its carbon. It also tests for the presence
of citrate permease. To detect for a pH shift, which is an indicator of the enzyme,
bromthymol blue is added to the medium. The agar changes from a green to blue color if
the enzyme is present which is what I observed in my test. The fifth test I conducted was
the Urease test. The urease test detects the degradation of proteins, and the utilization of
this as a nitrogen source. If urea is present, alkaline ammonia accumulate and rises the
pH turning it from salmon-orange to fuchsia which is what I observed. The last test
conducted for my gram negative organism was the Nitrate Reductase test. This test was
looking for the utilization of the enzyme nitrate reductase. This enzyme allows for the
organism to undergo the nitrogen cycle and utilize ammonia as their nitrogen source. The
nitrogen broth used contains nutrients that contain KNO3 as the source of nitrate.
Durham tubes also trap the nitrogen gas produced during the reaction. The medium is
evaluated for the presence of reduced forms of nitrates by adding the nitrate reagent A
and B. If nitrates are present, the broth turns a red color and produces a red precipitate.
This is the reaction that I found. This test was also incubated for 48 hours compared to 24
hours because it was a broth and it allowed for the extra time for the reactions. It was still
incubated at 37C. From all of these tests I concluded that my gram negative bacillus
organism is, Pseudomonas Aeruginosa
The purpose for conducting this experiment was obtained. Both organisms were
correctly identified and all of the skills learned throughout the semester in lab were put to
use in determining the individual bacteria of the mixed culture. Some sources of error
that may have occurred would definitely be in the aseptic techniques performed. Any
number of contaminants may have gotten into the tests and slightly messed up the results.
Another possible source of error would be that lab was only held once a week and results
were not readily available to look at and describe by the specified incubation periods.