Faith Wagner

Section 8
April 30, 2010

Identification of Unknown Mixed Culture

Introduction
In this experiment, each student was given a mixed bacteria culture. Each culture
contained one unknown Gram positive bacteria and one unknown Gram negative
bacteria. The purpose of the experiment was for each student to “identify the genus and
species of [these two unknown bacteria]” by “utiliz[ing] the techniques and information
learned in previous exercises” (p. 241). The possible Gram positive bacteria included:
Corynebacterium xerosis, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus
epidermidis, and Clostridium perfringens. The possible Gram negative bacteria included:
Enterobacter aerogenes, Pseudomonas aeruginosa, Neisseria flavescens, Proteus
vulgaris, and Moraxella catarrhalis. The mixed culture tested in this report was #19.

Materials and Methods

During the entirety of the experiment, aseptic techniques as detailed in exercise 4
of the lab manual (p. 37-41) were used. First, a Gram stain as described in exercise 6 (p.
53-57) was performed on the mixed culture in order to identify the morphologies of the
bacteria. A smear of the culture was prepared using the techniques described in exercise 5
(p. 47-50). The smear was stained with crystal violet for 30 seconds, washed with water,
stained with Gram’s iodine for 60 seconds, washed with water, washed with 95% ethanol,
washed with water, stained with safranin for 60 seconds, and washed with water. This
slide was observed under a light microscope. Then, two Tryptic Soy Agar plates were
streaked with the mixed culture. The streak plate technique as described in exercise 7 (p.
61-67) was followed. One of these plates was incubated in aerobic conditions while the
other plate was incubated in anaerobic conditions. Both plates were incubated at 37°C for
24 hours. After these plates were incubated, a Gram stain (exercise 6 p. 53-57) was made
using the colonies from each plate. The purpose of these Gram stains was to find out if
only one or both of the bacteria grew in aerobic and anaerobic conditions. The next step
was to make streak plates of the mixed culture using differential media. A plate of
Columbia CNA agar with 5% sheep blood was streaked, and a plate of MacConkey agar
was also streaked. Both plates were streaked again using the technique from exercise 7
(p. 61-67). They were incubated at 37°C for 24 hours. Because it selects for Gram
positive organisms, the CNA plate served as a pure culture of the unknown Gram positive
bacteria for the rest of the tests. Similarly, because it selects for Gram negative
organisms, the MacConkey plate was used as a pure culture of the unknown Gram
negative bacteria. Next, the catalase test from exercise 28 (p. 217-219) was performed on
both the Gram positive and the Gram negative bacteria. In order to perform the test, a
sample of the Gram positive bacteria from the CNA plate was placed on a slide.
Hydrogen peroxide was added to the bacteria, and it was observed whether or not
bubbling occurred. This same procedure was done using a sample of the Gram negative
bacteria from the MacConkey plate. Later, the oxidase test was performed on the Gram
negative bacterium. Exercise 22 (p. 179-181) outlines this procedure. A drop of water
was placed on a slide, and a loopful of bacteria was transferred from the MacConkey
plate to the water. This bacterial suspension was transferred to the gray area of the
oxidase strip. The strip was observed for a change in color. The next four tests were part
of the IMViC series detailed in exercise 25 (p. 197-200). These tests were performed on
the Gram negative bacteria. First, a sulfide-indole-motility agar deep was inoculated with
bacteria from the MacConkey plate. This agar deep was inoculated at 37°C for 24 hours.
Following its incubation period, five drops of Kovac’s reagant were added to the SIM
tube. Next, a tube containing methyl red-Voges Proskauer broth was inoculated with the
Gram negative bacteria. This tube was incubated at 37°C for 48 hours. Following its
incubation period, two tests were performed on the MRVP broth culture. The first test
required the addition of 0.6 ml of α -naphthol solution and 0.2 ml of 40% potassium
hydroxide to 2.5 ml of MRVP broth culture. The next test required the addition of five
drops of methyl red reagant to the tube of remaining MRVP broth. Third, a Simmon’s
citrate agar slant was streaked with the Gram negative bacteria. This slant was incubated
at 37°C for 24 hours, and it was observed for the presence of a blue color following
incubation. The next test was the nitrate reductase test, and it was also performed on the
Gram negative bacteria. Exercise 27 (p. 207-212) outlines this procedure, in which a
loopful of bacteria from the MacConkey plate was added to a tube of nitrate broth. This
tube was incubated at 37°C for 48 hours. Afterwards, 5-10 drops each of nitrate reagant A
and nitrate reagent B were added to the tube. It was noted if this resulted in a color
change. Then, the urease test was performed on the Gram negative bacteria. This
procedure is detailed in exercise 26 (p. 203-204). Bacteria from the MacConkey plate
were streaked on the surface of a urea slant, which was incubated at 37°C for 24 hours.
The coagulase test was next. This test was performed on the Gram positive bacteria for a
change. Exercise 30 (p. 229-230) lays out this procedure. One loopful of bacteria from
the MacConkey plate was transferred into a coagulase tube. This tube was incubated at
37°C for 24 hours. This test concluded the experiment.

Results
When the Gram stain was done on the mixed bacteria culture, the microscope
showed that the Gram positive bacteria was coccus while the Gram negative bacteria was
bacillus. Then, samples from the TSA aerobic plate and from the TSA anaerobic plate
were also Gram stained in order to determine if they had Gram positive bacteria, Gram
negative bacteria, or both. The Gram stain from the TSA aerobic plate showed both
bacteria that stained purple and bacterial that stained pink. This meant that both the Gram
positive bacteria and the Gram negative bacteria grew under aerobic conditions.
Interestingly, it was noted during this Gram stain that the Gram positive bacteria seemed
to always appear in pairs. This led to the revision of the prior morphology. The Gram
positive bacteria were not simply coccus; they were diphlococcus. The Gram stain from
the TSA anaerobic plate was a bit confusing. When it was viewed under the microscope,
there were clearly bacteria that were pink. There were also dots of purple scattered
throughout. It wasn’t obvious whether these purple dots were leftover purple violet stain
that had not been washed off well enough or whether they were truly Gram positive
bacteria. So, this test was not conclusive for whether or not the Gram positive grew in
anaerobic conditions. There was definitely growth on the CNA aerobic plate. The culture
was observed, and it was determined that the hemolysis pattern of the Gram positive
bacteria was gamma. There was also growth on the MacConkey plate. The Gram
negative bacteria that grew on this plate were translucent. This led to the conclusion that
the bacteria that grew on this plate did not ferment lactose. When the catalase test was
performed, both the Gram positive bacteria and the Gram negative bacteria bubbled when
they contacted the hydrogen peroxide. This was a positive result. In the oxidase test, the
oxidase test strip did not show any color change when the Gram negative bacteria
suspension was added to it. This meant that the Gram negative bacteria was negative for
the presence of oxidase. In the IMViC series, which was performed on the Gram negative
bacteria, the SIM agar deep turned red when Kovac’s reagent was added. This meant a
positive result for the Tryptophanase (Indole) test. Next, when the α -naphthol solution
and the 40% potassium hydroxide was added to the MRVP broth culture, no rose color
developed. This was a negative result for the Voges Proskaer test. Similarly, when methyl
red reagent was added to the MRVP broth culture, no red color developed. This also was
a negative result for the Methyl Red test. Lastly, the citrate agar slant was green in color.
This indicates a negative result for the presence of citrate permease. For the nitrate
reductase test (performed on the Gram negative bacteria), when the nitrate reagent A and
nitrate reagent B were added to the tube of nitrate broth, a red color resulted. This was a
positive result. In the urease test, the Gram negative bacteria caused the urease tube to be
a bright pink color following incubation. This was a positive result. Lastly, the coagulase
test was performed on the Gram positive bacteria. This test was interesting because
following the incubation period, the coagulase tube looked empty. Clearly, no clot had
formed, so it was counted as a negative result. These results are summarized in a table
below:

Gram positive bacteria Gram negative bacteria

Gram Stain positive negative
Morphology diphlococcus rods
TSA aerobic positive positive
TSA anaerobic negative positive
Growth on Columbia aerobic positive negative
Hemolysis gamma
Growth on MacConkey aerobic negative positive
Lactose Fermentation negative
Oxidase negative
Tryptophanase (Indole) positive
Methyl Red negative
Voges Proskaer negative
Citrate Permease negative
Nitrate Reductase positive
Urease positive
Catalase positive positive
Coagulase negative

Discussion
As mentioned in the introduction, the purpose of these tests was to identify the
unknown Gram positive bacteria and the unknown Gram negative bacteria. The purpose
of the Gram stain on the mixed unknown was to determine the morphologies of each
bacteria. The Gram positive bacteria first appeared to be coccus. Upon further
observation in a different Gram stain, the Gram positive bacteria appeared to be
diphlococcus. The morphology of the Gram positive bacteria was telling because C.
xerosis was the only bacteria with diphlococcus morphology. The Gram negative bacteria
appeared to be bacillus. The purpose of the TSA aerobic and anaerobic plates was to
determine whether or not the bacteria grew in these conditions. It was concluded that
both the Gram positive bacteria and the Gram negative bacteria grew readily in aerobic
conditions. However, only the Gram negative bacteria grew in anaerobic conditions.
Note: this was a helpful result because C. xerosis was the only Gram positive bacteria
that does not grow in anaerobic conditions. The CNA plate and MacConkey plate were
streaked in order to provide a sources of pure bacteria cultures for the rest of the tests.
The fact that the hemolysis pattern of the Gram positive bacteria was gamma was not
conclusive because there were other bacteria with this same pattern. The fact that the
Gram negative bacteria did not ferment lactose ruled out the possibility that it could be E.
aerogenes. For both the Gram positive bacteria and the Gram negative bacteria, the
catalase test was positive. This ruled out the possibilities of C. perfringens and E.
faecalis, both of which are negative for the presence of catalase. The oxidase test yielded
a negative result. This meant that the Gram negative bacteria was not M. catarrhalis or
N. flavescens. The SMViC series was performed on the Gram negative bacteria to further
narrow down the possible Gram negative bacteria. From the results of the SMViC series,
it was clear that the Gram negative bacteria was not E. aerogenes or P. aeruginosa. These
bacteria would have yielded very different SMViC results. At this point, the Gram
negative bacteria possibilities were narrowed down to only one option: P. vulgaris.
However, more tests were performed in order to lend credibility to this guess. The nitrate
reductase test and the urease test both confirmed the identity of the Gram negative
bacteria; it was in fact P. vulgaris. Now, for the Gram positive bacteria, the coagulase test
had been a bit confusing. This made sense taking the other tests into account. First, the
Gram positive bacteria had a diphlococcus morphology. Also, it did not grow in
anaerobic conditions. These two facts seemed to indicate that the bacteria was C. xerosis.
The gamma hemolysis pattern fortified this conclusion, and the fact that there was no
result listed on the Complete Biochemical Chart for a coagulase test on C. xerosis
explained why the coagulase tube looked empty following incubation. In conclusion, it
was determined that unknown mixed culture #19 contained C. xerosis and P. vulgaris.