Microbiology (Bi410)
Systematics Exercise
Unknown Identification Report
Course: BI 410 01
Name: Abby Soltis
Date Submitted: 12/11/11
Unknown Number: 17
Unknown Microorganisms (Genus & species)
#1: Streptococcus lactis
√
#2: Proteus vulgaris
√
Grade (20 points possible) ___17/20_____
Instructor’s comments:
Abby all in all you have a pretty good report. However, some careful editing would have
caught some spelling errors and the incorrect indication of pH. See my comments in the
margin and if you have questions, see me.
Abby Soltis
Microbiology
12/11/2011
The Identification of Two Unknowns
Introduction:
It is useful to be able to identify bacteria in order to determine a
disease-causing organism or bacteria that produces products for
human consumption, such as vitamins or medicines. Due to the size
and lack of distinct morphology, bacteria are difficult to identify with
only a stained specimen and a microscope. Therefore, it is necessary
to use physiology to determine the genus and species of bacteria.
Physiology is determined by manipulating the bacteria’s environment
through the use of differential and selective medias, as well as a
variety of tests.
A mixture of two unknown bacteria has been provided and
through the separation of the bacteria in T streak plate and the use of
selective media, the two bacteria were separated and gram stain
reactions were identified. The KIA, Catalase, lactose, and MR-VP
tests further determined that the mixture of unknown bacteria
contained Streptococus lactis and Proteus vulgaris.
Materials and Methods:
Isolation of Bacterial Species
First, a gram stain was performed on the broth culture in order
to confirm the presence of gram-positive and gram-negative bacteria.
Secondly, a T streak plate was made from the original broth culture
onto a Tryptic soy agar, TSA, plate. After the plate was incubated for
48 hours, one of the bacterial colonies was isolated. A gram stain
was performed to confirm the presence of only gram negative or
gram-positive bacteria. This isolated colony of bacteria was pigs-tail
streaked onto a TSA slant culture and incubated for 48 hours. A gram
stain was performed on the culture after the 48-hour incubation
period.
In order to isolate the gram-positive bacteria, a T streak plate
was made from the original broth culture onto a plate with Phenylethyl
alcohol agar, PEA, which is a selective media for gram-positive
bacteria. This plate was incubated for 48 hours. After 48 hours,
isolated colonies formed on the plate and were confirmed by a gram
stain. An isolated colony of bacteria was pigs-tail streaked onto a
TSA slant culture and incubated for 48 hours. A gram stain was
performed on the culture after the 48-hour incubation period.
Identification of the Bacterial Species
From the gram-negative TSA slant culture a lactose test and a
Methyl Red ‐ Voges Proskauer, MR‐VP, test were performed. In order
to confirm the results, an Enterotube was also used. From the grampositive TSA slant culture a Kligler's Iron Agar, KIA, and a Catalase
test were performed.
A lactose test was used on the gram-negative bacteria because
a negative result would indicate Proteus vulgaris. The MR-VP test
would differentiate between Enterbacter aerogenes and Escherichia
coli in the event of a positive lactose test. The MR-VP test would
result in a negative/positive for Enterbacter aerogenes and a
positive/negative for Escherichia coli. This rationale is depicted in
table 1.
A Catalase test was used on the gram-positive bacteria
because a negative test, with no bubbling, would indicate
Streptococcus lactis. The KIA test would differentiate between
Bacillus subtilis and Staphylococcus epidermidis. The KIA test would
yield a Yellow slant and butt for Staphylococcus epidermidis and a
red slant and yellow butt for Bacillus subtilis. This rationale is
depicted in table 3.
Results/Data:
A gram stain was performed on the original unknown culture
showing the presence of a gram-positive coccal shaped bacteria and
gram-negative bacillus shaped bacteria depicted in figure 1. These
two types of bacteria were isolated and pure cultures were created.
The gram stains of each of these pure cultures resulted in either a
coccus, gram-positive or bacillus shaped, gram-negative bacteria
(results not shown).
The two tests performed on the gram-negative bacteria from
the pure culture included the lactose test and the MR-VP test. The
possible results of these tests are shown in table 1. When the lactose
test was performed, the lactose test media remained its original
purple color and no gas was formed in the Durham vial, indicating a
negative result. When the MR test was performed the media changed
from a yellow color to a red, indicating a positive test. When the VP
test was performed the media stayed a yellow color, indicating a
negative VP test. These results are described in table 2. An
Enterotube was also performed on the gram-negative bacteria. The
results coded for Proteus vulgaris (results not shown).
The two tests performed on the gram-positive bacteria from the
pure culture included the KIA test and the Catalase test. The possible
bacteria and the results of the two tests are listed in table 3. The KIA
test indicated a yellow butt and a yellow slant after a 48-hour
incubation period. The Catalase test resulted in small amounts of
bubbling, but not enough to call this a positive Catalase test.
Therefore, the results for the Catalase test are indicated as negative.
The results from these tests on the gram-positive, pure culture are
shown in table 4.
Figure 1: This photo is a gram stain done from the original broth culture with
two unknowns depicting gram-negative bacillus shaped bacteria in red and
gram-positive coccus shaped bacteria in blue.
Gram-negative
Bacteria
Enterobacter
aerogenes
Escherichia coli
Proteus Vulgaris
Lactose Test
MR-VP Test
Acid and Gas
-/+
Acid and Gas
Negative Test
+/+/-
Table 1: This table represents the possible results of the Lactose and MR-VP tests for
each of the possible types gram-negative bacteria.
Test (Gram-negative)
Results
Lactose Test
Media remained
purple and no gas
was formed (negative
test)
MR-VP Test
MR- turned red
(positive), VPremained yellow
(negative)
Table 2: This table shows the results from the lactose and MR-VP tests on the
bacteria from the gram-negative, pure culture.
Gram-positive
Bacteria
Staphylococcus
epidermidis
Streptococcus lactis
KIA test
Catalase Test
Yellow slant and butt
+
Yellow slant and butt
-
Bacillus subtilis
Red slant and yellow
butt
+
Table 3: This table shows the possible results for the KIA and Catalase tests on the
gram-positive, pure culture.
Test (Gram-positive)
Results
KIA
Yellow slant and butt
catalase
No significant
bubbles present
(negative)
Table 4: This table represents the results of the KIA and Catalase tests on the
pure, gram-positive culture.
Discussion/Conclusions
Based on the results from Lactose, MR-VP, and Enterotube
tests from the gram-negative, pure culture, Proteus vulgaris has been
isolated. At each stage of the isolation process a gram stain has been
performed, each time indicating the presence of only gram-negative
bacteria with similar morphology. The result of the lactose test was
negative, indicating that the organism does not ferment lactose. If the
organism did ferment lactose, the PH of the lactose would change
and the color would change from yellow to purple. The negative result
is consistent with Proteus vulgaris, which does not ferment lactose.
A negative lactose test shows that the bacteria must be Proteus
vulgaris, but a MR-VP and Enterotube test were also performed to
confirm the results. An MR-VP test was used to determine which
major fermentation pathway the bacteria uses. A positive MR test
indicates a mixed acid fermenter, while a positive VP test indicates
the use of a butanediol fermentation pathway. When the MR test was
performed the methyl-red was added to the solution and the color
remained red, which indicates a lowering of PH, which occurs due to
the mixed acids produced. The VP test was negative, confirming the
positive MR test results indicating that the organism is a mixed acid
fermenter, which is true of Proteus vulgaris. The Enterotube was read
after a 24-hour incubation and coded, which confirmed our results:
the isolated gram-negative bacteria were shown to be Proteus
vulgaris.
Based on the results form the KIA and Catalase test from the
gram-positive, pure culture, Streptococcus lactis has been isolated.
The first test was a KIA, which indicates the type of sugars fermented
by the organism. The KIA solution contains 1% lactose and 0.1%
glucose and the PH indicator phenol red. The result of this test was a
yellow butt and slant, indicating that much acid was produced, the PH
was lowered and the organism ferments both lactose and glucose.
With this result, the organism isolated could be either Streptococcus
lactis or Staphylococcus epidermidis.
The negative results of the Catalase test differentiate
between Streptococcus lactis and Staphylococcus epidermidis. The
Catalase test is used to determine the presence of catalase, which
breaks down hydrogen peroxide into water and oxygen in a bubbling
reaction, which disposes of toxic peroxide from the cell. This is
indicated by the addition of hydrogen peroxide to the culture. This test
result was largely negative (a little bubbling did occur) implying that
this organism does not use catalase to get rid of hydrogen peroxide.
This implicates Streptococcus lactis as the isolated, gram-positive
organism.
Based on the tests performed, the original mixture included
Streptococcus lactis and Proteus vulgaris.