Chelsea Smith
Microbiology 313 Section 2
Ball State University
Bacterial Unknown #26 = Staphylococcus aureus
1
Introduction
The purpose of this assignment was to be able to identify and confirm the identity of an
unknown bacterial sample using simple biochemical tests learned previously in laboratory
exercises. The bacterium would be identified down to its genus and species levels from a group
of sixteen bacteria selected by the instructor. The unknown bacterium given to each student
could have been one of the following: Bacillus subtilis, Bacillus thuringiensis, Citrobacter
freundii, Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Klebsiella
pneumoniae, Lactobacillus acidophilus, Micrococcus luteus, Proteus mirabilis, Proteus vulgaris,
Pseudomonas aeruginosa, Pseudomonas fluorescens, Serratia marcescens, Staphylococcus
aureus, or Staphylococcus epidermidis.
The relevance of this assignment is that bacterial identification is used every day in many
sectors (clinical, food industry, research, etc.). Using biochemical tests for identifying an
unknown bacterium is especially important in the clinical sector. The technicians want to
quickly identify if the organism is pathogenic, and if so, determine what the specific species is
(Willey et. al.). This way proper treatment can be given to a sick patient.
Differential tests are used to eliminate possible species and determine the correct
bacterium, and these biochemical tests work because each species has their own set of
biochemical characteristics that makes them unique (Leboffe). Bacteria are classified by many
characteristics, including gram reaction, cell morphology, motility, and nutritional requirements
(Willey et. al.). The main advantage of using phenotypic techniques for identification is that
there are many simple biochemical tests that can be performed to reach the conclusion. It may
take some time (mostly for incubation periods), but most of these tests are accurate and cheap. A
disadvantage of this technique is that some tests require a young, pure culture in order to proceed
2
properly, and this could produce a false negative. There is also the risk of contamination of
cultures or media used for the tests, which could produce false results.
Materials & Methods
There are many biochemical tests that can be performed in the process of identifying an
unknown bacterium. These tests can utilize different media that may be in the form of a broth,
an agar slant, or an agar plate. Inoculations for all media are used with aseptic techniques. Broth
tubes are inoculated by inserting the inoculating needle with bacteria on it down at least one
centimeter into the broth. The agar slant can be inoculated by stabbing the butt portion at least
one centimeter straight down and/or streaking the slant with the bacteria laden needle. Agar
plates can be spot inoculated in one spot at the center of the plate or streak inoculated across the
entire surface using the inoculating loop.
The following tests were used to either identify or confirm the identity of the unknown
bacteria given. The use of each identification test was decided based on the dichotomous key
(see Figure 1 attachment) made prior to testing the unknown. The Gram Stain is a differential
test based on how thick the peptidoglycan layer is around a cell. The theory and procedure for
this test was found in pages 105-109 in the lab manual (Leboffe). At the conclusion of the test,
gram-positive bacteria have purple cells and gram-negative bacteria have red/pink cells. Cell
morphology can be determined at this point of the test as well.
The Motility Test was used to differentiate between motile and non-motile bacteria. The
theory and procedure was found on pages 224-225 in the lab manual (Leboffe). For the SIMmotility medium in a test tube, a non-motile bacterium has white growth only along the stab line,
and a motile bacterium has white growth diffused throughout the entire tube. This growth
occurred after 24-48 hours of incubation at 37 degrees Celsius.
3
Determining the oxygen requirements for a bacterium was found using the OxidationFermentation (O-F) Test, which differentiates bacteria based on their fermentative or oxidative
metabolisms. The theory and procedure of this test was described on pages 155-157 in the lab
manual (Leboffe). After O-F Glucose tubes were incubated for 48 hours at 37 degrees Celsius,
the oxygen requirements were decided based on whether or not either tube changed a yellow
color.
One test used to determine the ability of bacteria to ferment mannitol is the Mannitol
Salts Agar (MSA) Test. The mannitol makes the medium differential, and the high sodium
chloride concentration makes the medium selective because it will dehydrate and kill most
bacteria. More information about this medium and the procedure used for this test was found on
pages 137-138 in the lab manual (Leboffe). The inoculated plate incubated for 48 hours at 37
degrees Celsius before observations were taken.
The Catalase Test was used to determine if the bacterium produced catalase enzymes, and
it also helped to confirm the oxygen requirements for the organism. It is a differential test that
determines if a bacterium is catalase-positive or catalase-negative. The theory and procedure for
this test was found on pages 165-166 in the lab manual (Leboffe). The Slide Test method was
chosen, and observations were taken immediately after the test was completed.
The Nitrate Reduction Test differentiates bacteria based on their ability to convert
(reduce) nitrate into other forms of nitrogen compounds. The theory and procedure was found
on pages 171-174 in the lab manual (Leboffe). After incubating the inoculated tubes for 48
hours at 37 degrees Celsius, reagents were added before results could be determined.
Sugar Fermentation Tests were used to determine if the bacterium could ferment sucrose,
lactose, and mannitol. Phenol Red Broth was used to differentiate between organisms that could
4
and could not ferment a specific carbohydrate. The theory and procedure for this test was found
on pages 158-160 in the lab manual (Leboffe). Observations of gas production and/or color
change in each tube were taken after 48 hours of incubation at 37 degrees Celsius.
Results
After each test was performed, the observations and results were recorded. The first test
was the Gram Stain, and this showed the gram reaction as well as cell morphology. The given
unknown was stained purple at the conclusion of the test, meaning it was gram-positive. Further
examination under the microscope showed that this bacterium was coccus shaped, and the cells
tended to group together. Table 1 (shown below) gives the results for the rest of the biochemical
tests performed to identify and confirm the identity of the unknown bacterium.
Table 1. Results of Biochemical Tests Performed for Three Similar Species.
Test
Gram Stain
Cocci Shape
Motility
O2 RequirementsFacultative anaerobe
Mannitol [MSA]
Catalase
Nitrate Reduction
(Confirming)
Sucrose Tube
(Confirming)
Lactose Tube
(Confirming)
Mannitol Tube
(Confirming)
Unknown #26
Enterococcus Staphylococcus
"Staphylococcus aureus"
faecalis
epidermidis
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
-
5
Discussion
Throughout the process of determining the identity of the given unknown bacterium, the
bacterial dichotomous key (Figure 1) was used to eliminate species and choose the appropriate
tests. The key was created with information researched about each of the sixteen possible
bacteria (Bergey & Holt). Since gram stain reaction was at the top of the key, this was the first
test performed on the unknown. The gram-positive result eliminated nine possible bacterial
species, and it was also observed that the cells were cocci shaped. The group of gram-positive
bacteria remaining was next divided by motility; therefore a motility test using SIM medium was
stab inoculated. After 48 hours of incubation, a white growth was seen along the stab line where
the bacterium was inoculated. There was no diffusion throughout the medium, therefore it was
determined that this species was non-motile.
Following the dichotomous key, there were only five bacteria that were gram-positive
and non-motile: Enterococcus faecalis, Lactobacillus acidophilus, Micrococcus luteus,
Staphylococcus aureus, and Staphylococcus epidermidis. The next division in the key was
determining whether or not the organism was a facultative anaerobic species. The OxidationFermentation Test using O-F glucose was used to determine the organism’s oxygen
requirements. Two tubes were inoculated, one with a top layer of mineral oil and one without
the oil. After incubating for 48 hours, both tubes had turned yellow from the starting green color
of the broth. According to the lab manual, this was interpreted as an oxidative and fermentative
organism, which means it was a facultative anaerobic organism (Leboffe). This result eliminated
Micrococcus luteus since it was not a facultative anaerobe.
The next step on the key was cell shape, and this had already been determined from the
gram stain. Only three species (Enterococcus faecalis, Staphylococcus aureus, and
6
Staphylococcus epidermidis) remained as possible choices that the unknown could be. The next
step was to determine whether the bacteria could ferment mannitol, and the MSA plate was used
for this test. After the plate was spot inoculated in the center and incubated for 48 hours, a
yellow halo had formed around the growth on the plate. This indicated that this bacterium
produced acid when it fermented the mannitol in the agar, meaning it was mannitol-positive.
Now the identification of the unknown was between two species: Enterococcus faecalis and
Staphylococcus aureus.
The biochemical characteristic that separated these two organisms was their ability to
produce the catalase enzyme. The Catalase Test was conducted, and the inoculating loop with
the bacteria on it was smeared into a few drops of hydrogen peroxide. Bubbles formed
immediately in the mixture, which meant that the bacterium was catalase positive. This allowed
for an initial identification of the organism to be Staphylococcus aureus. There were many other
biochemical characteristics found for S. aureus that were not included in the dichotomous key,
and some of these were used to confirm this identification.
Previous research found that S. aureus was positive for nitrate reduction, and it was
positive for sucrose and lactose fermentation. The Nitrate Reduction Test was conducted, and
after the inoculated broth incubated for 48 hours, there was no gas bubble in the Durham tube.
After reagents A and B were added to the broth, a red color change occurred. This meant that
the organism had the ability to reduce nitrate to nitrite, which confirmed that the organism was
positive for nitrate reduction.
Three separate sugar fermentation tubes (sucrose, lactose, and mannitol) were inoculated
to confirm the organism’s ability to process these specific carbohydrates. The tubes were
inoculated and incubated for 48 hours, and observations were taken based on gas production and
7
color change from the starting red color. The sucrose tube did not have any gas production, but
it did change to a yellow color, confirming it was positive for sucrose fermentation. The same
results occurred for both the lactose and mannitol tubes, confirming that the organism was
positive for lactose and mannitol fermentation as well. Taking all of the biochemical tests into
consideration (results shown in Table 1), the unknown bacterium was confirmed to be
Staphylococcus aureus.
It is important to understand the S. aureus species because it is a known human pathogen.
The food industry and clinical sector are the two areas that are most vigilant toward this
pathogen. S. aureus is a major cause of food-poisoning, and this is an even larger concern
because the bacteria can attach to food and form biofilms that are difficult to remove (Gutiérrez
et. al.). It has also been found to contaminate rural drinking water, which can be transferred to
food cooked in this water or consumed from the well directly (LeChevallier & Seidler). S.
aureus can produce cytotoxins and enzymes that convert the host cells into nutrients that help the
organism grow, which is how it causes disease in the host (Dinges et. al.). The more that is
understood about Staphylococcus aureus, the better industries can protect humans against it and
ward off diseases that it causes.
Literature Review
Bergey, D.H. and Holt, J.G. 1994. Bergey's manual of determinative bacteriology. pp 1-787,
Williams & Wilkins, Baltimore
Dinges, M.M., Orwin, P.M. and Schlievert, P.M. 2000. Exotoxins of Staphylococcus aureus.
Clinical Microbiology Reviews, 13: 16-34.
Gutiérrez, D., Delgado, S., Vázquez-Sánchez, D., Martínez, B., López Cabo, M., Rodríguez, A.,
Herrera, J.J. and García, P. 2012. Incidence of Staphylococcus aureus and Analysis of
8
Associated Bacterial Communities on Food Industry Surfaces. J. Applied and
Environmental Microbiology, 78: 8547-8554.
Leboffe, M.J. and Pierce, B.E. 2010. Microbiology Laboratory Theory & Application. pp 1-424,
Morton Publishing Company, Colorado.
LeChevallier, M.W. and Seidler, R.J. 1980. Staphylococcus aureus in Rural Drinking Water. J.
Applied and Environmental Microbiology, 30: 739-742.
Willey, J.M., Sherwood, L.M. and Woolverton, C.J. 2011. Prescott’s Microbiology. 9th ed. pp 11014.
9
10
Legend for Figure 1
11