Auriell Frederick

11/26/12 Pathogenic Microbiology Laboratory 4415

Identifying a Mixed Unknown Pathogenic Bacterial Specimen
Procedure # 1
A catalase test was performed. The purpose of the test is to determine if the unknown
organisms are catalase negative or positive. If an organism is catalase positive it will result in the
formation of bubbles during the reaction which is indicative of its ability to convert hydrogen
peroxide in to water and oxygen. Samples of both Gram-negative and Gram-positive organisms
were subjected to catalase testing.
The gram-negative organism was catalase positive, which reinforces the hypothesis that
E coli has been properly identified as the Gram-negative organism.
The unknown Gram-positive organism was not reactive to the catalase test. Members of
the Staphylococcus family are catalase positive, therefore, may be ruled out. B. cereus should be
eliminated from the list as a gram-positive candidate, also, as it is bacillus shaped, not spherical.
The remaining unidentified organisms possible are: Streptococcus penumoniae,
Streptococcus pyogenes and Enterococcus faecalis.
Procedure #2
A citrate test was performed to confirm the hypothesis of the Gram-negative organisms
identity as E.coli. This tests functions to determine if the organism is utilizing citrate as a carbon
source for energy. The test used in this procedure is Simmons Citrate Agar, containing sodium
citrate as the sole source of carbon. The pH indicator is bromthymol blue. If there is a color
change on the growth medium, the organism is citrate positive. This test is helpful in
differentiating from other Enterobacteriaceae coliforms. When Simmons Citrate agar is
inoculated with Escherichia coli, the medium remains green which is a negative result for the
citrate test.
A normal loop (not heavy) was used to inoculate the medium. A color change occurred
where the stab took place but not on the streak of the surface, therefore no growth was seen and
the test further served to reinforce the hypothesis of the Gram-negative organisms identity as E.

coli.
Procedure # 3
A Blood Agar test is performed to distinguish Streptococci from Enterococcus faecalis,
based on the hemolysis of their clearing. When streaked onto this medium, E faecalis will result
in a gamma clearing (no clearing), whereas S. pneumonia is alpha hemolytic and S. pyogenes is
beta hemolytic. Alpha hemolysis occurs when the iron from hemoglobin oxidizes and beta
hemolysis results by the complete lysis of erythrocytes.
Both Gram-negative and Gram-positive organisms were subjected to a Blood Agar test.
No identifiable clearing resulted in either organism, though it was questionable whether the
results were correctly interpreted. No clearing on the agar is indicative of a gamma clearing,
which indicates that the Gram-positive organism is E. faecalis.
Procedure #4
The final test performed was to ensure that the appropriate Gram-positive organism was
correctly identified. This test used was the Optochin test and is useful to determine the presence
of S. pneumoniae, which has a partial hemolytic clearing (alpha). In the event that the hemolysis
test is incorrectly read, this test will correctly identify S. pneumoniae from E. faecalis, as S.
pneumoniae is Optochin sensitive.
The results of the test were negative. This served to instill confidence that the correct
unknown organism was, in fact, E. faecalis.

References
Holt, John G., et. al., Bergey's Manual of Determinative Bacteriology, Ninth Edition, 1994.
Leboffe, M.J., and Peirce, B. E., A Brief Microbiology Laboratory Theory and
Application, Second Edition, 2012