Unknown Organism # 3B Report

November 19, 2014

BIOL 4415-1
Courtney S. Zahn

Introduction

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Zahn Unknown Organism # 3B

The case study regarding Unknown Organism #3B involved a 64-year

old female who complained of pain near her hip region. She had received a
prosthetic hip replacement 2 months prior. She was diagnosed with
bacteremia. Blood culture indicated susceptibility to novobiocin and
polymyxin B on P agar.
Interpretation of the case study began with a search regarding bacteria
that are commonly associated with prosthetic hip implants in humans.
Staphylococcus and Streptococcus organisms are commonly seen in
prosthetic hip implant infections. Escherichia coli is also seen in infected
hips following replacement surgery, but less commonly than Streptococcus
or Staphylococcus (Rafiq, Gambhir, Wroblewski, & Kay, 2006). A search was
then performed to determine which of these bacteria were susceptible to
novobiacin and polymyxin B, which was observed on P agar, and noted in the
case study. It became evident that Staphylococcus epidermidis bacteria are
susceptible to novobiacin as well as polymyxin B (Rowlinson, LeBourgeois,
Ward, Song, Finegold, & Bruckner, 2006).
Methods
Using Bergeys Manual of Determinative Bacteriology as a guide, the
process of identifying Unknown Organism # 3B began. The first step in the
identification process was a Gram stain. The stain showed a Gram positive
organism with coccoidal morphology. The Gram stain was repeated for
accuracy. Each subsequent Gram stain gave the same results. Escherichia
coli is Gram negative, so was quickly eliminated from the suspects list due to
the Gram positive result of the Gram stain. This narrowed the potential
suspects down to S. aureus and S. epidermidis.
Following Gram staining, the organism was streaked via inoculating
loop on blood agar for hemolysis. Gamma hemolysis was observed following
streaking and incubation. The organism was also streaked onto plates of
nutrient agar, Mannitol Salt Agar (MSA), Phenylethyl Alcohol (PEA) Agar, and
MacConkey agar. After incubation, results were gathered from each plate.
Growth was seen on the nutrient agar and PEA agar plates. Growth on the
PEA plate confirmed a Gram positive coccoidal organism. The MSA plate
showed growth, but did not turn yellow, which eliminated Staphylococcus
aureus from the suspects list, but provided evidence that Unknown Organism
3B was in fact a Staphylococcus bacteria. No growth was observed on the
MacConkey agar, which further confirmed the Gram positive result of the
Gram stain.
Once the plates were observed and data was collected, motility tests
were performed. The organism was observed in a microscopic wet mount,
and it appeared to be nonmotile. For certainty, a second motility test was
performed with a Motility Agar stab. This test confirmed the nonmotile
result. A Triple Sugar Iron (TSI) agar test was also performed, and turned
yellow in color following incubation. This provided evidence that the
unknown organism was positive for acid production. A glucose fermentation
test also turned yellow in color, indicating the organism to be positive for

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Zahn Unknown Organism # 3B

glucose fermentation. A urease test showed positive results, indicating

urease production. Catalase and oxidase tests were performed, both
showing negative results. Staphylococcus epidermidis remained at the top
of the list of possible suspects.
Results
Table 1 lists all microbiological tests, results, evidence confirming the results,
and images (if available) of the results relating to Unknown Organism # 3B.
Table 1
Test

Result

Gram Stain

Positive

Morphology

Cocci

Viewed with microscope

following Gram stain

Catalase

Negative

No bubbles; indicative of
anaerobes and facultative
anaerobes mainly
Streptococci

Oxidase

Negative

Motility

Negative

No color change; no
production of cytochromec oxidase; cannot reduce
oxygen; indicative of
anaerobic bacteria
Only Brownian motion; no
self-propelled motility

MSA

Positive
(growth)
Negative
(no color

Evidence Confirming
Result
Purple

Well-defined growth along

stab line with no cloudy,
diffuse growth away from
stab line; indicative of no
flagellated cells
Pink; no color change/no
acid; intolerance for high
salt environment; cannot
ferment mannitol;
indicative of

Image

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Zahn Unknown Organism # 3B

change)

Staphylococcus spp., but

no S. aureus
Non-hemolytic; no lysis of
Red Blood Cells

Blood Agar

Gamma
Hemolysi
s

Nutrient
Agar

Positive

Growth

PEA

Positive

Growth indicative of
Gram positive coccoidal
bacteria

Lactose
(MacConkey
)

Negative

No growth; indicative of
Gram positive bacteria

Urea

Positive

Phenol red turned fuchsia;

indicative of the alkaline
environment seen from
the production of
ammonia following urea
degradation

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Zahn Unknown Organism # 3B

Glucose
Positive
Fermentatio
n

Phenol red turned yellow;

indicative of carbohydrate
fermentation and acid
production

TSI

Turned yellow; indicative

of the ability to ferment
sugars and produce
hydrogen sulfide

Positive

Discussion and Conclusion

After performing several tests and following Bergeys flow charts, it
was concluded that Unknown Organism # 3B was Staphylococcus
epidermidis . There were no problems encountered as we performed our
tests on the organism. All tests were important in narrowing down the list of
possible suspects, but the MSA test was the determining factor, because it
confirmied the organism was Staphylococcus and ruled out S. aureus due to
the ability of the organism to grow on the medium, but the inability to
ferment mannitol.
Additional tests could have been performed to assist in the
identification of Unknown Organism 3B. A DNase test with negative results
would have further confirmed our results. DNase tests are used to measure
the DNase exoenzyme which breaks down nucleotides. This exoenzyme is
present in Staphylococcus aureus, but not in Staphylococcus epidermidis.
Staphylococci are Gram positive, facultative anaerobes that often
occur in clusters resembling grapes. Staphylococcus epidermidis is gamma
hemolytic, non-pigmented, coagulase-negative, and does not ferment
mannitol. It is a common S. epidermidis is a significant cause of
opportunistic infections, and commonly forms biofilms on catheters, shunts,
artificial heart valves, and many other surgical devices. Endocarditis and
sepsis can occur as a result of these infections (Murray, 2013). The biofilm
increases the virulence of the bacteria and makes it difficult to treat the
infection. Antibiotic-resistance is a common problem associated with S.
epidermidis (Fox, 2016).
Appendix A
Tests
Gram Stain
Morphology
Catalase

Results
Positive
Cocci
Negative

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Oxidase
Motility
MSA
MacConkey
Agar
PEA
Blood Agar
Nutrient
Agar
Glucose
Fermentatio
n
TSI
Urea

Zahn Unknown Organism # 3B

Negative
Negative
Negative
Negative
Positive
Positive
Positive
Positive

Positive
Positive

Work Cited
Bergeys Manual of Determinative Bacteriology. Bergeys Manual Trust.
Springer, 26. May 2014. Web. 26 November 2014.
http://www.antibmcrobe.org/b93.org/.
Fox, A. Bacteriology chapter 13 Part 2: Staphylococci. Microbiology and
Immunology Online: University of South Carolina School of Medicine.
2010. Web. 26, November 2014.
http://pathmicro.med.sc.edu/fox/streptococci.htm.
Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. Staphylococcus. Medical
Microbiology. Elsevier Saunders. 7th edition. 2013.
Rafiq, I., Gambhir, A. K., Wroblewski, B. M., & Kay, P. R. The microbiology of
infected hip arthroplasty. International Orthopaedics. December
2006. 30(6). 532-535. Published online Aug 1, 2006. Doi:
10.1007/s00264-006-0125-8. Retrieved from web 11/26/14.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172734/
Watson, R. Summary of biochemical tests. General and Medical Microbiology.
Web 26, November 2014.
http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm.