Unknown Organism # 2 Report

November 7, 2014
BIOL 4415-1
Courtney S. Zahn

Introduction

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Zahn Unknown Organism #2

The case study regarding Unknown Organism #2 involved a 60-year-old man

who was admitted to the hospital with acute pain of the right lower quadrant and
periumbilical abdomen. He had no previous history of alcohol abuse, cholelithiasis,
abdominal trauma or surgery, nor ingestion of raw food or medications. On
admission, hematological tests revealed normal hematocrit and platelet counts, and
an increased white blood cell count of 23,800 mm-3 with 40% lymphocytes. A
computed tomographic (CT) abdominal scan was performed revealing necrosis over
20% of the pancreas. The pancreatic parenchyma showed a slightly abnormally
structured and irregular mass-like aspect on the head, compatible with an
inflammatory process. The pancreatic body reflected a homogeneous pattern with
an irregular and slightly dilated main pancreatic duct. A gallbladder with
hyperechogenic foci without an acoustic shadow floating inside, which is compatible
with microlithiasis was described. Samples obtained were submitted for
cytohistological and microbiological evaluation. Pathology results showed small
fragments of pancreatic parenchyma without evidence of malignancy surrounded by
areas of necrosis and inflammation.
Interpretation of the case study began with a search regarding bacterial
infections that commonly cause acute pancreatitis with necrosis. Gram negative
enteric bacteria have been the most common causative organisms of acute
bacterial pancreatitis. Enteric bacteria are also a common cause of gallbladder
infections (Dionigi, Rovera, Dionigi, & Cuffari, 2006). From this knowledge, suspects
from the list of potential unknown organisms were narrowed down to Enterobacter
aerogens and Citrobacter freundii.
Methods
The first method used to identify the unknown organisms was to streak the
organisms for isolation via inoculating loop. Gram stains were performed, which
showed both Gram-positive and Gram-negative results. The Gram stains were
repeated several times for accuracy. Each subsequent Gram stain gave the same
results. Following Gram staining, the organisms were streaked on blood agar for
hemolysis. Gamma and beta hemolysis was observed following streaking and
incubation. Samples were taken from each colony and were streaked via
inoculating loop onto two plates of nutrient agar for isolation of the species.
Following incubation, isolation of the organisms allowed for Gram staining
them individually to determine which organism was involved in the case study. The
organism that showed gamma hemolysis was labeled Colony 1. The Gram stain of
colony 1 provided evidence of a Gram-negative, rod-shaped organism. After the
Gram stain was completed, an oxidase test was performed to determine if the
organism was indeed part of the Enterobacteriaceae family. The result was
negative, which confirmed our theory.
Following the oxidase test, a lactose fermentation test was performed by
streaking the colony onto plates of Eosin Methylene Blue (EMB). Dark blue colonies
were observed following incubation, which proved the organism does ferment
lactose. This allowed for elimination of Enterobacter aerogens since it does not
ferment lactose. Citrobacter freundii does ferment lactose, so this test was
solidifying this organism as our unknown, but further testing was needed for
conformation. An indole test was performed with positive results. Following the
indole test, a citrate test also gave positive results. These tests confirmed C.
freundii as the unknown related to the case study.

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It was determined that the second colony was not related to the case study.
Colony 2 was isolated on a plate of nutrient agar, and then a Gram stain was
performed. The Gram stain resulted in a Gram-positive, diplococcic organism.
Following the Gram stain, the organism was streaked onto a Mannitol Salt agar
(MSA) plate for possible elimination of Staphylococcus. No growth was seen on the
MSA plate, so Staphlococcus was eliminated from our list of possible unknown
organisms. A negative Catalase test and the beta hemolysis observed following
streaking on blood agar allowed for the potential suspects to be narrowed down to
Streptococcus pyogenes and Streptococcus agalactiae. An endospore stain was
then performed with positive results. This concluded that the second unknown
organism was Staphylococcus agalactiae.
Citrobacter freundii is part of the Enterobacteriaceae family. It is an
opportunistic pathogen, which causes many infections of the respiratory tract,
urinary tract, blood, and other sterile sites in immunocompromised individuals.
Citrobacter freundii and other members of the Enterobacteriaceae family can reach
the pancreas or other areas of the body that do not normally contain normal flora,
by translocating from the gut (Murray, Rosenthal, & Pfaller, 2013).
Streptococcus agalactiae is part of the Streptococcus family and is in the
normal flora of the gastrointestinal tract, upper respiratory tract, and urethra of
many humans. It is the most common cause of neonatal sepsis and postpartum
infection (WebMD, 2014).
Results
Table 1 and 2 list all microbiological tests, results, evidence confirming the results,
and images (if available) of the results. Table 1 relates to Unknown Organism #1.
Table #2 relates to Unknown Organism #2.
Table 1
Test
Gram Stain
Morpholog
y
Catalase

Result
Negative
Rods in
chains
Positive

Evidence Confirming Result

Pink

Oxidase

Negative

Motility

Negative

No color change; no production of

cytochrome-c oxidase; cannot reduce
oxygen; indicative of anaerobic
bacteria
No movement from stab; indicative of
no flagellated cells

Citrate

Positive

Blue; indicative of Gram negative

Image

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Zahn Unknown Organism #2

bacteria capable of utilizing citrate as

the sole carbon and energy source
Red rim on top of broth; indicative of
the ability to produce tryptophanase
Normal-looking colony; no hemolysis

Indole

Positive

Blood Agar

Gamma
Hemolysis

Nutrient
Agar

Positive

Growth

EMB

Positive

Indicative of Gram negative bacteria

Lactose

Positive

Dark pink colony

Morpholog
y
Gelatin

Rods in
Chains
Positive

DNAse
Plate

Positive

Viewed with microscope following

Gram stain
Change from semisolid to liquid;
indicative of presence of gelatinase
enzyme and therefore the use of
gelatin as a source for carbon and
energy
Clear halos around colonies; indicative
of the ability to use DNA as a source of
carbon and energy for growth due to
presence of the enzyme DNase

Table 2
Test
Gram Stain

Result
Positive

Evidence Confirming Result

Purple

Image

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Zahn Unknown Organism #2

Morpholog
y
Catalase

Diplococci
Negative

No bubbles; indicative of anaerobes

and facultative anaerobes mainly
Streptococci

Oxidase

Negative

Motility

Negative

No color change; no production of

cytochrome-c oxidase; cannot reduce
oxygen; indicative of anaerobic
bacteria
No movement from stab; indicative of
no flagellated cells

Citrate

Positive

MSA

Negative

Blood Agar

Beta
Hemolysis

Nutrient
Agar
EMB

Positive

Growth

Negative

Lactose

Positive

No growth indicative of Gram

negative bacteria
Dull pink colony

Sporeforming
Stain

Positive

Blue; indicative of Gram negative

bacteria capable of utilizing citrate as
the sole carbon and energy source
Pink; no color change/no acid;
intolerance for high salt environment;
cannot ferment mannitol
Clear, colorless zone around colonies
indicative of complete lysis of Red
Blood Cells

Red; Indicative of endosporeformation

Discussion and Conclusion

After performing several tests and following Bergeys flow charts, it was
concluded that our unknown organisms were Citrobacter freundii and Streptococcus
agalactiae. Some problems were incountered as we ran our tests on the organisms.
Our Gram stains were perfomed multiple times due to contamination. Thankfully,
the control organism allowed for additional Gram stains, which provided accurate
results. We also had errors with our catalase test due to contamination, and had to
perform the test a second time with the control organism to achieve accurate
results. All tests were equally important in determining the identification of the

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unknown organisms. In hindsite, the lactose fermentation test performed on Colony

1 was critical in distinguishing C. freundii from E. aerogenes due to the positive
result. The endospore stain performed with Colony 2 was critical in distinguishing S.
pyogenes from S. agalactiae due to the ability of the bacteria to form spores.
Additional tests that could have been perfromed on Colony 1 to differentiate
between C. freundii and E. aerogenes include a sulfide production test and a VogesProskauer test. An additional test that could have been performed on Colony 2 to
distinguish between S. pyogenes and S. agalactiae is the L-pyrrolidonyl-betanaphthylamide (PYR) test, which would have had a positive result in S. pyogenes
and a negative result in S. agalactiae (Todar, 2012).

Appendix A

Tests

Results

Unknown
Organism #1

Unkown
Organism #1

Gram Stain
Catalase
Oxidase
Motility
Citrate
Indole
Blood Agar
Nutrient Agar
EMB
Lactose
Morpholgy
Gelatin
DNAse Plate

Negative
Positive
Negative
Negative
Positive
Positive
Gamma
Positive
Positive
Positive
Rods in chains
Positive
Positive

Appendix B

Tests

Results

Unknown
Organism #2

Unknown
Organism #2

Gram Stain
Catalase
Oxidase
Motility
Citrate
MSA
Blood Agar
Nutrient
EMB
Lactose

Positive
Negative
Negative
Negative
Positive
Negative
Beta
Positive
Negative
Positive

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Morpholgy
Spore-forming
Stain

Zahn Unknown Organism #2

Diplococci
Positive

Work Cited
Bergeys Manual of Determinative Bacteriology. Bergeys Manual Trust. Springer,
26. May 2014. Web. 07 November 2014. http://www.antimicrobe.org/b93.org/.
Dionigi, R., Rovera, F., Dionigi, G., Diuri, M., & Cuffari, S. Infected pancreatic
necrosis. Surgical Infections (Larchmt). 2006;7 Suppl 2:S49-52. Web. 07
November 2014. http://www.ncbi.nlm.nih.gov/pubmed/16895505.
Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. Enterobacteriaceae. Medical
Microbiology. Elsevier Saunders. 7th edition. 2013.
Todar, Kenneth. Streptococcal Pyogenes and Streptococcal Disease. Todars
Online Textbook of Bacteriology. Kenneth Todar, PhD. 2008-2012. Web. 07
November 2014. http://www.textbookofbacteriology.net.
Streptococcus Group B Infections Overview. Medscape Drugs and Diseases.
WebMD. 1994-2014. Web. 07 November 2014.
http://www.emedicine.medscape.com.