Mystery Microbe Project
Title: To identify Mystery Microbe #_ using a system of identification tests, including: API20E
Enterobacteriaceae Identification system, Bergey’s Manual of Determinative
Bacteriology, identification flow charts, and experimentation log sheets.
Procedure:
1. Identify top unknown bacteria candidates using API20E Enterobacteriaceae
Identification System.
2. Identify additional, or eliminate, bacteria candidates using identification flow charts
from lab manual and handouts.
3. Utilize Bergey’s Manual and web resources to make final selection.
4. Write an argument to defend identification methodology.
5. Complete Descriptive Chart for identified bacteria
Observations: Refer to lab notebook log sheets.
Analysis:
AP120E
 The possible genus of the Mystery Microbe is: Escherichia, Citrobacter, Serratia,
Hafnia, and Enterobacter. These are the only bacteria that will be considered
further.
 The API20E showed negative for VP, but we also had a negative MR test, so the
VP test will be no longer be considered.
 API20E results used were for: ONPG, ADH, LDC, ODC, CIT, H2S, URE, TDA, IND,
GEL, GLU, MAN, INO, SOR, RHA, SAC, MEL, AMY, and ARA.
 Bacteria considered from the list of possible bacteria which correctly matched at
least 14 out of 20 are:
o Serratia liquefaciens
17 of 20
o Citrobacter amalonaticus
16 of 20
o Escherichia coli
15 of 20
o Serratia odorifera 2
15 of 20
o Enterobacter sakazakii
15 of 20
o Serratia marcescens
15 of 20
o Hafnia alvei
14 of 20
 Ruling out at this point:
o Our unknown Mystery Microbe was found to be MR-, and CIT +, whereas
E. coli is MR +, and CIT-. In light of these two differences E. coli will no
longer be considered.
o Our unknown Mystery Microbe was MR-, H2S -, and an optimal
temperate of 22 degrees Celsius. Citrobacter amalonaticus is MR+, H2S+,
and an optimal temperature of 37 degrees Celsius. These three
differences will eliminate Citrobacter amalonaticus.
o Our unknown Mystery Microbe was found to be positive for GEL, and
LDC. According to our API20E test result chart, Enterbacter sakazakii is
never positive for both GEL and LDC. These two reasons will eliminate
Enterbacter sakazakii.
Flow Charts
 The dichotomous flow chart (given out in lab) was used to determine possible
bacteria. Both the Citrate Utilization Test and the Mannitol fermentation can
show inconclusive results, so both paths in the flow chart will be considered.
 Lab handout flow chart from lab for Facultative Anaerobes gave us these
bacteria choices:
o Facultative Anaerobe  Motile Citrate +  Lactose +  MREnterobacter
o Facultative Anaerobe  Motile  Citrate -  URE -  E. coli
o Facultative Anaerobe  Motile  Citrate + URE +  Serratia
o Because of the three essential differences stated above, E. coli is no
longer considered, and two differences ruled out Enterbacter sakazakii.
 The flow chart yields the following possibility:
o Serratia
 The API20E test also listed the genus Hafnia as a possibility, and is still being
considered.
Comparison of MM#4 to Hafnia and Serratia using Bergey’s Manual of Determinative
Bacteriology
 According to Bergey’s Manual, bacteria morphology is compared as follows:
o Hafnia- straight rods, 2-5μm in length, gram-negative, motile
o Serratia-straight rods, 0.9-2.0μm in length, gram-negative, motile
o Mystery Microbe #4- straight rods, 1-2μm in length, gram negative,
motile
 Using Bergey’s Manual, bacteria temperature preference is:
o Hafnia- optimal temperature 30-37 degrees Celsius
o Serratia-optimal temperature 30-37 degrees Celsius
o Mystery Microbe #4- grew at both 22 and 37 degrees Celsius
 Ruling out at this point:
o Bergey’s manual indicates that Hafnia alvei is 2-5μm in length, and our
MM#4 is 1-2μm in length. Also, Hafnia alvei colonies grow as clusters,
are smooth, convex, and are off white and shiny when grown on agar.
Our MM#4 grows in small, single colonies, which are flat, semi-mucoid,
and range from red to orange in color. Based on size, color, colony
growth, and texture Hafnia Alvei will no longer be considered.
We now know that Serratia is our genus:
 As listed above, we have three possible species of Serratia:
o Serratia odorifera 2
o Serratia liquefaciens
o Serratia marcescens
 Ruling out at this point:
o Our Mystery Microbe #4 is positive for positive for ODC and negative for
RHA. Serratia odorifera 2 is never positive for ODC, and positive for RHA
95% of the time. These two strong differences eliminate Serratia
odorifera 2.
Bacteria
Serratia liquefaciens
Serratia marcescens
MM#4
Pigment
Colorless
Red-orange
Red-orange
Hemolysis
Some strains hemolytic
ά hemolytic
ά hemolytic
Conclusion:
Based on the API20E Enterobacteriaceae Identification results, the flow charts, and utilization
of Bergey’s Manual of Determinative Bacteriology, both Serratia liqufaciens and Serratia
marcescens are strong candidates for the identity of our Mystery Microbe. However, when we
compare the pigment and hemolysis properties of both candidates, we have decided that the
unique red color of our Mystery Microbe makes it evident that its identity is Serratia
marcescens.
Error Analysis:
We found this assignment to be fairly challenging, yet completely rewarding when coming (at
last!) to a conclusion. There were quite a few test results that could be fairly questionable, and
if given the chance, we would chose to perform them again. Some test results readings on the
API20E test strip were subjective, and we didn’t analyze colors exactly in the same way.
Inositol, Sorbitol and Sucrose results were difficult to analyze, so we were forced to be flexible
in which way to go with the data. All three tests were positive if the color was yellow and
negative if the color was blue or blue-green. Ours were all green (not blue-green). After
conferring with our professor, we decided to count all three tests as positive results. Green is in
between yellow and blue on the color spectrum, and we believe that the tests were closer
towards yellow then blue.
Test Analysis:
The two tests that we found to be the most helpful indentifying our Mystery Microbe were the
Liquefaction of Gelatin test and the Rhamanose test.
o Liquefaction of Gelatin test: We placed a lot of weight on this test because in the
possible given choices of microbes, only a small amount of bacteria listed ever
have positive readings. Our result was total liquefaction, shown by an evenly
disbursed black liquid. In the list, 3 of the 7 bacteria known to have positive
readings were in our possible choices.
o Rhamanose test: These test results are the opposite of the GEL test. Ours was
negative, and a large percentage of the listed possible bacteria were positive.
Our final choice, Serratia marcescens was found to be positive only 2% of the
time.
The two tests we feel could be deleted to save time and expenses are the Glucose test, and the
Trytophane Deaminase test.
o Glucose test: At least in this situation with the listed possible bacteria, Glucose is
positive across the board 100% of the time. This could be eliminated and the
data results from this test given to students.
o Trytophane Deaminase: Our reason is similar to the Glucose test, however the
listed possible bacteria are found to be positive for this 0% of the time, with the
exception of the two organism listings. If the organisms were not used in future
Mystery Microbe projects, this test could be eliminated.
We were fortunate to not have to repeat any tests, however in the case of the API20E
Enterobacteriaceae Identification test, it would have been helpful to do another run of it to be
compared and contrasted to our original test.
Summary of the Mystery Microbe Project:
While sometimes feeling overwhelming, this project still manages to teach some very important
life skills. The first lesson to be learned is that sometimes it takes several steps to come the
right answer, and even if you think you know what something might be, you have to back it up
with cold, hard facts. This project is directly tied to our future professions, as well as lives in
general, because of the essential skills you must learn to complete it. You must be observant,
determined, and patient. We are both headed for the nursing profession, and these skills will
help us to take all of a patient’s symptoms, and one by one using deductive logic, break it all
down and come to a diagnosis.
All about Serratia marcescens:
S. marcescens is a facultative anaerobe part of the
Enterobacteriaceae family. It can grow in several
common natural habitats including soil, water, and the
intestines; as well as bathtubs, sinks, and primarily
starchy foods. S. marcescens is a gram-negative,
bacillus shaped bacteria that is involved in nosocomial
infections. It is the most common cause of pathogens
within its Serratia genus. This particular bacterium is
known for its association with catheter associated
bacteremia, urinary and respiratory tract infections,
wound infections, and meningitis. Ways in which
transmission can occur is by direct contact, droplets, in
saline irrigation solutions, and in other “sterile” solutions. Also, poor hand washing and using
contaminated medical equipment have contributed to the spread of pathogens. Treatment for
S. marcescens includes cephalosporins, gentamicin, and amikacin. Unfortunately for humans,
most strains are resistant to several antibiotics, including penicillin and ampicillin. Individual
strains of Serratia marcescens have the ability to build immunities to previously effective
antibiotics. S. marcescens is a motile organism and can grow in temperatures ranging from 5–
40°C. When grown on TSA plates and incubated at room temperature, 22°C, it exhibits a distinct
red color called prodigiosin, while incubation at body temperature results in a tan-orange color.
Photograph of Serratia marcescens:
http://www.microbelibrary.org/ASMOnly/details.asp?id=2566&Lang=
This website is very informative of what Serratia marcescens is:
http://www.serratia-marcescens.org/
This source is more of an overview of Serratia marcescens:
en.wikipedia.org/wiki/Serratia_marcescens
NOTICE THIS STUDENT DID NOT CORRECTLY SITE SOURCES ACCOURDING TO APA FORMAT!