Discovery of the Identity of Unknown Bacterium #48
By Rebecca Laper
Biology 208L
Introduction
Being able to distinguish bacteria from each other is an extremely important skill for
scientists, doctors, and many other professions. Using the skills that were taught throughout the
previous labs students were given an unknown bacterium and told to identify it. The skills that
needed to be used for this lab were things like staining, microscopy, inoculating the media, and
use of enterotubes. All of these skills were necessary for the students to showcase their deductive
reasoning skills to discover the identity of their bacterium on their own (Matthews, 2013).
Materials and Methods
To start this lab each student was given a nutrient agar slant with an unknown bacterium
growing on it. The number for my bacterium was #48. The first step for this lab was to
aseptically inoculate two agar slants to be incubated at two different temperatures. The lab ran
out of enough agar slants so for my bacterium one nutrient agar slant was inoculated and one
nutrient agar plate was inoculated. The slant was then incubated at 37◦C and the plate was
incubated at 25◦C. This was done so that the temperature preference of the bacteria would be
shown. Whichever one showed the most growth was used as the reserve stock and the other was
used as the working stock. After determining the working stock, it was used to inoculate several
other tests, such as a tube with Fluid Thioglycollate Medium, a nutrient agar plate, and a tube
with Litmus Milk. The nutrient agar plate was then used to inoculate an Enterotube II System,
which allowed the student to look at twelve different tests at once. Other things that were done
with the bacteria were things like making hanging drop slides to see if the bacterium were
motile, gram staining, acid-fast staining, and endospore staining. The cell shape and pigment
were also observed. All the tests and observations for the bacterium were recorded in a chart.
Discussion
To be able to identify bacterium #48, all the results were used to eliminate all the other
bacteria that it could not be. The first thing that eliminated many of the other bacteria was the
oxygen preference of #48. When the tube with Fluid Thioglycollate Medium was inoculated the
bacterium clearly grew throughout the tube showing that it was a facultative anaerobe and that it
could live with or without oxygen. The next test that allowed for elimination of other possible
bacteria was the gram staining test. Bacterium #48 had pink rods showing that it was gram
negative which made it unnecessary to do the Acid Fast stain and the Endospore stain; those only
need to be done if the bacterium has gram positive rods. After these two test results it narrowed
the choices to four bacteria, Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, and
Serratia marcescens. Serratia marcescens was then eliminated because it had a temperature
preference as 25◦C where bacterium #48 had a temperature preference of 37◦C.
So with three choices left the next test that was looked at was the litmus milk test.
Bacterium #48 was looked at 3, 5, and 7 days after being inoculated in the litmus milk. The
results showed that the litmus milk had turned a very light pink which means the lactose was
fermented, lactic acid was produced and the pH of the tube dropped (Matthews, 2013). The
litmus milk also showed coagulation at the bottom of the tube, called a “curd”. This means that
“the protein became denatured and precipitated out of the solution” (Matthews, 2013). The
litmus test also showed litmus reduction which turned the bottom of the tube white. All three of
these results on the litmus test narrowed my choices to either Escherichia coli or Enterobacter
aerogenes.
To find out which of the two bacteria #48 was the Enterotube system was the last thing
looked at. After looking at the results of the Enterotube, it was clear that the bacterium #48 had
to be Escherichia coli (E. coli). Bacterium #48 matched ten out of the eleven tests for E. coli,
where it matched only six out of eleven for Enterobacter aerogenes. Bacterium #48 tested
positive for Glucose, Lactose, Lysine, Indole, Arabinose, and Sorbitol. It tested negative for
Ornithine, Adonitol, Voges-Proskauer, Citrate, and Urea. The one test that bacterium #48 didn’t
test the right way if it is indeed E. coli was the Ornithine test, E. coli normally tests positive for
this test so it is probable that bacterium #48 read as a “false negative”.
Conclusions
All of these test results together allowed the student to determine that bacterium #48 was
E. coli. Many of the characteristics listed in Bergey’s Manual of Determinative Bacteriology
(1974) and in Table 8.1: Selected Characteristics of Some Bacteria Used as Unknowns
(Matthews, 2013) matched up with the characteristics determined in class of bacterium #48. The
tests that proved the most significant for determining the identity of the bacterium was the Gram
stain, litmus milk test, oxygen preference, temperature preference, and the Enterotube system.
E. coli are a very large and varied collection of bacteria. Most E. coli live in the intestines
of humans and animals and are harmless. However there are some strains of E. coli that can
cause a person to get extremely sick. They would have symptoms such as severe abdominal
cramps, vomiting, and bloody diarrhea (Staff, 2011). A strain that could cause symptoms like
this is called E. coli O157:H7. This can come from a variety of places such as “contaminated
water or food, especially things like raw vegetables or undercooked ground beef” (Staff, 2011).
So while most E. coli is harmless it is important to watch out for strains that can make a person
sick.
References
Holt, J. G., Kreig, N. R., Sneath, P. H. A., Staley, J. T., & Williams, S. T. (Eds.). (1974).
Bergey’s manual of determinative bacteriology (8th Ed.). Baltimore, MD: Williams &
Wilkins.
Matthews, D. M. (2013). Microbiology lab manual. Unpublished manuscript, Russell
Sage College, Troy, NY.
Staff, M. C. (2011, July 28). E. coli. Retrieved from Mayo Clinic:
www.mayoclinic.com/health/e-coli