Identification of Unknown #28
Science and Research
94%
Introduction
Bacteria are typically considered dangerous, a health hazard, something that causes sickness.
Most people who group bacteria into this stereotype do not realize that bacteria are actually ubiquitous,
meaning they're commonly found throughout the earth. Not only that, but they are necessary for normal
life cycles and evolution to continue, and for humanity to progress on many levels, including
biomedical, food-related, natural resources, alternative energy sources, and more (Tortora 2013).
In a population that is constantly increasing, the studies above become even more important. An
increase in population with the same land size results in increased exposure to disease and increased
competition and demand for commodities. Since bacteria can be used as a solution to these concerns, it
is imperative that the professionals in these fields have a consistent, reputable reference manual for
identification, classifications, and descriptions. The most trusted of this literature is Bergey's Manual of
Determinative Bacteriology (Holt, Kreig, Sneath, Staley, & Williams, 1974).
In this experiment, an unknown, numbered bacterial sample was assigned to each student of the
BIO208 lab. The student then had to classify the organism's genus and species through a process of
tests, gradually eliminating possible organisms (Matthews, 2013). This experiment tested the student's
ability to apply learned knowledge from lecture and laboratory in terms of practical laboratory skills
and techniques with deductive reasoning abilities, and familiarity with bacteriology reference manuals.
Materials and Methods
A nutrient agar slant containing an unknown bacterium was coded #28 and assigned to the
student. Two more agar slants were aseptically inoculated from this original culture, one to be
incubated at 25 degrees C and the other at 37 degrees C. Through the differences of environmental
Identification of Unknown #28
temperature during incubation, the bacteria's optimal growth temperature can be determined, further
aiding in identification. From these two agar slants, the one exhibiting the most growth was selected for
the reserve stock, as a backup culture source, and refrigerated, while the lesser growth slant was
utilized for further inoculations as the working stock. These further innoculations of the unknown
organism included test tubes of Fluid Thioglycollate Medium (FTM), litmus milk, and a nutrient agar
plate (NAP). After culturing the NAP, various characteristics of the bacterial colonies can be observed,
further classifying and identifying them. Odors can also be observed from NAPs, since some bacteria
produce a foul smell. The NAP was also used for innoculation of an Enterotube II system. This system
utlizes 12 characteristic-specific tests with little media in a fast, efficient manner, all contained in one
labelled tube (Matthews, 2013).
A Gram-stain was conducted to determine the morphological characteristics of the bacteria. No
further staining was required. A slide of the bacteria was viewed under the video microscope and an
image was captured and recorded with the rest of the data obtained in this experiment. This data was
recorded in a descriptive chart and evaluated against the table, Selected Characteristics of Some
Bacteria Used Unknowns (Matthews, 2013) and the Bergey's Mannual of Determinative Bacteriology
(1974).
Results
Below, recorded in Table 1 are the biochemical properties and the cultural and morphological
characteristics of organism #28, observed from the agar slants, plates, and innoculations.
Identification of Unknown #28
Table 1. Biochemical, Cultural, and Morphological Characteristics of Organism #28
Slant properties
Moderate translusent, colorless effuse growth
Colony characteristics
Small, round with raised smooth margins and
donvex elevated colonies
Fluid Thioglycollate Medium (FTM)
Facultative aerobe
Optimum Temperature
37° C
Litmus Milk
Acid production and litmus reduction observed
from a creamy, purple/blue texture throughout
(on Day 7)
Glucose
Positive (yellow)
Lysine
Positive (purple)
Ornithine
Positive (purple)
H2S/Indole
Negative
Adonitol
Negative (red)
Lactose
Negative (red)
Arabinose
Negative (red)
Sorbitol
Negative (red)
Voges-Proskauer
Negative (beige)
Dulcitol/PA
Negative (green)
Urea
Negative (purple)
Citrate
Positive (blue)
Cell Shape
Rod
Arrangement
Chains
Gram Reaction
Negative
Motility
Non-motile
Identification of Unknown #28
Below in Figure 1 is a Gram stain of the unknown bacteria #28.
Figure 1. Unknown #28: Gram negative rods in chain-like arrangment
Discussion
After the innoculations were completed, an Acid-fast stain was done. This Acid-fast stain had to
be prepared twice, because the first prepared slide was too dye-soaked to differentiate cells. The second
test turned out sufficient, but still dye-heavy, despite careful timing and conservative dye-application.
From this, a Gram-negative bacteria was observed, and captured in image, as above in Figure 1. The
innoculations prepared, especially the Litmus milk test, are most informative when observed
throughout the innoculation period. This is because certain chemical reactions can occur that re-alter
the environment of the culture, possibly altering or omitting important observations (Matthews, 2013).
The innoculations prepared in this experiment were only viewed once, seven days after innoculation.
Although this did not seem to have an impact on final identification of the organism, if observed
throughout incubation, more information and a firmer identification could have been formed.
Identification of Unknown #28
Another test, the Enterotube II system, innoculated many medium at once with one swab of
culture, then allowed to incubated for a week. From this test, three of the results (lactose, arabinose,
and sorbitol) were inconsistent with the organism's final identification and characteristics in the table of
Selected Characteristics of Some Bacteria Used as Unknowns (Matthews, 2013). These results are
italicized in Table 1. Reasons for this are unknown, but most likely attributed to contamination of the
medium. After all other tests were completed, the final identification was between two organisms, both
of which had identical characteristics of these three tests. Therefore, the altered results did not affect
the outcome of the identification, and the Eneterotube II System did not have to be redone.
Conclusions
After compiling and analyzing results and notes, the student was able to identify unknown
organism #28 as Enterobacter aerogenes. Other than the three tests mentioned above, and italicized in
Table 1, all test results and observations of characteristics were consistent with what was outlined in the
table of Selected Characteristics of Some Bacteria Used as Unknowns (Matthews, 2013) and in
Bergey's Manual of Determinative Bacteriology (1974). The FTM tube, cell shape, Gram-stain, and the
Indole, Voges-Proskauer, and Citrate test in the Enterotube II System proved to be the most significant
in determining the organism's identity. The three Eneterotube tests were what differentiated
E. aerogenes from Escherichia coli bacterium.
The Enterobacterium are a common bacteria who reside in the instestinal track. They attack
glucose producing acid or any acid, as well as gas (H2), and sometimes they break down pectin and
alginate, as well. This aids in digestion and health of the gut. They can be motile with peritrichous
flagella or non-motile, as was this culture of E. aerogenes. This species is non-pathogenic and
common, as well as a beneficial cohabitant of the gastrointestinal tract (Holt, Kreig, Sneath, Staley, &
Williams, 1974).
Identification of Unknown #28
Reference List
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.
Tortora, G. J., Funke, B. R., & Case, C. L. (2013). Microbiology: An Introduction (11th Ed.).
Boston, MA: Pearson.