DISCOVERY OF THE IDENTITY OF UNKNOWN BACTERIUM #44

Devi Dinghoor
Russell Sage College
BIO 208 L Microbiology Labs
Professor Peters
April 27, 2016

DISCOVERY OF THE IDENTITY OF UNKNOWN BACTERIUM #44

Introduction

Microbiology is defined as the study of microorganism. One the few organisms studied in
the course of microbiology is bacteria. Bacteria are single cells organisms that are found in air,
soil, water and on our body. Bacteria are very simple cells that falls under the category of
prokaryotic cells. Meaning they do not have a nucleus or a membrane bound organelles, but they
do contain a cell wall. Bacterial cells wall are composed of a macromolecular network known as
peptidoglycan. A thick peptidoglycan cell wall are found in gram positive bacteria. While, a thin
peptidoglycan cell wall are found in gram negative bacteria (Tortora, Funke, Case, 2013). The
identify of an unknown bacteria can be determine by performing a gram stain to figure out if the
bacteria is a gram positive and gram negative.
All students including myself was given a sample of an unknown bacteria in
Microbiology lab. Using the knowledge and skills gained from microbiology lab, I was required
to identify an unknown bacteria independently. This was done by finding out the morphological,
cultural and biochemical characteristics of the unknown bacteria assigned. Figuring out the
identity of the unknown bacteria involved creating different kinds of gram staining procedures
and inoculation of the organism. The unknown bacteria is one of the 16 possibilities of organisms
found on Table 8.1 of Selective Characteristics of Some Bacteria Used as Unknowns (Matthews,
Rea, 2016). Results from conducting the tests are used to compare to results on Table 8.1 of
Selective Characteristics of Some Bacteria Used as Unknowns (Matthews, Rea, 2016) to identify
the unknown organism. The Bergeys Manual of Determinative Bacteriology (1994) was also
used to verify the identity of the unknown bacteria.

DISCOVERY OF THE IDENTITY OF UNKNOWN BACTERIUM #44

Materials and Methods

An unknown sample of a bacteria growing on an agar slant was distributed to me in lab.
The unknown bacteria was labeled # 44. To prevent contamination, I used aseptic techniques by
inoculating two agar slants from the original agar slant culture. I incubated one agar slant at 25
C, while I incubated the other agar slant at 37 . This was done to determine the temperature
preference of the bacteria by assessing the amount of growth in the tube. A week later, the tubes
were views to see which tube had the greatest growth. The tube with the less growth were used
as my working stock, the tube with the greatest growth was used as my reserve stock. The
working stock was used to make slides and inoculate tests. The reserve stock was stored in the
refrigerator to be used as a backup plan in case any problems occurred with the working stock. I
used the reserve stock to observe characteristics of my bacteria. I used my mystery organism
tube to inoculate a tube of fluid thioglycollate medium, litmus milk and nutrient agar plate. I did
a gram staining procedure to determine if my bacteria is a gram positive or gram negative as well
as the shape of the cells and arrangement of it. I also performed an endospore staining procedure
to check for endospores. I used the nutrient agar plate to view the culture characteristics of my
bacteria and to inoculate the Enterotube II. A week later, I used the Enterotube II to determine the
biochemical characteristics of my unknown bacteria # 44 (Matthews, Rea, 2016).
Results from performing all of these procedures were recorded in table one. I use the
results from these procedure to compare it to Table 8.1 of Selective Characteristics of Some
Bacteria Used as Unknowns (Matthews, Rea, 2016). I also used the results to compare to it to the
information in Bergeys Manual of Determinative Bacteriology to verify the identity of the
unknown bacteria (Bergey, Holt, Kreig, Sneath, Staley &Williams, 1994)

DISCOVERY OF THE IDENTITY OF UNKNOWN BACTERIUM #44

Results
Table 1. Characteristics of organism # 44
The morphological, cultural, and biochemical characteristics as well as the temperature
preference and oxygen preference of organism # 44 are shown below.
Slant properties
Colony characteristics

Translucent, creamy yellow, beaded growth

Small, round shaped, wavy (undulated)

Fluid Thioglycollate Medium (FTM)

Optimum Temperature
Litmus milk

margin, flat elevated colonies

Facultative anaerobe
25 Celsius
Litmus stayed purple, watery purple layer on
top of tube, curd formation on bottom layer of

Glucose
Lysine
Ornithine
H2S/Indole
Adonitol
Lactose
Arabinose
Sorbitol
Voges-Proskauer
Dulcitol/PA
Urea
Citrate
Cell Shape
Arrangement
Gram Reaction
Motility

tube.
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Positive
Rods
Chains
Positive
Motile

DISCOVERY OF THE IDENTITY OF UNKNOWN BACTERIUM #44

Figure 1. Gram stain results for

unknown bacteria # 44

As shown above is a picture of a gram positive, rod shaped bacteria arranged in chains.

DISCOVERY OF THE IDENTITY OF UNKNOWN BACTERIUM #44

Figure 2. Endospore stain results for unknown bacteria # 44

Figure 2. illustrates a picture of the endospores found in my gram positive bacteria. The image
displays round shape spores that are stained light green.

Figure 3. Fluid thioglycollate medium results for unknown bacteria # 44

Figure 3. illustrates the results of my FTM tube inoculated with unknown bacteria #44 for one
week. The tube shows the oxygen preference for unknown bacteria # 44.
Discussion
Upon performing a Gram stain, I was able to narrow down a few possibilities of my unknown
bacteria. Unknown bacteria # 44 turned out to be a Gram positive bacteria arrange in chains of
purple rods as shown in figure 1. After performing the gram stain, it narrow down my unknown

DISCOVERY OF THE IDENTITY OF UNKNOWN BACTERIUM #44

bacteria from sixteen to five choices. My five possible choices of unknowns was
Mycobacterium smegmatis, Mycobacterium vaccae, Bacillus cereus, Bacillus megaterium, or
Bacillus subtilis because these were all gram positive rod shaped organisms. Additional staining
such as endospore stains was require for organisms that were gram positive. After performing an
endospore staining, Mycobacterium smegmatis, Mycobacterium vaccae were eliminated because
they did not test positive for endospores.
The fluid thioglycollate medium (FTM) helped me to discover the oxygen requirement of
my organism. Based on the results from the FTM, the temperature preference of unknown
bacteria # 44 was 25 Celsius. While, the oxygen preference of my unknown was determine to
be a facultative anaerobe. The oxygen preference of my bacteria led me to speculate that the
unknown may be Bacillus Cereus. This eliminated the two other bacteria, which were Bacillus
megaterium, and Bacillus subtilis.
The litmus milk test tube was observed days three, five and seven after it was incubated
with my mystery organism. The litmus milk aid in testing for acid production, protein
coagulation, peptonization, alkaline reaction and litmus reduction in my unknown bacteria. The
protein coagulated in my litmus milk because curd formation was found at the bottom of tube
when tilted to a 45 angle. Peptonization also occurred due to the watery translucent layer at the
top of the tube. Alkaline reaction occurred because the litmus milk remained purple. Based on
the Table 8.1 Selected Characteristics of Some Bacteria Used as Unknowns (Matthews, Rea,
2016), the results matched Bacillus megaterium, Bacillus subtilis, and Bacillus cereus.
Therefore, my results from the litmus milk test was very useful in helping me determine the
identity of my unknown bacteria.

DISCOVERY OF THE IDENTITY OF UNKNOWN BACTERIUM #44

The last test due to determine the identity of my unknown bacteria was the Enterotube II.
According to Table 8.1 Selected Characteristics of Some Bacteria Used as Unknowns
(Matthews, Rea, 2016), Bacillus cereus displayed a positive result for glucose, while Bacillus
subtilis displayed negative for glucose. Therefore, the glucose test eliminated Bacillus subtilis.
The two choice was between Bacillus megaterium Bacillus cereus, which both tested positive for
glucose and citrate. They both tested negative for lactose, lysine decarboxylase, ornithine,
adonitol, indole, arabinose and sorbitol.
Conclusion

After concluding various tests, I was able to detect that the sample of organism # 44
was Bacillius cereus because thats the only organism that is facultative anaerobe and contains
endopsores. The description of Bacillus cereus written in Bergeys Manual of Determinative
Bacteriology (1994) supports the results from Table 8.1 Selected Characteristics of Some
Bacteria Used as Unknowns (Matthews, Rea, 2016). The procedures that helped me identify my
unknown organism the most were the gram stain, endospore stain, oxygen preference, and the
Enterotube II.
According to Bergeys Manual of Determinative Bacteriology (1994), the genus Bacillus
are gram positive, motile organisms that contains rod shaped cells. These rod shaped cells tend to
appear in chains or pairs. Bacillus organisms have endospores that look oval, cylindrical or
round, this means they can survive in any harmful environmental conditions. This genus tend to
have an oxygen preference of aerobic or facultative anaerobe. They are resistant to head and ph.
Bacillus cereus is widespread in soils and food such as vegetables, fish, milk, rice and
leftovers. Bacillus cereus is associated with food poisoning because it produces a toxin which

DISCOVERY OF THE IDENTITY OF UNKNOWN BACTERIUM #44

can cause illness when ingested. People can get sick from eating contaminated foods that have
Bacillus cereus because it contains spores that can survive any environment. And it is resistant to
heat, therefore, it can survive in cooked foods. It causes two food-borne illnesses, which are
diarrhea and vomiting (Maczulak, 2011).

References

Bergey, D. H., & Holt, J. G., Kreig, N.R., Sneath, P. H. A., Staley, J.T., & Williams, S.T. (1994).
Bergey's manual of determinative bacteriology (9th ed.). Baltimore: Williams & Wilkins.
Maczulak, Anne. "Bacillus." Encyclopedia of Microbiology. New York: Facts on File, 2011. 7376. Facts on File Science Library. Gale Virtual Reference Library. Web. 26 Apr. 2016.
Matthews, D. M. (2016). Microbiology lab manual. A. Rea (Ed.) Unpublished manuscript,

DISCOVERY OF THE IDENTITY OF UNKNOWN BACTERIUM #44

Russell Sage College, Troy, NY.

Tortora, G. J., Funke, B. R., & Case, C. L. (2013). Microbiology: An introduction (11th ed.).
Boston: Pearson.

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