Unknown Experiment
BI 303
Danion Terauds
and
Brian Turek
3/31/05
Structure:
The layout of this lab report will be in chronological order to show test and
deductive patterns. Our lab report will also include photographs, where possible, and
other graphical representations of our results.
Procedure:
Given the choice of 13 possible bacteria, we created a spreadsheet (Table 1.1)
with the bacteria and their reactions to particular tests, according to Bergey’s Manual of
Determinative Bacteriology, 9th Edition. We then compared our unknown bacteria test
results with the spreadsheet in order to eliminate the possible bacteria from the list. This
was done only when it did not meet two separate tests or characteristics of our unknown.
We chose that possible bacteria had to fail two criteria before being eliminated because of
the possibility a test failure or inconclusive results.
As a procedural constant, in the beginning of every lab period we made it a point
to inoculate fresh nutrient broth and/or inoculate a nutrient agar slant with our unknown.
A fresh culture is imperative for the Gram’s stain, and redundancy is important incase of
contamination.
Tests/Results
Unknown Sample: Tube #7

Streak Plate (BI 303 lab manual pg. 64-67)
o Date Inoculated: 3/15/05
Results Recorded: 3/17/05
o Sample Age: < 24 hours (original stock)
o Media: Nutrient Agar
o Notes: We ran this procedure first to isolate colonies of the bacteria, to
determine if the culture was pure. Also, we can determine visible
characteristics of the colonies, to serve as a preliminary identifier of the
bacteria.
o Results: Yellow colonies and uniform
colonies. (Fig. 1.1)
o Discussion: With our results, we can
determine that the culture is pure. Also,
the yellow colonies point toward M.
luteus, M. phlei, S. epidermidis, and S.
aureus.
Fig. 1.1 – Streak plate results.
(Note: Thinning yellow colonies.)

Simple Stain (BI 303 lab manual pg. 7-8)
o Date: 3/15/05
o Sample Age: < 24 hours (original stock)
o Stain: Methylene Blue
o Notes: We decided to run this test because of its fast results and we can use
the physical attributes, size, and grouping to help identify the bacteria. Also,
we can compare the physical attributes to the future Gram’s stain for
consistency.
o Results: Blue spherical clusters (grape like)
throughout the slide. Cells are cocci and
~1.0µm in size. (Fig. 1.2)
o Discussion: Our results indicate a defining
structure of the Staphylococcus species.
Grape-like cluster
Fig. 1.2 – Simple stain results.
(Note: Image transcribed onto paper
from the slide sample.)

Gram’s Stain (BI 303 lab manual pg. 13-16)
o Date: 3/15/05
o Sample Age: < 24 hours (original stock)
o Notes: The Gram’s stain is a definitive test for determining and classifying
bacteria.
o Results: Cells are stained purple, cocci shaped, tetrads and clusters, and cells
are ~ 1.0µm in size.
o Discussion: The purple stained cells are indicative of Gram positive bacteria.
The physical attributes and groupings were similar to the simple stain, but
revealed more of a tetrad arrangement.

Hanging Drop Method (BI 303 lab manual pg. 23-26)
o Date: 3/15/05
o Sample Age: < 24 hours (original stock)
o Notes: Due to the instant results and the inexpensiveness to perform, we
decided that this would be an efficient way of gathering more data.
o Results: No motility was detected.
o Discussion: Because of no motility, we decided that further tests needed to be
performed to validate non-motility.

Motility Stab (BI 303 lab manual pg. 23-26)
o Inoculation Date: 3/15/05
Results Recorded: 3/17/05
o Sample Age: < 24 hours (original stock)
o Media: Soft agar (~0.4% agarose)
o Notes: Because of the hanging drop test results,
we thought that it would be necessary to perform a
more definitive test to determine motility.
o Results: No motility. No growth within the agar,
but growth was abundant on a surface streak. (Fig.
1.3)
o Discussion: These results confirm the assumption
that our unknown is in fact non-motile, and is a
possible aerobe.
Fig 1.3 – Motility stab.
(Note: brown spot on picture
is a glass stain, not motility or
growth.
 3/15/05 Results Synopsis
The key test results taken on 3/15/05 were from the Gram’s stain and simple stain. The
clustered pattern of bacterial arrangement and the positive Gram’s test eliminates many
of the possible bacteria (Table. 1.2), and pointing our tests in the direction of M. luteus,
S. epidermidis, and S. aureus.

Mannitol Salt Agar Test (BI 303 lab manual pg. 66-67)
o Inoculation Date: 3/17/05
Results Recorded: 3/22/05
o Sample Age: 48 hours (cultured slant – 3/15/05)
o Notes: The MSA test was performed to differentiate S. aureus from S.
epidermidis and M. Luteus.
o Results: Low growth of bacteria and no color
change to agar.
o Discussion: Due to the lack of Mannitol
fermentation (positive fermentation would yield a
yellow hue); we can determine that the unknown
bacterium is not S. aureus. The low growth on the
MSA, points at the possiblility of the bacteria being
halotolerant.
Fig. 1.4 – MSA test.
(Note: No yellow color
change.)
 3/17/05 Results Synopsis
On 3/17/05 we were able to observe the results from the streak plate and the motility stab,
both inoculated on 3/15/05. The streak plate results were indicative of M. luteus (bright
yellow colonies). The motility stab (negative result) does not differentiate between the
possible bacteria that are left (refer to table 1.2), but only validates them.

Oxidase Test (BI 303 lab manual pg. 101-103)
o Date: 3/22/05
o Sample Age: 5 days (cultured slant – 3/17/05)
o Notes: Due to the unknown bacteria’s inability to ferment mannitol, it left us
to differentiate between the possibility of M. luteus and S. epidermidis. The
oxidase test differentiates M. luteus, which produces a positive result, and the
Staphylococcus species which is considered to be an oxidase negative species.
o Results: The oxidase test strip turned purple in the presence of the unknown
sample.
o Discussion: A purple result in indicative of an oxidase positive bacterium,
Microccocus luteus.

Catalase Test (BI 303 lab manual pg. 97-98)
o Date: 3/22/05
o Sample Age: 5 days (cultured slant – 3/17/05)
o Notes: We decided to run the Catalase test, because of its inexpensiveness,
quick results, and its further criteria for validation.
o Results: An effervescent reaction occurred. (Fig. 1.5)
o Discussion: Bubbles are indicative to a Catalase
positive reaction.
Fig. 1.5- Catalase test.
(Note: bubbles forming on
the slide.)
 3/17/05 Results Synopsis
On this day of results, we found that the incubated MSA did not ferment, indicating that
the unknown bacteria is not S. aureus. From the oxidase test, we were able to rule out the
Staphyloccous species. Thus we conclude that our unknown species is Micrococcus
luteus.
Conclusion
We conclude that our unknown species (#7) is Micrococcus luteus. From our
results and discussions, that we have already stated, reasonably justify our assumption.
Post Experiment Disscussion
Reviewing our tests and results we found that some tests were unnecessary or not
performed in the correct chronological order. If we were to run the experiment again, we
would:
1.) Create a streak plate.
2.) Perform a Gram’s stain,
3.) Perform an oxidase test.
Using this pattern we could reasonably identify M. luteus.
We feel that this experiment helped hone our laboratory skills, and improved our
deductive laboratory techniques.
Resources
Benson, Brown, et al. (2005). BI 303 General Microbiology. United States: McGrawHill
Holt, J. (Ed.), et al. (1994). Bergey's Manual of Determinative Bacteriology. 9th ed.
Baltimore: Williams & Wilkins.