Final Unknown Experiment
BI 303
Danion Terauds
4/29/05
Structure:
The layout of this lab report will be in chronological order to show test and
deductive patterns. My lab report will also include photographs, where possible, and
other graphical representations of my results.
Procedure:
Given the choice of 13 possible bacteria, I 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. I then compared my 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.
I 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 I made it a point to
inoculate fresh nutrient broth and/or inoculate a nutrient agar slant with my unknown. A
fresh culture is imperative for the Gram’s stain, and redundancy is important incase of
contamination.
Tests/Results
Unknown Sample: Tube #11

Streak Plate (BI 303 lab manual pg. 64-67)
o Date Inoculated: 4/19/05
Results Recorded: 4/21/05
o Sample Age: < 24 hours (original stock)
o Media: Nutrient Agar
o Objective: To isolate colonies and determine purity of the unknown sample.
Also, to determine visible characteristics of the colonies, to serve as a
preliminary identifier of the bacteria.
o Results: White and semi-transparent colonies
with irregular edges. (Fig. 1.1)
o Discussion: From these results, I determined
that the culture is in fact pure. The colony
attributes do not stand out as a tell tale sign of a
particular bacteria. Post test note (4/25/05):
Upon further research the semi-translucent
colonies with irregular edges is a determinate
characteristic of Pseudomonas fluorescens.
Fig. 1.1 – Streak plate results.
(Note: Semi-translucent colonies.)

Simple Stain (BI 303 lab manual pg. 7-8)
o Date: 4/19/05
o Sample Age: < 24 hours (original stock)
o Stain: Methylene Blue
o Objective: I decided to run this test because of its fast results and I can use the
physical attributes, size, and grouping to help identify the bacteria. Also, I can
compare the physical attributes to the future Gram’s stain for consistency.
o Results: The slide consisted of small cocci-bacillus cells 1µm x 2-3 µm. The
organization of the cells was not consistent, with cells occurring in pairs,
groups, chains, and singularly. There were
Green Cell
also a few cells that did not match trend of
the slide; they looked liked small green
algae.
Pair
Chain
o Discussion: The appearance of the green cell
on the slide raises questions to the purity of
Single
the culture. Also, because of the variety of
blue cell
cell groups, narrowing criteria can not be
defined.

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: 4/19/05
o Sample Age: < 24 hours (original stock)
o Objective: Determine if the unknown bacterium is Gram positive or negative,
in order to greatly narrow the scope of my search.
o Results: Very light purple color cells grouped singularly and in pairs.
o Discussion: In my opinion, the test was not conclusive. It was very hard to
differentiate if the color was a very light purple or an off colored pink.
 4/19/05 Results Synopsis
From a definitive scientific aspect, I could not draw any firm conclusions from today’s
simple stain and Gram’s stain results. I personally question the purity of the sample that I
had chosen. I am waiting for my bacterial streak plate results to settle the question of
purity. Also, I believe that the inconclusive Gram’s stain results could be due to an older
sample then 24 hours. Today, I have inoculated fresh NA broth, so that I can use that for
a new Gram’s stain.
 4/21/05 Results Synopsis
I was able to observe the unknown bacteria streaked onto nutrient agar. The cells are
uniform, and there does not appear to be any foreign bacterium. I would have to
conclude that the sample then is pure for growth, and the broth created 4/19/05 would be
pure as well. The green bacteria noted in the 4/19/05 simple stain could have been a
contaminant in the immersion oil, on the microscope, or non-sterile microscope slides.
Note: due to time constraints, I was unable to perform a gram’s stain on the fresh
bacterium. So, I will prep a fresh batch of the unknown bacteria on 4/25/05, and perform
the gram’s stain on 4/26/05.

Gram’s Stain (BI 303 lab manual pg. 13-16)
o Date: 4/26/05
o Sample Age: < 24 hours (nutrient broth – 4/25/05)
o Objective: Determine if the unknown bacterium is Gram positive or negative,
in order to greatly narrow the scope of my search. Because this test was
inconclusive the first time, this second test with a fresh culture is necessary.
o Results: Pink bacillus shaped cells occurring signally and in pairs.
o Discussion: This very obvious in determining that the unknown bacterium is
Gram’s negative. This narrows the possibility of my unknown to six bacteria,
as noted in Table 1.2.

KOH Test (BI 303 lab week 2 – Gram & Acid Fast.ppt)
o Date: 4/26/05
o Sample Age: < 24 hours (cultured slant – 4/25/05)
o Objective: To back up the validity of the Gram’s stain test results (i.e. double
check).
o Results: When KOH was mixed with the unknown
bacteria, the mixture became viscous. When
lifting the mixing loop out of the mixture, strand of
the mixture stuck to the slurry and the loop,
forming bead like structures on the strand.
o Discussion: This simple test concludes that the
previous Gram stain was correct in identifying the
bacteria as Gram negative.
Fig. 1.3 – KHO test.
(Note: strand connecting the
mixture with the loop.)

Oxidase Test (BI 303 lab manual pg. 101-103)
o Date: 4/26/05
o Sample Age: < 24 hours (cultured slant – 4/25/05)
o Objective: To test for the ability of the unknown bacteria to produce
cytochrome oxidase. Thus, this test is to determine possibility of A. faecalis or
P. fluorescens as the unknown bacteria.
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, narrowing the
bacterial possibilities to A. faecalis or P.
fluorescens.
Fig. 1.4 – Oxidase test.
(Note: purple coloring of test
strip.)

Catalase Test (BI 303 lab manual pg. 97-98)
o Date: 4/26/05
o Sample Age: < 24 hours (cultured slant – 4/25/05)
o Objective: I 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. Although both A. faecalis and P.
fluorescens are catalase positive, this test supports the
assumption that the unknown bacteria may be one of
the two.
Fig. 1.5 - Catalase test.
(Note: bubbles forming.)
 4/26/05 Results Synopsis
With finally begin able to get a clear Gram stain result and the positive oxidase result; the
possible bacteria were limited to two bacteria: A. faecalis or P. fluorescens. The changes
are noted in Table 1.2. Both A. faecalis and P. fluorescens are very similar in size, shape,
and growth (Rhodes - online). Upon further research, I found that to differentiate
between the two, two test can be performed: nitrate test (Bergy pp. 94 & 131) and a
flagella stain (BI 303 lab manual pg. 58).

Flagella Stain (BI 303 lab manual pg. 58-61)
o Date: 4/28/05
o Sample Age: 2 days (cultured slant – 4/26/05)
o Objective: Differentiate between A. faecalis or P. fluorescens. Both bacteria
have flagella for motility, but A. Faecalis has peritrichous flagella, where as P.
fluorescens has polar flagella.
o Results: Clusters of 1 x 2-3µm bacteria with the appearance of very faint polar
tails. No evidence of peritrichous flagella on the side of the bacteria.
o Discussion: Polar flagella are indicative of P. fluorescens.

Nitrate Test (BI 303 lab manual pg. 107-110)
o Date: 4/28/05
Results Recorded: 4/29/05
o Media: Nitrate broth
o Sample Age: 2 days (cultured broth – 4/26/05)
o Objective: To further conclude that the assumption of P. fluorescens being the
unknown bacteria. I would expect growth in the nitrate broth, and a
conversion of the NO3 to NO2, resulting in a positive test.
o Results: A thick layer of growth near the top of the solution (fig. 1.6). After
reagents A&B were added, there was not color change. Also, with the addition
of Zinc there was no color change.
o Discussion: The heavy growth near the surface of the nitrate broth is
indicative of the bacteria’s ability to use nitrogen for growth. The non-reaction
after zinc was added shows the bacteria’s ability reduce nitrate completely to
molecular nitrogen (½ N2). This evidence is supportive of P. fluorescens.
Fig. 1.5- Growth in Nitrate broth
(Note: thick white growth band at top)
Fig 1.6 – Broth after Zinc added
(Note: no red pigment)
 4/28/05 Results Synopsis
With the flagella stain indicating polar flagella, I would assume that the bacterium is P.
fluorescens. Yet, because the flagella stain was light, I will wait for the nitrogen test
results to make my final determination.
 4/29/05 Results Synopsis
The nitrogen test results (as described above) were very supporting of P. fluorescens. I
feel that I can reasonably separate P. fluorescens from A. Faecalis on the basis of:
positive nitrate test result, polar flagella, and grayish semi-translucent colony formation
(as stated with streak plate results).
Conclusion
I conclude that my unknown species (#11) is Pseudomonas fluorescens. I feel that
I have reasonably justified my assumption with the results and discussions as stated in the
body of my experimentation.
Post Experiment Discussion
At the beginning of my experiment I was receiving very inconclusive results. As I
stated earlier in the paper, this could have been due to various factors including
contaminates or culture age. I feel that I was correct in going back and re-doing tests,
when I was 100% sure of my controllable variables: culture age, purity, and sterility of
materials.
Reflecting on the time it took to complete the tests. I feel that if the first day’s test
results had been very conclusive, then I could have finished the unknown determination
within the 5-7 days. But because of test inconsistency and questionable purity, the
unknown determination took 10 days to complete. But I feel that being confident in
results outweighs the time it takes to complete the experiments.
Resources
Benson, Brown, Harley, Kleyn, and Bicknell, ed. Microbiology. McGraw-Hill, 2005.
*The above contents were taken from the following sources:
Benson and Brown, Microbiological Applications Lab Manual: Complete
version, 9th edition. McGraw-Hill, 2004.
Bicknell and Kleyn, Microbiological Experiments: A Health Science Perspective,
4th edition. McGraw-Hill, 2003
Harley, Laboratory Exercises in Microbiology, 6th edition. Mc-Graw Hill, 2004.
Holt, J. (Ed.), et al. Bergey's Manual of Determinative Bacteriology. 9th ed. Baltimore,
MD: Williams & Wilkins, 1994.
Rhoads, T. Southern Georgia College,
http://www.sgc.peachnet.edu/users/trhoads/WWW/MICROLAB/unktips.pdf.
Apr 2005.