LAB REPORT OF 7 POSSIABLE
UNKNOWN BACTERIA
(Proteus vulgaris, Staphylococcus epidermis, Bacillus
subtilis, Escherichia coli, Streptococcus agalactiae,
Moraxella catarrhalis, or Enterobacter aerogenes
UNKNOWN #29
HARRY BROWN
6/11/2012
Dr. Ann Sylvia
Microbiology-Bi 234
Spring 2012
The purpose of this lab was to determine an unknown bacteria by preforming different test to
the unknown and other known bacteria as controls for the testing. Determining unknowns is
important in the process of treating different genes of bacteria when they invade the body.
I choose unknown # 29, which was prepared by our teacher. It was in broth form and from
there nutrient agar slants where used to determine what the optimal growth was for the
unknown bacteria. After inoculating two agar slants with the unknown bacteria, one was
incubated at 25 degrees Celsius, and the other at 37 degrees Celsius. These two agar slants
where incubated for 48 hours and then examined to determine morphology of the bacteria.
After that, one tube was chosen to be the reserve stock specimen, which was placed in the
refrigerator to be examined at a later date for further growth, and the other was used as the
working specimen. The reserve stock specimen had no further growth once placed in the
refrigerator which shows that the unknown do not grow in psychrophilic conditions.
Gram staining was the next test to be preform on the unknown by using the culture that was
grown and or the broth that we first were given of our unknown. Gram staining is an essential
step in narrowing down the possibilities of the unknown bacteria. Bacteria are either Gram + or
Gram –, which once that could be determined; other biochemical test could be performed to
further narrow down the possibilities of the unknown. For example:
Test used to identify Gram positive bacteria
Catalase test
Mannitol Salt agar
Blood agar plates
Starch hydrolysis test
Nitrate broth
Coagulase test
Motility agar
CAMP test
Spirit blue agar
Bile Esculin Agar
Test used to identify Gram negative bacteria
Oxidation test
Sugar broths with Durham tubes
Urease test
Simmon’s Citrate agar
Nitrate broth
Sulfur Indole motility media (SIM)
Motility agar
Kliger’s Iron agar
Methyl Red/ Voges-Proskauer (MR/VP)
MacConkey agar
After gram staining the unknown #29, twice, with cultures from both original broth and
nutrient agar slant, it was determined to be a gram – bacteria. With both gram stains
performed, the bacteria seemed to resemble both coccus and bacillus morphology. The first
assumption was that it was a diplococcus gram negative bacteria, but with further examination
the bacteria where similar to shorten bacillus rods which are common in two of the unknown
Gram – bacteria (Esherichia coli and Enterobacter aerogenes).
At this point multiple tests were performed to determine which of the two possible
bacteria that had been selected from the gram staining. Table 1 has a list of the biochemical
test that were performed and the significants of each test.
TABLE 1
Test
Gram staining
Optimal growth
Reason for test
Gram +/-, morphology, characteristics
What temp. is best for growth of bacteria
O/F glucose test
Determining if bacteria is fermentative and
oxidative metabolism
Fermintation of glucose w/ Durham tube
Urease
Hemolysis
Oxidase
Acid and gas production
Capability to hydrolyze enzyme urease
Capability to hemolysis blood
Identify if microorganism contains enzyme
cytochrome oxidase which is important in the
electron transport chain.
Determine if bacteria are capable to migrate.
Determine if bacteria can use citrate as its sole
carbon source.
Oxygen requirements
Capability to produce organic acids
Fermentation of glucose to produce 2,3
fermentation, which is a more neutral end
product organic acid
Production of proteases, enzymes that
degrade proteins.
Organisms ability to reduce Nitrate to Nitrite
Ability of bacteria to produce enzyme,
catalase, which detoxifies hydrogen peroxide
by breaking it down into water and oxygen
gas.
Motility (SIM agar slant)
Citrate
FTM
MR (Methyl Red test)
VP (Voges-Proskauer test)
Gelatin deep
Nitrate reduction
Catalase
The results of these tests are listed on Unknown chart 2 on the
next page
Conclusion of unknown #29
Multiple tests were performed on the unknown bacteria using multiple media to determine
growth of the bacteria. Grams stain was perform twice on the unknown bacteria due to the fact
that the first gram stain that was preformed looked more like Gram- coccus or coccibacillis.
When the first few test that were ran did not have the correct reactions for Moraxella catarrhia
which is a gram- coccibacillis bacteria, a second gram stain was performed to verify the correct
reaction to the gram stain. The unknown was definitely gram- but the bacteria looked more like
shorten rods. With this and the prior results, tests were performed in the direction of two
known bacteria that are from the same family and that have similar reactions such as optimal
growth, fermentation of glucose, motility, catalase just to name a few. The two bacteria being
Escherichia coli and Enterobacter aerogenes do have several significant differences for
example; E. aerogenes has a positive test for Voges-Proskauer which means it carries out 2,3butanediol fermentation were as E. coli does not, E. coli is positive for methyl red test were as
E. aerogenes is usually, but not always, negative, E. aerogenes grows on a Simmon’s citrate test
were as E . coli does not. When the MR/ VP test were performed, the unknown #29 bacteria
had the same results as E. coli, but when the citrate test was performed the unknown #29
bacteria had a positive reaction like the reaction that E. aerogenes has. There was one test that
could be performed which was a very reliable test to determine E. aerogenes vs. E. coli which is
called the indole test. This test determine if the bacteria can breakdown the amino acid
tryptophan into indole by using the SIM’s media which was used when determining motility of
the bacteria. E. coli is a positive test, were as E. aerogenes is not. This test could not be
performed due to not having Kovac’s reagent available for the testing.
Further research has shown that E. coli does have natural variants that cause a positive reaction
to citrate, although it is not common. The test performed with the unknown had a citrate agar
tube that was half and half. The top half of the tube was bromthymol blue and the other half
stayed a green color even after multiple days of incubation. For this reason and the correlation
of tests performed on the unknown bacteria #29, I conclude that this unknown bacteria is
Escherichia coli.