IDENTIFICATION OF UNKNOWN BACTERIA
It is virtually impossible to identify bacteria based on physical characteristics alone. This is
due to the fact that there are only a few basic shapes and physical features commonly seen
in the prokaryotic world. Instead, biochemical testing has been used to make bacterial
identification down to the “species” level. These schemes are based on creating and
matching biochemical profiles of the production of enzymes, acids and gases by isolated pure
cultures of a given microorganism. Identification schemes and flow charts can be found in
reference texts such as “Bergey’s Manual of Determinative Bacteriology” or “The
Each group of ...view middle of the document...
NOT write in the above ID books
1. Maintain a pure culture of the unknown organism provided
2. Determine physical characteristics of the organism provided
3. Inoculate various biochemical tests and be able to read and understand the
significance of each test, whether positive or negative.
4. Use the information generated by testing, along with the given reference flow chart
and identification texts, to deduce the Genus and Species designation of the unknown
Fall 2011 - Jackie Reynolds, Richland College, BIOL 2420
5. Hand in a report of the testing performed (Unknown identification sheet), a journal of
how you arrived at the identification you indicated and a TSA plate containing the
unknown organism streaked to demonstrate isolated colonies. Each student will hand
in their own separate report even though you have performed the work together as a
group. Remember it is import to keep your own journal and not to plagiarize other
students in the group.
Clean glass slides
Gram stain reagents
Oxidase strips and reagent
Various biochemical media (distributed in sets during subsequent lab periods)
SCHEMATIC OF IDENTIFICATION PROCEDURE
TSA slant/TSB oxidase test
1. Inoculate a TSA slant using an inoculum from the original culture you have been given.
2. Inoculate a TSB broth from the original culture.
3. Streak a TSA plate for isolated colonies using the 3 section method, using the original
agar slant culture. You will check for purity of the culture with this plate as well as use it
to characterize the colony structure.
4. Incubate all cultures at 25 c or 37 C as directed by your instructor.
5. Gram stain your unknown organism using the TSB broth culture. Note the shape,
arrangement and Gram reaction of your organism.
6. In order to confirm the oxygen requirements of your unknown you will perform the
oxygen requirements exercise using the known organisms provided and your unknown
organism. (see Oxygen Requirements exercise).
7. Place the original stock slant labeled with your group names and instructor in the
1. Observe your new slants looking carefully for signs of contamination. Compare to the
original slant noting the color (note pigmentation), texture, opacity and odor.
2. Describe the colony morphology displayed by isolated colonies observed on the
streaked plate (refer back to the Colony Morphology experiment).
3. Run the catalase test (CATALASE exercise in your lab manual)
4. Run the oxidase test (OXIDASE exercise in your lab...