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Biology 2261 : Identification of an Unknown/Practicum*
Mark: 20 points
The basic skills learned in the microbiology lab will be put to the test. In order to get all points for this exercise, you
must correctly identify the two unknown bacteria given in a mixed culture. To do this, you must be able to
effectively streak plate, perform stains, use a microscope and apply and interpret biochemical tests.
You may use course materials and reference books; however, you are required to do your own work. You are NOT
permitted to get help from other students in the class, the instructor, student lab prep workers or other persons. You
are NOT permitted to help other students in the class. No cultures are to be taken out of the class. Any student who
is caught attempting to gain an unfair advantage from others, give an unfair advantage, or use laboratory resources
outside the teaching lab, will receive a zero for the entire lab grade. All the skills needed to be successful on this
assignment have been used over the course of the semester. Be confident in your ability.
A broth culture containing any two organisms from the following list will be given to each student. BE SURE TO
RECORD THE CODE NUMBER OF YOUR UNKNOWN. The student will be given two TSA plates on which
to streak the broth in order to separate the two organisms. You have two plates to do it correctly. No additional
plates will be provided for this step.
Bacillus subtilis
Corynebacterium xerosis
Enterobacter cloacae
Enterococcus faecalis
Escherichia coli
Micrococcus luteus
Salmonella enterica
Serratia marcescens
Staphylococcus aureus
Staphylococcus epidermidis
If you successfully separated your unknowns on the TSA plates, you will then need to establish pure cultures and
identify the unknowns using any of the following supplies:
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First Period: 2 TSA plates (for establishing pure cultures by streak plating)

Second Period: gram stain reagents and slides, endospore stain reagents and slides, 1 mannitol salt agar
plate, 1 eosin methylene blue agar plate, oxidase reagent and cotton swabs or filter paper, 3% hydrogen
peroxide, 1 phenol red glucose fermentation tube, 1 phenol red sucrose fermentation tube, 1 phenol red
lactose fermentation tube, 1 MR-VP broth, 1 sulfide-indole-motility agar tube, 1 starch agar plate, 1
Salmonella-Shigella agar plate, and 1 nutrient gelatin tube. If your isolation was not successful, you may
request isolated cultures to complete the lab, but with a deduction of ⅓ credit. You may not re-streak for
isolation. Also, you may have only ONE set of biochemical test media. If you take more than that, you will
be penalized.

Third Period: Stains and slides, reagents to complete biochemical tests.
Many of these tests are appropriate for only one type of organisms or another. For example, you are wasting your
time and are heading in the wrong direction if you plate a Gram positive bacterium on an eosin methylene blue agar
plate. Much of your success in this exercise hinges on good isolation using the streak plate technique and good
gram staining. On the basis of those results, come up with a strategy to effectively use the supplies available. Also,
you should note that doing a biochemical test on an incompletely isolated organism (especially a test that uses broth
medium) is likely to give incorrect results.
Once you have identified your organisms, you will submit a typed report. The report must have your name and
unknown code number. You then have the following options of how detailed you want your report to be. Once
you have turned in a report with a particular option, you may not go back and change your option. For
example, if you choose option 1 and then miss both organisms, you get a zero. Simple as that!!! No matter
how close you got to the right organisms…. just one contaminated biochemical test messes up your whole
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unknown. Too bad!!!! You have the opportunity to do option 2 or option 3, with the chance of partial credit,
but you are just too busy to do a report.
OPTION 1:
All or Nothing ( Type your report)
 Name of first unknown. If correct full credit (10 points), if wrong NO CREDIT.
 Name of second unknown. If correct full credit (10 points), if wrong NO CREDIT.
OPTION 2:
Table of Results ( Type your report)
 Name of first unknown and a table of tests run and results for first unknown. If correct full
credit, if wrong up to ⅓ partial credit (1-3 points)
 Name of second unknown and a table of tests run and results for second unknown. If correct
full credit, if wrong up to ⅓ partial credit (1-3 points)
OPTION 3:
Well Defended Report ( Type your report and include pictures if any)
 State the objectives of the experiment.
 A brief introduction to identifying bacteria.
 Name of first unknown and a table of tests run and results for first unknown. Explain your
strategy and reason for each step you used to identify the unknown. If correct full credit, if
wrong up to ⅔ partial credit (1-6 points).
 Name of second unknown and a table of tests run and results for second unknown. Explain
your strategy and reason for each step you used to identify the unknown. If correct full credit,
if wrong up to ⅔ partial credit (1-6 points).
*Practicum is considered as an exam for lab work, hence you are requested to maintain complete silence
Refer to the syllabus or ask the instructor as to the time and date the reports are due. In your case
( Biol 2261- Spring 2010), report for unknown is due Monday April 26 in class, lecture time.
*************Good luck**********