The Final Project: Isolation and identification of ONE unknown bacteria
A LAB PRACTICAL
Each student will receive a TSA slant containing two bacteria. Each student is
responsible for streaking the bacteria for isolation on the provided TSA plate and for
conducting a variety of tests to describe the biological properties of ONE of the purified
bacterial species. In most cases each student will be able to identify the organism based
on the biological properties. In the event that the (properly performed) tests do not
distinguish between 2 or more organisms, the students will be responsible for listing the
possible organisms that may represent what they were working with.
Furthermore, each student is responsible for planning a time-line for each
experiment and documenting the results. Each student will be supplied with sufficient
material to conduct all of the required tests. If a student has made a mistake and requires
additional media or reagents then that student MUST PURCHASE the additional
material with points that will be subtracted from the final score.
THIS FINAL EXERCISE IS A LAB PRACTICAL. The students are allowed to come
into the lab at anytime during the week to work independently and ARE allowed to
consult with their peers (fellow students). However, the students are NOT to ask the
instructor for help unless there is a problem that has occurred due to the fault of the
instructor. (If the latter is true then at least 2 additional students will experience the same
problems). If the student is having a problem they MUST trouble shoot the reasons for
the problem themselves (with the help of their peers) and then approach the instructor. If
the instructor has to solve a problem that is the fault of the student then that will cost the
student 5 points in consulting fees.
Table 1. Cost of media, reagents and consulting fees, etc.
Media, reagent, consulting
Consulting
Stapling and collating report
TSA plate
TSA slant
Phenol red broth with glucose
Phenol red broth with lactose
Simmons Citrate Agar slants
Tryptone Broth for Kovak’s test
MRVP broth for MR and VP tests
Hecktoen Enteric Agar plate
MacConkey Lactose plate
Starch Agar plate
Mannitol Slat agar plate
Kligler Iron Agar slant
FEE in points subtracted from final score
5 points (see above)
5 points (you can staple papers in departmental office, library, etc.)
2.5 points
2.5 points
2.5 points
2.5 points
2.5 points
2.5 points
2.5 points
2.5 points
2.5 points
2.5 points
2.5 points
1 point
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Each student is responsible for CLEARLY labeling their plates and tubes such
that they can keep up with the 37oC incubation time, the age of the culture and the
location of their tubes. The instructor will not be removing tubes and placing them in the
refrigerator during the weekends; therefore, (AGAIN) it is important that each student
create a time line for their work before they start the tests such that the media are not over
or under incubated.
Table 2. Required incubation times at 37oC for each media, age of culture before
gram stain and catalase test or before performing tests with reagents
Media incubation time/ age of culture
TSA slants/plates
Gram stain
Catalase test
Simmon’s Citrate Agar slant
Kligler Iron Agar slant
Incubation Time (hours)
24-48 hours depending on growth rate
Culture no older than 24-48 hours
Culture no older than 24-48 hours
48 hours
No more or less than 24 hours (do not overinoculate) (exactly 24
hours)
Phenol Red broth with Durham tubes/sugars
exactly 24 hours
MRVP broth for MR and VP tests
Tryptone broth for Kovac’s test
Hectoen Enteric Agar plate
Starch Agar plate
MacConkey Lactose plate
Mannitol Salt Agar plate
48 hours
At least 24 hours but no more than 48 hours
No more than 24 hours
No more than 24 hours
No more than 24 hours
At least 24 hours but no more than 48 hours
Independent project:
Day 1: Each student will receive a TSA slant containing two unknown bacteria. Each
student will streak the bacteria for isolation on a supplied TSA plate.
Day 2: Each student should pick one (and only ONE) colony from the TSA plate and
inoculate this one colony onto a TSA slant. One colony is sufficient for obtaining
copious growth on the slant. You should check your slants for growth after 24 hours, if
the growth is slight you can incubate your slants for another 24 hours. If you have
enough growth; place the slant in the refrigerator or start working immediately with the
culture. After you have obtained sufficient growth you should refrigerate your slant until
the end of your project.
Troubleshooting guide: Make certain that your inoculating loop is sufficiently cooled
such that you do not kill all of the bacteria that you are sub-culturing from the TSA plate
to the TSA slant. Use asceptic technique such that you do not introduce bacteria from
external sources. When sub-culturing the colony from your plate onto the TSA slant
make certain that you only sample one colony. In many cases the colonies will look
alike but may represent two different bacterial species—this will “mess up” your results
and cost you points if you require new media.
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Day 3 or 4: (Suggested—Gram stain/catalase test). Keep your TSA slant until the end
of the exercises as you will be using this as the source of your inoculations onto or into
other media. Since it is important that the cultures are relatively young on the TSA slants
it is suggested that you immediately sample the bacteria from the slant for the Gram
staining and catalase tests. Otherwise, your cultures will remain viable on the slant for
sub-culturing into or onto the other media for at least a week.
After this point each student is responsible for developing their own time line for subculturing the bacteria from the slant onto or into the media that will be used to determine
additional biological properties of the unknown organism.
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Final Project: Identification of one unknown bacteria (50 points)
1. Make a timeline for when you will be conducting the various experiments. That
includes the dates that you plan to inoculate media, incubate the media and stop the
incubations, to store the media and/or to conduct experiments and to observe the results.
Use the suggested incubation times in Table 2 above and use a calendar to record dates
for each exercise. Keep in mind that you will not have access to your incubated cultures
over the weekend and/or during holidays. Work around your own personal schedule. For
example, if you will not be in on a given day then you should not inoculate cultures that
only require 24 hours incubation on the previous day!!!!! (5 points)
DATE:
Experiment(s) Streak unknowns on the slant for isolation on a TSA plate
DATE:
Experiment(s) Pick one colony and inoculate onto the surface of a TSA slant
DATE:
Experiment(s)
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2. Describe (completely) the characteristics of the ONE colony that you isolated from the
TSA plate and inoculated onto the TSA slant. Use your lab handout as a guide for
describing the colony characteristics. (2.5 points)
3. Describe the Gram staining reaction of the organism that you tested. Also describe the
bacterial shape, relative size and bacterial arrangement. (You may also draw pictures, but
you must describe in writing what you have observed.) (2.5 points)
4. Describe the appearance of the bacteria that grew on the MacConkey-Lactose,
Mannitol Salt, and Hecktoen Enteric Agar plates. What do these growth characteristics
and reactions with the media imply? If you did not observe growth you must describe
why that was so. (12 points)
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5. Describe what happened to the starch agar plates when the inoculated plates were
flooded with Gram’s Iodine. What does the reaction on that plate imply? (2.5 points)
6. Describe the appearance of the inoculated Simmon’s Citrate Agar slant. What does
the appearance imply? (2.5 points)
7. Describe the appearance of the inoculated Kligler’s Iron Agar slant. What does the
appearance imply? (5 points)
8. Describe the appearance of the inoculated Phenol Red Broth with glucose and Durham
tubes. What does the appearance imply? (2.5 points)
9. Describe the appearance of the inoculated Phenol Red Broth with lactose and Durham
tubes. What does the appearance imply? (2.5 points)
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10. What happened to the inoculated Tryptone broth when Kovak’s reagent was added?
What do those results imply? (2.5 points)
11. What happened to the inoculated MRVP broth when methyl red was added? What
do those results imply? (2.5 points)
12. What happened to the inoculated MRVP broth when Vogues-Proskauer reagents
were added? What do those results imply? (2.5 points)
13. What happened when you added a loopful of the unknown bacteria to a glass slide
and then added Hydrogen peroxide. What does that imply? (2.5 points)
**14. What was your unknown micro-organism(s)? This should be based on the
reactions above and the chart of unknowns that the instructor will give to you personally
when you have answered all of the questions above and shown your answers to the
instructor. (3 points) **1
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** Once you have received your table of known bacteria and their characteristics you will no longer able
to ask the instructor for help. You must use your data to find the best possible match for your unknown
bacteria. You can ask your student peers for help.
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