BIOL 3702L: Microbiology Laboratory
Skills Test: Bacterial Unknown
This skills test will examine your ability to i) isolate two different bacterial from a mixed
culture and ii) identify each bacterium using pertinent diagnostic characteristics. You
have already performed or are presenting performing these diagnostic tests. Therefore,
you should be familiar with how they are conducted.
To assist you, expected diagnostic test results have been provided in several tables in
the web page document entitled “Diagnostic Features of Selected Bacteria” (available at
the following URL - http://www.as.ysu.edu/~crcooper/DFSB.pdf). Note that not every
test and every test result is listed for each bacterium. The reason is simple. Some of
these tests are irrelevant and others are not routinely used to identify certain microbes.
In addition, the idea of this exercise is to make you think – not to give away the
answers!
You will receive a mixed culture unknown containing two different bacteria. They can be
of any combination from among those bacteria listed on the attached sheet entitled
“Diagnostic Features of Selected Bacteria”. You are to follow the initial instructions as
described below to begin this skills test. In addition, you are to keep a log of your
activities and observations in this test. Use the “Skills Test Log Sheet” which available
at the following URL - http://www.as.ysu.edu/~crcooper/STLS.pdf. The third page of the
log sheets may be photocopied as needed. Furthermore, you will need to submit by the
stated deadline (as listed in the laboratory syllabus or as provided by your laboratory
instructor) the “Mixed Culture Unknown Answer Sheet and Flow Chart” (available at the
following URL - http://www.as.ysu.edu/~crcooper/UASFC.pdf). Submit both of these
forms stapled together. Finally, students may assist one another so far as providing
advice and encouragement. Any collaboration beyond this level this stands precariously
at the precipice of academic dishonesty. In short, each individual is to perform his/her
own work with regard to this test.
To begin, read the following instructions carefully. Begin working immediately. The
“Mixed Culture Unknown Answer Sheet and Flow Chart” and the “Skills Test Log Sheet”
must be submitted as a stapled unit to your laboratory instructor by the stated deadline.
If any of the items is not submitted by the deadline, a 25% penalty will be applied. If any
item is submitted more than 24 hours late, but less than 48 hours late, a 50% penalty
will be applied. If any item is submitted more than 48 hours late, then the student will
receive a 100% penalty, i.e., a score of “0” will be recorded.
Day 1
1. The course/laboratory instructor will provide a rack of mixed culture unknowns
that have been randomly labeled with a code number. Select one tube and
immediately record the number of that unknown on the report sheet that will also
be provided by your instructor.
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BIOL 3702: Bacterial Unknown Skills Test
2. Obtain one (1) of each of the following agar plate media:
= MacConkey’s Agar (MAC)
= Mannitol Salts Agar (MSA)
= Tryptic Soy Agar (TSA) [Nutrient Agar (NA) may be substituted for TSA]
3. Using your very best technique, separately streak a loopful of the mixed culture
unknown onto the MAC, MSA, and TSA plates. Incubate these plates at 35°C37°C overnight.
4. [OPTIONAL] Prepare a heat-fixed smear from your mixed culture unknown on a
glass slide. Gram stain your smear. Observe and record your results. These
observations may give you an idea of what to expect upon isolating your
unknown bacteria. Note, however, it is conceivable that you may isolate two
bacteria having the same Gram-stain reaction and morphology, in which case it
may be difficult to distinguish between the different isolates.
Day 2
4. Observe your streak plates for isolated colonies and record your results.
NOTE 1: MAC medium is a differential and selective growth medium. Only Gramnegative bacteria grow on this medium. In addition, bacteria that ferment lactose
appear as pink colonies (occasionally purplish) or as white colonies with a pink
center. Non-lactose fermenters appear clear, colorless, or white only.
NOTE 2: MSA medium is also a differential and selective growth medium. Only
Gram-positive bacteria grow on this medium. In addition, those Gram-positive
bacteria that ferment mannitol grow as white colonies and cause the surrounding
medium to appear yellow. Gram-positive, non-mannitol fermenters appear clear,
colorless, or white only with the exception of Micrococcus luteus. The latter
microbe appears naturally as a yellow colony.
NOTE 3: TSA and NA media are not differential or selective growth medium.
Both Gram-positive and Gram-negative bacteria grow on this medium. Hence,
the purpose of this medium is to select bacteria based upon differences in colony
morphology.
6. From your different media, identify two different bacteria. On the underside of
your medium, mark the location of two distinct, and well-isolated colonies. Using
a loop, pick a small portion of these two colonies and inoculate each onto TSA or
NA slants. Incubate the slants at 35°C-37°C. These slants will serve as your
stock cultures to inoculate your diagnostic media.
NOTE: Depending upon the nature of your mixed culture unknown, two different
colony types may not appear on MAC or MSA plates. Remember, these media
are selective. If you have two Gram-negative bacteria, they will grow on the MAC
plate, but not the MSA plate. Conversely, if you have two Gram-positive bacteria,
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BIOL 3702: Bacterial Unknown Skills Test
they will grow on the MSA plate, but not the MAC plate. If you have one Grampositive and one Gram-negative bacterium in your mixed culture unknown, then
you should see the growth of only one colony type on the MAC and only one
colony type on the MSA plate. In most cases, two colony types should appear on
the TSA (or NA) plate.
If you cannot find two different types of bacteria based upon their growth patterns on
the selective media or by their colony types on the TSA (or NA) plate, see your
instructor immediately. Due to technical problems, it is possible that your
particular mixed culture unknown was not capable of supporting the isolation of
two different bacteria. In this case, you will be given another mixed culture
unknown or you will be provided the isolated unknown bacteria (this is at the
option of the instructor). No penalty will be assessed.
However, if the two bacteria were not isolated due to your lack of technical
proficiency, the instructor (at his/her option) will give you another mixed culture
unknown or provide you two isolated unknowns. In either case, a penalty will be
assessed if isolated colonies are not obtained because you are not capable of
performing a quality streak plate.
7. You may now Gram stain your isolated bacteria by one of the following two
options:
Option A: From the location marked on the plates from which you isolated your
unknown bacteria, remove some of the colony with a loop and make a heat-fixed
smear on a glass slide. Gram stain your smear. Observe and record your results.
Option B: Incubate your stock cultures for 12-18 hours (i.e., until Day 3), then
from each tube separately remove some of the growth of the unknown bacterium
with a loop and make a heat-fixed smear on a glass slide. Gram stain your
smear. Observe and record your results.
Day 3
8. If you did not Gram stain your isolated bacteria on Day 2 (Step 6, Option A), do
so today (Step 6, Option B). Observe and record your results.
9. Based upon the Gram-stain results of your unknown bacteria, select one or two
diagnostic tests to begin to elucidate the identity of these bacteria. Also begin to
diagram a flow chart of the steps you took to identify each of your unknown
bacteria. Your flow charts should start with the type of media upon which they
were isolated.
By way of example, suppose one of your bacteria was a Gram-negative bacillus
that formed pink colonies on MAC, i.e, a lactose fermenter. This would eliminate
quite a few of the possible bacterial unknowns found on the attached sheet
entitled “Diagnostic Features of Selected Bacteria”.
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BIOL 3702: Bacterial Unknown Skills Test
Alcaligenes faecalis – eliminated; non-lactose fermenter
Citrobacter freundii – still under consideration; lactose fermenter
Enterobacter aerogenes - still under consideration; lactose fermenter
Escherichia coli - still under consideration; lactose fermenter
Klebsiella pneumoniae - still under consideration; lactose fermenter
Proteus vulgaris– eliminated; non-lactose fermenter
Pseudomonas aeruginosa- still under consideration; lactose fermentation unknown
Salmonella typhimurium– eliminated; non-lactose fermenter
Shigella dysenteriae– eliminated; non-lactose fermenter
Now, you must chose a diagnostic test that will help eliminate others not having
characteristics common to your unknown bacterium. One possibility (though not
necessarily the best or only choice of tests), based upon the above bacteria still
under consideration, would be the SIM test. Let’s suppose you did perform this
test and the results were “-, -, +”. This would suggest that your unknown is now
one of two possible bacteria as indicated below (see “Diagnostic Features of
Selected Bacteria” sheet):
Enterobacter aerogenes - still under consideration; “-, -, +” reaction
Pseudomonas aeruginosa- still under consideration; “-, -, +” reaction
Again, you must choose a diagnostic test that will help eliminate one of these
bacteria. A flow chart of these actions might look similar to the one presented in
your laboratory manual.
NOTE: Before proceeding to actually performing some of these tests, you should realize
that you will not have access to the microbiology laboratory over the weekend. Since
some of the tests you might perform require that they be examined after 24 or 48 hours
of incubation, it may be wise to wait until next week to proceed (Day 6). Nonetheless,
you may dive right in and carry out some of the tests, but you must be aware that
certain tests results may not be valid after the specified observation period.
Grading: This skills test will be graded based upon the following criteria: i) submission
of your work by the deadline, ii) isolation of two different unknown bacteria, iii) correct
identification of these unknowns, iv) use of the fewest possible diagnostic tests to obtain
the correct answer, and v) proper completion of the log sheet and flow charts.
DO NOT PERFORM MORE DIAGNOSTIC TESTS THAN IS NECESSARY TO
OBTAIN THE CORRECT IDENTIFICATION!!! EFFICIENCY DOES COUNT!!! Based
upon your log sheets and flow charts, the course/laboratory instructor will be able to
determine if you performed your work in an efficacious manner. If you did not, points will
be deducted from your score. In addition, should you be observed using an abundance
of media to perform virtually every test possible, then you will be penalized!
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