Biology 205
Species Identification
of a Bacterial
Unknown
(30 points)
What’s
my name?
Directions for Work on Your Bacterial Unknown
The following pages contain the General Directions for getting started on the
identification of your unknown bacterial species. Also included is a Summary of Tests
for a Bacterial Unknown. You will use these tests to help identify the culture,
morphological, and physiological characteristics of a bacterial species.
General Directions
1. Write down the number of your
unknown somewhere very safe. No
number at the end of the project – no
points.
8. For the physiological tests, you will have
only one opportunity to perform the test.
9. All the tests on the following pages and
described in detail in the lab manual.
Look them up and read about them
before doing the test.
2. Use good aseptic technique! Remember
other bacteria can get into your culture
tubes and plates if you do not use good
aseptic and sterile technique. If they do
contaminate your unknown, you will
have to clean up the culture. Clean up
could take weeks.
10. HAVE FUN! Look at this project as a
mystery. You must identify the unknown
bacterial species. Remember: this project
stresses the importance of the method
and process, not solely identifying the
correct bacterial species.
3. Make sure you know what you are doing
before doing a test. There is not sufficient
material to redo many tests.
Summary of Tests for a Bacterial
Unknown
4. Use small pieces of tape to label all tubes.
Always put your name (initials?) and the
number of the unknown organism on all
tubes and plates.
Over the next few weeks, you will need to do
23 observations and/or physiological tests to
identify the species of your unknown
bacterium. The following information will
help you in your investigation.
5. Use the indicated containers to store
your tubes for incubation.
6. When you are finished getting results
from a tube or plate:
 Place any plates in the biohazard bin.
 Place any culture tubes on discard
bench by the prep room door. When
placing tubes on the bench, remove
any tape used to label the tube.
7. Place culture plates in the biohazard bag.
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Summary of Tests for a Bacterial Unknown
I. CULTURE
CHARACTERISTICS
II. STAIN, SIZE, AND
MORPHOLOGICAL
CHARACTERISTICS
Appearance of Nutrient Broth
Observe your nutrient broth culture
according to the drawings on
laboratory handout [see Lab 9].
 Results: Record your observations
on your Lab Report sheet by
checking the appropriate boxes.
Gram Stain
 Do a Gram stain with a young broth
culture (see Lab 4).
 Results: Record your observations
on your Lab Report sheet by
checking the appropriate box.
Better Growth Temperature
Cell Size
 Inoculate two T-soy agar slants each
with a loopful of unknown.
 Place one slant on the 22°C tray and the
other on the 37°C tray.
 Results: Next week record your
observations on your Lab Report
sheet by checking the appropriate
box.
 Determine cell size (rods = length;
spheres = diameter) by using several
cells to establish a “typical” cell size.
 Results: Record your observations
on your Lab Report sheet.
Cell Shape
 Determine the shape of the bacterial
cells.
 Results: Record your observations
on your Lab report sheet by
checking the appropriate box.
Oxygen Requirement
 Inoculate a loopful of your unknown
into a thioglycollate tube (see Lab 6).
 Place the tube in the rack on the 22°C
tray.
 Results: Next week record your
observations (see Lab 7) on your
Lab Report sheet by checking the
appropriate box.
Cell Arrangement
 Determine the cellular arrangement(s)
for the bacterial cells.
 Results: Record your observations
on your Lab Report sheet.
Colony Appearance on Agar
 Inoculate a T-soy agar plate with a
loopful of your unknown by making a
four-way streak plate (see Lab 1).
 Place the plate USD on the 20°C tray.
 Results: Next week record your
observations on your Lab Report
sheet by checking the appropriate
boxes.
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III. PHYSIOLOGICAL CHARACTERISTICS
Carbohydrate Fermentation Tests
Other Tests
Catalase Test
 Procedure: Inoculate each sugar
fermentation broth with a sample of
unknown from agar.
 Place all broth tubes on the 22°C tray.
 Results: Next week observe for color
change and bubble (see procedures
manual); Record your observations
on your Lab report sheet.
 Procedure: Use your 4-way streak plate
and follow the procedure outlined in the
procedures manual.
 Results: Record your observations
on your Lab Report sheet.
Oxidase Test
 Procedure: Use your 4-way streak plate
and follow the procedure outlined in the
procedures manual.
 Results: Record your observations
on your Lab Report sheet.
IMViC
Methyl Red/Voges-Proskauer Tests
 Procedure: Inoculate one MRVP broth
with a sample of unknown from agar.
 Place the broth tube on the 22°C tray.
 Results: Next week pour half of the
MRVP broth into a separate tube and
use the two tubes for testing.
 Methyl red (MR) test: Follow
procedure in the procedures
manual. Record broth color on
your Lab report sheet.
 Voges-Proskauer (VP) test:
Follow procedure in the
procedures manual. Record
broth color on your Lab Report
sheet.
Hydrogen Sulfide Test
 Procedure: Use the inoculated SIM agar
deep (from indole production test).
 Results: Follow the procedure in the
procedures manual and record agar
deep coloration on your Lab Report
sheet.
Urease Test
Procedure: Inoculate a urea slant by
streaking and stabbing with a sample of
unknown from agar.
 Place the agar slant on the 22°C tray.
 Results: Follow the procedure in the
procedures manual and record agar
slant color on your Lab Report sheet.
Indole Production Test
 Procedure: Inoculate a sample of
unknown from agar with your needle
into a SIM agar deep.
 Place the agar deep on the 22°C tray.
 Results: Follow procedure in the
procedures manual and record your
observations on your Lab Report
sheet.
Starch Hydrolysis Test
 Procedure: Do a simple streak on a
starch agar plate using a sample of
unknown from agar.
 Place the agar plate USD on the 22°C
tray.
 Results: Follow the procedure in the
procedures manual and record agar
color surrounding the streak on your
Lab Report sheet.
Citrate Utilization Test
 Procedure: Inoculate by streaking and
stabbing a citrate agar slant with a
sample of unknown from agar.
 Place the agar slant on the 22°C tray.
 Results: Follow the procedure in the
procedures manual and record agar
slant color on your Lab Report sheet.
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Identifying the Unknown Bacterial Species
Now that you are finishing with the
physiological tests, you are ready to begin
identifying your unknown.
3. Some characteristics and tests are more
important and reliable than others.
 The gram stain is very important.
 The carbohydrate fermentation and
catalase tests usually are reliable.
1. Look over the selected characteristics for
each species as provided in the tables on
the following pages.
 Your organism is on one of these pages,
so use these condensed descriptions to
eliminate as many bacteria as possible.
4. A few characteristics and tests may be less
reliable.
 Some bacterial species give variable
test results for citrate utilization and
urease tests.
2. You will discover a table lists only the
results for most physiological tests. Most
bacterial descriptions state that a test gives
a reaction (+) or no reaction (−) for a
particular bacterial species. That means
that the majority (>80%) of bacterial
strains of the species will give that result.
However, you are working with only one
strain, so there is the possibility you may
have a bacterial species that will give a
variable (V) result for one or more tests.
Therefore, your test results may not
completely agree with the species
description. That is ok! You must look at
the overall character profile of your
results to select the species that your
unknown most closely fits.
5. In preparing your lab report, please include
the actual results you see. Be sure to
include if the test was positive or negative.
This is essential in evaluating the results.
Remember: You are trying to find the bacterial
species that most closely matches the
characteristics you have found for your
unknown. Make sure you consider the overall
biochemical profile.
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Biochemical/Physiological Characteristics
TABLE 1. Gram Negative Bacilli and Cocci
Carbohydrate
Fermentations
Species
gIu
IMViC Tests
Acid/ CO 2 from
lac
suc
man
ind
MR
Other tests
VP cit
H 2S urease starch cat
oxi
Aeromonas sobria
+/+
–/–
+/+
+/+
+
–
–
I
–
–
+
+
+
Alcaligenes faecalis
–/–
–/–
–/–
–/–
–
–
–
+
–
–
–
+
+
Citrobacter freundii
+/+
+/–
+/I
+/+
–
+
–
+
+
+
–
+
–
Enterobacter aerogenes
+/+
+/I
+/+
+/+
–
–
+
+
–
–
–
+
–
Escherichia coli
+/+
+/+
+/I
+/+
+
+
–
–
–
–
–
+
–
Pseudomonas myxofaciens
+/+
–/–
+/+
–/–
–
I
+
+
+
+
–
+
–
Pseudomonas fluorescens
–/–
–/–
–/–
–/–
–
–
–
+
–
+
–
+
–
Xanthomonas campestris
+/–
+/–
+/–
+/–
–
–
–
+
+
–
+
+
–
TABLE 2. Gram Positive Bacilli and Cocci
Carbohydrate
fermentations
IMViC Tests
Other Tests
Species
Acid/ CO 2 from
gIu
lac suc man
ind
MR
VP
cit
H 2 S urease starch cat oxi
Bacillus cereus
+/–
–/–
+/–
–/–
–
+
–
I
–
+
+
+
–
Bacillus megaterium
–/–
–/–
–/–
–/–
–
–
–
–
–
–
+
+
–
Bacillus subtilis
–/–
–/–
–/–
–/–
–
–
–
+
–
+
+
+
–
Enterococcus faecalis
+/–
+/–
+/–
+/–
–
+
–
–
–
–
–
–
–
Micrococcus luteus
–/–
–/–
–/–
–/–
–
–
–
–
–
+
–
+
+
Micrococcus roseus
–/–
–/–
–/–
–/–
–
–
–
–
–
I
+
–
–
Sarcina aurantiaca
–/–
–/–
I/–
–/–
–
–
–
–
–
–
–
–
I
Staphylococcus
epidermidis
+/I
+/I
+/–
–/–
–
+
–
–
–
+
–
+
–
glu = glucose; lac = lactose; suc = sucrose; man = mannitol; ind = indole; MR = methyl red;
VP = Voges-Proskauer; cit = citrate; H 2 S = hydrogen sulfide gas; cat = catalase; oxi = oxidase;
+ = positive reaction; − = no reaction; I = intermediate reaction.
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