Expansion Module: Selected Bacterial Tests
INTRODUCTION
As you are learning, many types of media are available for growing bacteria. Recall that selective
media inhibit the growth of certain bacterial species, while other species have enzymes that allow them
to cope with the selective agents. Also, differential media contain chemicals that produce visible
differences among different groups of bacteria, but those differences are unrelated to how well the
organisms grow on the medium. Both selective and differential media can help a microbiologist to
determine the identity of an unknown organism.
Today we will perform three new tests that can be useful to a microbiologist when he/she is faced with
identifying an unknown organism.
The following is adapted from:
Leboffe, M.J. and B.E. Pierce. 2005. A Photographic Atlas for the Microbiology Laboratory. 3rd ed.
Morton Publishing Company, Englewood, Colorado, USA.
DAY ONE ACTIVITIES (Performed by pairs of students)
Activity 1: TSI (Triple Sugar Iron) Agar Slant.
The TSI test differentiates bacteria principally on the ability to reduce sulfur, which produces
hydrogen sulfide (H2S); secondarily, this medium can be used to determine if the organism can
ferment glucose, lactose, and sucrose (or a combination of those carbohydrates). However, the PRCarbohydrate broth tests are a better choice to use if you are investigating whether or not a
particular organism has the ability to ferment a single specific carbohydrate. The TSI agar is
prepared as a slant with a deep butt, and contains a variety of nutrients (in addition to the
carbohydrates above), as well as sodium thiosulfate as the principle source of sulfur. Phenol red is the
pH indicator in the agar. (Yellow below pH 6.8, red from pH 6.8 to 7.4, and pink above pH 7.4).
The production of hydrogen sulfide (visible as a black precipitate) indicates sulfur reduction.
If there is no fermentation after inoculation and an appropriate incubation period, then there should be
no change in the agar slant (except for growth on the slanted surface).
If inoculated with a glucose-only fermenter, the entire medium will turn yellow within a few hours.
However, because there isn’t much glucose in the medium, it will be used up within ~12 hours. The
organism will then begin metabolizing peptone (which is also present in the medium) producing NH3
(an alkaline by-product) and raising the pH. After 18-24 hours the alkaline products will be sufficient
to turn the slant red but will not overcome the acidic conditions in the butt, which will remain yellow.
Therefore, a red slant and a yellow butt indicates that glucose was the only carbohydrate fermented.
If glucose and lactose, or glucose and sucrose are fermented, the entire medium will turn yellow and
stay that way for at least 24 hours, because the concentrations of lactose and sucrose in the medium are
much higher than that of glucose.
If gas is produced, the agar may be lifted off the bottom of the test tube and it may crack/break apart.
Materials needed:
Test tube rack
Bunsen burner
Inoculating pick
Inoculating loop
E. coli stock culture
P. vulgaris stock culture
Two TSI slants (per pair of students)
Procedure:
You will inoculate one TSI slant with E. coli and the other with P. vulgaris.
1. Inoculate the TSI slant with a stab in the butt. Do not use a loop for this inoculation. You
must use a pick. Straighten the pick as much as possible before you make the inoculation.
Remember to use good aseptic technique. The stab should pass through the center of the agar
down the length of the tube until the pick is about 1-1.5 cm from the bottom of the tube.
2. Carefully pull the pick out of the agar and cap the tube.
3. Using aseptic technique, streak the surface of the agar slant with the same inoculum you used
to make the stab. You will need to use a loop in order to streak the agar surface.
4. Incubate at 37 degrees C for 24 to 48 hours. Do not exceed 48 to 72 hours for this test.
Activity 2: Motility Agar.
The motility test is used to detect bacterial motility, which is an important differential characteristic
of Enterobacteriaceae. This test medium has a very low concentration of agar, which allows for the
movement of motile bacteria. Cells are stab-inoculated into the agar. Non-motile bacteria will only
grow where they were inoculated. Motile bacteria will grow along the stab and will also swim out
away from the stabbed area.
Materials needed:
Test tube rack
Bunsen burner
Inoculating pick
E. faecalis stock culture
P. vulgaris stock culture
Two Motility Agar tubes (per pair of students)
Procedure:
You will inoculate one Motility Agar tube with E. faecalis and the other with P. vulgaris.
1. Inoculate the Motility Agar tube with a stab in the butt. Do not use a loop for this
inoculation. You must use a pick. Straighten the pick as much as possible before you make
the inoculation. Remember to use good aseptic technique. The stab should pass through the
center of the agar down the length of the tube until the pick is about 1-1.5 cm from the bottom
of the tube.
2. Carefully pull the pick out of the agar and cap the tube.
3. Incubate at 37º C for 24 to 48 hours (do not exceed 72 hours for this test).
Note: This test only works with organisms that can grow anaerobically (facultative anaerobes).
Obligate aerobes will grow on the top of the agar but will not grow in the stab.
Activity 3: Catalase Test.
This test is used to identify organisms that produce the enzyme catalase. Aerobic and facultatively
anaerobic bacteria produce enzymes capable of detoxifying hydrogen peroxide (H2O2) and superoxide
radical (O2). Superoxide dismutase catalyzes conversion of superoxide radicals (the more lethal of
the two compounds) to hydrogen peroxide. Catalase converts hydrogen peroxide into water and
molecular oxygen (O2) which escapes as a gas. Here’s the chemical reaction: 2 H2O2  O2 + 2 H2O.
When hydrogen peroxide is added to a bacterial culture, if oxygen gas bubbles immediately form, the
organism is catalase positive (+). If no gas bubbles form, the organism is catalase negative (-).
Materials Needed:
Test tube rack
Bunsen burner
Two clean glass slides for each student
3% hydrogen peroxide from the refrigerator
Any bacterial stock culture
Inoculating loop
Procedure:
1. Put a drop of hydrogen peroxide on a clean slide.
2. Mix cells from your stock culture into the hydrogen peroxide drop. Be sure to use aseptic
technique. (Between you and your partner, you will do this 4 times. Use a different stock
culture each time).
3. Observe the presence/absence of bubbling.
4. Record your observations regarding your catalase tests below:
Name of organism 1:
Name of organism 2:
Name or organism 3:
Name of organism 4:
____________________________.
____________________________.
____________________________.
____________________________.
Catalase reaction:
Catalase reaction:
Catalase reaction:
Catalase reaction:
______________
______________
______________
______________
Note: A more sensitive test can be run by growing the organism on a TSA plate or slant (densely
inoculated). After incubating for a sufficient amount of time, you would then put one or two drops of
hydrogen peroxide directly on the growing organisms on the TSA plate/slant. Again, you would
observe for the presence/absence of bubbling.
DAY TWO ACTIVITIES
Activity 1: Observation of TSI Agar Slants.
Results for the TSI agar slant test can vary widely, depending on the bacterial species present. The
table below (Table 1) summarizes the potential results.
Table 1. TSI Results and Interpretations.
Slant/Butt/Gas
Glucose
Red/Red/None
Red/Yellow/None
A
Red/Yellow/Gas
AG
Yellow/Yellow/Gas
AG
Yellow/Black/Gas
AG
Red/Black/Gas
AG
Lactose
AG?
AG?
-
Sucrose
AG?
AG?
-
H2 S
+
+
Record your results for your TSI tests below:
TSI slant inoculated with E. coli:
TSI slant inoculated with P. vulgaris:
Activity 2: Observation of Motility Agar.
Results: Observe your cultures by holding them up to a light source.
1. A positive (+) test for motility is evidenced by a cloudy, diffuse growth throughout the
medium, but especially at the top and bottom of the stab.
2. A negative (-) test for motility is evidenced by well defined growth along the stab only.
3. V = variable.
Record your results for your Motility Agar tests below:
Motility Agar tube inoculated with E. faecalis:
Motility Agar tube inoculated with P. vulgaris:
QUESTIONS
1. What bacterial biochemical characteristics can be discerned by using a TSI slant?
2. What role does the pH indicator, phenol red, serve?
3. Describe a circumstance when the results from a TSI slant could be misleading.
4. If you were trying to determine the identity of an unknown organism that you suspected was an
obligate aerobe, would the Motility Agar test be a good test to perform? Why or why not?
5. The catalase test may be useful in differentiating between the beta hemolytic bacteria. Which ones
are they? What are the possible catalase results for these beta hemolytic bacteria? (You will need to
consult your “Unknown Organisms List” to figure this out).