Expansion Module: Selected Bacterial Tests

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1 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 A Photographic Atlas for the Microbiology Laboratory. 3 rd 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 (H 2 S); 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 PR-Carbohydrate 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 NH 3 (an alkaline by-product) and raising the ph. After 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. 1

2 Materials needed: Inoculating pick Inoculating loop Escherichia coli stock culture Staphylococcus epidermidis stock culture Two TSI slants (per pair of students) You will inoculate one TSI slant with E. coli and the other with S. epidermidis. *** NOTE: the instructor will also demonstrate the TSI test with Proteus 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 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: Inoculating pick Enterobacter cloacae stock culture Staphylococcus epidermidis stock culture Two Motility Agar tubes (per pair of students) You will inoculate one Motility Agar tube with E. cloacae and the other with S. epidermidis. 2

3 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 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 (H 2 O 2 ) and superoxide radical (O 2 ). 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 (O 2 ) which escapes as a gas. Here s the chemical reaction: 2 H 2 O 2 O H 2 O. 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: Two clean glass slides for each student 3% hydrogen peroxide from the refrigerator Any bacterial stock culture Inoculating loop 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: Catalase reaction: Name of organism 2: Catalase reaction: Name or organism 3: Catalase reaction: Name of organism 4: 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 3

4 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 Lactose Sucrose H 2 S Red/Red/None Red/Yellow/None A Red/Yellow/Gas AG Yellow/Yellow/Gas AG AG? AG? - Yellow/Black/Gas AG AG? AG? + Red/Black/Gas AG Record your results for your TSI tests below: *** NOTE: do not open tube lids *** TSI slant inoculated with E. coli: TSI slant inoculated with S. epidermidis: 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. Record your results for your Motility Agar tests below: Motility Agar tube inoculated with E. cloacae: Motility Agar tube inoculated with S. epidermidis: 4

5 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). 5

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