Lab 18 KIA SIM

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Feb 20, 2024

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KIA (Kligler’s Iron Agar) & SIM (Sulfide - Indole - Motility) OBJECTIVES: Students should be able to 1. describe the possible results of growth on KIA 2. explain how the medium composition relates to each growth outcome 3. explain why it is critical to read the results at 48 hours and not earlier or later 4. perform and interpret KIA tests 5. describe the possible results of growth on SIM 6. explain how the medium composition relates to each growth outcome 7. perform and interpret SIM tests INTRODUCTION Kligler’s Iron Agar (KIA) KIA is a medium intended to help identify Gram negative bacteria. It identifies bacteria based on their abilities to ferment glucose and lactose . In addition, the medium demonstrates the ability of the bacteria to produce gas from glucose fermentation and to produce H 2 S. Composition of Kligler’s Iron Agar Ingredients per liter of deionized water: Beef extract 3.0 g Ferrous sulfate 0.2 g Yeast extract 3.0 g Sodium chloride 5.0 g Peptone 15.0 g Sodium thiosulfate 0.3 g Proteose peptone 5.0 g Agar 12.0 g Lactose 10.0 g Phenol red 0.024 g Glucose 1.0 g Final pH 7.4 Note that lactose is present in 10 times the concentration of glucose and that phenol red is present as an indicator dye . Sulfur-Indole-Motility (SIM) SIM media is used to differentiate gram negative enteric bacteria based on three different characteristics of bacterial metabolism. The Sulfur test is looking for the ability to reduce sulfur compounds in the media, which creates hydrogen sulfide gas (H 2 S). The Indole test is for the ability to breakdown tryptophan, an amino acid found in peptone, which creates the product indole. Organisms with this ability has the enzyme tryptophanase. The Motility test indicates the presence of flagella, which the bacteria can use to move around the medium. Ingredients: • Beef extract • Peptone • Agar (3.5 gram per liter compared to 15 grams per liter in TSA) • Ferrous ammonium sulphate • Sodium thiosulfate
ACTIVITY 1: KIA MATERIALS – per student pair KIA slants Inoculating needle Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, and Citrobacter freundii METHODS 1. Inoculate the KIA by stabbing with the inoculating needle and then streaking the surface of the slant. This is accomplished with one transfer from the reference culture. 2. Incubate the tubes at 37 o C for 48 hours. 3. In addition, you may inoculate your unknown into a KIA tube if it is a gram negative rod. You may refer to this video for inoculation technique: https://youtu.be/wiB_odmZ3TQ RESULTS The following results are possible. https://www.youtube.com/watch?v=NW4dFIgBM74 • Organisms that ferment both glucose and lactose will produce acid that will turn the medium yellow. • Organisms that ferment only glucose will show as a yellow butt and a pink slant. This is because when the organisms run out of glucose they switch to an aerobic process in the slant (not possible in the anaerobic butt) using peptones in the medium producing alkaline amines. • Organisms that are non-fermenters – do not ferment either sugar but may use the peptones in the slant in an oxidative process. The medium will be a red color with a pink slant. • Gas production from glucose fermentation is detected by cracks or bubbles in the agar. • H 2 S production is seen as a black butt. The H 2 S reacts with the ferrous sulfate in the medium to produce iron sulfide – the black precipitate. If the slant is yellow, then the H 2 S is masking an acid butt. If the slant is red, then the organism only ferments glucose.
Organism: E. coli Pr. vulgaris Ps. aeruginosa C. freundii Draw the results for each organism Slant color Butt color H 2 S production Gas production Sugars fermented Shorthand symbols 1. Record the results for each organism and indicate what those results mean metabolically. slant (lac fermentation) / butt (glucose fermentation) / black precipitate ( H 2 S production) / cracks ( gas from glucose) Shorthand: Slant and butt: Acid = A ; alkaline or no change = K ; H 2 S & Gas = +/- DISCUSSION QUESTIONS 1. What is the meaning of a color change of phenol red? 2. Why are there no organisms that ferment lactose and not glucose? 3. Why are there different concentrations of glucose and lactose in the medium? How do they contribute to the interpretation of this test? 4. What may happen if this test is read too early? If it is read later than the recommended incubation time?
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ACTIVITY 2: SIM MATERIALS – per student pair SIM tubes - The medium comes as a “deep” meaning that it is not slanted. Inoculating needle Enterococcus faecalis, Proteus vulgaris, and Escherichia coli Kovac’s reagent METHODS 1. Stab the agar with the inoculating needle straight down about 2/3 through the middle of the tube. 2. Incubate at 37 o C. 3. After incubation, add several drops of Kovac’s reagent to test for indole. RESULTS Observe the tubes for sulfide production and motility. To test for indole, add Kovac’s reagent. Record the results in the table below for each organism and indicate what those results mean metabolically. • Sulfide (H 2 S production) is observed as a black precipitate (iron sulfide) in the medium. Any H 2 S produced will react with the iron in the medium to produce iron sulfide. If an H2S-producing organism is motile, the resulting black precipitate becomes diffuse as the microbe spreads through the medium. • Indole production from tryptophan in the medium. If the organism has the enzyme tryptophanase, it will break down the amino acid in the peptone to use as a carbon and energy source. A byproduct of this reaction is the production of indole, which can be detected by adding Kovac’s reagent. The reagent will turn pink in the presence of indole. • Motility , the ability of the bacteria to swim through the medium, which has a reduced agar concentration, from its site of inoculation. If the organism is not motile growth will only occur along the stab line.
Organism Name: Ent. faecalis E. coli Pr. vulgaris Draw the results for each organism SULFUR H 2 S production INDOLE production MOTILITY DISCUSSION QUESTIONS 1. What is the source of tryptophan in this medium? 2. Why is the agar concentration reduced? 3. If the organism produces H 2 S, how will the medium appear? 4. What does motility imply about the structure of cells? 5. Fill out the following table: What does a positive reaction look like? If positive, what characteristic or ability does the bacteria have? Sulfur Indole Motility

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