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Figure 1: Motility Test. The tube on the left, inoculated with the motile bacterium Proteus mirabilis, shows a positive test for motility. The tube on the right, inoculated with the nonmotile bacterium Staphylococcus aureus, shows a negative test for motility. (Laura Cathcart, Deanna Swartzfager, Patricia Shields, University of Maryland, College Park, MD)

Figure 2: Motility Test Showing Nonmotile Phenotype. A test tube containing motility test medium that was needle inoculated with the nonmotile bacterium Staphylococcus aureus, then incubated at 35°C for 26 hours. This medium is semisolid, allowing bacteria to move in it. The addition of a tetrazolium salt allows tracking of bacterial movement through the medium, as bacterial metabolism reduces the colorless soluble tetrazolium salt to its insoluble, dark red form. The clear, sharp red line of bacterial growth in this tube shows the lack of movement of S. aureus from its initial inoculation along a single stab line. Motility testing is very useful in the differentiation and identification of bacteria such as the enterics and gram-positive rods. (Janelle Hare, Morehead State University, Morehead, KY)

Figure 3: Motility Test Showing Nonmotile Phenotype (Labeled view). A test tube containing motility test medium that was needle inoculated with the nonmotile bacterium Staphylococcus aureus, then incubated at 35°C for 26 hours. This medium is semisolid, allowing bacteria to move in it. The addition of a tetrazolium salt allows tracking of bacterial movement through the medium, as bacterial metabolism reduces the colorless soluble tetrazolium salt to its insoluble, dark red form. The clear, sharp red line of bacterial growth in this tube shows the lack of movement of S. aureus from its initial inoculation along a single stab line. Motility testing is very useful in the differentiation and identification of bacteria such as the enterics and gram-positive rods. (Labeled view) (Janelle Hare, Morehead State University, Morehead, KY)

Figure 4: Motility Test Showing Motile Phenotype. A test tube containing motility test medium that was needle inoculated with the motile bacterium Proteus mirabilis, then incubated at 35°C for 26 hours. This medium is semisolid, allowing bacteria to move in it. The addition of a tetrazolium salt allows tracking of bacterial movement through the medium, as bacterial metabolism reduces the colorless soluble tetrazolium salt to its insoluble, dark red form. The fuzzy, indistinct red line of bacterial growth shows that the motile P. mirabilis has moved from its initial inoculation along a single stab line outward in all directions. Motility testing is very useful in the differentiation and identification of bacteria such as the enterics and gram-positive rods. (Janelle Hare, Morehead State University, Morehead, KY)

Figure 5: Motility Test Showing Motile Phenotype (Labeled view). A test tube containing motility test medium that was needle inoculated with the motile bacterium Proteus mirabilis, then incubated at 35°C for 26 hours. This medium is semisolid, allowing bacteria to move in it. The addition of a tetrazolium salt allows tracking of bacterial movement through the medium, as bacterial metabolism reduces the colorless soluble tetrazolium salt to its insoluble, dark red form. The fuzzy, indistinct red line of bacterial growth shows that the motile P. mirabilis has moved from its initial inoculation along a single stab line outward in all directions. Motility testing is very useful in the differentiation and identification of bacteria such as the enterics and gram-positive rods. (Labeled view) (Janelle Hare, Morehead State University, Morehead, KY)

Figure 6: Klebsiella pneumoniae, a Nonmotile Organism, in a Sulfide Indole Motility Deep. Klebsiella pneumoniae, a nonmotile organism, in a sulfide indole motility (SIM) deep.  Note that the medium is very clear and the stab line is evident. SIM medium is used to test for hydrogen sulfide production, indole production, and motility. An uninoculated SIM deep is a transparent yellow semisolid (3 g/liter of agar) medium. Inoculate the SIM deep with an inoculating needle making the stab perpendicular to the agar surface. If the organism can produce hydrogen sulfide from sodium thiosulfate, then ferrous ammonium sulfate, an iron indicator, combines with the hydrogen sulfide to produce a black precipitate in the agar. SIM medium contains tryptophan. If the organism can produce tryptophanase, it can degrade tryptophan to indole, pyruvate, and ammonia. After inoculation and incubation, Kovács reagent is added to the test tube. If indole is present, a dark pink-red liquid layer will be evident on top of the agar. In the absence of indole the reagent layer remains yellow. If the organism is nonmotile, the agar remains clear and the stab line is evident. Motile organisms grow outward from the stab line and produce turbidity in the medium that obscures the stab line. It is important that the motility and hydrogen sulfide reactions be recorded prior to adding Kovács reagent. Kovács reagent contains an aldehyde that "bleaches out" the black color and makes the medium cloudy. (Diane Hartman, Baylor University, Waco, TX)

Figure 7: Enterobacter aerogenes, a Motile Organism, in a Sulfide Indole Motility Deep. Enterobacter aerogenes, a motile organism, causes turbidity in the sulfide indole motility (SIM) deep.  The stab line is obscured. SIM medium is used to test for hydrogen sulfide production, indole production, and motility. An uninoculated SIM deep is a transparent yellow semisolid (3 g/liter of agar) medium. Inoculate the SIM deep with an inoculating needle making the stab perpendicular to the agar surface. If the organism can produce hydrogen sulfide from sodium thiosulfate, then ferrous ammonium sulfate, an iron indicator, combines with the hydrogen sulfide to produce a black precipitate in the agar. SIM medium contains tryptophan. If the organism can produce tryptophanase, it can degrade tryptophan to indole, pyruvate, and ammonia. After inoculation and incubation, Kovács reagent is added to the test tube. If indole is present, a dark pink-red liquid layer will be evident on top of the agar. In the absence of indole the reagent layer remains yellow. If the organism is nonmotile, the agar remains clear and the stab line is evident. Motile organisms grow outward from the stab line and produce turbidity in the medium that obscures the stab line. It is important that the motility and hydrogen sulfide reactions be recorded prior to adding Kovács reagent. Kovács reagent contains an aldehyde that "bleaches out" the black color and makes the medium cloudy. (Diane Hartman, Baylor University, Waco, TX)

Figure 8: Motility Test. Enterobacter aerogenes exhibits motility in the sulfide indole motility (SIM) deep on the left. Klebsiella pneumoniae, a nonmotile organism, is in the SIM deep on the right. SIM medium is used to test for hydrogen sulfide production, indole production, and motility. An uninoculated SIM deep is a transparent yellow semisolid (3 g/liter of agar) medium. Inoculate the SIM deep with an inoculating needle, making the stab perpendicular to the agar surface. If the organism can produce hydrogen sulfide from sodium thiosulfate, then ferrous ammonium sulfate, an iron indicator, combines with the hydrogen sulfide to produce a black precipitate in the agar. SIM medium contains tryptophan. If the organism can produce tryptophanase, it can degrade tryptophan to indole, pyruvate, and ammonia. After inoculation and incubation, Kovács reagent is added to the test tube. If indole is present, a dark pink-red liquid layer will be evident on top of the agar. In the absence of indole, the reagent layer remains yellow. If the organism is nonmotile, the agar remains clear and the stab line is evident. Motile organisms grow outward from the stab line and produce turbidity in the medium that obscures the stab line. It is important that the motility and hydrogen sulfide reactions be recorded prior to adding Kovács reagent. Kovács reagent contains an aldehyde that "bleaches out" the black color and makes the medium cloudy. (Diane Hartman, Baylor University, Waco, TX)

Figure 9: Proteus vulgaris, a Motile Organism, in a Sulfide Indole Motility Deep Proteus vulgaris, a motile organism, in a sulfide indole motility (SIM) deep. The black precipitate indicates hydrogen sulfide production by the organism. SIM medium is used to test for hydrogen sulfide production, indole production, and motility. An uninoculated SIM deep is a transparent yellow semisolid (3 g/liter of agar) medium. Inoculate the SIM deep with an inoculating needle, making the stab perpendicular to the agar surface. If the organism can produce hydrogen sulfide from sodium thiosulfate, then ferrous ammonium sulfate, an iron indicator, can combine with the hydrogen sulfide to produce a black precipitate in the agar. If the organism is nonmotile and produces hydrogen sulfide, the black precipitate is observed only along the stab line. Motile organisms that produce hydrogen sulfide will produce a black precipitate throughout the tube, wherever the organism grows. SIM medium contains tryptophan. If the organism can produce tryptophanase, it can degrade tryptophan to indole, pyruvate, and ammonia. After inoculation and incubation, Kovács reagent is added to the test tube. If indole is present, a dark pink-red liquid layer will be evident on top of the agar. In the absence of indole the reagent layer remains yellow. If the organism is nonmotile, the agar remains clear and the stab line is evident. Motile organisms grow outward from the stab line and produce turbidity in the medium that obscures the stab line. It is important that the motility and hydrogen sulfide reactions be recorded prior to adding Kovács reagent. Kovács reagent contains an aldehyde that "bleaches out" the black color and makes the medium cloudy. (Diane Hartman, Baylor University, Waco, TX)

Figure 10: Sulfide Indole Motility Medium. Sulfide indole motility (SIM) medium tubes were inoculated with a nonmotile Staphylococcus (left) and a motile member of the family Enterobacteriaceae (right) by stabbing with an inoculating needle.  Both tubes were incubated at 37°C for 48 hours. Motility is evidenced via the diffuse growth of the Enterobacteriaceae throughout the medium as contrasted with Staphylococcus growth restricted to the stab line. (Clarissa L. Kaup and J. L. Henriksen, Bellevue University, Bellevue, NE)

Figure 11: Sulfide Indole Motility Medium. Sulfide indole motility (SIM) medium is a semisolid agar used to determine hydrogen sulfide (H2S) production, indole formation, and motility.  SIM medium is used to differentiate members of the family Enterobacteriaceae.  Haziness that spreads from the stab line indicates a positive test for motility.  Tubes must be compared to an uninoculated tube to discriminate between faint haziness and motility.  A red color development after addition of Kovács reagent indicates indole production.  A black precipitate indicates H2S production.  Test tubes:  (A) uninoculated tube, (B) contains the nonmotile and indole-negative bacterium Klebsiella pneumoniae, (C) contains the motile and indole-positive bacterium Escherichia coli, and (D) contains the motile, indole-negative, and H2S-producing bacterium Proteus mirabilis.  (Renee Wilkins, University of Mississippi Medical Center, Jackson, MS)

Figure 12: Sulfide Indole Motility Medium for Sulfide Reduction, Indole Production, and Motility. Sulfide indole motility (SIM) medium is used to detect sulfide reduction, indole production, and motility.  The left tube is H2S negative and contains a nonmotile organism.  The right tube is H2S positive, indicated by the black streak, and contains a motile organism based on the diffuse growth.  The indole test was not performed on these tubes. (Ken Van Horn, Focus Diagnostics, Inc., Cypress, CA)