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Indole Test Send Print

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Created: Tuesday, 08 December 2009
Last update: Wednesday, 06 July 2011
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Indole test on Escherichia coli and Enterobacter aerogenes
FIG. 1. A positive and a negative result from an indole test done in tryptone broth. The positive result given by Escherichia coli is  indicated by the red layer at the top of the tube after the addition of Kovács reagent. The negative result given by Enterobacter aerogenes is indicated by the lack of color change in the top of the tube after the addition of Kovács reagent. (Mary G. Miller, Southeastern Louisiana University, Hammond)   
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Indole test on Escherichia coli and Klebsiella pneumoniae
FIG. 2. A positive and a negative result from an indole test done in tryptone broth. The positive result given by Escherichia coli in the positive control (tube 1) and the test (tube 2) is indicated by the red layer at the top of the tube after the addition of Kovács reagent. The negative result given by Klebsiella pneumoniae in the negative control is indicated by the lack of color change in the top of the tube after the addition of Kovács reagent. (Shashidhar Vishwanath, Kasturba Medical College, Karnataka, India)
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Uninoculated tryptone medium
FIG. 3. Uninoculated tryptone medium.  Tryptone medium is a liquid medium that can be used to screen for tryptophanase activity via the indole test. (Maria MacWilliams, University of Wisconsin—Parkside, Kenosha)  
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Indole test on Escherichia coli
FIG. 4.  The positive result of an indole test in tryptone broth done on Escherichia coli.  The red reagent layer indicates the presence of indole, a by-product of tryptophan metabolism.  The culture was incubated for 48 hours at 37°C.  Several drops of Kovács reagent (p-dimethylaminobenzaldehyde) were added. This chemical reacts with the indole in the culture to produce the red product, rosindole. (Maria MacWilliams, University of Wisconsin—Parkside, Kenosha)
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Indole test on Enterobacter aerogenes
FIG. 5. The negative result of an indole test in tryptone broth done on Enterobacter aerogenes.  The Kovács reagent does not change color when added to the culture; it appears as a thin, yellowish liquid on the surface of the bacterial culture. The culture was incubated for 48 hours at 37°C prior to the addition of the detection reagent. (Maria MacWilliams, University of Wisconsin—Parkside, Kenosha)
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Indole test on Escherichia coli and Enterobacter aerogenes

FIG. 6. The indole test.  (left) An uninoculated tube of tryptone broth.  (center) The 48-hour Escherichia coli culture grown at 37°C tests positive for the presence of indole as indicated by the red reagent layer after the addition of Kovács reagent.  (right)  The 48-hour Enterobacter aerogenes culture has not broken down the tryptophan in the medium and thus no color change occurs upon addition of the Kovács reagent.  The reagent appears as a thin yellow layer on top of the culture medium. (Maria MacWilliams, University of Wisconsin—Parkside, Kenosha)

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Indole test on Enterobacter aerogenes
FIG. 7. Results of a negative indole test in tryptone broth done on Enterobacter aerogenes after a 24-hour incubation at 37°C.  A negative result is indicated by the lack of color change in the top of the tube after the addition of Kovács reagent. (Anne Hanson, University of Maine, Orono)  
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Indole test on Escherichia coli
FIG. 8. The positive results of an indole test in tryptone broth done on Escherichia coli after a 24-hour incubation at 37°C. A positive result is indicated by the red layer at the top of the tube after the addition of Kovács reagent. (Anne Hanson, University of Maine, Orono)
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Indole test on Escherichia coli and Enterobacter aerogenes
FIG. 9. A positive and a negative result of the indole test done in tryptone broth after a 24-hour incubation at 37°C. Tube A shows a positive result given by Escherichia coli as indicated by the red layer at the top of the tube after the addition of Kovács reagent. Tube B shows a negative result given by Enterobacter aerogenes as indicated by the lack of color change in the top of the tube after the addition of Kovács reagent. (Anne Hanson, University of Maine, Orono)
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Sulfur-indole-motility test results from various microbes

FIG. 10. Sulfur-indole-motility test results from various microbes. From left to right:  (A) Escherichia coli, (B) Staphylococcus aureus, (C) Salmonella arizonae, (D) Enterobacter aerogenes, and (E) Proteus vulgaris. After addition of Kovács reagent, a pink ring at the top of the tube indicates a positive indole result (A and E). Blackening of the media indicates hydrogen sulfide production (C and E). Growth feathering away from the stab line creating a cloudy appearance in the media indicates motility (A, C, D, and E). Growth strictly along the stab line indicates a nonmotile organism (B). (Tasha L. Sturm, Cabrillo College, Aptos, CA)

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