Indole Production Test


What is the purpose of the test? 


This test determines whether the microbe produces indole from the amino acid tryptophan.



How is the indole production determined? 


If indole is produced, it will react with a chemical reagent added after incubation to produce a color change.



What medium is used? 


There are two media that are used for this test: Sulfide-Indole-Motility (SIM) medium and Tryptone broth medium. This program uses the Tryptone broth medium.


SIM is a nutrient medium which allows the detection of three different traits in bacteria: it contains sulfates to serve as the substrate for detecting sulfide (H2S) production; abundant tryptophan as a substrate for indole production; and its content of 0.5% agar limits bacterial swimming, thereby allowing detection of motility.


Tryptone broth serves the same purpose as SIM as far as indole production is concerned, and the means for conducting the test and interpreting results remain the same as SIM.



How is the test performed? 


An inoculum from a pure culture is transferred aseptically to a sterile tube of SIM or tryptone broth. An inoculating wire should be used for inoculating SIM and a loop or wire for tryptone broth. SIM should be stabbed all the way to the butt carefully to disturb the medium as little as possible. The inoculated tube is incubated at 35-37 C for 24 hours.



What reagents are added? 


Five to ten drops of Kovac's reagent are added to the tube. The reagent does not mix with water and forms a thin layer above the agar or broth. The reagent reacts with indole to produce a ring that is cherry red in color.



To perform this test in VirtualUnknown™ Microbiology, complete the following steps:


Inoculation of Medium


1. Select the tryptone broth medium.  

2. Start your Bunsen burner.  

3. Select the inoculating loop tool.  

4. Flame your inoculating loop to sterilize it.  

5. Remove the caps from your test tubes.  

6. Flame the mouths of your test tubes.  

7. Use the sterile inoculating tool to pick up an inoculum from the culture tube of the unknown bacterium.

8. Immediately transfer the inoculum into the fresh, sterile medium.  

9. Flame the mouths of your tubes once again.  

10. Replace the caps on the test tubes.  

11. Re-flame the inoculating tool.  


Incubation of the Inoculated Medium


12. Place the inoculated tube into the 35-37 C incubator.  

13. Press the New Day button to move forward 24 hours.  

14. Incubate for the appropriate length of time. This test is properly interpreted after 24 hours.  

15. Retrieve desired incubated culture from the incubator.  


Addition of Reagents


16. Select the dropper tool and the appropriate reagent needed from the chemical shelf. For this test, select Kovac’s.  

17. Remove the cap from the tube.  

18. Place the end of the dropper into the tube and add the reagent to the culture.  


Determination of Test Results


19. Observe test result. For this test, addition of the appropriate reagent produces a cherry-red ring floating above the culture medium if the test is positive. If negative, the ring that forms is yellow or amber.  

20. Record test result.

21. Dispose of the culture.