CAROLINA LABSHEETS
In this lab students perform a differential test to distinguish bacteria based on the production of indole. Bacteria are grown on media containing tryptophan and then treated with Kovac’s reagent. If they possess the enzyme tryptophanase, the bacteria can metabolize tryptophan into indole, pyruvic acid, and ammonia. Kovac’s reagent then reacts with indole to produce a red color that will be concentrated in a layer at the top of the media. In the absence of indole, Kovac’s reagent remains yellow.
This activity assumes that students have previously worked with bacteria and are familiar with sterile technique. If this is not the case, we recommend that students complete the activities described in our “Introduction to Sterile Technique” LabSheet before beginning this activity
Pre-poured media tubes: either 1% tryptophan solution tubes (827402), SIM medium (826922), or Motility Indole Ornithine (MIO) medium (825862)
inoculating loops (for 1% tryptophan broth medium) or needles (for SIM or MIO media)
Disinfectant (e.g., 70% ethanol, Lysol®, bleach solution)
Tape and/or markers to label tubes
The 1% tryptophan solution is a broth that can be inoculated with a loop. SIM and MIO media are semi-solid agar and should be stab-inoculated with a needle. MIO media also can be used for motility testing. For details, see our “Bacterial Motility” LabSheet.
If you have an autoclave, you can prepare your own SIM media tubes using SIM Dehydrated Medium (787020). One hundred grams will make about 500 tubes.
Caution: Kovac’s reagent is flammable and toxic. Keep the bottle away from heat and open flame. Consult the MSDS and your institution’s safety regulations and use appropriate personal protective equipment.
Ensure that students understand and adhere to safe laboratory practices when performing any activity in the classroom or lab. Demonstrate the protocol for correctly using the instruments and materials necessary to complete the activities, and emphasize the importance of proper usage. Model proper laboratory safety practices for your students and require them to adhere to all laboratory safety rules.
This activity requires that students work with bacterial cultures and open flames. Have students use sterile technique at all times and wear appropriate safety equipment (such as safety glasses or goggles, gloves, and aprons).
Have them wipe down all work surfaces with disinfectant before and after the lab, and wash their hands after entering and before exiting the lab. Disinfect cultures and any contaminated items remaining at the end of the lab by autoclaving them or by flooding them with disinfectant overnight before proper disposal.
Bacteria must be incubated for 24–48 hours between the initial inoculation and the addition of Kovac’s reagent.
If you are not using pre-poured media tubes, you will need to prepare tubes the day before the lab.
Students can work singly or in pairs. The E. coli and Enterobacter cultures can be shared between groups, with one tube used for up to 10 inoculations. However, this additional handling could increase the risk of contamination.
Each student or group will need access to a Bunsen burner as well as the items listed below. On Day 2, you may prefer to have students bring their tubes to a central location stocked with the Kovac’s reagent and droppers.
Optional
In addition to indole production, SIM media is also used to assess hydrogen sulfide production and bacterial motility. E. coli and Enterobacter are both hydrogen-sulfide negative and motile. If you wish to examine the other characteristics, recommended bacteria include Citrobacter freundii (154941; hydrogen sulfide +, indole –, motility +) and Staphylococcus epidermidis (155556; hydrogen sulfide –, indole –, motility –).
MIO media is also used to assess motility and ornithine decarboxylase activity. E. coli and Enterobacter aerogenes are ornithine-positive. Klebsiella pneumoniae (155095A; pathogenic) is negative for these traits.
To explore other tests for bacterial identification, consider the Bacterial Fermentation Kit (154710), Bacterial Biochemical Identification Kit (154715), and Enteric Biochemical Detective Kit (154717).
| Positive/Negative | Is indole present? (yes/no) | |
| E. coli | + | yes |
| Enterobacter aerogenes | - | no |
| Control | - | no |
*Next Generation Science Standards® is a registered trademark of Achieve. Neither Achieve nor the lead states and partners that developed the Next Generation Science Standards were involved in the production of this product, and do not endorse it.
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