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

Student Lab Sheet

Needed Materials*

Escherichia coli, slant culture (155065)

Enterobacter aerogenes, slant culture (155030)

pre-poured media tubes: either 1% tryptophan solution tubes (827402), SIM medium (826922), or Motility Indole Ornithine (MIO) medium (825862)

Kovac’s reagent, 30 mL (871389)

droppers

test tube rack

Bunsen burners or alcohol lamps

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

incubator

Optional Materials
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.

Safety

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.

Procedures

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.

Day 1

3 tubes of 1% tryptophan, SIM, or MIO media per group
inoculating loop (for broth) or needle (for semi-solid media)
tape and marker to label tubes
E. coli tube culture
Enterobacter aerogenes tube culture

Day 2

inoculated tubes from Day 1
Kovac’s reagent
dropper

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).

Answer Key to Questions Asked on the Student LabSheet

	Positive/Negative	Is indole present? (yes/no)
E. coli	+	yes
Enterobacter aerogenes	-	no
Control	-	no

If the media did not contain tryptophan, what result would you expect to see in each vial?
We would expect a negative result, an absence of indole, in all the vials.

Why did you incubate the cultures for 24–48 hours before adding Kovac’s reagent?
This was done to allow time for the bacteria to grow and break down the tryptophan.

What is the purpose of the control vial?
It is a negative control to show that indole is not already present or that there is not bacterial contamination in the media.

This test lacks a positive control. Why might this be an issue? What could you use as a positive control?
If the Kovac’s reagent did not work, the media did not contain tryptophan, or there was some other problem, we might incorrectly conclude that E. coli does not produce indole. Alternatively, we might have trouble interpreting the results without knowing exactly what a positive result looks like. A useful positive control could be a bacterium that we already know does produce indole, or media that has indole already added.

E. coli is exclusively an intestinal bacteria, while Enterobacter is naturally associated with plants and soil. Suppose you wanted to determine whether a local lake is contaminated with feces. You used the following procedure, with the following results.

1. You collected a water sample and spread it on an agar plate. Numerous colonies grew.
2. You picked one colony from the plate and subcultured it onto fresh media.
3. You conducted a series of tests from that subculture, narrowing the bacteria to either E. coli or Enterobacter.
4. You conducted an indole test on bacteria from that subculture.

Will the results of this test indicate whether the water has fecal contamination? Why or why not? If not, what additional actions could you take to be certain it is or is not contaminated?
The result of this test would not necessarily indicate an absence of contamination. The original sample will have many different bacterial species and numerous colonies, and this is only a test of a single colony. If the test shows that this bacterium is Enterobacter, it does not rule out the presence of E. coli (or other fecal bacteria) in other colonies from the sample. To be certain, we would need to test many more colonies to determine whether fecal contamination is present.

If the test indicates E. coli, then we would be able to say that fecal contamination is likely, although with only a single colony we still would not know the extent of the contamination.

Student Lab Sheet