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In this lab students do a differential test to distinguish bacteria based upon the manufacturing of indole. Bacteria space grown ~ above media containing tryptophan and then treated through Kovac’s reagent. If they possess the enzyme tryptophanase, the bacteria deserve to metabolize tryptophan right into indole, pyruvic acid, and ammonia. Kovac’s reagent climate reacts v indole to produce a red color that will be focused in a layer in ~ the optimal of the media. In the lack of indole, Kovac’s reagent stays yellow.

This task assumes that students have previously functioned with bacteria and also are familiar with sterile technique. If this is no the case, us recommend the students complete the tasks described in ours “Introduction to Sterile Technique” LabSheet prior to beginning this activity

Student lab Sheet

Needed Materials*

Escherichia coli, slant society (155065)

Enterobacter aerogenes, slant culture (155030)

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

Kovac’s reagent, 30 mL (871389)


test tube rack

Bunsen burners or alcohol lamps

inoculating loops (for 1% tryptophan broth medium) or needles (for center or MIO media)

tape and/or markers to label tubes


Optional products The 1% tryptophan systems is a broth that deserve to be inoculated v a loop. SIM and also MIO media are semi-solid agar and also should be stab-inoculated with a needle. MIO media likewise can be offered for motility testing. For details, watch our “Bacterial Motility” LabSheet.

If you have actually an autoclave, you can prepare your own SIM media tubes making use of SIM Dehydrated medium (787020). One hundreds grams will certainly make about 500 tubes.

You are watching: Using an indole test, red indicates that the bacteria


Caution: Kovac’s reagent is flammable and also toxic. Store the party away indigenous heat and open flame. Top the MSDS and your institution’s safety regulations and also use appropriate personal protective equipment.

Ensure that students understand and also adhere to safe activities practices when performing any task in the class or lab. Show the protocol for properly using the instruments and materials necessary to complete the activities, and also emphasize the importance of proper usage. Model appropriate laboratory safety practices for her students and require them come adhere to every laboratory security rules.

This task requires the students work with bacterial cultures and also open flames. Have students usage sterile technique at every times and wear ideal safety equipment (such as safety glasses or goggles, gloves, and aprons).

Have castle wipe under all occupational surfaces through disinfectant before and also after the lab, and wash your hands after ~ entering and before exiting the lab. Disinfect cultures and any contaminated items remaining at the finish of the laboratory by autoclaving them or through flooding them through disinfectant overnight before appropriate disposal.


Bacteria need to be incubated because that 24–48 hours between the early stage inoculation and the enhancement of Kovac’s reagent.

If you are not using pre-poured media tubes, friend will have to prepare tube the day prior to the lab.

Students have the right to work singly or in pairs. The E. Coli and Enterobacter cultures deserve to be shared in between groups, with one tube supplied for up to 10 inoculations. However, this added handling could increase the danger of contamination.

Each student or group will need accessibility to a Bunsen burner as well as the items provided below. On work 2, you may prefer to have actually students carry their tubes come a main location stocked through the Kovac’s reagent and droppers.

Day 1

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

day 2

inoculated tube from work 1 Kovac’s reagent dropper

Optional In addition to indole production, sim media is also used to evaluate hydrogen sulfide production and bacterial motility. E. Coli and Enterobacter are both hydrogen-sulfide negative and motile. If you great to study the other characteristics, recommended bacteria encompass Citrobacter freundii (154941; hydrogen sulfide +, indole –, motility +) and also Staphylococcus epidermidis (155556; hydrogen sulfide –, indole –, motility –).

MIO media is likewise used to evaluate motility and ornithine decarboxylase activity. E. Coli and Enterobacter aerogenes are ornithine-positive. Klebsiella pneumoniae (155095A; pathogenic) is negative for this traits.

To explore other tests for bacterial identification, think about the bacter Fermentation Kit (154710), bacterial Biochemical to know Kit (154715), and also Enteric Biochemical Detective Kit (154717).

Answer vital to inquiries Asked ~ above the college 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 an outcome would you expect to watch in each vial? We would intend a an adverse result, an absence of indole, in all the vials.

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Why did girlfriend incubate the cultures for 24–48 hrs before including Kovac’s reagent? This to be done to permit time because that the bacteria to thrive and malfunction the tryptophan.

What is the purpose of the regulate vial? It is a an unfavorable control to display that indole is not currently present or that there is not bacterial contamination in the media.

This check lacks a positive control. Why could this it is in an issue? What might you use as a confident control? If the Kovac’s reagent did no work, the media did not contain tryptophan, or there to be some various other problem, we could incorrectly conclude that E. Coli does not develop indole. Alternatively, we could have trouble interpreting the outcomes without knowing exactly what a positive an outcome looks like. A valuable positive control could it is in a bacterium that we already know does develop indole, or media that has indole already added.

E. Coli is specifically an intestinal bacteria, if Enterobacter is naturally associated with plants and also soil. Suppose you want to recognize whether a local lake is contaminated v feces. You offered the adhering to procedure, v the following results.

You built up a water sample and spread it on one agar plate. Numerous colonies grew. You choose one colony from the plate and also subcultured the onto new media. You conducted a series of exam from the subculture, narrowing the bacteria to either E. Coli or Enterobacter. You carried out an indole check on bacteria from the subculture.

Will the outcomes of this test indicate whether the water has actually fecal contamination? Why or why not? If not, what added actions could you take to be certain it is or is no contaminated? The an outcome of this test would certainly not necessarily indicate an absence of contamination. The initial sample will have plenty of different bacterial types and countless colonies, and also this is only a check of a solitary colony. If the test mirrors that this bacterium is Enterobacter, that does not dominion out the visibility of E. Coli (or other fecal bacteria) in other nests from the sample. To be certain, us would have to test many much more colonies to determine whether fecal air pollution is present.

If the test suggests E. Coli, then us would have the ability to say that fecal contamination is likely, back with just a single colony we still would not know the extent of the contamination.