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Chapter 7 - Identification of Bacteria using Diagnostic Media

7 - 1 Putting a name to a microbe

You are running a temperature of 103 °F, with aches chills and general malaise. A red, puss-filled, welt is present on the sole of your foot and red streaks are starting to appear in the veins of your leg. The microbe growing in the welt has been cultured. Attending physicians know they need to act fast to save you, but what is the identity of the microbe is causing the illness? And what antibiotic do they use to treat it?

You are trying to understand the fate of a pollutant in the environment. You trace its concentration and find it disappears in one plot of land much faster than at any other site. What microbe is doing this?

Reports abound of the mailing of anthrax spores to various media outlets and politicians. The general public is in a panic and worries that every white powder they see is actually anthrax spores. A rapid test is needed to identify actual anthrax spores while calming the public, but what?

Bacterial identification is essential for answering these types of questions. There are several ways to identify microbes and this chapter discusses the most often used methods. The oldest method is biochemical testing. Here the absence or presence of growth of a microbe in a battery of test media is used to create a biochemical fingerprint. By matching the reactions of the test strain to the reactions of known species, it is possible to determine the identity of the microbe. A second method of strain identification is antibody tests. A specially designed antibody, that reacts with an antigen on a test species, is used to probe a sample. If the target species is present, a detectable reaction takes places, usually a color change. A third method of identification is by the use of a DNA probes. When short pieces of DNA are used to amplify a sequence, most often by PCR, they are called a primers. In this case a unique DNA sequence is created that binds specifically to the species being tested for. If the target microbe is present, a detectable reaction takes place. In this chapter we demonstrate these methods for identifying microbes. There are more methods for identification of bacteria, but the three we will describe are commonly in practice.

7 - 2 Protocol for inoculation of medium

Below is listed the typical classroom protocol. Read through it to get an idea of the steps necessary to perform the tests.


3 of each of the following medium per student:

Glucose Fermentation Broth (5 ml/tube)

Lactose Fermentation Broth (5 ml/tube)

Tryptone Broth (5 ml/tube)

Starch Agar Plates

Heart Infusion Agar (HIA) plates

Motility Agar Medium

Nitrate Broth


Escherichia coliEnterococcus faecalis
Klebsiella planticolaLactobacillus plantarum
Pseudomonas fluorescensBacillus cereus
Micrococcus luteusBacillus subtilis
Staphylococcus epidermidisBacillus polymyxa
Chromobacterium violaceumSerratia marcescens

The class will work in groups of 3 with each student receiving 3 cultures to investigate. Each student will be responsible for observing each test on every organism! It is usually most convenient to record data on the characteristics of microorganisms in tabular form.

  1. Perform a Gram stain on each of the pure cultures. Note the cell morphology and Gram reaction and be sure to examine the Gram stains prepared by other members in your group.
  2. Inoculate both of the fermentation broths with the cultures. Avoid shaking the tubes since this may trap an air bubble in the Durham tube, confusing the results.
  3. Streak a single line of each culture onto the middle of a plate of starch agar. Do not streak for isolated colonies.
  4. Streak each culture onto a plate of HIA for isolated colonies.
  5. Inoculate each culture into a tube of tryptone broth to test for the production of indole from tryptophan. Tryptone is a peptone that contains high concentrations of tryptophan.
  6. Nitrate reduction. Inoculate a tube of Nitrate Broth.
  7. Incubate all tubes and plates at 30°C for 2-5 days.

Period 2


3 of each of the medium listed in Period 1 per pair

Dropper bottles of 3% H2O2 and Kovac's reagent

One test bacterium from your instructor

Remember to observe all the test results for every culture examined, not just for the bacteria that you tested.

  1. Observe the fermentation broths. First, look for turbidity in the tube, record this as growth. Second, examine the color of the medium. The original color was purple (alkaline), the fermentation of the sugar to acid causes the medium to turn yellow (acidic). Record whether your organism ferments the sugar to acid. Finally look for a bubble in the Durham tube; if present it signifies the production of gas.
  2. Observe the streak on the starch agar plate. Then gently flood an area around growth with Gram's iodine. Allow the plate to react for about 2 min. and pour off the iodine solution into the sink. Starch will react with the iodine to form a blue complex. If the starch has been hydrolyzed by the extracellular enzyme amylase, a clear zone will be seen around the streak. Record this as a positive result. If the blue color runs all the way to the edge of the growth record this as a negative result.
  3. Observe the colonies on HIA and record your results. Are all the colonies of the same general type? If they are not, what does this mean? Describe the pigmentation, opacity, form, elevation and margin of the colonies. (Refer to Figure 7-4 for the correct terms to use.) After observing all nine cultures, perform the catalase test by adding one-half dropperful of 3% H2O2 to an area of the plate. Place the lid back on the plate and watch for the production of bubbles. The appearance of a constant evolution signifies the presence of the catalase enzyme.
  4. Carefully layer half a dropperful of Kovac's reagent onto the surface of the tryptone broth cultures and let sit for a few minutes. The development of a red ring at the broth reagent interface is a positive test for indole production. (Note that only one of the known cultures is expected to give a positive test for indole.)
  5. Nitrate reduction. To observe for the reduction of nitrate in the Nitrate Broth all the way to nitrogen gas (i.e., denitrification), look for a bubble within the Durham tube. To test for the reduction of nitrate only to nitrite, add a dropperful of each of the two nitrite reagents - sulfanilamide and N-(1-naphthyl)-ethylenediamine - to each of the tubes. Mix well. Any appearance of a red color (which may or may not persist) indicates the presence of nitrite. If no red color was seen, add a few more drops of each reagent, mix well, and observe again. If no indication of gas or nitrite was seen, the nitrate may not have been reduced, or it was reduced to a product for which we are not testing. Before discarding the tubes, one can check for unreduced nitrate by adding a small amount of granulated zinc to any tube not showing a positive reaction for nitrite or gas. Mix the tube well. If nitrate is present, it will be reduced by the zinc to nitrite which will react with the reagents already added, forming a pink or red color. (Disregard any gas generated with the addition of the zinc.) Do not record for the organism any reaction you see only upon addition of the zinc.

7 - 3 Typical results for biochemical tests

Below are pictured typical results for the 12 test species. Because of variation in incubation time, size of inoculum and variable skill in the experimenter, results for each species may be slightly different. With that caution, familiarize yourself with these. Pay special attention to the colony morphology, as that can be very distinctive for each species.

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Figure 7.8. Gram stains of isolates. The Gram reactions of the 9 isolates.

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Figure 7.9. Colony morphology of test strains. Note the margin, shape and elevation of the test strains. Also record the color of each strain.

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Figure 7.18. Catalase reactions for test strains. Note the catalase reactions of each of the test strains. Look for evolution of bubbles.

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Figure 7.10. Indole reactions of isolates. Record the indole tests for each microbe. Note that only E. coli is positive in this test.

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Figure 7.11. Reactions in glucose fermenation broth. Note the presence of growth, color and presence or absence of a gas bubble for each strain.

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Figure 7.19. Reactions in lactose fermenation broth. Note the presence of growth, color and presence or absence of a gas bubble for each strain.

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Figure 7.12. Starch hydrolysis of test strains. Observe for zones of clearing around starch plates that have been flooded with iodine. Note that the zone of clearing should be larger than 3 mm.

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Figure 7.20. Nitrate Broth reactions. Reaction in nitrate broth for the various test species.

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Figure 7.21. Motility of selected strains. The motility test using semi-solid medium.

7 - 4 Determination of unknowns

In this part of the lab, you will be given two unknown cultures. Each is one of the ten microbes that you observed results for in the previous sections. By clicking on the button below, reactions in the various media will be presented to you. Interpret the reactions and then using a table, or dichotomous key that you generated from know results, determine the identify of your unknown.

Get Unknown Isolate

7 - 5 Summary of identification of bacteria

In many cases scientists and health professionals need to identify a microbe in a given environment. Learning the members of a population of microbes present in nature can give us new insights into biochemical processes that are taking place. This leads to a better understanding of environments and the role microbes play in them. Identifying a microbe growing in a patient will identify the disease and the treatments that are effective at eliminating it. Identification tests can also be used to monitor and eliminate microbes present in the food supply, increasing food safety by eliminating pathogens and decreasing spoilage. These are just a few of the many reasons for identifying microbes.

For decades, microbial identification had dependent upon determining the biochemical capacity of the microbe by growing in various test media. These tests probe the metabolic capacity of the strain under study, determining what the microbe could use as a carbon source (fermentation broth), its relationship to oxygen (thioglycollate medium), cell wall structure (Gram stain), and many other properties. Hundreds of media and tests have been developed to help identify microbes. These tests can be fairly accurate, but because many depend upon growth of the microbe, they often require a one day incubation before they can be read. This can be a serious detriment, especially in the food and health field, rapid diagnosis is especially critical.

A search for more rapid methods lead to the development of tests based on antibodies and DNA methods. Antibody methods depend upon the reaction of a specially prepared antibody against an antigen that is unique to the target microbe. The most common DNA methods utilize the short DNA sequences called primers that hybridize to distinct sequences in the target microbe. The primers are then used as a template in PCR reactions, producing a detectible PCR fragment, that indicated the presence of the target microbe. Antibody and DNA-based methods are more rapid than classic biochemical tests, but they tend to be specialized for the presence of specific microbes.

Due to the large body of information that has been gathered on most cultured micorbes, it is now possible to design a set of tests to determine the identity of almost any microbe.