Please note, you must be an educator in higher ed or maybe high school to qualify to recieve the MCI
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.
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
|Escherichia coli||Enterococcus faecalis|
|Klebsiella planticola||Lactobacillus plantarum|
|Pseudomonas fluorescens||Bacillus cereus|
|Micrococcus luteus||Bacillus subtilis|
|Staphylococcus epidermidis||Bacillus polymyxa|
|Chromobacterium violaceum||Serratia 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.
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.
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.
Figure 7.8. Gram stains of isolates. The Gram reactions of the 9 isolates.
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.
Figure 7.18. Catalase reactions for test strains. Note the catalase reactions of each of the test strains. Look for evolution of bubbles.
Figure 7.10. Indole reactions of isolates. Record the indole tests for each microbe. Note that only E. coli is positive in this test.
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.
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.
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.
Figure 7.20. Nitrate Broth reactions. Reaction in nitrate broth for the various test species.
Figure 7.21. Motility of selected strains. The motility test using semi-solid medium.
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.
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.