Examine the characteristics of cell and colony morphology,Gram reaction,and perhaps most importantly,metabolic/biochemical test results.

The identification of microorganisms found in a patient sample

Experiment 21 IDENTIFICATION OF UNKNOWN MICROORGANISMS

Introduction There are myriad strategies and procedures that have been developed to separate and identify microorganisms. Some are based on cell morphology, others on metabolic ability, and still others on molecular (DNA-based) and serological (antibody-based) characteristics. Such tests have countless benefits, from aiding in classification to identifying environmental contaminants to diagnosing diseases. In this experiment, you will be given a “patient” sample containing two different unknown bacterial species that you will isolate and identify.

You will examine the characteristics of cell and colony morphology,Gram reaction,and perhaps most importantly,metabolic/biochemical test results. (Molecular identification of the species will be performed in Exp. 25.) Through identification of the unknowns, you will learn about the concept of bacterial identification and further refine your isolation, pure culture, microscope, and staining techniques. Although identifying bacteria typically starts with an enormous list that is narrowed down, it would be difficult to do all of the testing needed to completely identify all bacteria. Thus, a smaller list is used in this class. Because you are now an “expert” on using differential media, you are given info about various tests (which you may or may not have used before) that may or may not need to be performed depending the given unknown, as well as identification tables and notes.

The following principles will influence the identification of the unknown bacteria:

• The first step in identification must always be to isolate the different types of bacteria in the sample—i.e., to obtain a pure culture of each bacterial type.

• Deciding which tests are the most informative for identifying a genus/group or species.

• Interpretation of positive and negative results. Any test result for an organism is based on the probability (10% to 100%) that a given isolate will produce reactions similar to other isolates of the same genus or species. The natural variation seen between organisms may be due to mutations, recombination and selection. The tests that have a high probability of being positive or negative for a given species are recorded in the Exp. 21 tables and notes.

• First Stage tests (Table 1) and then Second Stage tests (Tables 2-7) will be performed in order to identify the unknown bacteria. Successful testing will be based on obtaining pure cultures and accurate results (e.g., note experimental details like time and temperature).

• Students are to always keep a “working” pure culture of their unknown bacteria. Working cultures should be streaked at the start of each class to avoid any contamination that may occur.
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