17-1 Instructions

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During the course of this semester, you have been observing and perfecting many basic bacteriological skills and techniques. You have studied some of the physiology of bacteria as they relate to culture media and biochemical testing. You probably know where in this manual you can find information on specific bacterial characteristics and the procedures for the various tests. The purpose of this exercise is to correlate the various bits of information to strengthen your understanding of, and familiarity with (1) the media and tests used this semester and (2) the major characteristics of the genera of bacteria with which you have had some experience. We have been using typical species of these genera throughout the course.

Dichotomous Key

The exercise will consist of two parts. First, you will prepare a Figure 17-1 and Figure 17-2. Use tests such as the primary tests considering any of the secondary tests!

Some things to consider in making your key:

  1. If you believe that either a primary test (e.g., catalase) or a secondary test (e.g., lactose fermentation) will differentiate between two genera, by all means go for the primary test! The only instance where lactose fermentation was (for us) of realistic importance in differentiating between genera was in Experiment 14 for the enteric bacteria.
  2. Can glucose fermentation be used to differentiate Bacillus from anything else (such as Lactobacillus)? Go back to Section 11-3 and note that we studied several species of Bacillus - some of which were facultative anaerobes. That should answer the question.
  3. Where you may have had some experience with an organism having a variable reaction or a weakly (or delayed) positive reaction (e.g. - our strain of Citrobacter was very weakly lactose positive in Lactose Fermentation Broth and negative in KIA), would you want to attach great importance to such a characteristic to help differentiate that organism? Use something more definitive!
  4. Where you may not know a reaction for a particular organism (H2S for Bacillus? Glucose fermentation for Neisseria?), you need not consider it for this exercise. One does not have to know all of the reactions for the 12 organisms to make a dichotomous key.

With these things in mind, you can prepare a key which will be of use in characterizing typical members of these genera, even outside of the course. This key should be submitted no later than the two weeks before the course ends. It will then be graded and handed back in time for you to consider which tests you will need to identify your unknowns.

Isolation and Identification of Unknowns

The second part of this endeavor involves the actual isolation and identification of the three components of the mixed unknown. One typical representative of each of three genera on the list below will be found in this mixture. Naturally you should want to start off by checking the mixture with a gram stain and then streaking for isolated colonies. Note that you have two media for this purpose. One - Heart Infusion Agar - is an all-purpose medium which will support the growth of all 12 genera. The other - MacConkey Agar - is a selective-differential medium which will (in this experiment) support the growth of only the gram-negative rods and will differentiate between any two or more different organisms growing on it.

Hints concerning temperatures of incubation can be derived from previous experiments, but all of the organisms on the list can grow at 37°C, 30°C or room temperature. (Which may be best to prevent "overgrowth?") Regarding the times of incubation, all of the test media may be incubated for 1, 2 or more days unless you are using KIA to check glucose and lactose fermentation (in which case the tube is incubated for just one day at 37°C).

Remember to record your unknown number.

Figure 17-1 Possible organisms in the test

Bacillus Staphylococcus Neisseria Micrococcus
Morganella Enterobacter Pseudomonas Klebsiella
Citrobacter Lactobacillus Escherichia Enterococcus

The table shows the possible genera of the species of microbes that you may find in this test. You must design a dichotomous key to help you work out the identity of your microbe.

Figure 17-2 Media and reagents available

Heart Infusion Agar plates MacConkey Agar plates
Heart Infusion Agar slants Glucose Fermentation Broth (with Durham tube)
Lactose Fermentation Broth (with Durham tube) Motility Medium
Kligler Iron Agar (Remember to use only for gram-negative rods!) Tryptone Broth and Kovac's Reagent (for indole test)
Simmons Citrate Agar 3% Hydrogen Peroxide
Oxidase Reagent (not to use if dark blue!)

You may use any of the media or reagents listed here to identify your isolate. This is more than sufficient and please don't ask for media or reagents that are not on this list.

Don't forget the Gram stain! How old should the culture be? Can a reliable Gram stain be made directly from a MacConkey Agar colony? What about oval cells? Are they rods or cocci? Remember that the Gram stain and the catalase and oxidase tests should be run only on cultures growing on all-purpose media.

A word of caution: Do not identify anything as Neisseria unless (1) it is oxidase-positive and (2) it looks exactly like the Neisseria slide from Experiment 12

The teaching personnel will be available if you hit a snag. Do not expect them to lead you through the procedure! You are on your own! Answers to most or all of your questions will be found in your notes and experience or somewhere in this manual.

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