Identification of an Unknown Enterobacteriaceae. The purpose of the experimental determination of an unknown was to demonstrate the utility of many tests that indicate the metabolic behavior of the unknown.

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Identification of an Unknown Enterobacteriaceae

Introduction

This lab experiments serves as an application of all the lessons that were taught and to test if the student understood and remembered the various tests that were mentioned in the laboratory and lecture classes in bacteriology. The purpose of the experimental determination of an unknown was to demonstrate the utility of many tests that indicate the metabolic behavior of the unknown.  In this experiment, my unknown #14 was determined to be Proteus vulgaris.

Proteus vulgaris is one of the most commonly isolated members of Proteus species. The genus Proteus is a member of a large gram negative bacilli family, Enterobacteriaceae. Proteus organisms are known to be one of those to cause serious infections in humans, along with Escherichia, Klebsiella, Enterobacter, and Serratia species. (Nester et al. 2008)

The bacterium is a gram-negative rod with flagella.  As a gram-negative rod, it has an extracytoplasmic outer membrane.  It creates an endotoxin, which can cause a deadly systemic inflammatory response in 20 to 50 percent of its victims. It has been shown that its optimal growth temperature was at 37 C. P. vulgaris is a chemoheterotroph, which means it uses carbon sources like glucose for energy and carbon. As a chemoheterotroph, it ferments glucose but not lactose or mannitol.  However, because it is a facultative anaerobe, the glucose fermentation only occurs in anaerobic conditions. If placed in non-ideal, aerobic conditions, the microbe will use a variety of organic molecules to survive. (Struble, et al., 2009)

When identifying the microbe, several tests can be used.  It will test positive on the citrate test and urease test.  Because it ferments glucose but not mannitol or lactose, it will only test positive in the glucose tests. Likewise, when observing the plated colonies, it will be noticed on non-selective media a "swarming" behavior, where the microbe grows in waves.  The bacterium grows and stops in waves, creating what appear to be distinctive rings.  This feature is the result of the microbe’s flagella, which allow it to be extremely motile. (Alachi, P. 2007)

Materials & Methods

A broth containing an unknown microbe and labeled with #14 was obtained from the lab instructor. A gram stain was initially performed to determine if the unknown microbe was gram positive or gram negative.  If the bacterium was gram positive, it retained the purple color; if it was gram negative, it retained a pink or red color.

MacConkey agar acting as a visual pH indicator, distinguishes those Gram-negative bacteria that can ferment the sugar lactose (Lac+) from those that cannot (Lac-).  The test was performed using streak method and incubated at 35 C. If the colonies are pink, they are Gram- lactose-fermenting bacteria. These pink colonies are typically coliform bacteria in the family Enterobacteriaceae, inlcuding the genera Escherichia, Klebsiella, Enterobacter, Hafnia and Citrobacter. Non-lactose fermenting (yellow), non-coliform members of Enterboacteriaceae include the genera Proteus, Morganella, Providencia, Edwardsiella, Salmonella and Shigella. (Alachi, P. 2007)

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Triple sugar iron agar (TSI) is a differential medium that contains lactose, sucrose, a small amount of glucose (dextrose), ferrous sulfate, and the pH indicator phenol red.  It is used to differentiate enterics based on the ability to reduce sulfur and ferment carbohydrates. As with the phenol red fermentation broths, if an organism can ferment any of the three sugars present in the medium, the medium will turn yellow (A/A).  If an organism can only ferment dextrose, the small amount of dextrose in the medium is used by the organism within the first ten hours of incubation at 35 C. After ...

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