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Bacilli Lab Report

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Identification of Bacilli

Nursing

Sheila Power

December 8, 2003

BIO U122

Introduction:

During the "Identification of Bacilli" lab, bacilli cultures are isolated in order to be identified. When the bacillus is isolated in pure culture, the Gram stain and several biochemical tests will be performed in order to determine its identity.

Materials:

* 1 Shigella sonnei culture

* 2 sterile cotton swabs

* 1 MacConkey agar plate

* 1 Triple Sugar Iron (T.S.I.) tube

* 1 Sodium Citrate Plate

* 1 Simmons Citrate plate

* 1 Urease plate

* 1 Phenylalanine deaminase (P.A.D.) plate

* 1 S.I.M. (sulfide, indole, motility) tube

* Loop

* Bunsen Burner

* Lighter

* 2 Glass slides

* Drying tray

* Bottle of distilled water

* Bottle of hydrogen peroxide (H202)

* 1 syringe

* Bottle of crystal violet

* Bottle of safranin

* Bottle of iodine

* Bottle of 95% ethyl alcohol

* Book of blotting paper

* Microscope

* Bottle of oil

* Lens cleaning wipes

Methods:

In order to confidently identify our assigned bacilli species, many tests can be used to decipher certain reactions and activities of the bacilli characteristics. Such tests of elimination include gram stain, catalase test, MacConkey agar, Triple Sugar Iron (T.S.I.), Simmons Citrate, Urease, Phenylalanine deaminase (P.A.D.), and S.I.M. tube tests.

The gram stain is known as the preliminary differential test that colors the cell walls according to the biochemical properties in order to divide the bacteria into gram positive and gram negative groups. As most of the complex is washed away from gram negative cells, the color displayed is the reddish pink tone of the safranin dye. Gram positive cells, however, will reveal the deep purple color of the trapped crystal violet iodine (CVI) complex. Observing the slides under the microscope will lead to the deciphering between the displayed colors and reveal the bacteria's morphology.

A catalase test is an examination of the metabolism of the bacteria. Catalase is the enzyme used in the following reaction: 2H202 => 2 H20 + 02. Aerobic metabolism leads to the accumulation of hydrogen peroxide, which is illustrated by a bubbling reaction when it comes into contact with Bacillus.

MacConkey agar is a selective-differential agar which is based primarily on lactose fermentation in the media. The selective agents include bile salts and crystal violet. If there is lactose present in the media, the agar will ferment to a pink/brick red color as negative lactose fermentation results in a colorless agar.

The Triple Sugar Iron (T.S.I.) tube is a multiple test medium which has both a slant and a deep or "butt". Glucose, lactose, and sucrose and phenol red acid/base color indicator are the carbohydrates typically contained in the T.S.I. agar. Sodium thiosulfate is incorporated in order to metabolize a possible sulfur source. There are several combinations possible with the T.S.I tube as it tests for more the presence of several substances at once. If the slant of the agar is red and the butt is yellow (K/A), this illustrates that glucose is the only sugar that was fermented. If both the slant and butt are yellow (A/A), the glucose has been fermented along with either lactose or sucrose. If sulfur is present, the medium will metabolize the sodium thiosulfate to produce H2S which combines with ferrous sulfate in the medium, resulting in an insoluble black precipitate usually in the butt of the T.S.I. agar. Any cracks or bubbles within the medium demonstrate the production of gas within the agar.

Simmons citrate is used to detect the presence of sodium citrate, the sole source of carbon in the bacilli species. Microorganisms that metabolize citrate into alkaline end products will grow as those without the enzymes for citrate utilization will not grow. For detection of the sodium citrate growth, brothymol blue is used as the color indicator. A positive citrate culture results in a royal blue color and illustrates growth on the medium as a negative culture results in the agar remaining green and no growth present.

The urease test allows the determination of the hydrolization of urea to ammonia and carbon dioxide (urea => (urease) NH4+ + C02). Phenol red is the pH indicator in the medium that detects the alkaline condition of the medium. If ammonia was produced, the medium turns bright pink. If the agar remains salmon pink then there is no color change, indicating a negative result.

The phenylalanine deaminase (P.A.D.) test detects the degradation of phenylalanine into

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