Unknown Identification Laboratory Report Assignment

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UNKNOWN IDENTIFICATION LABORATORY REPORT
Introduction
The Gram Stain test: This is a very important preliminary test used in initial classification as well as characterization of bacteria. It is used to differentiate bacteria based on staining differences in biochemical and physical characteristics of bacterial cell walls. The Gram Stain test therefore groups bacteria into two major categories: Gram-positive and Gram-negative bacteria. Not only is the test important in distinguishing between the two classes of bacteria, but also helpful in staining the bacteria for clear observation of their morphology under a light microscope. Gram-positive bacteria are identified by the purple color retention after the staining procedure while the Gram-negative bacteria give pink coloration on their cell walls. The differences in color retention are attributed to difference in physical and chemical composition in their cell walls. For example, the Gram-positive have a relatively thick cell wall composed of 90% peptidoglycan complex and teichoic acid cross-links. On the contrary, Gram-negative bacteria have thin cell walls composed of only 10% peptidoglycan complex and high lipid content. The thick cell walls in Gram-positive bacteria make them retain the purple color of crystal violet dye, even after being treated with ethanol. However, the crystal violet dye is quickly decolorized by ethanol, and they only retain the color of the second dye, Safranin. Based on the morphological characteristics of bacteria, the Gram stain test can identify different bacteria based on their sizes and shapes. Bacteria vary in size ranging from 0.5 to 10 microns. As for the shapes, they can be rod-shaped (or bacillus); helical (or spirilum); curved (or comma-shaped); and spherical (or coccus).
Sugar Fermentation Test: some bacteria are known to ferment carbohydrates, especially the simple sugars such as glucose, lactose, xylose, maltose, and sucrose. Each category of bacteria can only ferment some, and not all, of the sugars. As a result, this test is used separately and differentially to identify bacteria that are capable of fermenting different sugars. Bacteria that ferment a specific sugar can be identified by noting color changes when the bacteria are put into the sugar-phenol red broth. Positive results are noted when the phenol red broth changes color from red to yellow, indicating the presence of acidic end products as a result of sugar fermentation. On the contrary, negative changes are detected when there is no color change, indicating that the organism cannot ferment the sugar in question.
The Catalase test: Certain groups of bacteria are known to produce the enzyme catalase due to their use of oxygen in respiration. The enzyme catalase is meant to protect these classes of bacteria from harmful and destructive oxygen radicals and metabolites released during respiration. A classic example of a metabolite released during oxygen respiration is hydrogen peroxidase, which is bactericidal. To prevent themselves from such bactericidal metabolites, these bacteria produce catalase which breaks down hydrogen peroxide to water and oxygen molecules. Catalase-positive bacterial isolates will give effervescence immediately hydrogen peroxide is added to the bacterial culture. On the other hand, catalase-negative isolates do not give any effervescence.
The Urease test: Some bacteria possess the ability to breakdown urea, the main product in amino acid decarboxylation, to its constituent products – ammonia and carbon dioxide gas. For these bacteria, this is an indication that they possess the enzyme urease. On urea agar medium, urease-positive bacteria will give a strong reaction which is shown by an intense color change from yellow to bright pink. Contrarily, urease-negative bacteria do not give any color change; they retain the original yellow color of the urea agar medium.
Methyl Red test: this is a microbiological test used to detect the ability of bacteria to produce stable acidic end products from glucose fermentation (mixed-acid fermentation). These acidic end products are of different types and proportions. Normally, methyl red-positive organisms give a red coloration when incorporated in a methyl red broth containing glucose. Methyl red-negative strains do not change the color of the methyl red broth; it therefore remains peachy-yellow.
The Voges-Proskauer test: The test is used to determine the ability of some groups of bacterial organisms to give neutral or non-acidic end products from the metabolism of glucose. In these organisms, glucose metabolism gives products such as acetyl methyl carbinol (or acetoin) from resulting organic acids in the metabolism chain. In this test, a Methyl red/Voges-Proskauer broth is used. Voges-Proskauer positive organisms give pink coloration, while Voges-Proskauer negative strains do not give any color. Color change is due to the presence of acetoin confirming that the organisms under test produce neutral products from glucose metabolism.
Objective
The only objective in this experiment is to identify the unknown organism.
Hypothesis
Through application of differential tests, the unknown organism will be clearly identified. All negative tests will be eliminated, step by step, until a conclusive identification of the organism is made.
Materials and Procedure
For the Gram Stain test, the r...