Biochemical: Escherichia Coli & Salmonella Typhimurium

The first step in the identification process is generally obtaining isolation. A usual isolation technique for isolation is the streak plate. With this method a small droplet of culture is spread over the surface of the medium in a pattern that gradually thins the sample out and separates the cells spatially over the sections of the plate. Streaking is used to isolate single cells in order to separate different types of bacteria in a mixed sample. The plates used are sterile and provide nutrients to sustain growth. Gram’s Stain is a widely used method of staining bacteria as an aid to identification.
It is used to determine the identity of a sample. Gram staining is used to differentiate bacterial species into two large groups Gram-positive and Gram-negative based on the chemical and physical properties of their cell walls. Gram’s stain differentiates between two major cell wall types. Bacterial species with walls containing small amounts of peptidoglycan and lipopolysaccharide, are Gram-negative and bacteria with walls containing relatively large amounts of peptidoglycan and no lipopolysaccharide are Gram-positive.
The Gram stain also allows for cell size, shape, and arrangement to be determined. Biochemical testing also helps to identify organisms. One type of biochemical test is fermentation tests. Fermentation is the formation of gas, acid, and other products by the action of bacteria on pyruvic acid. PR Glucose, PR Lactose, and PR Sucrose fermentation detection can be seen as broth color change and the presence or absence of a bubble. Making use of a mannitol salt agar growth can help determine and/or isolate gram positive cocci, interpretations are made by growth and color results.

Citrate and Malonate tests are based on differentiating organisms based on ability to grow when an essential nutrient is available in a limited number of forms. The results are interpreted based on color change and growth, with any change resulting as positive which means citrate is utilized. Malonate is utilized and positive if the liquid changes to dark blue. Hydrolytic enzymes that are used in reactions that use water to split complex molecules, these enzymes are detected by the Urease and Bile Esculin tests, which produce identifiable color changes in the medium. A positive Bile Esculin test esults when the medium is darkened,a nd negative when there is no color change. A positive Urease test occurs when the medium is pink and means a strong urease production and orange/yellow is negative with no production of urease. The catalase test detects an organism’s ability to produce catalase, an enzyme that that detoxifies hydrogen peroxide. When catalase is present the reaction is positive and bubbles are formed, absence of catalase is negative and with no bubbles. SIM is a combination media, which includes core tests to differentiate members of a specific bacteria and can be used to replace a sequence of individual tests.
SIM tests for sulfur reduction and indole production. Sulfur is reduced when the media is black(+) and sulfur is not reduced when the medium is not black(-). Indole is formed from Tryptophan when there is red in the alchol layer of Kovac’s agent and not formed when the reagent’s color is unchanged. Making use of biochemical test, Gram’s staining, and streak plate isolation are tools that can be utilized in order to determine the identity of bacteria. Using changes in media to confirm an organism. Each organism has its own characteristics that makes it differ from others, thus the identity can be found.

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