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The Gram stain is used to rapidly identify organisms in a patient specimen and classify them as either gram-positive or gram-negative. This assay can identify in minutes a pathogenic organism if it is present in sufficient number and the test is performed correctly. This test requires microscopic examination of a stained slide, and recognition that gram-positive organisms are purple and gram-negative organisms are red. The specimen from a patient which may contain pathogenic organisms is applied to a glass slide, and then fixed and stained in a series of steps that ultimately leads to the differential staining of gram-positive and gram-negative organisms.


Plating a microbiologic specimen from any of a variety of sites with a potential infection involves placement of the sample onto a bacteriologic plate containing solid medium or into a liquid medium that enhances bacteriologic growth. When applying a sample onto solid culture medium, the initial sample is spread onto the plate in a specific way to improve the separation of colonies which grow on the plate. The separation of colonies also permits subculturing of specific colonies, if necessary, for identification and antimicrobial susceptibility. The colonies that grow on the plate can be tentatively identified as a specific microorganism based upon the size, shape, color, hemolytic activity, and rate of growth of the colony. Confirmatory identification of genus and species can be established using mass spectrometry or other more traditional biochemical methods. The inoculation of a sample into liquid medium simply requires addition of the sample to the liquid. If bacterial growth occurs, changes in the appearance of the medium occur.


There is great concern for the presence of a pathogen in the circulating blood because sepsis is associated with high mortality. Blood samples are collected into specific bottles which promote the growth of microorganisms either aerobically or anaerobically. A specific amount of blood is added to a blood culture bottle immediately after the blood is collected in such a way that contamination with organisms from the skin or other site does not occur. The bottles are then placed in a specially equipped incubator which detects carbon dioxide generated within individual blood culture bottles. A positive blood culture is identified when there is generation of carbon dioxide associated with the growth of microorganisms. When a blood culture bottle becomes positive, it is then processed for identification of the microorganism and assessment of its antimicrobial sensitivity.


In the version of the assay called the dilution method, increasing concentrations of antimicrobial agents are added to separate tubes. ...

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