The microbiology section in the laboratory identifies pathogenic organisms in specimens.

Biochemical Activities of Bacteria:

In many distinct areas of microbiology, the ability to identify microorganisms has important application. For example, in food microbiology it is important to be able to accurately identify food spoilage contaminants. In microbial ecology, the identification of microorganisms helps us characterize biodiversity. In the field of medical microbiology, a branch of microbiology that investigates pathogenic microorganisms, the primary focus is to isolate, identify, and study microorganisms responsible for infectious disease.

Many microorganisms are permanent residents, or normal flora, of the human body. Bacteria that are normal flora are important symbionts of the human body, most of which cause no ill effects and some, which are actually beneficial to human health. Only a small percentage, less than 10%, of all known bacteria are pathogenic, or able to cause disease in a susceptible host. In order to identify an unknown in the clinical laboratory, a sample must be collected from the patient. This could be a sample of urine, feces, saliva, or a swab of the throat or skin. Because the clinical samples will most likely contain many microorganisms, both normal flora and pathogens, it is important to isolate the pathogen in a pure culture using various types of selective and differential media. Following isolation, one of the first steps in identifying a bacterial isolate is the Gram stain, which allows for the determination of the Gram reaction, morphology, and arrangement of the organism. Although this information provides a few good clues, it does not allow us to determine the species or even genus of the organism with certainty. Thus, microbiologists use characteristic biochemicalactivities to more specifically identify bacterial species. A Few Biochemical/Physiological Properties Used for identification of bacteria include: nutrient utilization (carbohydrate utilization, amino acid degradation, lipid degradation), resistance to inhibitory substances (high salt, antibiotics, etc.), enzyme production (catalase, coagulase, hemolysins, etc.) and motility.

This series of lab exercises will introduce many of the physiological characteristics/biochemical activities of bacteria commonly encountered in a clinical microbiology laboratory. Knowledge of these key characteristics will enable the identification of unknown bacterial isolates. It is important to thoroughly understand the basis for each biochemical test and know the key physiological characteristics of the bacterial genera and species presented in these labs.

GRAM STAIN

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.

MICROBIOLOGIC CULTURE AND ORGANISM IDENTIFICATION

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.

BLOOD CULTURES

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.

ANTIMICROBIAL SENSITIVITY TESTS

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

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