Chapter 9 - Microscopic Techniques

Abstract

Publisher Summary Microscopy plays an integral role in the study of microorganisms and can provide extremely useful information about them. This chapter describes the basic techniques used in microscopy, various types of microscopy, and their applications in the field of environmental microbiology. By varying the type of lenses and light sources, five types of light microscopy can be defined: bright-field, dark-field, phase-contrast, differential interference, and fluorescence. Bright-field microscopy is most commonly used to examine morphology and perform Gram stains, while phase-contrast microscopy is used to view the living cells. Methods of sample preparation vary with the type of microscopy used and can be as simple as placing a drop on a glass slide or as complex as thin sectioning and mounting on a copper grid as with transmission electron microscopy. The number of microorganisms can be determined by the direct count method and includes viable, dead, viable but nonculturable (VBNC), and viable but difficult to culture (VBDC) organisms. Fluorescence in situ hybridization (FISH), a kind of fluorescence microscopy, involves the use of fluorescently labeled nucleic acid probes to target DNA or RNA sequences within an organism; therefore, can be used to detect all bacterial cells or a genus or even a single population of cells. The confocal laser scanning microscope (CLSM) can be used to generate three-dimensional views; therefore, commonly used in bright-field, dark-field, and fluorescence microscopies. The electron microscope produces high resolution images, and the two most common types of electron microscopy are scanning electron microscopy and transmission electron microscopy. Scanning probe microscopy deals with imaging surfaces on a very fine scale, and one of most common types of this technique is atomic force microscope (AFM) that can view single atoms.