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Chapter 2 : Sampling and Staining for Light Microscopy
Specific morphological details are required to characterize a microorganism; these are usually determined by means of light microscopy, but some require more sophisticated techniques such as laser scanning microscopy or electron microscopy. This chapter concentrates on the general means of characterizing bacteria by light microscopy because it includes the cytological approach to making the best of preparations for study and photomicrography. The field of aerobiology encompasses both indoor and outdoor components and focuses on a wide range of microorganisms, including pathogenic and nonpathogenic varieties. Although the numbers in a unit of volume may be quite small, the simplest approach to air sampling is the use of open petri dishes containing a suitable nutrient agar near a suspected contamination source and is applicable when the concentration of organisms is relatively high. Translational movement of bacteria by flagellar propulsion (swimming) may be observed in wet mounts of specimens by use of in most cases, the low-power or high-dry objectives. To ascertain the presence of flagella in doubtful cases, as well as to determine flagellar distribution (polar, peritrichous, or lateral), staining procedures and electron microscopy may be required. Gram staining is the most important differential technique applied to bacteria. Mature, dormant endospores of bacteria, when viewed unstained, are sharp edged, even sized, and strongly refractile, shining brightly in a plane slightly above true focus. A great number of techniques based on light microscopy are special to particular areas of bacteriology. The chapter gives examples of some special methods which are not routinely used.