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United States Department of Agriculture

Agricultural Research Service

Title: Advantages of Low Temperature Scanning Electron Microscopy for Bacteriological Studies

Authors
item Wergin, William
item Roy, Stephane - UNIV MASS., DEPT. BIOL.
item Babic, Isabelle
item Watada, Alley

Submitted to: Scanning
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 20, 1997
Publication Date: N/A

Interpretive Summary: Bacteria are economically important pathogens that not only affect plants while they are growing but also cause spoilage during shipment and on the store shelf. To gain a better understanding of what types of bacteria are involved in food spoilage and how these organisms interact with the plants, scientists have used powerful instruments called scanning electron microscopes to observe the bacteria at very high magnifications. Unfortunately, the procedures that are used to prepare the bacteria for observation with these instruments frequently result in: 1) loss of the bacteria from the plant; 2) elimination of their secretions; or 3) shrinkage and rearrangement. Recent studies in our laboratory have allowed us to avoid these problems by freezing the bacteria and observing them in their frozen state with a modified scanning electron microscope. The present study, which documents the advantages of observing bacteria by this smethod, indicates that this technique is a valuable new research tool that will enable research scientists to gain useful information about the structure of disease organisms that are important in agriculture.

Technical Abstract: Recently, several studies alerted bacteriologists to the artifacts associated with the aqueous fixation procedures that are normally used for conventional EM studies. To avoid the problems, investigators have evaluated several alternative procedures to prepare bacteria for EM studies: 1) osmium vapor fixation minimized the effects of aqueous chemical fixation and preserved the extracellular polysaccharide (EPS) matrix; 2) cryofixation and freeze substitution improved preservation of ultrastructural features: 3) a synthetic polymer created replicas from frozen hydrated specimens; 4) unfixed samples were directly embedded in water-soluble Nanoplast resin; and 5) a variable pressure SEM was used to image directly the hydrated microorganisms. Recent studies in our laboratory have allowed us to avoid aqueous fixation by observing frozen, fully hydrated biological specimens with the low temperature SEM. The present study documents the advantages of imaging bacteria with this technique and also demonstrates several unique advantages that are applicable to studies in bacteriology.

Last Modified: 7/28/2014
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