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United States Department of Agriculture

Agricultural Research Service

Title: Use of Low Temperature Scanning Electron Microscopy to Observe Bacteria in Planta

Authors
item Roy, Stephane
item Babic, Isabelle
item Watada, Alley
item Wergin, William

Submitted to: Journal of Bacterial
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 1, 1995
Publication Date: N/A

Interpretive Summary: Microorganisms hasten the deterioration of fresh-cut fruits and vegetables, and although chlorine wash is used to kill the microorganisms, many prevail and grow after the wash. To develop an improved method of control, information is needed on the identity and location or site where the microorganisms continue to grow after the chlorine wash. In this study, a low temperature scanning electron microscopy method was developed which allowed the visualization of the site where the microbial growth occurred and the method did not affect the viability of the microorganisms, thus they could be recovered for the identification analysis. This low temperature scanning electron microscopy method will be helpful to other scientists who need to determine the site of microorganisms growth and need to recover the microorganisms for identification.

Technical Abstract: Electron microscopy (EM) has greatly helped to elucidate bacterial structure and function. However, several recent studies caution investigators about artifacts that resulted from the use of conventional EM preparation procedures. To avoid these problems, low temperature scanning electron microscopy (LTSEM) was evaluated for examining frozen, fully hydrated specimens. Spinach leaves (Spinacia oleracea L., cv. New Jersey) which were naturally infected with bacteria, were used as the experimental material. One cm segments of the infected leaves were plunge frozen in liquid nitrogen, transferred to a cryochamber for sputter coating and then moved onto a cryostage in an SEM. After observation, some of the frozen, hydrated leaf segments were transferred onto Agar medium to determine whether observation in LTSEM was nondestructive for the bacteria. Other tissue segments were chemically fixed by freeze substitution. The results indicated that the LTSEM procedure: i) preserved the surface glycocalyx o the bacteria, ii) did not appear to affect the viability of the bacteria, which could be recovered and cultured on Agar medium, and iii) allowed recovery of the observed sample so that freeze substitution, embedding and TEM observations could be made. In conclusion, bacteria can first be observed using LTSEM and then either processed for TEM observation to obtain fine structural details or recovered and cultured to taxonomically identify the bacteria.

Last Modified: 10/25/2014
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