plant surface microbiology.pdf

27 Applications of Quantitative Microscopy in Plant Surface Microbiology 531
damine isothiocyanate-labeled monoclonal antibodies) could be easily seen.
They also utilized the noninvasive optical sectioning ability of the confocal
microscope to locate the Azospirillum cells within the root mucigel layer.
More recently, we have used optical sectioning by LSCM to document the
entry of neptunia-nodulating rhizobia into crevices at lateral root emergence
of the aquatic legume Neptunia natans (Subba-Rao et al. 1995), and azorhizobia
colonized on the root surface and within cortical cells of intact rice
roots (Reddy et al. 1997).
6 CMEIAS: A New Generation of Image Analysis Software for
in Situ Studies of Microbial Ecology
6.1 CMEIAS v. 1.27: Major Advancements in Bacterial Morphotype
Classification
A major challenge in microbial ecology is to develop reliable methods of
computer-assisted microscopy that can analyze digital images of microbial
populations and complex microbial communities at single cell resolution,
and compute useful ecological characteristics of their organization and
structure in situ without cultivation. To address this challenge, we are developing
customized semi-automated image analysis software capable of
extracting the full information content in digital images of actively growing
microbial populations and communities. This analytical tool, called CMEIAS
(Center for Microbial Ecology Image Analysis System) consists of plug-in
files for the free downloadable program UTHSCSA ImageTool (Wilcox et al.
1997) operating in a personal computer running Windows NT 4.0/2000. The
first release version of CMEIAS was developed primarily to perform morphotype
classification of bacteria in segmented digital images of complex
microbial communities (Liu et al. 2001). This CMEIAS version 1.27 uses pattern
recognition algorithms optimized by us to recognize bacterial morphotypes
with an overall classification accuracy of 97 %, and a sensitivity that
can classify morphotypes present in the community at a frequency as low as
~0.1 % (Liu et al. 2001). CMEIAS v. 1.27 can recognize 11 major morphotypes,
including cocci, spirals, curved rods, U-shaped rods, regular straight
rods, clubs, ellipsoids, prosthecates, unbranched filaments, rudimentary
branched rods, and branched filaments, representing a complexity level of
morphological diversity equivalent to 98 % of the genera described in the 9th
edition of Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994).
An interactive edit feature is included in CMEIAS v. 1.27 to revise the output
of automatic classification data if necessary (occurring at a 3 % error rate),
and add up to five additional morphotypes not included in the automatic
classification routine (Liu et al. 2001). Our first major application of CMEIAS
v. 1.27 was to contribute data on dynamic changes in community structure,