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New Zealand Plant Protection 63 (2010): 15-23

Microscope methods for observation of the phylloplane flora

I.C. Hallett, K.S.H. Boyd-Wilson and K.R. Everett

ABSTRACT

Microscope-based observation of surface microbes can support indirect techniques, such as culturing or DNA analysis of surface washings, by illustrating microbial distribution patterns, inter-relationships and the presence of unculturable or non-recovered organisms. Comparisons have been made between techniques of contrasting complexity. For example, surface replicas of the leaf made from transparent materials and scanning electron microscopy (SEM) were compared for their ability to present an accurate picture of the leaf surface and microbial populations. “Environmental” SEM (ESEM) and cryo-SEM minimise change and provide the most realistic and detailed images of the surface but have logistical difficulties. Conventional, critical point dried SEM samples, even with extra processing and some physical change, usually provided similar results and had advantages in handling. The simpler replica techniques retained microbial number and distribution when compared to ESEM but were poor with rough surfaces. Microbial material on replicas could be stained or labelled with antibodies to improve identification.

Keywords: light microscopy, scanning electron microscopy, ESEM, cryo-SEM, surface replica, microbial ecology, immune-labelling, fungi, bacteria.

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