Histopathological methods for the investigation of microbial communities associated with disease lesions in reef corals.

AIMS: To determine the spatial structure of microbial communities associated with disease lesions of reef corals (Scleractinia). METHODS AND RESULTS: Agarose pre-embedding preserved the structure of the disease lesion and surrounding tissues prior to demineralization of the carbonate exoskeleton and embedding in resin. Fluorescence in situ hybridization (FISH) was used to localize bacteria in the lesions of various diseases. CONCLUSIONS: The techniques successfully preserved the in situ spatial structure of degenerated coral tissues. In one case (white plague disease), significant bacterial populations were found only in fragmented remnants of degenerated coral tissues at the lesion boundary that would not have been detected using conventional histopathological techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the composition, spatial structure and dynamics of microbial communities within the disease lesions is necessary to understand the process of disease progression. The methods described may be applicable to a wide range of diseases involving necrotic lesion formation and requiring extensive tissue processing, such as skeleton demineralization.

Fluorescence and Brightfield Cytology of Live M. tuberculosis Cells.

Although light microscopy fell out of favor as a research tool in prokaryotic biology in the 1980s, advances in the reagents available for cell labeling (staining) and in the user-friendliness of microscopes were underpinning a revolution in eukaryotic cell biology. The development of epifluorescence hardware, particularly confocal microscopy and low-light imaging systems, and computational deblurring and video enhancement methodologies, substantially extended the range of potential applications. These developments now enable us to detect weaker signals at higher levels of resolution than was previously possible. Finally the personal computer and related software developments have brought image analysis within affordable range for many laboratories and facilitate quantitation of cellular properties on an objective basis. We have sought to apply these advances across a range of prokaryotic applications and here we describe the methods we have applied to live Mycobacterium tuberculosis cells. Although we have principally been concerned with two applications, the determination of viability at the cellular level (see Note 1) and the nature and distribution of lipid domains, more general aspects of light microscopic cytological analyses are discussed below.

Relationships between culturability, activity and virulence in pathogenic bacteria.

While it is clear that the cells of many culturable pathogenic bacteria may become nonculturable but retain some cytological indices of activity and integrity, the potential for such cells to cause human disease is far from certain. Here we discuss both results and practical considerations relating to this issue. We conclude that there are no available cytological tests that satisfactorily predict whether cells have infective potential. In contrast, we recognize that nonculturable cells of pathogenic bacteria can retain substantial physiological activity, including the capacity to synthesize toxins. However, the clinical significance of these phenomena is yet to be established.

Solution and film properties of sodium caseinate/glycerol and sodium caseinate/polyethylene glycol edible coating systems.

The aim of this study is to determine the effects of plasticizer hydrogen bonding capability and chain length on the molecular structure of sodium caseinate (NaCAS), in NaCAS/glycerol and NaCAS/polyethylene glycol 400 (PEG) systems. Both solution and film phases were investigated. Glycerol and PEG reduced the viscosity of aqueous NaCAS, with the latter having a greater effect. This was explained in terms of protein/plasticizer aggregate size and changes to the conformation of the caseinate chain. In the film phase, glycerol caused more pronounced changes to the film tensile strength compared with PEG. However, the effect of glycerol on film water vapor permeability was smaller. These observations are attributed to the differences in plasticizer size and hydrogen bonding strength that controls the protein-plasticizer and protein-protein interactions in the films. Glass transition calculations from the tensile strength data indicate that the distribution of bonding interactions is more homogeneous in NaCAS/PEG films than in NaCAS/glycerol films.