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Improvements and important considerations of an ex vivo assay to study Candida albicans-splenic tissue interactions
M H Riesselman1, T Kanbe, J E Cutler
1Department of Microbiology, Montana State University, Bozeman 59717.
Abstract:
Improvements were made on an ex vivo assay to study adherence properties of Candida albicans to host internal organs. The assay is applicable to understanding mechanisms of C. albicans dissemination following a fungemia. Binding patterns of yeast forms to splenic tissue are intriguing and we found the following modifications to be especially relevant. Mice serving as spleen donors for the assay should be injected with 0.1 ml i.v. of a 10% concentration of luconyl blue, 5 min before killing. After collecting splenic sections on a glass slide, 100 microliters of a 1-2 x 10(8)/ml suspension of stationary yeast cells should be applied to the sections. The assay does not require a carbonate buffering system or serum supplements. Attachment of yeasts to host tissue occurs best if the interaction is allowed to proceed without agitation by rotation. Assessment of binding is facilitated by staining the slides with crystal violet and computer image analysis can be used for quantification of binding.
Insights
This study refines an ex vivo assay for Candida albicans adherence to host organs, crucial for understanding fungal dissemination after fungemia. The improved method enhances the study of yeast-to-tissue binding patterns in splenic tissue.
Area of Science:
- Medical Mycology
- Infectious Diseases
- Microbiology
Background:
- Candida albicans dissemination following fungemia is a significant clinical concern.
- Understanding yeast adherence to host tissues is critical for elucidating dissemination mechanisms.
- Existing ex vivo assays require optimization for studying C. albicans-host interactions.
Purpose of the Study:
- To improve an ex vivo assay for studying Candida albicans adherence to host internal organs.
- To investigate the binding patterns of C. albicans yeast forms to splenic tissue.
- To provide a reliable method for assessing fungal dissemination mechanisms.
Main Methods:
- Development of an ex vivo assay using splenic tissue sections.
- Injection of mice with 'luconyl blue' prior to spleen collection.
- Application of C. albicans yeast cell suspension to splenic sections without agitation.
- Staining with crystal violet and utilizing computer image analysis for quantification.
Main Results:
- Optimized protocol for C. albicans yeast adherence to splenic tissue.
- Demonstrated effectiveness of the assay without requiring carbonate buffering or serum supplements.
- Identified specific modifications for enhanced binding pattern assessment.
Conclusions:
- The refined ex vivo assay provides a valuable tool for studying C. albicans adherence and dissemination.
- The assay facilitates a deeper understanding of fungal-host interactions in splenic tissue.
- This method aids in the investigation of fungemia pathogenesis.