Methods for observing, culturing, and studying living ascospores

Ascospore morphologies provide important characters with which to diagnose and describe taxa in Ascomycota. Ascospore features such as size, shape, color, septation, wall thickness, and guttulation, among others, are provided in identification manuals and descriptions of new species. Yet, by tradition, ascospores are usually described from dead fungarium material, and unfortunately, occasionally from immature or overmature ones. However, living, mature ascospores display a wealth of taxonomically informative morphological features that are lost or obscured when they die. Examples of the severe morphological changes that ascospores undergo when they die are provided here. Data from living ascospores may not be observed and recorded by mycologists because field and laboratory practices do not prioritize the study of freshly collected specimens. In this review, we discuss how to assess ascospore maturity and describe methods to produce an ascospore deposit for the purpose of obtaining living, mature ascospores. Ascospores are ejected from living, mature asci onto a cover glass or growth medium. The ascospores collected on these surfaces can be used in microscopy and culture studies. Notes on a method for isolating conidia on growth medium are also provided. This guide is aimed at those who have a basic understanding of ascomycetes, including the various types of ascomata and mechanisms of ascospore liberation. Methods given in this paper are primarily applied to ascomycete fungi that have active ascospore discharge. Some methods may be adapted for use with other groups that have passive discharge. Our purpose is to promote standardized, accurate, and thorough morphological characterization of living ascospores, as well as to encourage the routine employment of culture-based methods.