Abstract Background Archegoniates (bryophytes, ferns and gymnosperms), such as the moss Physcomitrella patens, possess freely motile sperm cells (spermatozoids) which reach the egg cell via surface water. Although these motile flagellated sperm cells are a traditional botanical subject, they have not been thoroughly analysed in the flagship non-seed plant model species P. patens. Protocols are required to determine the behaviour of wild type sperms as a prerequisite for future research such as the characterization of mutants or factors that influence sperm number, morphology, viability and motility. Results Here, we present protocols for the observation of fixed, as well as live sperms utilizing a standard microscope at intermediate magnifications. Fixed samples can be used for the fast assessment of sperm number and morphology. To determine functionality, the observation of live sperms is required. Protocols for determining both sperm motility and viability are provided, allowing both parameters to be distinguished. Conclusions These step-by-step protocols are particularly useful for researchers so far not familiar with the analysis of motile gametes and are meant to aid the establishment and improvement of these analyses in order to stimulate research on spermatogenesis in the moss model species P. patens.
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