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Aims: Validation of stereology and three-dimensional reconstruction for monitoring the probiotic effect of Aeromonas hydrophila on the gut development of germ-free Artemia franciscana nauplii., Methods and Results: Germ-free Artemia nauplii were cultured using Baker's yeast and dead Aer. hydrophila. Live Aer. hydrophila were added on the first day to the treatment group. The gut length and volume were monitored on days two and four using stereology and three-dimensional reconstruction. Both methods showed comparable results. Stereology was least labour intensive to estimate volumes, while three-dimensional reconstructions rendered architectural and topographical data of the gut. Moreover, a positive effect of probiotic bacterium, Aer. hydrophila is likely., Conclusion: Slight increment in the growth of the digestive tract of A. franciscana nauplii exerted by probiotic bacteria could be detected using stereology and three-dimensional reconstruction., Significance and Impact of the Study: The gnotobiotic Artemia rearing system is unique to investigate the effects of micro-organisms on the development of nauplii. However, in the base of this model system, only survival counts and length measurements exist as monitoring tools. Therefore, additional tools such as stereology and three-dimensional reconstruction are prerequisite to obtain more powerful analysis., (© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)