DAPI staining and DNA content estimation of uncultivable microbial eukaryotes’ nuclei (Arcellinida and Ciliates)

Though acting as a major component of eukaryotic biodiversity, many microbial eukaryotes remain poorly studied, including the focus of the present work, testate amoebae of the order Arcellinida (Amoebozoa) and non-model lineages of ciliates (Alveolata). In particular, knowledge of their genome structures and changes in genome content over their often-complex life cycles remains enigmatic. However, the limited available knowledge suggests that microbial eukaryotes have the potential to challenge our textbook views on eukaryotic genomes and genome evolution. In this study, we developed protocols for DAPI (4’,6-diamidino-2-phenylindole) staining of Arcellinida nuclei and adapted protocols for ciliates. In addition, image analysis software was used to estimate the DNA content in the nuclei of Arcellinida and ciliates and to compare them to measurements of well-known model organisms. The results demonstrate that the methods we have developed for nuclear staining in these lineages are effective and can be easily applied to other microbial eukaryotic groups by adjusting certain stages in the protocols.