Abstract
Academic research on dinoflagellate, the primary causative agent of harmful algal blooms (HABs), is often hindered by the coexistence with bacteria in laboratory cultures. The development of axenic dinoflagellate cultures is challenging and no universally accepted method suit for different algal species. In this study, we demonstrated a promising approach combined density gradient centrifugation, antibiotic treatment, and serial dilution to generate axenic cultures of Karenia mikimotoi (KMHK). Density gradient centrifugation and antibiotic treatments reduced the bacterial population from 5.79 ± 0.22 log10 CFU/mL to 1.13 ± 0.07 log10 CFU/mL. The treated KMHK cells were rendered axenic through serial dilution, and algal cells in different dilutions with the absence of unculturable bacteria were isolated. Axenicity was verified through bacterial (16S) and fungal internal transcribed spacer (ITS) sequencing and DAPI epifluorescence microscopy. Axenic KMHK culture regrew from 1000 to 9408 cells/mL in 7 days, comparable with a normal culture. The established methodology was validated with other dinoflagellate, Alexandrium tamarense (AT6) and successfully obtained the axenic culture. The axenic status of both cultures was maintained more than 30 generations without antibiotics. This efficient, straightforward and inexpensive approach suits for both armored and unarmored dinoflagellate species.
Highlights
Academic research on dinoflagellate, the primary causative agent of harmful algal blooms (HABs), is often hindered by the coexistence with bacteria in laboratory cultures
The present study aims to develop a method using a combination of three simple techniques, a series of Percoll density gradient centrifugation, antibiotic treatment and serial dilution selection for the establishment of an axenic dinoflagellate culture of K. mikimotoi
There was no significant difference of the remaining total bacterial counts between algal samples centrifuged with 90% and 90%–50% Percoll gradients, but decreased significantly from 5.02 ± 0.2 log[10] CFU/mL to 4.38 ± 0.05 log[10] CFU/mL when dinoflagellate samples were centrifuged with 90–50–30% Percoll gradient with no further increase on adding another layer of 10% density to the gradient (i.e., 90–50–30–10%)
Summary
The primary causative agent of harmful algal blooms (HABs), is often hindered by the coexistence with bacteria in laboratory cultures. Bacteria attached firmly onto the algal surface cannot be removed efficiently with algal phototaxis a lone[12] Another group of researchers developed an axenic culture of A. tamarense by treating the algal cells with lysozyme/SDS and antibiotics[13]. Their method is straightforward and efficient, but the lysozyme/SDS treatment can damage the dinoflagellate cells. The present study aims to develop a method using a combination of three simple techniques, a series of Percoll density gradient centrifugation, antibiotic treatment and serial dilution selection for the establishment of an axenic dinoflagellate culture of K. mikimotoi (unarmored). The study tested the efficacy and applicability of the developed method for another dinoflagellate species, A. tamarense (armored)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
