This study outlines the cultivation of Saccharibacteria in a binary culture with host bacteria. The methodology emphasizes the importance of maintaining optimal growth conditions for both organisms.
Begin with a sterile tube containing a host-specific growth medium. Add an overnight-grown host bacterial culture.
Then, introduce a filtrate containing Saccharibacteria, a parasitic ultrasmall bacteria, to establish a binary culture.
Incubate the binary culture under microaerobic conditions that are optimal for the host bacteria.
The nutrients in the medium promote the growth and proliferation of host bacteria, making the culture turbid.
Due to their obligate parasitic nature, Saccharibacteria attach to the surface of the host bacterial cells.
These bacteria then form close membrane contacts with the host and extract nutrients essential for their survival and proliferation.
As infection progresses, the growth of the bacterial host slows down, resulting in reduced turbidity.
To support continued growth, transfer the binary culture into fresh medium at regular intervals.
This maintains host viability and promotes the long-term stability of the binary culture.
Prepare culture tubes by aliquoting two milliliters of appropriate growth media into the tubes.
Then, add 200 microliters of overnight culture of host organisms and 100 to 200 microliters of resuspended filtered sample to each tube. Incubate the combined samples under conditions appropriate for the host organism. Passage the cells every two to three days by transferring 200 microliters of the binary culture to two milliliters of fresh growth medium in a new tube.
If the passaged cultures show no growth at 200 microliters of uninfected host culture, when passaging the cells, return inoculated tubes to the incubator.
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