TITLE: Pure Culture and Characterization of bacterial symbiont of Anabeana sp.
AUTHOR: Nell Ament
SCHOOL: Falmouth Academy
SCHOOL ADDRESS: 7 Highfield Drive, Falmouth, MA. 02540
The purpose of this work was to pure culture and begin characterization of a bacterial symbiont of cyanobacteriaum Anabaena sp. Attached to its heterocysts, specialized nitrogen fixing cells among vegetative cells in the Anabeana sp’s filaments, are small rod-shaped bacteria.
To successfully pure culture the symbiont bacteria from the crude culture of Anabeana (as it was found in nature), an Anabeana filament was carried through a dilution series of ½ seawater solution to rinse off contaminants. The cleansed filament was added to a pure culture of Anabaena sp. which became colonized by the symbiont. This was inoculated onto a plate off of which colonies were picked and reintroduced to Anabaena, thus completing a test of Koch’s Postulate to show the symbiont had been successfully isolated. Subsequent experiments to characterize the bacteria included: comparing symbiont bacteria counts in the crude (including all the bacteria from the natural environment) and 2m (just Anabaena sp. and its symbiont) cultures; determing Anabaena’s health with and without the symbiont by counting the number of vegetative cells between heterocysts; determining if the relationship between the bacteria and Anabeana was specific by introducing colonies of the symbiont bacteria to seven other species of cyanobacteria completing the Gram-staining procedure in the symbiont; finding the bacteria’s vitamin requirements by looking at the number of bacteria when different vitamins were included in the media; and then determining if the bacteria would grow free from its host if nutrients were provided in the media.
The results of these experiments were as follows. The number of bacteria per heterocyst was not significantly different in the crude and 2m cultures so long as vitamins were provided; this shows that , other than vitamins, there was nothing the symbiont needed which it could not obtain from its host. The presence of the bacteria did not significantly affect Anabaena’s health. The bacteria did not attach to any cyanobacteria other than its original host Anabaena: further, no bacteria would attach to Anabaena other than the original symbiont. Therefore, evidence supports that this was a specific interrelationship. The bacteria was found to be Gram-negative. The bacteria did not grow free from Anabaena when nutrients were provided in the media nor when both vitamins and nutrients were provided. The bacteria may have had no reason to leave the heterocysts, or they could have needed nutrients other than those used. It was determined that the symbiont bacteria has a vitamin requirement, and that it most likely needs
Title: Pure Culture and Characterization of bacterial symbiont of Anabeana sp. Page 2
only Thymine and B12. Observations under both a light microscope with phase contrast and an electron microscope revealed that the bacteria branches, and that some of its cells have flagella. It was also observed that the bacteria attach to Anabaena’s akinetes, cells filled with nutrients made in harsh conditions, as well as the heterocysts. When the 2m. culture was put in media which could not support Anabaena and where its cells burst, creating a media of cell contents, the bacteria was able to grow free in the media.
Thus the bacterial symbiont of Anabaena sp. was pure cultured and its characterization begun. This is new knowledge relevant to the barely understood field of bacteria-cyanobacteria relationships.
.