STUDIES ON METHANOGENIC ENDOSYMBIONTS IN ANAEROBIC PROTOZOA, WITH REFERENCE TO THE CYTOCHEMICAL ASPECTS OF HYDROGENASE

The life cycle of amoebaflagellate Psalteriomonas lanterna includes two stages: flagellate and amoeba. When 2% oxygen was added to the headspace of the culture, the flagellate lost its symbiont metbanogenic bacteria. When flagellates without endosymbionts were cultured with 1% oxygen, small amoeba appeared. When cultured with 1% oxygen, amoeba cells grew quickly, the concentration of cells was about 1.2×10 ceUs/ml, and inhibition occurred at oxygen concentrations over 2.5%. The cells died when the oxygen level exceeded 7.%. Amoebae grew poorly in anaerobic culture. Addition of Methanobacterium formicicum Strain MsL to the amoebae resulted in higher concentrations of cells. The fluorescent endosyrnbionts in the sapropelic amoebaflagellate were proved to be methanogenic bacteria (species not identified). This was indicated by epifluorescence and electron microscopic observations, and the fact that methane was produced in the culture medium when the protozoa were washed in water. Cytochemical staining method revealed the presence of hydrogenase activity in the microbodies of P. lanterna without endosymbionts, suggesting that the microbody-like organelles are hydrogenesomes. After fixation of the cells in low concentrations of glutaraldehyde and incubation in the presence of hydrogen and the tetrazolium compound, 2-(2′-benzothizazolyl)-5-styryl-(-3-4-phthalhydrazidyl)-tetrazolium chloride, an electron-dense deposit was produced in the hydrogenosomes. In the control cells exposed to nitrogen, no deposit was found.