Mitochondria of Tetrahymena pyriformis: enumeration and sizing of isolated organelles using a Coulter Counter and pulse-height analyser.
An electronic particle counter (Coulter Counter ZBI) and pulse-height analyser (Channelyzer C1000) have been used to measure numbers and sizes of mitochondria isolated from the ciliated protozoon Tetrahymena pyriformis. Differential centrifugation of disrupted organisms, followed by single-step sub-fractionation of the mitochondrial fraction on sucrose gradients yielded a population of organelles extensively enriched in the activities of mitochondrial marker enzymes. Gradient-purified mitochondria (approx. 3 X 10(9) particles (mg protein)-1) were stable in electrolyte, exhibited unimodal volume distributions and were somewhat larger (0.93 +/- 0.13 (S.D.) microns 3; 19 preparations) than organelles in a crude mitochondrial fraction. Glutaraldehyde fixation of mitochondria in sucrose gradients decreased the apparent volume to 0.6 +/- 0.06 micron 3 (6 preparations). Based on the recovery in the mitochondrial fraction of mitochondrial membrane-bound cytochromes from a suspension of intact cells, the number of mitochondria per cell was estimated to be approximately 1440, representing 15% of the total cell volume. Isolated mitochondria were osmotically sensitive and exhibited an apparent marked contraction on adding Ca2+ (10 microM-10mM). Addition of chloramphenicol (500 micrograms ml-1) to exponentially growing Tetrahymena cultures resulted in an almost immediate cessation of cell division and a dramatic decrease in cell volume. Mitochondria purified from such cells were much smaller than control mitochondria (0.21 +/- 0.02 microns 3; 7 measurements); their population density was approximately 900 per cell, equivalent to 11% of total cell volume. The measurements of mitochondrial populations using the Coulter Counter and electron microscopy are in good agreement.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002701:Chloramphenicol / Q000494:pharmacology; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects / Q000648:ultrastructure; D013769:Tetrahymena pyriformis / Q000648:ultrastructure*
D002701:Chloramphenicol