PMID: 6411751May 1, 1983Paper

Mitochondria of Tetrahymena pyriformis: enumeration and sizing of isolated organelles using a Coulter Counter and pulse-height analyser

Journal of Cell Science
R K Poole

Abstract

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-1...Continue Reading

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