Rate of Escape of Haemoglobin from the Erythrocyte

Abstract

The technical problem of finding the rate at which pigment leaves the mammalian red cell was proposed to me by Dr. Hugo Fricke, with the idea that if the rate were known it might be possible to calculate whether the pigment leaves the cell through one or two holes in the membrane, or by diffusing across the membrane generally.

The method which I have used consists in taking motion pictures of the haemolysing cells.∗ The best cell to work with is the red cell of man, which is relatively large. A suspension is made by suspending the thrice-washed cells from 4 cc. of blood in 20 cc. of 1% NaCl, for a suspension of this strength, when diluted with equal volumes of a lysin solution, shows from 5 to 15 cells per frame of the film used (35 mm.). Saponin made up in 1.0% NaCl in such a dilution as produces complete lysis in about 3 min. is a convenient lysin. The optical system used should be one of the highest resolution practicable, e. g., an aplanatic condenser of N.A. 1.4 working at 2/3 cone and critically focussed, a 1/10 inch oil-immersion objective of N.A. 1.37, and a 10x eyepiece. If the illuminant is an arc used without color filters, the exposure is about 1/30 second. A film speed of 16 frames per sec. is quite sufficient.

To 0.5 cc. of the suspension is added 0.5 cc. of the dilution of lysin sufficient to give complete lysis in about 3 min. A drop of the mixture is rapidly placed on a slide and covered with a coverglass smeared with vaseline along the edges. The vaseline seal prevents the cells from drifting. The preparation is placed on the stage of the microscope, and the cells focussed through a side telescope which enables them to be seen while the film is in a position ready for exposure.