Effect of Methylene Blue on Respiration of Blocked and Developing Embryonic Cells. ∗

Abstract

We 1 have recently shown that actively developing embryos of the grasshopper, Melanoplus differentialis, have a respiratory rate 2 to 3 times higher than do embryos whose activity is to a large extent blocked as the result of diapause. The average respiratory rate for embryos immediately before and after diapause ranges between 0.1 and 0.15 mm. 3 per embryo per hour; the respiratory rate of blocked embryos averages approximately 0.05 mm. 3 per embryo per hour. In these 3 states of development the embryos are morphologically identical—moreover, the dry weight of the embryos is the same in each case, viz., ca. 0.07 mg.

In an attempt to gain some information regarding the nature of the block to respiration which occurs during diapause, the effect of methylene blue on the respiratory activity of blocked and developing embryos has been studied.

The embryos were freed from egg membranes and extra-embryonic materials and were suspended in a medium consisting of 0.9% NaCl, 0.02% each of KCl and CaCl₂ and 0.5% dextrose. The solution was buffered with phosphate (final concentration 0.01 molar) to pH 6.3—the pH of the perivitelline fluid of the egg.

Oxygen consumption of the embryos was determined by means of sensitive microdifferential manometers at 25° ± 0.02°C. Methylene blue solutions were prepared fresh daily in the saline-dextrose solution described above. In the use of cyanide the technique outlined by Huff and Boell 2 was followed.

The data in Table I show that methylene blue has only a slightly stimulating effect on the oxygen consumption of developing embryos, the average increase in respiration approximating 25%. On the other hand, the addition of methylene blue to blocked embryos practically doubles the rate of oxygen uptake. Nearly maximal stimulation is obtained with 3 × 10^-5 molar methylene blue. When the concentration of the dye is increased beyond 15 × 10^-5 molar, respiration is usually stimulated to a lesser extent. It is noteworthy that methylene blue raises the respiration of blocked embryos to the level which is characteristic for normally developing embryos. (Runnström. 3 )