. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 556 JOHN R. GREGG without phosphate or bicarbonate. Development was allowed to proceed at 10° C., the medium being renewed every two days or so. Immediately before using them as experimental subjects, the embryos were freed of their jelly-coats with the aid of jeweler's forceps. Chemical Anaerobiosis was obtained with the help of the apparatus depicted in Figure 1. Twenty jelly-free embryos, along with enough rear ing-medium to make a total volume of 2 ml., were placed in each of several 25-ml. Erlenmeyer flasks. The ground
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 556 JOHN R. GREGG without phosphate or bicarbonate. Development was allowed to proceed at 10° C., the medium being renewed every two days or so. Immediately before using them as experimental subjects, the embryos were freed of their jelly-coats with the aid of jeweler's forceps. Chemical Anaerobiosis was obtained with the help of the apparatus depicted in Figure 1. Twenty jelly-free embryos, along with enough rear ing-medium to make a total volume of 2 ml., were placed in each of several 25-ml. Erlenmeyer flasks. The ground glass neck of each flask was closed with a No. 1 two-hole rubber stopper bearing glass inlet and outlet tubes. The flasks were set in the clips of a Dubnof \ I \ / f f I \. FIGURE 1. Apparatus used to obtain anaerobic embryos. See description in section on chemical methods. Arrows indicate direction of gas flow. shaking bath running at 24° C., and connected in series with short lengths of rubber tubing. The shaking mechanism was then set in motion at a rate of 85 cycles per minute. At time zero, a source of washed 95% N2: 5% CO, was connected to the inlet tube of the first flask, and gassing was allowed to proceed at a rate of one liter per minute for the duration of the experiment. At the ends of chosen intervals, flasks were removed from the distal end of the train and their contents were emptied into 12-ml. graduated centrifuge tubes, each containing ml. of 30% trichloracetic acid. The volumes were adjusted with 6% trichloracetic acid to values depending on the expected amounts of lactic acid, and the tubes were placed in a Deepfreeze. After freezing and thawing, the embryos were homogenized with a ball-tipped glass rod, and protein-free extracts were obtained by centrifuging. The amounts of lactic acid in the protein-free extracts were estimated by a modi- fication of the method of Barker and Summerson (1941). One-mi, aliquots of protein-free extract were treated with
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