William H. Cole

A Perfusing Solution for the Crayfish Heart and the Effects of Its Constituent Ions on the Heart

A SATISFACTORY solution for nerves of the crayfish, Cambarus clarkii, was developed by van Harreveld (1936) based upon chemical analyses of blood. Since its composition varied considerably from solutions reported by others to be satisfactory for crayfish, especially with respect to calcium, it seemed desirable to test it on the crayfish heart, to compare it with those other solutions, and to study the effects of constituent ions on the heart.

Responses of the Barnacle to Some Strong Electrolytes and to Urea, Glucose, and Glycerol

P REVIOUS studies on the responses of various animals to mineral and aliphatic acids, sodium salts of mineral and fatty acids, and to alcohols have demonstrated several types of quantitative correlations between intensity of stimulation and the specific nature of the ions and molecules concerned (see Allison and Cole, 1933-34, and papers quoted therein; Sizer, 1935-36). New data have been secured from the barnacle on the sodium and potassium salts of the mineral acids and on urea, glucose, and glycerol, which have allowed: (i) comparison of stimulating effects of the salts with those of their corresponding acids; (2) description of two opposite types of responses, opening and closure, the occurrence of which depends upon the concentration of the substances used; and (3) determination that equally stimulating solutions of the strong electrolytes and of the nonelectrolytes have markedly different osmotic pressures.

Measurements of Fatal Doses of Chloroform in the Brains of White Rats.

Qualitative tests. The aqueous extracts of the brains of frogs, mice, rats, guinea pigs, cats, and dogs, killed by inhaling chloroform, have all shown the presence of chloroform by the pyridine test, 1 while similar extracts from animals killed by electricity, illuminating gas, CO2, blow on the head, and beheading, have all been negative for chloroform. In a series of 8 brains from rats killed by inhaling chloroform, the tests were made at the end of 1, 1 ½, 2, 3, 5, 6, 15 and 25 days respectively. In each case chloroform was indicated, in spite of the fact that considerable decay of the tissues had occurred. These results agree with those reported by Luedeking, 2 and Angiolani, 3 to the effect that chloroform in animal tissues persists unchanged for many days after death and decay. Quantitative tests. The chloroform present in extracts of brains from rats killed by inhaling chloroform has been measured by the pyridine colorimetric method. Into a 1-liter dish containing a rat, a known amount of chloroform was sprayed. At the moment of death (cessation of respiratory movements) the rat was removed and its brain extracted. A dose of 0.1 cc. was sub-lethal in 35 minutes; 0.2 cc. caused death in an average of 11.2 minutes; 0.4 cc. in 5.4 minutes; 0.8 cc. in 4.3 minutes; 1.0 cc. in 3.5 minutes; 2.0 cc. in 2.5 minutes; 4.0 cc. in 1.6 minutes and 5.0 cc. or more in 1.5 minutes. The time of death bore no clear relationship to size of the animal. In Table I are given the amounts of chloroform found in brains of killed and anesthetized rats.

The use of chloretone as an anesthetic for paramecium.

The described methods for quieting paramecia by using formaldehyde, quince seed jelly, cotton fibers, etc., are by no means satisfactory, if observations are to extend over more than a few minutes. They are of no use at all for observing a single animal, or a particular group of animals, over a period of several hours or days. Experiments have been made with chloretone∗ (trichlortertiarybutyl alcohol), and have shown it to be especially valuable for quieting paramecia for long periods of time, up to 8 days, with no undesirable effects. The paramecia used were extracted from wild cultures, and grown in hay infusion of known constant concentration. New cultures were started from time to time by adding a pipette full of the old to fresh infusions. These cultures contained a few of the smaller rotifers, many of the smaller ciliates, and a preponderance of Paramecium caudatum, a mixed population which seemed to be favorable for their growth and reproduction. It was found that a solution containing about 0.06 percent chloretone by weight would anesthetize paramecia in a few minutes, and keep them anesthetized for varying periods of time. The animals would recover if placed in fresh culture fluid previous to cytolysis. The technique consisted of placing 1 drop of culture fluid, densely populated with paramecia, in a shallow glass chamber on a slide, and then adding 1 drop of 0.12 percent chloretone solution. After measuring the drops of the two fluids from their respective pipettes it was found that such a mixture contained 0.056 percent chloretone. In all subsequent tests the same two pipettes were used. The size of the drops can be adjusted so as to yield just enough fluid to fill the chamber. The latter was then sealed with a cover glass using a mixture of bees wax and white vaseline, the consistency of which just allowed of its easy spreading at 20° C. (approximately 1 part of wax to 3 parts of vaseline). Gentle heat applied to the cover insured an air-tight seal. Vaseline alone did not give good results, since the animals always recovered within a few hours (from 2 to 24), a fact so far unexplained satisfactorily. It was noted that a few individuals are sometimes killed immediately upon the addition of the chloretone, due probably to the fact that they come in contact with the chloretone before it has been diluted with the culture fluid.

Gravity Shock in Rabbits. I. Lack of Correlation Between Plasma Protein and Specific Gravity.

The calculation of protein concentration from the specific gravity of human and dog serum or plasma 1 , 2 has been widely used in clinical and experimental laboratories especially since the development of the falling drop method for measuring specific gravity, 3 , 4 because it is quicker than analyses by the Kjeldahl method. It is important to know how the serum or plasma protein concentration changes in traumatic, hemorrhagic, and other kinds of shock, as well as in other pathological conditions, and much reliance has been placed on specific gravity determinations on the blood of such subjects.5 In order to determine whether the same relationship between specific gravity and portein concetration exists in rabbit plasma as in human and dog plasma, blood from 20 normal rabbits fed on Purina chow and later from the same animals suspended by their ears until the became unconsious from peripheral circulatory deficiency (“gravity shock”) 6 has been studied. The plasma of arterial heart blood was analyzed for protein by the micro-Kjeldahl method, for glucose by the Folin-Wu method as modified by Andes and Northup, 7 and the specific gravity was determined by the falling drop method. In Fig. 1 plasma protein before and during gravity shock is plotted against plasma specific gravity. The data for normal animals are well described by the line drawn through the open circles having the equation: P = 375 (Gp −1.0064), which is of the same order of magnitude as the equation reported for human 1 and dog 2 plasma. For rabbits in gravity shock, however, the constant relation between specific gravity and protein concentration did not hold, as indicated by the solid circles, showing even greater divergencies than those found by Moore and Van Slyke 1 in human nephritic patients.

Disappearance of Radioactive Phosphorus from Heart Blood of Limulus Polyphemus.

The disappearance of radioactive phosphorus (P32) from the heart blood of the horseshoe crab Limulus polyphemus, has been followed in 10 individuals, weighing from 94 to 245 g. From 0.1 to 0.2 ml of 0.001 molar Na2HPO4 solution was injected into the heart at the thoracic-abdominal junction. The amounts of radioactive phosphorus in the solutions and in the samples of blood withdrawn from the heart at frequent intervals following injection were measured with a Geiger-Müller counter, and were expressed as counts per minute per milliliter of solution or per milligram of dried blood. All counts were corrected for background and for decay. The amounts injected were calculated to give equal doses of radioactive phosphorus per gram of body weight, the average dose bearing 3.5 × 105 counts per minute. In every one of the 10 experiments, the logarithm of the concentration of radioactive phosphorus left in the blood was inversely proportional to time, the slope of the plot relating the two from zero time to 1 hour varying from −0.91 to −3.06. At the beginning of the experiment the average number of counts per milligram of dried blood was 83, and at the end of 1 hour 97.6% of the radioactive phosphorus had disappeared from the blood. A few experiments with the lobster, Homarus americanus, gave essentially the same results except that the rate of disappearance was much slower. These preliminary tests indicate that radioactive phosphorus will be useful in studying various problems in the physiology and biochemistry of the blood and tissues of Limulus and other invertebrates, just as it has been in the vertebrates. Several such problems are now being studied.

The Stimulating Efficiency of the Normal Primary Alcohols.

As reported recently, 1 the rhythmic movements of the cirri of barnacles are sensitive indicators of environmental stimulation. A study has been made of the stimulating efficiencies of the first 4 normal primary aliphatic alcohols on Balanus tintinabulum. † The threshold concentration of each alcohol was determined under constant conditions of illumination, temperature and rate of flow, and in the absence of mechanical stimulation. Under such conditions it may be assumed that the stimulating agent is furnishing the minimum amount of energy necessary to activate the receptor. The effects of any secondary processes initiated by excessive stimulation are thus avoided, and the interpretation of the stimulating process is less likely to be erroneous. From the data in Table I it is clear that the stimulating efficiencies of the alcohols increase about 3 fold as each CH2 group is added, beginning with methanol. This result is similar to that found by previous investigators in studying narcosis, toxicity and related effects. A review of the work and the theories on narcosis has been recently presented by Traube. 2 We have made similar studies on the frog, Rana pipiens. Protecting the animal from all external stimuli, except the alcohol being studied, it has been possible to determine equally stimulating concentrations of the first 5 members of the normal primary aliphatic alcohols. Constancy of reaction time was the criterion for judging equally stimulating solutions. The data, presented in Table II, show that the stimulating efficiencies increase with the number of CH2 groups. The agreement of these 2 sets of data with the so-called Traubes rule is striking, but we would like to point out that interpretations of stimulation based on surface tension effects alone are probably incomplete.