Experimental Biology and Medicine

Hitherto positively heliotropic Euglenœ have always been found to give the motor reaction when suddenly shaded, and not when suddenly illuminated. Conversely negatively heliotropic individuals were found to react only to sudden illumination and not to sudden shading. Upon this association Jennings has based his theory according to which heliotropic orientation in Euglena is by “trial and error.”

It has been found, however, that it is possible to obtain positively heliotropic Euglenœ which give the motor reaction when suddenly illuminated and not when suddenly shaded. It is also possible to obtain at will negatively heliotropic organisms which give motor reactions when suddenly shaded, and not when suddenly illuminated. Under certain conditions the motor reaction to shading is given by Euglenœ in which no heliotropism can be demonstrated at that light intensity. Under other conditions distinct negative heliotropism is obtained with a light intensity which does not bring about any motor reactions when allowed to shine suddenly on the organisms, or when they are suddenly shaded. These facts show that heliotropic orientation in Euglena does not depend upon the motor reactions, but upon a separate mechanism.

Galvanotropism, which has so far eluded observation in Euglena was obtained by using culture media containing citric acid. With this material it was found that the method of orientation is identical in both galvanotropism and heliotropism. Consequently the orientation to light is as direct as the locomotor mechanism of Euglena permits, and does not take place by “trial and error.”

Cells growing in unmodified blood plasma (Harrison's method) exhibit regularly an accumulation of fat droplets in their cytoplasm. In the case of the cells of the chick embryo this fat accumulation is quite marked after 24 to 48 hours, and reaches a maximum after five to seven days, at which time the cells are distended with fat droplets.

The experiments herewith reported were planned to determine the effect of a reduction in the fat content of the culture medium, brought about by dilution of the plasma, on the accumulation of fat by the cells. In the course of the experiments the influence of dilution on the length of life and morphology of the cells was also observed.

One part of plasma added to twenty or twenty-five parts of Ringer's solution forms a medium which coagulates satisfactorily in hanging drops. Studies were made with dilutions of 1:2, 1:5, 1:10, 1:15, and 1:20 of pigeon plasma in Ringer's solution, containing 0.9 per cent. NaCl. Pieces of chick embryo heart were used for cultivation. The tissue was finely divided into pieces of suitable size for cultures, which were washed in Ringer's solution for a half hour before using. Cultures from the various series were fixed in formalin at the end of two, three and four days and stained with hematoxylin and Sudan III.

The results may be briefly summarized:

In plasma diluted 1 : 2 the cells live practically as long (5 to 10 days without transfer), as in pure plasma, and show a similar accumulation of fat.

In a 1 : 5 dilution the fat content is slightly diminished; there is little or no effect on the length of life or on the morphology of the cells.

The swelling of frog's legs, in which the circulation of the blood is stopped, may be accounted for by osmotic pressure.

A low beaker is half filled with distilled water and a funnel inserted so that the stem extends to the bottom. A saturated solution of NaCl is slowly poured into the funnel and forms a layer beneath the pure water. About 1 c.c. of a mixture of 2 parts chloroform and 3 parts rancid olive oil is sucked up into a pipette and injected into the beaker so that it forms a drop suspended between the NaCl solution and the pure water. Two pipettes with capillary openings are filled with 1/10 normal NaOH solution and inserted into the beaker. The NaOH solution is allowed to flow onto opposite poles of the drop at the same time and rate. The drop quickly elongates toward the pipettes, i. e., toward the poles, and constricts along the equator, and sometimes divides into two. The smaller the drop, the more certain the division, provided the operator has sufficient skill.

The alkali forms soap which reduces the surface tension on the polar areas, and the hydrostatic pressure within the drop causes these areas to bulge, whereas the relatively higher surface tension of the equatorial region causes it to constrict until a barrel-shaped figure is formed, which rapidly becomes hour-glass shaped. The equatorial surface film contracts and the polar surface film spreads, causing vortex movements. The enlargement of the polar fields spreads the soap over larger areas, and the area of unaltered surface tension is reduced to a narrow equatorial band. This band, being partially released by reduction of tension at its edges, acts as a sphincter and constricts until it cuts the oil drop into two. This constriction of the oil drop may be considered as a rough model of cell division.

The relation of iodids to morphin was studied by Reid Hunt. He found that feeding mice, rats and guinea-pigs with potassium iodid increases their susceptibility to morphin poisoning. This may be considered as the chronic influence of iodids. In our experiments we studied their acute effects. Our experiments were made, in the first place, on rabbits and sodium iodid in 5 per cent. solution was the salt employed. Morphin was administered intravenously. The iodid solution was given intravenously, and sub-cutaneously, ten to thirty minutes before the morphin injection. The quantity of sodium iodid administered to each rabbit was quite large; 15 cubic centimeters intravenously and 30 cubic centimeters subcutaneously. Such injections of sodium iodid alone seemed to cause no ill-effects in rabbits.

Morphin, if not rapidly fatal, causes narcosis, paresis, tremors, convulsions and finally death. The fatal dose of morphin for rabbits is somewhat variable, it is therefore difficult to study the influence which other substances may exert upon the toxic action of morphin. The most definite results we obtained have been with doses of 300 and 250 milligrams of morphin per kilo body-weight. Eighteen rabbits received morphin alone; 11 of these animals received the drug (300 milligrams in each case) through the ear vein, while in 7 animals the morphin (300 or 250 milligrams) was injected through the jugular vein. Sixteen rabbits received sodium iodid besides morphin. In ten of these animals the injection (300 milligrams) was given through the ear vein and in six through the jugular vein. The difference in the results was quite striking. Of the eighteen animals which received morphin alone, ten died immediately after the injection, five lived less than 2 hours, one lived four days and two survived. Of the sixteen rabbits which received sodium iodid besides morphin, only one died immediately after the injection, three lived longer than three hours, i. e., between 3 and 20 hours (died in night), one lived eighteen, one twenty-three, and one thirty-six hours; one lived two days, one nine and one 16 days, and six rabbits survived.

The view that the fibers of striated muscle are surrounded by semi-permeable membranes has received a wide acceptance among physiologists, and there has been a good deal of speculation regarding the nature of these membranes. The hypothesis that they are composed of lipoids has received much attention. Artificial lipoid membranes, however, have been found to be either impermeable both to water and to dissolved substances or else nearly equally permeable to water and to dissolved substances. It is a general rule that artificial membranes composed of pure colloids are either impermeable to both water and dissolved salts; or else nearly equally permeable to water and salts, and impermeable only to colloids. The best known artificial membranes which are semi-permeable with regard to salts dissolved in water are composed of precipitates of insoluble salts such as copper ferrocyanide and calcium phosphate.

The animal body can present the conditions necessary for the precipitation of calcium phosphate, as, for instance, in the case of bone formation. The striated muscle fibers contain considerable amounts of dipotassium phosphate, and are surrounded by lymph which contains calcium chloride, so that it is far from inconceivable that thin layers of calcium phosphate might be precipitated at the surfaces of the muscle fibers.

I have examined some of the properties of celloidin membranes impregnated with calcium phosphate. Celloidin membranes free from precipitate are quite permeable both to water and to dissolved salts. Such membranes were filled with a dipotassium phosphate solution and immersed in a calcium chloride solution. Under these circumstances they become impregnated with calcium phosphate, and at the same time they become markedly semi-permeable with regard to salts dissolved in water. That is to say that if they separate salt solutions of different osmotic pressures, water passes rapidly from the less concentrated to the more concentrated solution.

It has been claimed that the most important factor in the causation of shock is diminution of CO₂ within the blood, and that this diminution is a regular consequence of all influences resulting in shock. That CO₂ possesses important physiological functions cannot be denied. An investigation therefore of the true significance of a diminution of its normal amount within the blood is important and bears a special relation to various methods of artificial respiration utilized in thoracic surgery. The present experiments were undertaken for the purpose of investigating the effects of acapnia and the relation of some factors concerned in its production to shock. In all of them dogs were used. The first series was performed for the purpose of studying the effect of variations in intrapulmonic air pressure upon the blood pressure. The thorax was opened laterally, a T-tube connected with a water manometer was tied in a small bronchus, and the heart enclosed in a Henderson cardiometer in series with a recording tambour. The blood pressure was recorded from the carotid artery. The thorax was closed and the animal was subjected to intratracheal insufflation from an apparatus provided with an exhaust valve which reduced the pressure to approximately zero about four (4) to twelve (12) times per minute. The blood pressure averaged 150 mm. Hg. when the intrapulmonic air pressure was not allowed to exceed 6 mm. Hg.

In one experiment, with an increase of intrabronchial pressure from 8 mm. to 30 mm. Hg, blood pressure fell from 122 mm. to 55 mm. Hg and the volumetric tracing indicated that the output from the heart had diminished about 44 per cent. These variations in blood pressure were completed within a few seconds after the change in intrabronchial pressure, and could be duplicated at will.

Leishman and other authors have shown that the anal and coxal excretions voided by ticks (Ornithodoros moubata), capable of transmitting spirochætes (Spirochæta duttoni), are infective and that susceptible animals inoculated with these fluids develop a spirochætal infection. It has been asserted that animals upon which infected ticks have fed will not become infected, unless these fluids are voided while the ticks feed, and it has been suggested that the infection is transmitted by the flowing of the fluids into the wounds made by the mouth-parts of the ticks in feeding. Spirochætes had not been seen in these fluids and it was suggested that they existed there in a coccoid form.

On several occasions, coxal and anal fluids, excreted by infected ticks, coming from Uganda and British Central Africa, have been examined. In every instance the fluid was taken while the ticks fed upon an uninfected animal. The fluid collected was free from blood and, in two instances, coxal fluid was collected apparently free from anal excretion. On six occasions, after the fluid had been centrifugalized, spirochætes were found in it; their morphology is not distinct from that of Spirochœta duttoni. Spirochætes were found in the fluid that apparently contained no anal excretion.

When the eggs of Toxopneustes variegatus were subjected to a 5/8 molecular NaCl, after the removal of the fertilization membrane, considerable numbers were subsequently fused together. I have counted as many as forty percentum, in the optimum solutions, of agglutinated and fused pairs, triplets, etc. Few of these reached the pluteus stage of development due to the early death of all fusions of more than three eggs, and to the large mortality of even the double embryos.

The plutei contain at least three characteristic tissues, namely, body wall, archenteron, skeleton. The first two of these behaved essentially as described by Driesch in various European species, and by the writer in the American species Arbacia punctulata; i. e., the body walls or the archentera of plutei derived from separate eggs were united either incompletely or so completely as to give little or no evidence of the original dual character of the larvæ.

It was supposed that the skeletal structures united in the same manner as the archentera. The evidence furnished by Toxopneustes however clearly shows that in this species at least no fusion of the skeletal parts occurred. Instead some very interesting changes took place which may be stated briefly as follows: One of the pair of fused larvæ developed normally in every detail, the other developed in nearly every instance, incompletely. An almost perfect series of fused larvæ were obtained in which the incomplete pluteus lacked more and more of the characteristic parts that constitute the perfect larval skeleton; and the order of their disappearance was in the reverse order of their appearance in ontogeny.

The union of the two larvæ, involved the approximation of their branched and complex skeletons, whose parts frequently overlapped but never fused.

The evidence presented by experimental diabetes seems to indicate that in the transformation of amino-acids into glucose lactic acid is an intermediary step. It has also been suggested that in the combustion of glucose lactic acid may arise from the cleavage of the sugar molecule. On account of this significant place held by lactic acid it appears of interest to investigate the relation it may hold to sugar production in human diabetes. Only cases of diabetes of considerable severity are suitable for the experiment, since in the milder degrees of the human disorder relatively large amounts of carbohydrate may be burned or stored and the sugar output is apt to fluctuate. In a patient it was ascertained that with a uniform diet for three days a fairly constant glucose excretion could be depended upon and lactic acid as the sodium salt was given on the second day of the experimental period with the result that there was a rise in sugar excretion shown by the following abstract from the protocol.

In a thirteen day metabolism study on an individual suffering from myotonia atrophica, we have studied the nitrogen metabolism, and urinary nitrogen partition, the sulphur metabolism and urinary sulphur partition, and the calcium, magnesium, phosphorous, chlorine and fat metabolism. The creatinine excretion was normal. The only striking metabolic anomaly noted in this study was the marked loss of calcium.

1. Depressor and pressor substances arise after vaccinia infection in the blood-serum of rabbits. 2. Aging tends to eliminate the depressor substance and a pressor substance then comes in evidence.

Note.-A serum obtained from a rabbit after streptococcus infection, which had 12 months previously given a profound depressor reaction was also tested (3-18-13) and was found to give no reaction. 8 C.C. of a saline extraction of the adrenals of a 30 day vaccinia rabbit, gave (3-28-13) no reaction. This extraction was made in 20 C.C. of physiological saline solution and was kept 6 days in the ice-box. 7 C.C. of a saline extraction of the adrenals of a two day vaccinia rabbit gave (3-8-13) a fine rise followed by a marked fall. This extraction was made in 20 C.C. and was kept 24 hrs. in the ice-box, i. e., since immediately after removal.

We have elsewhere shown 1 that cholesterol, when injected directly into rat carcinomas, causes a marked acceleration both of the primary and of the metastatic growth of the tumors.

This led us to form the opinion that cholesterol is probably a factor of importance in determining the incidence of carcinoma.

It has been shown by Dorée and Gardner, Ellis and Gardner, and others 2 that cholesterol is not synthesized by animals, the cholesterol in animal tissues being derived from their diet. This fact suggested the possibility that the incidence of carcinoma in inoculated animals might be diminished by feeding them for a considerable period prior to the inoculation upon a diet poor in cholesterol.

Accordingly twenty-two white rats, about two months old, were divided without exercising any selection into two lots. One lot of 15 were fed upon a diet composed exclusively of milk; the remainder were fed upon a mixed diet of oats and meat.

The content of cholesterol in milk, while by no means negligible, is extremely small in comparision with the content of cholesterol in other foodstuffs. Thus Tolmatscheff finds that human milk contains from 0.025 per cent. to 0.039 per cent. of cholesterol, 1 while Bömer and Kirsten find that the fats in cow's milk contain 0.5 per cent. cholesterol, corresponding to a content of less than 0.02 per cent. in the whole milk. 2 Meat, on the other hand, contains from 0.07 to 0.08 per cent. of cholesterol, 3 while the content of phytosterols in seeds is considerable. 4

Both lots of animals thrived well, the milk-fed animals presenting an especially well-nourished appearance.

At the end of two months both lots of rats were inoculated in the axillary region with portions of a Flexner-Jobling carcinoma. The diet of each lot of rats was maintained unaltered. At the end of twenty days the proportion of successful inoculations in each batch of animals was determined, with the following results:

It has been shown by Windaus 1 that digitonin combines with cholesterol to form a very insoluble and pharmacologically inactive compound. In view of the marked influence of cholesterol in accelerating the growth of carcinoma we have thought it of importance to ascertain the influence of digitonin upon the growth of Flexner-Jobling carcinoma in rats.

The digitonin employed was Merck's, stated to have no physiological action upon the heart. By heating the preparation to boiling in m/6 NaCl solution a soapy-looking fine suspension is formed which settles out in the course of several hours. We injected the digitonin, suspended either in m/6 NaCl, or in m/6 NaCl containing 1 per cent. of lecithin, directly into the tumors.

One hundred and sixty-six white rats were inoculated with Flexner-Jobling carcinoma in the axillary region. The number of successful inoculations, determined after 20 days, was 64, or 39 per cent.

On the 20th day after inoculation these animals were sorted, without selection, into three batches, of which one (consisting of 12 animals) served as controls, another (12 animals) received injections of digitonin, and the third (40 animals) received injections of digitonin together with lecithin.

We began by administering I C.C. of a 1 per cent. suspension of digitonin, suspended in m/6 NaCl and in m/6 NaCl + 1 per cent. lecithin respectively. The animals which received digitonin without lecithin evinced symptoms of severe local irritation, and one of the animals which received digitonin alone and two of those which received digitonin and lecithin died within a few hours after the treatment. Post-mortems showed that the heart had in each case stopped in extreme systole, the auricles being engorged. In subsequent treatments the dose of digitonin was reduced to I C.C. of a 0.5 per cent. suspension, and in the case of the animals which received lecithin, the concentration of the lecithin emulsion was increased to 2 per cent.

The publishing of negative results is, as a rule, a questionable proceeding. Occasionally however, a negative result may have a positive bearing upon a subject, of more or less value, in which case the objection does not hold. It is for this reason I present the following note.

In reviewing the subject of coagulation some time ago, I was struck by a certain similarity between thrombin and alexin. Both are inactivated by a temperature of 56 degrees; both are derived from leucocytes; 1 thrombin consists of thrombokinase and calcium, while alexin consists of two parts, as is well known. It occurred to me that there might be a closer relation between the two; that they might in fact, be identical. It has probably occurred to many another, but in a hasty glance over the literature, I can find no mention of it. At first sight the idea seems absurd, for alexin is contained in serum that has been collected over a clot, and hence contains no thrombin. The alexic potency of a serum, however, increases by standing for some hours in contact with the clot, and it is conceivable that during that time thrombin is excreted by the leucocytes, but is not apparent because of the absence of fibrinogen wherewith to combine. Having occasion to prepare some thrombin for another purpose, I determined to test the matter.

The immune serum used was rabbit serum immunized against ox corpuscles. Tested with guinea-pig serum it had a potency of 1-800. (Ox blood was used on account of the ease with which material could be obtained at any time.) Most of the experiments were carried out with a 1-100 dilution, although similar results were obtained with higher dilutions.

In continuing studies on the nature of alexin fixation by mixtures of unformed proteins and their antisera, it occurred to the writer to examine whether the alexin fixation which is exerted by specific precipitates was subject to the same conditions that prevail in the case of sensitized cell complexes in their relations to the alexin fractions as first obtained by Ferrata. It is well-known, of course, that by dialysis, by dilution with weak acid in distilled water and by a number of other methods of globulin precipitation, the alexin or complement can be divided into two functional parts, one which comes down with the globulins, the so-called “midpiece” and the other which remains in the albumin fraction, the so-called “endpiece.” Neither of these can produce hemolysis of sensitized cells alone. Together they functionate. The globulin fraction can be bound to sensitized cells, forming the so-called “persensitized cells” which are now hemolyzable by the endpiece alone. The albumen fraction does not become attached or fixed to the sensitized cells except in the presence of the globulin fraction. (The terms midpiece and endpiece are used for the sake of clearness since they are terms which have become etablished in the German literature. Owing to studies which are being made by Mr. Maltaner in this laboratory we feel that a definite nomenclature which assumes an intermediate function of the globulin, fraction, is premature.)

In experiments in which alexin fractions, produced by both the method of Ferrata and by that of Sachs and Altmann, were exposed to union with precipitates, formed in mixtures of beef serum and its antiserum, we have found that the conditions which prevail are entirely analogous to those which govern the attachment of the alexin fractions to the sensitized cells. A specific precipitate may fix the globulin fraction (midpiece) alone. It may fix the albumin fraction (endpiece) in the presence of the globulin fraction. It does not however, fix the albumin fraction (endpiece) by itself. The experiments were in every case controlled by titrations of the alexin fractions and the whole alexin in tubes set up parallel with the main experiment.

It has recently been shown by Churchman 1 in two interesting communications bearing on the subject that the aniline dye, gentian violet, has a selective bactericidal action on certain bacteria. The action of the dye has been spoken of as bacteriostatic, indicating that the growth of some species of bacteria is inhibited. In addition to this inhibiting influence, Churchman believes the substance has also a very definite bactericidal action. It was shown in the publications referred to that the action of gentian violet as a bacteriostatic or bactericidal agent presented in a general way a parallelism with the Gram stain. The majority of gram positive bacteria are inhibited by gentian violet, while the majority of gram negative bacteria are not. The action of gentian violet can be observed on the divided plates or by staining with gentian violet and determining subsequently whether the microörganisms so treated will grow on culture media. On divided plates, one half of an ordinary petri dish is covered with plain nutrient agar, the other half with nutrient agar to which has been added an aqueous solution of gentian violet; such a plate when streaked with gram positive bacteria will show a growth only on the side of the plate where there is no gentian violet, while gram negative bacteria usually grow equally well on the plain agar and on gentian violet agar.

Since it seemed possible that the differentiation of closely related species might be accomplished by the use of divided gentian violet plates, the method has been used in a further study of the Mucosus capsulatus group. In all, thirty-six strains of bacteria have been investigated; they include B. ozænæ, B. lactis aerogenes, B. enteriditis, B, pneumoniæ Friedlander, B. rhinoscleromatis, B. capsulatu, and B. Mucosus capsulatus.

Experiments in this laboratory have shown that light and other forms of radiant energy exert a marked influence on the rate of wandering in the electric current. Thus the rate of wandering of arsenic sulphide suspensions is about twice as great in the dark as under strong illumination in the sun, or in the carbon arc or in the Nernst lamp. On the other hand, the rate of wandering of mastic emulsions is increased under strong illumination, and that to the extent of about forty per cent. of the value in the dark. Ferric hydroxide is retarded in the light to the extent of about six per cent., while chlorophyll suspensions are accelerated in the light to the extent of about forty per cent. The effect of the radiant energy is not in general instantaneous, but requires a few minutes exposure to reach its greatest value. In general also the effect is reversible, that is colloids whose rates of wandering have been influenced by radiant energy, return to their original values if kept in the dark for some minutes.

These phenomena are very interesting in connection with photochemical reactions in general. Arsenic sulphide suspensions may be kept almost indefinitely in the dark. On exposure to light they oxidize rapidly. Knowing now that light also reduces the negative charge on the colloidal particles of arsenic sulphide, one can arrive at a comparatively simple electrical theory of photooxidation. If one assumes that oxidation takes place when two hydroxyl ions can give up their charges, forming water and nascent oxygen available for oxidation, and that subsequently the negative charges thus set free may react with free hydrogen ions and free oxygen to form new hydroxyl ions which may again discharge and oxidize, it will be readily seen that the rate of oxidation will primarily depend on the value of the negative charge which the oxidized particle itself carries.