Experimental Biology and Medicine

1. Although many experimenters have studied the regenerative power of different kinds of protozoa no one has observed the variations in this power at different age periods after division.

2. Uronychia transfuga is a marine hypotrichous ciliated protozoon about 100μ in length. It may be cut in any plane with a fine pointed scalpel and the pieces will live for several days, thus showing a hardiness which makes it a good subject for experimentation.

Under conditions of culture in the laboratory (at Roscoff, France) the cell divides about once in 36 hours, the period between divisions being marked by definite changes in size of the cell and in arrangement of the nuclear elements (macronucleus and micro-nucleus).

3. Experiments to test the regenerative power were made upon cells immediately after division; on cells from 12 to 18 hours after division; on cells immediately before division; and on cells during division. The cells, in all cases, were cut with a scalpel under a microscope, full records of 75 experiments being kept.

In experimental physiology where it is necessary that the animal be allowed to recover after the removal of an organ or the establishment of a lesion, the operative procedure is carried out under full anæsthesia and with aseptic or antispetic precautions as in human surgery. The preliminary disinfection of the skin by scrubbing with soap and water and the subsequent washing with bichloride, carbolic or alcohol, takes considerable time and in most cases must be done after the animal is anæsthetized; besides where the operative field includes the head or face there is always danger of the eyes being accidentally injured by the irritative fluids. Then again, after operation everyone has experienced the difficulty of keeping the dressing properly applied to the wound which must be protected from outside contamination unless the animal be kept in aseptic surroundings—a condition practically impossible in most laboratories. Any method, therefore, which will materially save time and trouble and at the same time not increase the risk is particularly desirable in animal surgery and such a method, I believe, is to be found in the use of iodine as a skin disinfectant.

The occurrence of abortive cases of poliomyelitis has already been established upon clinical and epidemiological ground. Netter and Levaditi 1 have given the only specific proof of an abortive case of poliomyelitis in a human being by demonstrating, in the serum of such a case, immune bodies capable of neutralizing the virus of poliomyelitis in vitro. Their case was a child which suffered a clinically obscure, mild illness about the same time that another child of the same family suffered a frank attack of poliomyelitis.

We have undertaken a similar demonstration in nine suspected abortive cases of poliomyelitis selected from a much larger number observed during an epidemic of acute anterior poliomyelitis in Iowa in the summer of 1910. Special interest is attached to these cases because of the mildness of their symptoms, the frequency of similar cases and their epidemiological relation to cases of frank poliomyelitis.

This work was begun with the attempt to obtain cultures of bacteria-free amebas in crushed rabies brains, the idea being either that the amebas might take up the rabies organisms as food or that they might free the rabies organisms from their host cells and thus bring about, in some way, a culture of the latter, as Clegg reports having done with the B. leprœ.

Four cultures of amebas were selected for a first trial, 3, presumably parasitic (type, Entameba coli), the fourth, a saprophyte (Ameba lirnax). Of these four, only one, a culture from a case of human amebic dysentery, grew with comparative ease and some abundance on agar plates streaked with rabies brains.

I then tried normal brains as controls and found that this strain of ameba grew with almost equal ease on them; so that the problem was changed for the time from that of a possible aid in determining the nature of the Negri bodies to that of the growth of pure cultures of amebas.

Only four investigators (Kartulis 1893, Casagrandi & Barbagallo 1897, Tsugitani 1898) have claimed to obtain amebas growing free from other living organisms and none of these have been quite clear in regard to their technic nor have they apparently grown their organisms for more than two or three culture generations, which, as controls show, does not rule out the factor of subsistence upon food stored up in the amebas themselves.

We have used in this study a rat sarcoma, a mouse sarcoma and a mouse carcinoma, transplantable tumors of a high degree of virulence. The technique has been practically the same as that employed by Burrows in the cultivation of the tissues of chick embryos and subsequently by Carrel and Burrows in the growth of various mammalian tissues; that is, plasma was obtained by centrifugalizing fresh blood under conditions that prevent coagulation and allowing it to clot in hanging drops which contained small pieces of tumor tissue. These preparations were incubated at 37° C. We have found that rat sarcoma grows in both rat plasma and mouse plasma, and that this is also true of mouse sarcoma. The growth in both instances, however, seems to be more vigorous and of longer duration when homologous plasma is used.

For the character of the growth in vitro, a description of the growth of mouse sarcoma in mouse plasma will suffice. The edges of a piece of sarcoma embedded in plasma are at first fairly uniform in thickness and the piece of tissue is dense and opaque. After twelve hours of incubation at 37° numerous elongated cells project from all sides of the tissue and these wander out into the surrounding plasma by amœboid motion. The throwing out of pseudopods and the associated streaming of the protoplasm can be seen quite beautifully under the microscope. As the cells wander from the original piece of tissue, it becomes less dense and we have observed very frequently the reduction of the original piece of sarcoma tissue to only a fraction of its initial size.

A dog weighing about 13.8 kilograms was given on different days, 700 grams of meat and 1,200 grams of meat at the noon hour. In the morning the metabolism was determined for an hour, while the dog slept quietly in the respiration calorimeter. The minimum or basal metabolism thus determined was found to be about 25 calories per hour. After the ingestion of meat at noon, the animal was again placed in the respiration calorimeter, and the hourly metabolism determined. The results are given in the following table.

It will be noticed that the metabolism rises from 25 calories in starvation, to 34 calories after the ingestion of 700 grams of meat, and from 25 calories to 40 calories, after the ingestion of 1,200 grams of meat. In general, the figures agree with Rubner's conception, that about 30 per cent. of the total energy of metabolized protein is wasted as free heat, within the organism. One new point stands out prominently in the above table, and that is, that for the first time, the direct and the indirect calorimetry in hourly periods have been found approximately to agree.

The apparatus demonstrated is in many respects a miniature of the calorimeters of this type which have been constructed at the Nutrition Laboratory in Boston. The writer wishes to acknowledge his great indebtedness to Dr. Francis G. Benedict, director of that laboratory, for his invaluable assistance in working out the problem of a small calorimeter.

In order to measure with a satisfactory degree of precision the gaseous and energy metabolism of infants and small animals, it has been necessary to introduce some special modifications. As a detailed description of these modifications with a report of the control tests of the apparatus will be communicated within a short time, particular mention need not be made at present.

Through the kindness of Dr. Lusk these experiments were made in conjunction with him, Dr. Williams and Mr. Riche.

As yet experiments have been made on only two children, but the results are given on account of the interest attaching to the first direct determination of heat produced by children.

The children were healthy, were gaining weight and were fed every four hours so as to keep their metabolism as constant as possible. They were put on the portable metabolism apparatus devised by Dr. Du Bois that allows great freedom, accurate collection of all the urine and feces and no discomfort. They were kept awake during the forenoon, fed at 1 p.m. and put at once into the calorimeter where they usually slept throughout the whole experiment.

Some of the results are given in the chart. It will be seen that the amount of heat produced varied considerably from hour to hour, and that during the same hours of different days with a constant diet the heat produced is not the same. The average for the hours in which the conditions were entirely satisfactory was 14.18 calories. The metabolism directly determined compared moderately with the metabolism as calculated in about half the cases. In the other half of the cases a low respiratory quotient indicated an erroneous oxygen determination, which invalidated the calculations.

Preliminary Report.

The object of this investigation was to determine the functional capacity of the kidneys of nephritics with regard to their power of eliminating nitrogenous material, water and salts, and to determine the influence of the “protective therapy” on the kidney efficiency.

We have up to the present the reports of nine cases that have been investigated for periods of two to ten weeks.

The patients after admission to the ward, except when an immediate change in diet was necessary, were placed on a general hospital diet for about three days. The urine and feces were collected and analyzed. After this preliminary test the patients were placed on a diet composed of two quarts of milk, with enough cream and sugar to supply the necessary calories. This diet contained 12 to 13 grms. of nitrogen, 3 grms. of NaCl and about 1,500 c.c. of water. A normal individual should reach nitrogenous equilibrium on about the third day of its administration. Several of our nephritic cases behaved in the following manner:

An aqueous solution of eosin will give with ferric chloride a precipitate which has a deep red color. If very dilute solutions are used, say 0.1 of 1 per cent. ferric chloride, and an eosin solution which is just barely pink, no precipitate will be visible on mixing the two, but the pale pink of the eosin changes to a deeper shade of red, and the fluorescence, which is quite noticeable even at this dilution, disappears. If the dilution of the eosin solution used be increased so that these color changes can no longer be distinguished with certainty, it is possible to demonstrate the reaction by the addition of a colloidal suspension of Witte's peptone, or of sodium oleate.

On the addition of such a solution, which should be sufficiently strong to cause a well marked opalescence, the red color of the iron-eosin stain will at once be apparent, especially where sodium oleate has been used, in which case a flocculent precipitate stained a rich red will appear. The examination under the microscope of such a precipitate, reveals an appearance which, in respect to color is apparently identical with that of oxyphilic granulation. It would appear that we have here not a true stain, in the sense of solution, but rather a case of condensation of the stain on the surface of colloidal particles.

Our experiments have reference to statements in literature, some of which, for a better understanding of our results, may be here quoted very briefly. On the basis of analyses of the contents of the gastro-intestinal canal of seven rabbits, Fischer and Moore make the statement that “the small intestine of the rabbit contains no sugar, when an animal is killed shortly after having consumed several hundred grams of carrots and carrot tops or cabbage.” J. B. MacCallum reported that in a few experiments with intravenous injection of sodium chloride which, as is well known, produces glycosuria he found some dextrose in the stomach and intestines the quantity of which was slightly increased when, previous to the infusion of the sodium chloride, both kidneys were ligated. MacCallum thought that the presence of glucose in the intestines under these circumstances is due to the hyperglycemia and looked upon it as a sort of “intestinal diabetes”; he believed further that in the absence of the kidneys the intestines assume a supplementary excretory function. Fischer, on the other hand, draws the conclusions from some experiments that hyperglycemia alone does not lead to a secretion of glucose into the intestines. This occurs only, he maintains, when the infusion of sodium chloride is administered to hyperglycemic animals as was the case in Mac-Callum's experiments on account of the use of morphine.

Soon after the introduction of the method of intratracheal insufflation, experiments were begun to study the control of strychnine poisoning by means of this method. So far three series of investigations were carried out. In the first series Shaklee and Meltzer employed, besides insufflation, curare and intravenous infusions of Ringer's solution. In these experiments it was first established that in intravenous injections, 0.4 mgr. of strychnine represents a reliable fatal dose per kilo dog. When using insufflation, curare and Ringer's solution quite a large percentage of dogs were saved which received even more than the fatal dose. Out of 6 dogs which received 0.5 mgr. per kilo, 5 were saved, and out of 22 dogs which received 0.8 mgs. p. kilo 13 were saved.

In a second series of experiments, carried out by the present authors and communicated at the last meeting of the American Pharmacological Society, ether was substituted for curare. Twenty dogs which received 0.8 mgr. strychnine per kilo, that is, twice the fatal dose, were subsequently treated by intratracheal insufflation, ether and intravenous injections of Ringer's solution. All these dogs survived, none died later from after effects and when finally killed the autopsy revealed no abnormal conditions in any of the animals.

In the first of two demonstrations, the authors lowered a cylindrical rubber bag, one and one half inches in diameter and eight inches long, into an oiled muslin bag of about the same dimensions. The rubber bag was then filled to overflowing with olive oil. The rubber bag expanded, as the oil filled it, to the full length and width of the muslin sheath. The sheath prevented further extension of the rubber bag and imparted rigidity to the osmometer that was ultimately constructed. The full double bag, with its mouth wide open, was then raised so as to enclose about an inch of the lower end of a long glass tube which was firmly supported vertically above the demonstration table. The glass tube was 5 feet long and its bore was 4 mm. in diameter. Ligatures were tightly secured around the neck of the double bag against the immersed lower end of the vertical tube. The bag then hung directly from the end of the tube. The bag and its sheath were in a tightly distended condition and a stationary column of oil an inch high in the tube was visible above the protruding edge of the sheath. The tube and bag were then lowered into a salt mouth liter bottle on the table until the bag almost touched the bottom of the bottle. The height of the bottle and the length of the bag were nearly equal. The tube was then marked with a label on the plane of the oil meniscus just above the neck of the bag, and enough ether was poured into the bottle to provide immersion for the bag to the depth of an inch. For a moment no change in the volume of oil was apparent, and the lateral pressure of the ether was obviously without mechanical effect. But in a minute or two diffusion currents were visible along the surface of the bag and oil rose rapidly in the tube.

The author has found that many fat-soluble pigments, such as Sudan III and Scarlet R, readily diffuse from solid and liquid fats through rubber into various solid and liquid media, among them both solid fat and oil. Thus, when Sudan III is dissolved in melted lard, the red liquid poured into a rubber bag, the bag supported in melted lard in a bottle, and the apparatus promptly immersed in ice water—the fatty matter will congeal before any sign of pigmentary diffusion occurs, but, in a few hours, a reddish tinge will develop outside of the bag, and on each successive day for several weeks further extension of the pigmented matter may be witnessed, until the whole of the external lard is deeply suffused with red. This process takes place quite rapidly when the lard and apparatus are kept in a thermostat at 40° C.

The demonstrations were intended to exhibit a few instances of such pigmentary diffusions as occur speedily enough at room temperature to yield positive results within an hour. The appended summary indicates briefly the precise nature and results of the demonstrations (including two control tests—4 and 5), which were made with thin rubber bags in ordinary glass bottles.

When dry bags of rubber, gold beaters'skin, parchment and collodion, each containing olive oil and Sudan III, are separately immersed in olive oil, and the remaining conditions of the environment are uniform, diffusion of the pigment promptly occurs through rubber, but does not take place at all through any of the other three membranes. When the bags are lifted from the oil, washed externally with ether, and then immersed in ether, 2 the pigment quickly passes through the rubber, but diffuses very slowly if at all through the remaining membranes.

Successive immersions of moist impermeable membranes (gold beaters'skin and parchment) in alcohol and ether, for different periods of time, fail to render the treated membranes more permeable to Sudan III under the conditions of the experiments already described.

The authors demonstrated the general facts in this connection pertaining to rubber and gold beaters'skin.

Experiments along these lines, with additional membranes, pigments and liquid media, are in progress, in an effort to obtain further knowledge of the functions of membranes in diffusion.

In view of the importance of the occurrence of spontaneous lesions in the kidneys and in the livers of rabbits and of guinea pigs in reference to the experimental work on these organs, Dr. E. C. Dickson and myself have made a careful study of these organs and incidentally of the heart and of the aorta in fifty rabbits and in one hundred guinea pigs. The animals used were partly fresh animals from the market, partly animals raised at the laboratory.

Many of the rabbits had been used in the physiological laboratory and had been killed immediately after the experiments; some died of coccidiosis; a few had been injected with material supposedly containing pneumococci without result; others had never been used. Several old rabbits that had been in the laboratory for a year or longer were especially selected on account of the greater likelihood of the existence of renal or hepatic lesions in them. Twenty-eight rabbits, among them some of the old ones, had entirely normal kidneys, nine showed slight parenchymatous lesions, three a few small areas of cellular infiltration. In ten we found scattered small areas in which were marked interstitial lesions with the formation of small depressions on the surface. Four of these proved to be radially arranged chronic septic foci which extended from the vicinity of the papilla to the outer surface.

Among the very many rats examined in San Francisco for plague only very few were found that showed evidences of dropsy and of renal disease. One such rat was carefully examined at our laboratory through the courtesy of the U. S. Public Health and Marine Hospital Service. This rat showed a very marked general oedema. The kidneys are small, distinctly granular. The measurements are 26 × 14 × 10 mm. The heart is moderately but distinctly enlarged, measuring in the formalin hardened specimen from base to apex—23 mm., the largest transverse diameter is 19 mm. and the largest antero-posterior diameter 14 mm. There are no gross lesions of the aorta. In sections of the kidneys the blood-vessels are found in a normal condition. The glomeruli are also quite normal except a few which show slight fibrous thickening of capsule and slight cystic dilatation. The epithelium in practically all tubules is very markedly and extensively degenerated, showing granular and fatty degeneration. There are many casts. Large bunches of peculiar unidentified, uncolored needle-shaped crystals are also found in the tubules. Large areas in the sections show collapse of tubules with much cellular infiltration and new formation of cellular fibrous tissue between them. The appearances are those of a chronic parenchymatous nephritis. It is possible that these lesions are the result of one or the other poison, such as arsenic or phosphorus, which were used in destroying rats in this city. In this connection it is noteworthy however how rarely such lesions were observed in spite of the very extensive use of such poisons.

Dreyer's demonstration that splanchnic stimulation increased the content of adrenal secretion in blood from the adrenal veins has been confirmed by several observers. Adrenal secretion therefore is under control of the sympathetic system.

Major emotional disturbances indicate the dominance of sympathetic impulses. In the cat, for example, fright causes dilation of the pupils, inhibition of the stomach and intestines, rapid heart, and erection of the hairs of the back and tail. Do not the adrenal glands share in this widespread subjugation of the viscera to sympathetic control?

To try this suggestion the inhibition of contraction in strips of longitudinal intestinal muscle, sensitive to suprarenin 1:20,000,000, was used as a biological test. Blood was obtained from the cat when quiet, and again after the animal was excited by the presence of a barking dog, by introducing, through the femoral vein, into the inferior vena cava to the region of the liver, a small vaselined catheter. The blood thus obtained was defibrinated and applied to the intestinal strip at body temperature.

Between June, 1908, and August, 1910, a number of tests upon the feces of cattle were made at the Illinois Agricultural Experiment Station to determine the presence or absence of tubercle bacilli. These tests were of two kinds, (1) by direct microscopic examination and (2) by inoculation of animals.

The microscopic method included the examination of stained slide preparations of (a) mucus on the exterior of the fecal mass, (b) the mixed feces, and (c) watery mucus scraped from the rectum, each stained slide being searched fifteen minutes with the aid of a mechanical stage. If organisms indistinguishable in appearance from bovine tubercle bacilli were found upon any of these slides the examination was recorded as positive. One hundred and nineteen samples from 53 cows which had reacted to tuberculin were examined in this way, with a positive result 53 times in samples from 34 of the cows. Thirty-five other samples from these same cows were negative. Nineteen cows gave negative results at all examinations, 31 in number. Thus, of the 53 cows tested, 34, or 64.2 per cent., gave a positive result at one or more tests, and 19, or 35.8 per cent., gave only negative results. Forty samples from 18 non-reacting cows were tested in the same way, with 23 positive and 17 negative results. Fifteen of the 18 cows, or 83 per cent., gave a positive result at one or more tests, while only 3, or 17 per cent., were negative at all tests. From this it appears that no significance in respect to the presence of tubercle bacilli can be attached to the finding of a few acid-fast bacteria in the feces of cattle.

Renewed investigation concerning the phenomena provoked by intravenous administration of Witte's peptone has demonstrated the production of a marked glycosuria, following such injections. The appearance of sugar in the urine is accompanied by hyperglycæmia. The experiments were carried out for the most part upon dogs. When Witte's peptone is injected into the rabbit glycosuria is not in evidence, an observation which is in entire accord with the failure of this substance to induce certain other phenomena in this animal which are brought about in the dog.

The tentative hypothesis is advanced that the presence of sugar in the urine is induced as a result of the respiratory disturbances set up by the “peptone” injection.

The phenomena connected with the injection of “peptone” mixtures are being subjected to further investigation.

Males and females of the fly Drosophila amphelophila occur in nature in about equal numbers. A long series of experiments failed to induce any change in the sex-ratio by adding different sugars, salts, acids or alkalies to the food on which the larvæ live. A remarkable alteration of the sex-ratio took place, however, in the second generation of certain crosses between two races that arose as mutations. When a male of a race with rudimentary (“short truncated”) wings was crossed to a female of a race with short proportionate wings all of the females had normal wings and all of the males short proportionate wings, like the mother. When these (F₁) were inbred, there were produced in the second generation three classes of individuals according to the character of the wings; namely, long (normal), short proportionate, and short rudimentary distributed according to sex as shown below: