Experimental Biology and Medicine

In a previous work 1 we were able to show that regularly constant sensitization of guinea pigs could be produced through the inoculations of heat-killed agar cultures of different moniliae. A comparative study of the sensitizing capacity of the Monilia pinoyi and Monilia psilosis demonstrated that while the former fungus could sensitize guinea pigs even after a single injection of a moderate dose of the culture, it was necessary to use repeated injections of the latter in order to obtain a successful sensitization of the animals. In view of the close serological and morphological relationship between these two species such a difference in the sensitizing capacity was difficult to explain.

On the basis of the above study the suggestion occurred to us that the sensitizing capacity of different organisms such as moniliae may represent an independent function of the fungus protein. If such be the case, it would seem reasonable to expect that this function of the fungus may be diminished or completely destroyed through the use of a suitable procedure. It is known that the addition of formaldehyde to bacterial products, such as toxins, or to bacterial vaccines brings about a considerable diminution or destruction of the toxic principle. Of especial significance is the fact that within certain limits, the removal of the toxic principle by formaldehyde is not associated with the decrease or disappearance of the antigenic property of bacterial toxins or vaccines.

X-rays are absorbed by a substance through which they pass in proportion, first, to a power of the atomic weight and, second, to the mass of the substance penetrated. This principle is fundamentally concerned in clinical roentgenology, for it permits certain tissues to be distinguished from others on the roentgen-screen and film. The normal lung is a case especially in point. Its average atomic weight is less than that of bone and its mass per unit thickness is much less than that of the neighboring soft tissues, owing to its infiltration with air. The organ is, therefore, more radiolucent than its environs. When air leaves the lung completely and the tissues shrink together, as in atelectasis, the mass per unit thickness becomes approximately the same as that of other soft tissues and the organ may not be distinguishable by x-ray examination from them. However, during certain roentgenologic observations by one of us on specimens of atelectatic lungs, the contrast between the density of atelectatic and air-containing lobes seemed greater than it should be, if atomic weight and mass alone were responsible. The matter was put to the following tests:

A dog was operated upon surgically and the main bronchus of the accessory lobe was ligated. The animal was sacrificed 24 hours later. The lobe was removed and found to be totally airless and collapsed. It was packed into one end of a cardboard cylinder, 3.7 cm. in diameter and 12 cm. in length, and the cylinder was placed upon one quadrant of a roentgenographic film-casset with the lobes next to the casset. The other quadrants were covered with a lead sheet. The ensemble was brought beneath, and 65 cm. away from, a linical x-ray tube, so that the rays could pass longitudinally through the clinder onto the film, and an exposure was made.

“Thorotrast” 20 is a fluid which is said to contain thorium dioxide, 25% by weight, in colloidal suspension and which was developed as an injection medium for clinical pyelo-cystography. Kadrnka 1 also administered the drug intravenously in man and animals and found that the thorium was taken up by the liver and spleen in such quantity as to render these organs radiopaque and plainly visible in the roentgen-film. He formulated a routine method for the purpose which required total dosage of 0.8 to 1 cc. per kg. body weight, given in fractions on several consecutive days. He saw no ill effects from this in most cases—in man occasional vomiting, and once in rabbits persistent hemoglobinuria. The only alterations in the blood were slight transient anemia and leukocytosis, although changes in coagulability were apparently not sought. Except for the presence of granules, believed to be metallic thorium, in the reticulo-endothelial cells of the liver and spleen, no gross or minute lesions were discovered. We are studying further the action of this drug in laboratory animals and report production of a purpuric disease.

Eight rabbits were used. Four were given intravenous injections of “Thorotrast”, 0.5 cc. per kg., on 2 consecutive days, and 4 were given larger doses. Three of the latter received a single injection of 3 cc. per kg., and the fourth received 3 cc. per kg. each day for 3 consecutive days. Three of the first 4 and 2 of the second had blood tests immediately before the initial injection and again 4 to 6 hours after the last injection, consisting in determination of bleeding time and clotting time, volumetric measurement of the thrombocytic content of the blood, and observations of clot contractility.

A highly satisfactory and differential medium for the study of “S” and “R” forms of pneumococci is described. The medium consists of ordinary beef infusion agar pH 7.8 to which 5 to 10% defibrinated blood of the dog is added. To obtain the optimum result, the organisms are plated on such a medium and incubated for 16 to 18 hours. On examination with reflected light the “R” colonies present an extremely rough surface in distinct contrast to the smoothness of “S” forms.

Evidences point to the dog blood cells as furnishing the important constituent of such a medium.

It is well known that muscles, like blood corpuscles, contain more potassium than sodium, whereas blood plasma contains more sodium than potassium. The unequal distribution of the cations between the corpuscles and the plasma has been shown experimentally 1 and theoretically 2 to be due to the impermeability of the corpuscle membrane to cations. Although a similar explanation may be offered in the case of muscle, no conclusive proof is available. Mauriac, Aubel and Boutiron 3 found that muscle immersed in experimental edema fluid gained Na but lost K, indicating that the muscle membrane is permeable both to Na and K. The permeability of K was confirmed by Wojkczak 4 who found that muscle in nutrient solution containing no glucose lose K. Mond and Amson 5 also found that resting uninjured muscle was permeable to K but impermeable to Na. Callison 6 in a study on the so-called “bound potassium” of muscle found that varying amounts of potassium went into the Ringer solution in which cut pieces of muscle were placed. Thus, while there is general agreement as to the permeability of the muscle to K, the evidence is conflicting with regard to Na. Monauni 7 had reason to believe that even K does not really penetrate through the cells but only through the interstices.

A general criticism which may be raised against all the previous work on the subject is the fact that they are in vitro experiments. In the course of a study on the swelling of muscle we had occasion to test the permeability of muscle in vivo to sodium, and we have obtained results which, we believe, are conclusive.

Rabbits were anesthetized with morphine hydrochloride. The abdominal wall was slit open, the ureters were tied, and the abdominal wall closed. 20 cc. of 25% NaCl solution were injected into the plèural cavity.

Nutritional edema is apparently another type of edema in which the mechanism of its production is related to the low level of plasma proteins, similar to that of nephrosis and plasmapheresis. In nutritional edema, however, the lowered plasma proteins are brought about by a deficiency in protein in diet. Denton and Kohman 1 produced edema in a large proportion of rats fed largely on carrots. This work was confirmed by Frisch, Mendel and Peters, 2 who in addition showed that the plasma proteins were low in their rats. Recently Shelburne and Egloff 3 succeeded in producing edema in a dog fed on a low protein diet. We add the following preliminary report of a study on the nitrogen balance, plasma proteins and extent of edema in 2 cases of nutritional edema under the influence of different dietary regimes.

The 2 patients, aged 11 and 20 respectively, were inmates of an orphanage in which they subsisted for a long time on a diet deficient in protein. They presented evidence of undernutrition with marked edema. There was no evidence of cardiac or renal disease. Case 1 had a total plasma protein value of 3.46% and albumin 1.43%. Case 2 had 4.01% for total proteins and 2.17% for albumin. Throughout the 21 periods of 4 days each their total caloric intake was kept at 1400, salt at 6 gm., and water at 1500 cc, while the level and character of the proteins in the diets were varied. Each diet was given for 2 to 5 periods. Diet 1 contained 23.5 gm. (Case 1) and 27.5 gm. (Case 2) of vegetable proteins, estimated intake of protein for them while in the orphanage. With this diet their nitrogen balance was barely maintained, and their plasma proteins showed a slight tendency to increase, and their edema after an initial decrease did not completely subside.

The 2 patients reported in the previous paper were studied from the standpoint of the effects of displacements of their blood acid-base balance on nitrogen balance, plasma proteins and edema. They were given known diets containing 1400 calories and enough vegetable proteins to secure nitrogen balance. The salt and water intake was kept approximately constant. Alkalosis was produced by the administration of sodium bicarbonate 12 to 20 gm. per day for one to two 4-day periods, and acidosis by 10% hydrochloric acid 30 cc. per day for 3 days or ammonium chloride 8 gm. per day for 4 or 5 days. There were altogether 3 experiments with alkali, one experiment with hydrochloric acid and one experiment with ammonium chloride with each individual. The results can be summarized as follows:

With each alkali administration, there was definite increase in edema with increase in body weight reaching a maximum within 3 or 4 days. Thereafter the edema began to subside and weight began to decrease in spite of continued alkali ingestion. With hydrochloric acid edema showed a slight diminution in Case 1 and remained unchanged in Case 2. The ingestion of ammonium chloride, however, resulted in a decrease of edema and weight in both cases.

The nitrogen balance in either case remained undisturbed with alkali or acid administration. The plasma proteins showed a slight lowering during several of the periods of alkali administration, but practically no change during the remainder. The variations in plasma proteins during acid periods were slight and inconsistent.

Since the introduction of the use of prophylactic typhoid vaccine, confusion has risen in interpreting the Widal agglutination reaction in the diagnosis of typhoid fever. Recently Felix 1 introduced the technic of demonstrating the presence of small-flaking or “O” agglutinin, which he claimed to occur only in infection but not after subcutaneous inoculation of the vaccine. Gardner 2 found that while “O” agglutinin can be produced by subcutaneous inoculation of the vaccine, it very rarely reaches a titer higher than 1:200. We wished to know whether this “O” agglutinin can be produced by repeated intravenous injections of the vaccine and whether its titer would be higher than that occurring after subcutaneous injections. After this work was begun, we noticed that Pijper and Dau had reported the finding of high titer “O” agglutination after oral ingestion of typhoid vaccine. 3

Large-flaking or “H” antigens were prepared by the usual method, namely, agar washings in saline or broth of B. typhosus, B. paratyphosus A and B. paratyphosus B preserved with 0.1% formalin, and used with suitable dilution. Small-flaking or “O” antigen was prepared from B. typhosus (Rawlin's strain) treated with alcohol; a modified Bien's method 4 was used which will be described in detail later.

In the complement fixation test for the diagnosis of syphilis or other diseases, it is usual to use an anti-sheep hemolysin for the production of whiclv rabbits have been employed almost exclusively. Numerous methods have been proposed for the immunization of these animals to produce a high titer serum, but we have found in our routine immunization of such animals that they vary considerably in their individual response to the injections of sheep erythrocytes.

For a routine diagnostic laboratory handling a large number of Wassermann tests where a constant and reliable supply of hemolysin is required, it seems desirable to immunize a larger animal which will yield a supply of hemolysin sufficient to last for a number of years. This will obviate the necessity of immunizing rabbits for this purpose at short intervals and will result in an economy in time and money. Attempts have been made to produce anti-sheep hemolysin on horses but Gilbert 1 was unable to produce it in a horse showing no natural hemolysin in the blood, but was successful with a mule having a small amount of hemolysin before immunization. As these animals are rather costly, it appears to us worth while to find a less expensive experimental animal.

Our choice has accordingly fallen on the donkey, a common beast of burden, which is relatively inexpensive, costing about 20 times the price of a rabbit here, and is of a convenient size for our purpose. Before immunization the blood of the donkey was first tested for the presence of natural anti-sheep hemolysin, but not a trace of it was present. Immunization was then begun by a series of inoculations given through the ear vein. All series of injections were given on 3 successive days at 4-day interval between each series and trial bleedings were taken each day before injection.

The technique for the preservation of the supravital stain of neutral red in paraffin sections has been described by Mcjunkin 1 and Forkner. 2 Their methods enable one to observe most of the neutral red present in the cells; but a certain amount of the stain is inevitably lost in the process of preparation, and they require rapid dehydration and strict limitation of the size of the tissue blocks. The method described below has the advantage over those previously described, in that the entire neutral red stain within the cells is faithfully preserved, that the size of the blocks can be reasonably large, and that the different steps of staining can be carried out in a more leisurely and comfortable manner.

In this technique, advantage is taken of the fact that neutral red is only slightly soluble in aqueous or alcoholic solution containing mercuric bichloride. After the minimal amount of the bichloride required in each solution for the maximal insolubility of the neutral red is determined, any solution which will check further dissolution of neutral red from the tissue can be easily prepared from it.

The tissue to be studied is best stained by the direct injection method described by Forkner with only a slight modification through replacement of his Zenker-formalin by solution A (see below).

For carrying out this technique we use the following 4 different special solutions:

Solution A Fixing solution, prepared as follows:

Dissolve a small quantity of neutral red in 15 cc. of 40% formalin in a large flat dish. Add to it 85 cc. of Zenker's fluid. Most of the neutral red previously dissolved in formalin is now precipitated out. After a few minutes, during which time the mixture may be stirred with a glass rod, the mixture is filtered for use.

The fact that different authors vary in their opinion concerning the rôle of the spleen as a reservoir for red blood cells (Radosaljevic and Sekulic, Feldberg and Lewin, and others 1 ) has induced us to study this problem.

In the first group of experiments, we studied the effect of intravenous injection of adrenalin on the peripheral blood picture of normal anesthetized dogs. Chloralose was the narcotic chosen because of its dilating effect on the spleen. In 16 dogs the curve of hematocrit determinations (femoral vein) showed an average maximal increase of 13.4%. In a second series of experiments we compared the curves of the hematocrit values of the splenic vein, portal vein and vena cava (or vena femoralis) of dogs after the injection of adrenalin. It was found that a marked but brief increase of the hematocrit numbers takes place in the splenic vein incident to the rapid contraction of the organ. On the other hand, the co-incidental hematocrit values of the portal vein or of the vena cava increase gradually. The curves in Fig. 1 demonstrate clearly the fallacy of single determinations.

In a third group of dogs the tests were repeated under similar conditions but following splenectomy. No or only a slight increase in the hematocrit numbers was found after the injection of adrenalin. In a fourth series of experiments the liver of dogs was completely excluded from the portal circulation by means of a reversed Eck fistula. No differences were noted in the curves of the hematocrit values as compared with normal dogs.

The purpose of the experiments was to determine the regulatory mechanism responsible for splenic contraction and dilatation. Tournade and Chabrol 1 believe in a combined nervous and hormonal control of the splenic contractions and Izquierdo and Cannon 2 observed no erythrocytosis after exposure to low oxygen tension in an animal in which the medullary substance of the adrenal glands had been destroyed.

In 20 normal dogs under chloralose narcosis, the hematocrit curve ol the peripheral blood was studied after the injection of adrenalin, after short asphyxia and after bleeding. The dogs were then adrenalectomized (2 stage operation) and on 11 of the animals which survived adrenalectomy for more than 2 days, the same tests were repeated and the results compared. It was found that the hematocrit curves after adrenalin injection show no or only a slight decrease in their maximal elevation after adrenalectomy. In contrast to this, the hematocrit values after brief asphyxia and after bleeding did not show the elevation found in normal dogs. In Fig. 1 the curves of hematocrit determinations of an adrenalectomized dog after adrenalin injection and after short asphyxia are shown. Fig. 2 records hematocrit curves obtained after bleeding of adrenalectomized and normal dogs.

The results seem to point to the assumption that adrenalin is the significant factor responsible for splenic contraction and the resulting erythrocytosis. As it is possible that the polycythemia and the change in the size of the red blood cells after adrenalectomy may be factors which influence the hematocrit curves, one of us is repeating the experiments and studying the contraction of the spleen of adrenalectomized animals in vivo by skiagraphic visualization methods.

From these results, it is suggested that glucose alone should not be used postoperatively and certainly not in the presence of intestinal obstruction, because glucose alone in the majority of instances exerted an inhibiting effect on intestinal activity. Insulin, OS the combination of insulin and glucose produced an increase in intestinal activity. The best results were obtained when insulin preceded the administration of glucose by about one-half hour's time. There was no correlation betn-een the intestinal activity and the hlood sugar findings.

The reactions described here bear a relationship to the subject of nonspecific protein therapy although there is no intention at this time to consider this phase of the subject. In connection with the study of nonspecific protein reactions, many observations have been reported upon the leucocytic response especially in the human being, and particularly following the intravenous injection of B. typhosus. A full review is given by Petersen. 1 Ling 2 reported various blood reactions both in the human and in the rabbit, employing several substances for injection, including milk, peptone, horse serum and B. typhosus. Most of the injections were administered intravenously. He obtained only a very slight response in his leucocytic reactions.

We wish to report the comparative leucocytic reactions produced in the rabbit by the subcutaneous, intraperitoneal and intravenous routes of injection for 8 different substances.

Twenty-four full grown rabbits, chiefly males, were used and the following antigens employed: Cysteine, peptone, globulin, market milk, Staphylococcus aureus, Streptococcus hemolyticus, B. typhosus and the supernatant portion of a sero-purulent exudate obtained from a streptococcal arthritis. The latter was centrifugalized and the supernatant portion used as such and also after filtration through a Seitz filter. The unfiltered portion was found to contain a few scattered chains of living streptococci.

Many reports describing the normal leucocytic findings in the abbit have been published. (Pearce and Casey, 1 Bushnell and Bangs, 2 Kleineberger and Carl. 3 ) The estimations of these workers are tabulated below:

The findings of Scott and Simon and of Cunningham, Sabin, Sugiyama and Kindwall are cited by Pearce and Casey.

In our observations upon the normal count of rabbits, the blood from the marginal ear vein of 27 normal full grown rabbits, mostly males, was employed. Two hundred counts were made from these animals, and the following estimations obtained :

While estimating these total leucocytic counts, it was noted after several counts had been made on successive mornings that an afternoon check made on the third day showed in some of the animals a very marked increment in the number of white blood cells. Without any apparently sufficient reason, the count made in the afternoon from the ear which had been used to collect the blood, would rise from 15,000 to 25,000 cells per cu.mm. The blood collected from the opposite ear at the time of these increased counts would show a normal result, i. e., a difference of 7,000 to 17,000 cells, with no change in the differential count.

While it is to be expected that any frank inflammatory reaction in a given area will yield a local accumulation of leucocytes with a neutrophilia, it is to be noted that in these animals the ears appear normal and present no cardinal signs of inflammation, and that the blood was collected from the circulatory venous system and not from the more diffuse capillary network.

In a preliminary study undertaken to ascertain the changes produced in excitability of the excised turtle ventricle by rest and activity, 11 hearts were used from Chelydra serpentina and Emys sp. The Lapicque potentiometer and chronaximeter were used. The stimuli were applied by way of non-polarizable electrodes.

The results, confirming and extending those previously reported by Garrey and Ashman 1 and by Ashman and Hafkesbring, 2 were variable, but certain conclusions clearly emerge.

1. Excitability, expressed either as rheobase, chronaxie or least time is greatly influenced by activity.

2. In hearts in good physiological condition when excised (3 examples) there was a treppe in excitability, comparable to the treppe in contractility, and the treppe was demonstrable both in rheobase and “least time.” Our evidence shows, although less clearly, that there was a treppe in chronaxie. Expressed in terms of quantity of electrical energy required to excite the tissue, activity definitely increased the excitability.

3. In 6 other hearts, also in good or fair physiological condition, it was impossible to demonstrate a definite treppe in rheobase, yet in these the treppe in least time and probably, therefore, in chronaxie was definite or striking. Compared with the resting heart, after several responses the least effective duration of a current of constant strength may be decreased by 20 to 25%.

4. In 2 ventricles the manifestations of fatigue completely masked the treppe. In these the rheobase was markedly raised by activity, although the chronaxia was shortened. In terms of the quantity of energy required to excite the tissue, activity here decreased the excitability. This again illustrates a point previously stressed, 3 i e., that the chronaxie is not necessarily a measure of excitability.

(1) Colon bacilli and staphylococci are commonly present in the urine of so-called normal dogs. (2) Organisms are very easily demonstrated in the glomerular capillaries, Bowman's capsule, and the tubules within the first 36 hours after injection of the renal artery with 24-hour cultures. (3) Areas in the kidney are found, following injection of the renal artery, in which there is complete necrosis of the tubules, but normal glomeruli and interstitial tissue are present showing that the tubules derive their main blood supply from the efferent vessel of the glomerulus. (4) The irijection of one renal artery in this investigation produced no lesions in the contralateral kidney. (5) Staplzylococcw aureus produced cortical abscesses after 22 hours, whereas the colon bacillus seldom produced abscesses, but resulted in degeneration of the distal tubules when from 4 to 6 agar slants were introduced into the renal artery. (6) Acute glomerulo-nephritis can be produced by injections of Staplzylococcus aureus and colon bacillus into the renal artery. (7) Chronic interstitial nephritis can be produced within a period of 3 weeks, especially following injection of colon bacilli.

Domogalla 1 and Surber 2 have used sodium arsenite to control vegetation in lakes and in sloughs respectively. Wiebe 3 has shown that concentrations of arsenic required to kill the vegetation are not harmful to our warm water fish: bass, bluegill, crappie, bullhead, golden shiner, and the common goldfish. The present investigation was undertaken to ascertain whether or not the fish when exposed to arsenic treated water, will acquire and store sufficient arsenic to make them unfit for human consumption.

The investigation shows: (1) That freshwater fish taken from natural waters contain appreciable quantities of arsenic; control fish from the Rock River (Ill.) gave arsenic values ranging from .100 mg. to .425 mg. of As₂O₃ per kg. (2) That fish acquire a certain amount of arsenic from arsenic treated water: the average amount of As₂O₃ per kg. from 9 control fish from the Rock River was .390 mg.; the average for 5 exposed fish was .886 mg. Domestic controls (9 fish) gave a negative test for arsenic. The treated fish (5 individuals) average .703 mg. As₂O₃ per kg.; the range being from .411 mg. to .965 mg. per kg. (3) That the arsenic content in any lot of fish is variable. This fact is brought out by the above figures. (4) There is not exact correlation between the arsenic content of the fish and the concentration of arsenic in the water and the time the fish are exposed; fish exposed to a total of 8 p.p.m. for 6 days contained more arsenic than fish that had been exposed to a total of 15 p.p.m. for 42 days. (5) That arsenic is eliminated by the fish. One bass was fed 3 mg. of As₂O₃on July 22, when analyzed on July 31, 9 days later, only .019 mg. of As₂O₃ were recovered.

This syndrome was first established by C. and O. Vogt, 1 who considered as its most characteristic feature the occurrence of quantitatively and qualitatively abnormal myelinated fibers in the striate body, giving it a peculiar marble-like appearance. They considered this condition as restricted to the striate body, and advanced the hypothesis that it was the result of a faulty development. Subsequent findings showed that the original assumptions were not justified. Bielschowsky 2 found similar changes in the cortex, showing that the condition was not restricted to the striate body; Anton 3 , Meyer 4 , Scholz 5 , and others suggested that the lesion might be the result of some acquired process (inflammatory or other vascular) rather than a developmental deviation.

Our investigations were particularly concerned with these 2 phases of the problem, and are based on a study of 4 cases. Histological examination of 3 of these left no doubt that the condition was that of “Status Marmoratus”, whereas the fourth case, still living, shows a clinical development and picture that makes this diagnosis quite probable. In all of these there was no hereditary predisposition to this disease, and the early development was such that in at least 3 of the 4 there was no reason to suppose that they were not normal to begin with. In all of these the condition seems to have developed following acquired diseases. Histologically too the condition appears to have developed on the basis of acquired processes, most probably inflammatory. In one of the cases, the “status marmoratus,” furthermore, was limited to the cortex, none of the lesions being found in the striate body. In this case the condition is chiefly localized around blood vessels which show signs of a previous inflammatory process.

Since Burns published his paper on “the sex of parabiotic twins in amphibia,” 1 it has been found difficult to reconcile his statements and his general conclusions with the results of other experimental studies in sex development of vertebrates. He alleged that everyone of 80 pairs of Ambystoma maculatum joined in parabiosis at the tailbud stage had developed into unisexual twins, 36 being of the female and 44 of the male sex. The absence of heterosexual combinations was said to suggest that embryomc sex differentiation is a hormone controlled reaction of the “all or none” type. Moreover, the approximate 1:1 ratio of male and female pairs was taken as an indication that there is no prepotency constantly favoring either sex, but that in genetically heterosexual pairs the first differentiating member governs also the development of its mate so as to develop into the identical sex. In contrast to this, Witschi 2 and Witschi and McCurdy 3 found in parabiotic frogs and newts a clear ratio of 1:2:1. In cases of secondarily induced sex reversal the male sex predominated. Later Burns 4 reported new experiments with Ambystoma tigrinum that gave results corresponding closely to ours. However, he still upholds the accuracy of his original statements with respect to Ambystoma maculatum. Differences of such fundamental character within one genus seemed extraordinary and since they were an obstacle to a rational interpretation of the whole body of experimental data, the authors were moved to repeat Burns' experiment with the same material—Ambystoma maculatum (Shaw), syn. A. punctatum (L) of New Haven, Connecticut. Though the investigation which was started in the spring of this year is not yet completed, it is already clear at this time that Burns must have misinterpreted his material.

In attempting to use serum rather than tissue extract for the coagulant in clotting heparin-plasma it was found that frequently a plasma rendered incoagulable with heparin could not be clotted with serum but could be clotted with tissue extract. This observation suggested that the coagulant in serum (thrombin) might be more sensitive to heparin than the coagulant in tissue extract (tissue fibrinogen).

One must consider the possibility of the concentration of coagulant in the tissue extract being greater than in the serum. This factor was controlled by rendering the 2 coagulants isocoagulant by dilution of the stronger of the 2 with physiological saline. They were regarded as isocoagulant when similar amounts of either shortened the coagulation time of a recalcified citrate plasma to the same extent.

The citrate plasma is from adult rabbits, titrated to 0.5%; serum is obtained from the clotted recalcified citrate plasma.

The tissue fibrinogen was kindly furnished by the Wm. S. Merrell Co. (“Fibrogen” for subcutaneous use).

In Experiment A. Table I, the plasma has a fairly long recalcification time of 13 1/2 minutes; this is reduced to 3 minutes by 4 drops of either coagulant. The heparin concentrations are high at the start being reduced by dilution, the clotting time becoming consistently shorter for both coagulants. In each set the serum is more inhibited than the tissue fibrinogen. Control at the end shows the coagulants to be isocoagulant and within 15 seconds of the original value.

Contrary to the general tendency of the hypophysis, and particularly the anterior lobe, to enlarge during pregnancy in many animals, it appears from the data on 86 albino rats tabulated below, that the organ, as a whole, does not enlarge. If anything, the whole organ is slightly smaller in the normal pregnant animals. No significant difference was found in the relative volume of the various lobes as determined by the paper weight method on serial sections.

All the animals used in these investigations were standard female albino rats reared under uniform conditions in the animal colony of the Department of Anatomy, University of Minnesota.

Group I—Normal non-pregnant controls were specifically chosen litter mates of the animals in Group II. They were all killed at approximately the same body weight of 185 gm.

Group II—Normal pregnant group-Litter mates of Group I were impregnated at about 175 gm. At least one animal was killed on each day of the gestation period, which in a rat is 22 days. Impregnation was done at this age on the assumption that they would have attained an average body weight similar to group I when killed if pregnancy had not been induced.

Group III—Consisted of a group of pregnant animals retained on a diet deficient in the anti-sterility vitamin E, after having passed through one gestation period with a diet deficient in vitamin E.

Group IV—Is a group of pregnant animals which was fed vitamin E after having passed through one gestation period with a diet deficient in vitamin E. This procedure did not apparently modify the relative volume of the lobes of the hypophysis as compared with group III.

In operations on the central nervous system in cats, we have found that Avertin has many advantages over such an anesthetic as ether. We have used it on about 100 animals and find it to be of special value in several respects.

1. Being given per rectum, the dangers of contamination by the ether mask are eliminated when operating on the head region.

2. The animals may be made to remain perfectly quiet through the entire operation even if lasting up to 5 hours, without the use of any other drug, thus reducing the number of assistants—an anesthetist not being needed. It is therefore especially valuable where long periods of immobility are required, as in experiments where electrodes are to be kept for a considerable time in exactly the same place.

3. The increase of flow of mucus and saliva which is often produced by ether is eliminated by the use of Avertin. This feature is especially desirable in operations such as those on the base of the skull, in which the trachea must be retracted to make good exposure.

4. The danger of tongue swallowing is eliminated since Avertin induces the tongue to drop forward.

5. After the use of Avertin the animal sleeps quietly for 24-48 hours. This period of rest encourages wound healing and reduces the danger of hemorrhage and infection, since the animal cannot open the wound.

Duran-Reynals 1 and others have shown that extracts from certain organs such as testicle greatly enhance the invasion of tissue by various bacteria and viruses. This can be demonstrated by injecting into the shaved skin of a rabbit a living suspension of pathogenic bacteria mixed with the organ extract to be tested: and at another site an equal number of bacteria in plain broth or saline. After 24 hours a comparison of the size of the lesions and the relative degree of necrosis furnishes an index of the enhancing role of the tissue extract.

We have investigated a similar effect exerted by secretions from the upper respiratory tract. In most of our experiments we have employed a virulent strain of B. lepisepticum (R. D.) as this organism is quite pathogenic for rabbits and produces a slowly progressing cutaneous lesion which has become familiar to us from previous experiments. The experimental material was obtained by irrigating with plain broth the nasal passages of various individuals both normal and those suffering from acute colds. This material was passed through Berkefeld or Seitz filters and tested aerobically for sterility. This was then concentrated by distillation in vacuo to approximately one-fifth the original value and preserved in the icebox with cystein 0.25% under vaseline seal. In other cases, fresh material was used without concentration. As control some of the broth was treated in an identical manner. In most of our experiments .2 cc. of the washings were mixed with .1 cc. fresh, thin saline emulsion of B. lepisepticum and .2 cc. of this mixture injected into one flank of a rabbit while in the opposite side an identical preparation of organisms and control broth was injected.

A study has been made of the biological differences in freshly isolated and old stock strains of the meningococcus. The majority of freshly isolated strains present colonies on blood agar which are pearly in color, lenticular and flattened in form, round in outline, and distinctly moist. While single colonies measure up to 4 mm. in diameter after 24 hours' growth, there is a tendency to coalesce with the formation of a sheet-like growth. By the end of 36 hours the original growth tends to become transparent and glassy, sometimes with the formation of minute, glistening crystals on the surface; new and opaque, yellowish white growth appears at the margin usually in the form of papillae, and this tends to grow over and obliterate the original glassy colony. While, save rarely, transplantation from the original growth is unsuccessful, subculture can be made from the marginal growth during the next 48 or 72 hours. The colonies so obtained are usually more opaque and domed than in the original culture. Individual colonies are mostly smaller and show less tendency to coalesce, but there are some larger, flattened colonies resembling those of the original culture, giving the plate a characteristically uneven growth. Further subculture accentuates those characteristics noted for the secondary growth—the colonies become smaller and more domed with less tendency to coalesce, Most of the older strains show opaque white colonies but in occasional stock strains the colonies are transparent. They remain viable on blood agar for 48 or 72 hours. In many the formation of crystals becomes well-marked at the end of 48 hours and subcultures are no longer successful. Rough forms have appeared in only one strain,—a stock Type IV culture with considerable yellowish pigment formation.

Having succeeded in producing potent antiserums to monkey poliomyelitis virus by the injection of horses with increasing doses of a suspension of infected spinal cords, the occasion of the recent epidemic of polio in New York City has made it possible to determine the potency of these antiserums as neutralizing substances for freshly isolated strains of virus. It has been shown 1 that concentrates from these antiserums were of very high titre and were effective in protecting monkeys already infected with poliomyelitis virus of monkeys as well as bringing about virus neutralizations (in vitro) in very high dilution. In this respect these serums were approximately 5 times as potent as human convalescent serum. With continued injection of horses and continued passage of the virus through monkeys a serum has been evolved which neutralizes monkey virus in a dilution upward of 1:500 in spite of the fact that the virus is now more infective for monkeys than it was previously (5% virus, M.L.D. 0.05 cc.) while human convalescent serum neutralizes

this virus in 1:20 only. On the other hand, human strains recently isolated fail to be neutralized in as low as 1:20 by the otherwise powerful serums. Convalescent serum from past epidemics neutralizes the new human strains in dilutions up to the neighborhood of 1:50. These relative values may be set down in tabular form. (See Table I.)

Although every part of the tobacco plant has been reported to contain nicotine, this alkaloid could not be detected by Vickery and Pucher in the fully ripened seed of Connecticut shade-grown tobacco by chemical methods. 1 Ilyin, 2 who has studied the distribution of nicotine in the plant, found that immature seed, and particularly the ovules at an early stage of development, contained small proportions, but that, as ripening progressed, the alkaloid content diminished until finally none could be demonstrated. In view of the toxic properties of nicotine it seemed that a simple physiological test for its presence in tobacco seed would consist in feeding trials on small animals. We therefore offered to albino rats a ration that consisted either of ground tobacco seed 98%, Osborne-Mendel salt mixture 3 2%, or ground tobacco seed 99%, sodium chloride 0.5%, calcium carbonate 0.5%; cod liver oil was administered as a supplement at the rate of 10 drops per day. The diet was consumed with avidity and without any evident untoward consequences; the animals grew at a satisfactory rate and appeared to be normal in every respect. This somewhat surprising outcome led to a detailed study of the nutritive properties of the tobacco seed.

The material for this study was supplied by tissue cultures of embryonic heart in which the cardiac muscle had grown out and become differentiated. The ventricles of 16 day rat embryos were cut into fragments 1 to 2 mm. in diameter and cultivated in a medium composed of rat plasma and embryo juice. In the course of 2 to 4 weeks a new growth of muscle appeared among the fibroblasts, taking the form of anastomosing bands similar to adult cardiac muscle. This new growth was closely applied to the under surface of the cover glass so that single cells could be observed with the highest powers of the microscope. Cross striations could be seen in many of the muscle cells of both explant and new growth after one month. The striated appearance in the living cell was produced by the arrangement of small brick-shaped bodies, apparently mitochondrial in nature, into more or less regular longitudinal and transverse rows. Since this picture was somewhat different from that presented by muscle from an adult animal it seems likely that we were dealing with a transition stage in which the differentiation was incomplete. The presence of myofibrillae with alternating dark and light bands and “membranes of Krause” in fixed preparations, however, indicated that the process was well advanced. Although the muscle in these cultures contracted spontaneously, its activity was intermittent, especially when exposed to room temperature for the making of photographs. This irregularity was overcome by substituting Ringer solution for the usual medium.

Since the intracellular movements were too rapid to be followed by direct observation through the microscope, an effect of slowing was accomplished by making “slow-motion” cinematographs. Exposures were taken at the rate of 32, 48 and 64 per second.

Following ether in man, there is a definite increase in blood amylase which does not occur after avertin or spinal anesthesia. According to the results obtained in dogs with fistulas, this is due to the fact that ether causes a cessation of the transport of pancreatic juice to the intestine while pancreatic secretory activity continues, resulting in a temporary accumulation of amylase in the blood.

During the course of experiments on the role of heredity in the resistance of rats to infection with S. enteritidis, some discrepancies from the uniform mortality rate to be expected in a highly inbred strain of animals were encountered. 1 An immunological study of the animals from this strain showed marked differences in the bactericidal power of the whole blood. Loeb and King 2 had previously found marked differences in reaction to tissue transplants in rats within each of the Wistar “A” and “B” strains.

The animals used in our experiments were descended from one pair of rats of the Wistar “A” strain, obtained in 1924 from Doctor Helen Dean King. These rats were then in the 48th generation of brother-sister matings. These brother-sister matings have been continued in our tests. All rats used have been shown not to excrete S. enteritidis in their feces and were carefully kept free from exposure to this infection. Blood taken from the heart of each animal was mixed with just sufficient sodium citrate to prevent clotting, and drawn into capillary pipettes previously coated with a thin layer of a saline dilution of an 18 hour culture of S. enteritidis, the method being that proposed by Heist and Solis-Cohen 3 for determining the pneumococcidal power of blood. After the open end of the pipettes had been sealed in the flame, they were placed in the 38° incubator for 4 hours. The contents of each pipette were then expelled into a Petri dish of fluid agar and incubated over night, then the number of colonies counted. Suitable control tubes were inoculated to demonstrate the viability of the organisms and to indicate the numbers of organisms present in each dilution.

Karrer, Euler and Rydbom 1 have demonstrated that carotene has provitamin A properties for the chicken but that xanthophyll is without effect in this regard. Evidence has been presented by Capper 2 that carotene is converted to vitamin A by the chicken.

The present experiment was an attempt to confirm the results of Karrer and his associates and to extend the study to include chlorophyll. In addition a study was made of the uric acid 3 content of the blood, it having been observed in earlier studies that this constituent increases in amount in the vitamin A deficient condition. Newly-hatched White Wyandotte chicks were placed upon a vitamin A deficient ration: 52.5% white corn, 20% wheat middlings, 10% dried skim milk, 10% meat scrap, 5% wheat bran, 0.5% sodium chloride, 1% calcite flour and 1% steamed bone meal. Sufficient irradiated ergosterol was used to protect against leg weakness. On this ration retardation of growth begins between 3 and 4 weeks of age. The pigments were dissolved in ethyl laurate and dilutions made with cottonseed oil. The daily allowance of each pigment was 0.03 mg. by capsule. Supplementation with carotene (M.P. 170°-173°) began at 26 days, with xanthophyll (M.P. 173°), at 27 and with chlorophyll at 31 days. An immediate growth response took place when carotene was fed but the birds which received the xanthophyll and the chlorophyll not only failed to grow, but lost weight and died within 2 weeks. Their behavior was essentially that of the non-supplemented control group. Nine or more individuals were used in each group.

The authors 1 have previously examined, from an arithmetical point of view, the Buchnerian hypothesis that antibody is a conjugate of body globulin and injected antigen. These speculations led us to undertake the following experimental investigation based upon the reactivity of artificial compound proteins in which the hapten chosen to confer the new specificity contains integrally an inorganic atom (As) for which a direct and very delicate chemical test is available. Two compounds, ovomucoid-diazo-arsanilic acid and casein-diazo-arsanilic acid, were prepared. The serum of a rabbit immunized against the first reacted with the second, showing the presence of an arsanilic-aeid-specific antibody. The optimal precipitation ratio by volumes of antiserum to casein-diazo-arsanilic acid was found to be about 150. Such a mixture is considered neutral— at least neither reagent is present in any large excess. It seems reasonable to suppose that within this zone, antibody and antigen enter respectively into the precipitate in the proportion of one molecule to one molecule, or one molecule to a small number of molecules. If it is assumed that the antibody is a globulin conjugate of the antigen, and contains a similar amount of arsenic (as would necessarily be true if there were no breaking up of the injected antigen), it can be shown from the amounts of arsenic and casein found in the casein-diazo-arsanilic acid solution, and the above ratio, that 10 cc. of antiserum should have contained about 20 mmg. (20 × 10_-6 g.) of arsenic, an amount easily detectable. None was found. This would seem to cast doubt on the Buchnerian hypothesis.

In certain regions neotonous individuals of Triturus viridescens are found in the same ponds with metamorphosed ones. Their gills persist after the body fin has been lost and the sex organs are well developed. 1 The object of these experiments was to find out whether such gills could be affected by environmental changes. If this occurred it would indicate that the hereditary background of these newts was different from that of those that lose their gills early. 2 Attempts to reduce the gills were made (1) by forced residence in a dry environment, (2) by implants of anterior pituitary lobe taken from gill-less adults from another region and (3) by implants of thyroid gland from the same source.

Gilled newts from Woods Hole, 6 to 10 cm. in length, and typical gill-less adults from South Hadley collected in October and November were used. Experiments were carried on through the winter and spring. Among 500 Woods Hole newts, 75% had gills on each side containing circulating blood. Their sex organs were partially or completely developed. The thyroids of 6 gilled newts suggested a state of rather low activity. 3 A slightly less inactive condition obtained in thyroids of gill-less individuals from Woods Hole. On the other hand, the thyroids of South Hadley adults appeared active (compare adult Ambystoma opacum, Uhlenhuth 3 ).

On October 14, 1930, 2 lots of Woods Hole newts with gills averaging 0.13 cm. in length were divided into 4 groups according to size. Twenty were kept in jars cushioned with moss occasionally sprinkled with water. Twenty others were kept in jars of water. Ten aquatic adults (South Hadley) were kept in moss-bedded jars and 10 in water. All were kept under the same conditions.

The prooestrous uterine bleeding of dogs and menstruation of primates have been considered by some physiologists to be very similar and to represent in 2 classes of mammals the result of similar causative factors. (Heape, 1 Marshall. 2 )

We have obtained data from experiments conducted on dogs which we believe strongly indicate that the prooestrous bleeding of the dog is not homologous with the menstruation of primates. The evidence for this belief is as follows: (1) Ovariectomy of 5 dogs in the metoestrous phase did not result in external or profuse microscopical bleeding. In all 5 cases microscopical bleeding occurred as early as the 7th day and as late as the 16th day after ovariectomy. This bleeding was only of one or 2 days' duration. No external or microscopical bleeding was observed after ovariectomy of one immature dog and one dog in oestrus. Ovariectomy of monkeys results in external bleeding of about the same quantity as that seen at the normal menstrual period. (E. Allen 3 ; Morrel et al 4 ; Saiki, in press.) (2) Injection of alkaline extracts of the anterior lobe of the hypophysis 22 which contained the gonad stimulating hormones into 2 dogs with ovaries intact resulted in external bleeding during the period of injection. This bleeding stopped soon after the injections had been discontinued. In monkeys profuse microscopical or external bleeding does not occur during the injection period. A latent period of some days intervenes between the cessation of injection and the appearance of blood. (Hartman 5 ; Saiki, in press.) (3) Control experiments in which castrated dogs were injected with quantities of the anterior lobe extracts sufficient to produce external bleeding in the intact animal did not result in profuse microscopical or external bleeding.

The question arises whether the marked increase in rate of heart heat which develops in hyperthyroid and in thyroid fed animals will appear in isolated hearts treated with thyroxin if a sufficient time is allowed for the action of the hormone. Lewis and Mc-Eachern 1 have shown that hearts removed from thyroid fed, or thyroxin treated rabbits maintain a 50% increase in rate for 8 to 10 hours, and that normal excised rabbits' hearts show no immediate effect of thyroxin.

We have kept excised turtle hearts at 20°C. ± .02° for 60 hours, recording automatically the rate during this period by means of the Loomis chronograph. 2 The heart was tied by the tips of the auricles to a heart lever and suspended in a modified Ringer's solution, containing 0.1% glucose and phosphate buffer to pH = 7.3, oxygenated by pure O₂ gas. Addition of thyroxin 20 , dissolved in a minimal amount of alkali as a stock solution of 1:1000, does not change the pH of the solution. The details of transmitting the heart beat to the chronograph are described in a previous paper. 2

The hearts beat about 10 times in 20 seconds at first, gradually slowing to 10 beats in 30 seconds in 2 hours when the thyroxin, 1:10⁵, is added. No immediate change in rate occurs, the heart gradually slowing, with some rhythmic variation in rate, over a period of 2 days, when the rate may be 10 beats in 80 seconds. The 4 hearts studied showed no tendency whatever to increase in rate and behaved in every way like normal hearts, which likewise continually slow over long time periods.

There is perhaps no other single criterion for the identification of the culture type that is so significant as colony appearance. It is . regrettable, however, that in many investigations slight or no effort is made to proceed beyond recording the colony appearance. That this procedure is ill-advised is evident to every investigator who finds discrepancies in the characteristics of apparently established types.

In connection with studies on the pathogenicity of certain strains of Bacterium coli, note was made of the fact that some dyes tended to cause the appearance of clearly defined rough colonies with marked consistency in cultures that had been quite uniformly smooth for many generations. Use was made of a series of dyes to ascertain whether this was a constant phenomenon, perhaps associated with specific dye radicals, and there were found 4 of 25 common dyes which gave definite results.

Methylene blue (1-1000) yielded 60% roughs on the first transfer, with no satisfactory increase toward the desired 100% on repeated transfer. Crystal violet (Grübler, prewar stock) yielded 70-80% roughs on the first transfer with some tendency to increase on repeated transfer but falling short of 100%. Neutral acriflavine (1-1000) produced no roughs but practically 100% intermediates. Acriviolet, a mixture of crystal violet and neutral acriflavine, averaged about 95% roughs on the first inoculation, while the second, or at most the third transfer, even of thoroughly stabilized smooths, yielded 100% dissociation into rough colony types. The base used in these dye experiments was plain beef extract 3% agar, with no enrichment, hence there was no conflict between elements tending toward roughness and smoothness. A later report will deal with the restoration of roughs to smoothness, with a discussion of the effect of certain enriching substances favoring smoothness.

It has been definitely shown that the normal structure of the thyroid gland is dependent upon an autacoid present in the anterior lobe of the pituitary gland. In amphibians its release at the time of metamorphosis stimulates the thyroid to excrete its content and induces the increased cellular activity characteristic of this period. In mammals Loeb and his coworkers 1 , 2 and Aron 3 , 4 showed that the injection of anterior pituitary extracts and emulsions produced also some characteristic changes in the thyroid suggestive of a higher activity. The follicular epithelium became higher, mitoses were present and colloid was excreted. The glands increased in size from 2 to 4 times and became more vascular. The metabolism of treated guinea pigs, as shown by Siebert and Smith 5 was markedly increased. As previous observations 6 had shown that young ducks were very sensitive to pituitary extracts and emulsions, it seemed worth while to investigate the possibility of inducing in this species functional and structural changes indicative of hyperthyroidism.

A series of light brown Leghorn cockerels ranging from 22 to 86 days in age were given daily subcutaneous homeoplastic hypophyseal implants. In the first experiment the entire hypophysis was utilized while in subsequent tests only the anterior lobe was employed. The number of daily implants per bird varied from 19 to 28. The treated birds, as well as controls, were weighed and the head furnishings measured at regular intervals, at first daily, later on alternate days. All treated individuals remained active and in excellent condition throughout the experiments so that they could not be distinguished from controls in this respect. In fact treated males were consistently somewhat heavier than their respective controls when treatments were discontinued.

One of the effects, quite apparent after 4 to 5 days' treatment, was a stimulation of head furnishings. These became turgid and reddish in color and revealed noticeable growth. In the longer treatments there was a noticeable slackening in growth toward the close of the experiment while in some of these there was a cessation followed by a slight regression in size. Bird No. 167, 59 days old when the experiment began, received 23 daily implants. On the day the experiment started its comb measured 4.7 cm. in length and 2.6 cm. in height, while 15 days later it measured 7.5 cm. in length and 4.5 cm. in height, the maximum size attained. On the day following the last implantation, 24th day of the experiment, when the bird was killed, measurements of comb were 6.8 cm. in length and 4.0 cm. in height. The best control comb showed a gradual increase during this period from 4.5 cm. in length by 2.6 cm. in height to 5.8 cm. in length by 3.5 cm. in height.

In a series of experiments daily subcutaneous homeoplastic hypophyseal implants were made on juvenile light brown Leghorn females. 1 These ranged in age from 28 to 59 days respectively when experiments began. In only one experiment was the entire hypophysis employed, while in all others only the anterior lobe was utilized. The number of implants received in any one experiment varied from 19 to 28. Weights were taken and measurements of head furnishings made at regular intervals as in experiments on cockerels. Treated females remained in excellent condition and were not adversely affected throughout any of the experiments. In none of the experiments was there a significant difference in weight between treated and control. In some the treated individuals were a trifle heavier than their respective controls, in others the reverse was true. This was not the case with treated males which were usually significantly heavier than their controls.

The first and most obvious effect to be noted was the phenomenal growth of head furnishings. These, pale and small when experiments began, became reddish and turgid after 5 to 6 days' treatment and revealed steady growth. Bird No. 178 was 22 days old when experiment started. It received 28 daily implants and was killed on the day following the last treatment. Its comb measured 1.3 cm. in length and 0.4 cm. in height when the experiment began, while on the day following the final implant it measured 4.0 cm. in length and 2.1 cm. in height, a creditable size for a pullet 4 months of age. The comb was quite stout, firm, and erect, resembling much more a male comb of similar size than the flabby, loppy comb of similar dimensions on an older female.

The purpose of this investigation was to determine whether any differences were to be observed in the relative anesthetic effects over widely different _pH ranges, comparing a series of general anesthetics with a series of local anesthetics and a series of hypnotics. Alcohols such as allyl and isoamyl, and also general anesthetics such as chloroform, ether, chloretone, etc., do not form salts and consequently their distribution between oil and water is not markedly changed by a change in the (H⁺) concentration of the water phase.

Local anesthetics like neothesin, cocaine and butyn are lipo-soluble organic bases with relatively low solubility as free bases in water, but in the presence of acids they form water-soluble salts. Consequently the oil-water distribution coefficient shifts from a high oil-solubility and low water-solubility on the basic side, to a low oil-solubility and high water-solubility on the acid side.

In the case of the barbituric acid hypnotics exactly the reverse relation is found. The barbituric acids are lipo-soluble organic acids possessing very low water-solubility but on addition of alkali to the water phase water-soluble salts of the barbituric acids are formed. Consequently the distribution coefficient shifts from a high oil-low water solubility on the acid side, to a low oil-high water solubility on the basic side.

It follows from the above that if the induction of anesthesia is in any measure dependent upon the lipoid-water distribution coefficient of the anesthetic a shift from the acid or neutral to the basic side should exert little or no effect on the alcohols and general anesthetics, should increase the effect of bases like cocaine and decrease the effect of pseudo acids of the barbituric acid type.

Information upon the amount of radiation needed to cure rickets with respect to the area exposed is very meagre. Maughan and Dye 1 have shown that in chickens only a small amount of radiation is necessary to cure rickets and that the area covered by the feathers receives very little or perhaps none of the beneficial rays.

Eighteen rats at 4 weeks of age were put upon the Steenbock rachitic diet 2965 and after 21 days, having developed a severe degree of rickets, were ready for treatment. Treatment was continued for 21 days and degree of healing was judged by radiographic examination. As a source of ultraviolet radiation the General Electric Sunlamp with the Type S1 bulb was used. Previous studies had shown that exposure of the whole rat 30 minutes per day to the G. E. Sunlamp at 3 feet brought about complete healing of rickets in 3 weeks' time.

It was found that very little radiation is effective through the hair of the rat as an exposure of 2 square inches of unshaved back of the rat for 40 minutes daily produced only a beginning healing. Irradiation of the skin of the rat is much more effective as an exposure of one-fourth square inch of shaved back of rat for 20 minutes daily at 3 feet brought about complete healing of rickets in 3 weeks. Irradiation of this area of the skin produces greater healing of rickets than exposure of the whole animal for the same length of time. According to recent work of Diack 2 on the surface area of rats, this area of one-fourth square inch amounts to about one-eightieth of total surface area. It is surprising that such a small fraction of the total surface area of the rat can absorb sufficient of the rays to heal rickets.

Many investigators have assumed that the calcium of heparin is not available for oxalate precipitation, as performed in the Clark-Collip 1 modification of the Kramer-Tisdall 2 method. Interest in this question was aroused by the difference of opinion of various workers regarding the concentration of calcium in serum and in heparin plasma. Cantarow, 3 in a series of 100 determinations, using heparin as an anticoagulant, found that the calcium level was 0.5 mg. to 1.0 mg. less in each 100 cc. of plasma than in the corresponding serum. Greisheimer and Arnold, 4 and Loucks and Scott 5 have published data on this question.

In the present study several different samples of heparin were used. Heparin was added to redistilled water, to calcium chloride solutions, to plasma, and to serum. These heparinized solutions, with corresponding non-heparinized solutions as controls, were analyzed for calcium by the Clark-Collip modification of the Kramer-Tisdall method.

Dogs' blood served as the source of plasma and serum. All reagents were tested and found to be calcium-free. A microburet whose outlet was ground and fitted with a hypodermic needle was used for titration.

Increase in the number of platelets after splenectomy or other operations, according to certain observers,¹ appear to be rather constant in rabbits. The behavior of platelets after splenectomy in humans is very different from that after other operations. After splenectomy there is usually an abrupt rise to a high level beginning immediately after operation. After other surgical procedures the variations are inconstant. The number of platelets may decrease or increase.

A study was made of the water and chloride metabolism of dogs during the development of low serum protein edema. The sites of deposition of water and chloride were determined by analyses of tissues obtained at autopsy.

Dogs were fed a diet of known amount and composition. After a control period of one to 2 weeks, blood was removed every day for 1 to 7 days, was deprived of its plasma, and the washed cells suspended in calcium-free Locke's solution were returned to the animal. Blood and urine analyses were performed during both the control and experimental periods.

At suitable junctures in the experimental periods, the dogs were sacrificed by carbon monoxide gas and immediately autopsied. Tissues were removed and analyzed for water and chloride content. The values found on 7 dogs were compared with the average values of tissues taken from 3 dogs that had been subjected to various control procedures, and sacrificed and autopsied exactly as the hypopro-teinemic animals.

Edema was found to occur consistently when the serum protein concentration was belowT 4.0%. The occurrence of the edema was associated with retention of sodium chloride and water, and the retained chloride and water were recovered with considerable accuracy in the tissues analyzed. Skin stored most of the water while most of the retained chloride was found in muscle. The retained water and chloride constituted an isotonic concentration in the skin and a hypertonic concentration in muscle.

The Arthus phenomenon was first observed as a skin reaction and subsequently shown to occur in various internal situations with interesting and valuable results. 1 The Shwartzman phenomenon has been studied as a skin reaction in great detail, 2 but little attention has been given to the finer features of the lesion produced or to its manifestations in internal viscera. The observation of Frisch 3 that the reacting factor may be given intraperitoneal gives no information as to the occurrence of the phenomenon in internal viscera.

The stomach was selected as the organ first to be studied, because of its accessibility, the fact that control areas can readily be obtained and thin sections easily prepared for detailed microscopic study. Barium sulphate starch mixture was applied as a depilatory 24 hours before operation, which was performed aseptically under ether anesthesia, and the stomach exposed by high left rectus incision. The concentrated toxic filtrate of B. coli was prepared according to the method of Ecker and Rimington 4 and was shown to be sterile before use. The injection into the stomach wall of 0.2 cc. concentrated filtrate was into the submucosa, except in 1 instance where it was into the muscularis. Intracutaneous injection of 0.2 cc. concentrated filtrate was given in the lower abdominal region. Of 16 rabbits so treated, 12 were given 2.5 cc. concentrated filtrate intravenously 24 hours after the local injection. The animals were killed by fracture of the neck at periods of 3 1/2, 5(2), 12, 24, 25(2), 72(2) hours, and 7 days (2) after intravenous injection. The 4 control animals, with local but no intravenous injections were killed at 7 and 24 hours and 7 days (2) after the local injection. After careful gross examination of stomach and skin, blocks were fixed in Zenker's fluid, embedded in paraffin and sections stained with hematoxylin and eosin.

The Shwartzman phenomenon affords a new experimental method for the study of the local reactivity of joints to bacterial products.

The bacterial filtrates employed in this investigation were (1) from B. coli prepared by the method described by Ecker and Rimington 1 and (2) from B. typhosus supplied us through the kindness of Dr. Gregory Shwartzman.

The preparatory factor was injected lateral to and below the patella with the knee flexed, in amounts varying from 0.01 to 0.40 cc. of filtrate. Simultaneous injections were made into the skin of most animals. The intravenous injection of the 'reacting factor' was given from 20 to 30 hours later and animals were sacrificed from 1 to 192 hours after receiving the reacting factor in amounts usually of 1 cc. per kilogram body weight.

Seven control animals received only the 'preparatory factor' and were sacrificed at intervals corresponding to those of the experimental series.

The Shwartzman phenomenon was elicited in the synovial membranes of the knee joints in 6 of 11 rabbits tested. The reaction occurred with less frequency and severity in the joints than in the skin. Attempts to elicit the reaction in pleural and peritoneal cavities were negative. In rabbits sensitized simultaneously in knee joint and skin a severe skin reaction was not necessarily associated with a severe synovitis and vice versa.

The criteria employed for the recognition of the reaction were histologic and based on the presence of endovascular damage, followed by thrombosis and necrosis of the vessel with exudation of leucocytes and hemorrhage.

Digitalis varies markedly in its pharmacological activity, even the alcoholic extracts deteriorating often within a comparatively short time. In view of the extreme differences of opinion held by pharmacologists and pharmacists in regard to the effect of ultraviolet rays on digitalis, the present investigation was undertaken. A standard tincture was kept in the ice-chest in the dark. Samples of this were irradiated with an Alpine Sun lamp, and with a water-cooled Kromayer lamp with and without various filters, and the pharmacological activity of the tinctures after assay was determined by 4 different methods: (1) the official one-hour frog method, 1 (2) the cat method of Hatcher and Brodie, 2 (3) the phytopharmacological method of Macht and Krantz, 3 and (4) the goldfish method of Pit-tenger and Vanderkleed. 4 The great majority of animal experiments were performed on cats because, in the author's experience, that is the most reliable method for assaying digitalis. About 100 cat experiments were performed in connection with the present investigation. Each sample or fraction of digitalis tincture was tested on at least 3 cats and in cases where the readings obtained showed a wide divergence, as many as 8 cats were sometimes used to obtain the average minimal lethal dose. The phytopharmacological method was found to be particularly adapted to comparative study of various samples of digitalis simultaneously and under identical conditions of light, temperature, barometric pressure, etc. At least one hundred sets of such experiments were performed and each control, as well as each sample of digitalis tincture, was tested on 8 or 10 healthy seedlings of Lupinus albus. The frog and goldfish methods were used only to corroborate the results obtained by the cat and phytopharmacological experiments. Each sample was tested on at least 10 goldfish and 12 frogs.

The senior author has demonstrated repeatedly the presence of a specific toxin in the blood of pernicious anemia, which is not found in the blood serum of any other form of anemia, and is therefore of diagnostic interest as well as of value in following the results of various therapeutic procedures. 1 Later studies by Macht have revealed that a similar specific phytotoxic reaction is exhibited by spinal fluid from pernicious anemia patients, as compared with normal human spinal fluid. 2 This was found to be of value in differentiating neurological complications of pernicious anemia from similar cases of different etiology. Employing Macht's phytopharmaco-logical methods, the authors made a systematic study of 125 patients in respect to toxicity of their stomach washings for the growth of living seedlings of Lupinus albus. A uniform procedure or technique was employed. A patient's stomach was washed in the morning, before food was taken, with 100 cc. of distilled water. The hydrogen ion concentration of the pumped-out fluid was then determined and the phytotoxic index was obtained. A 2% solution of the washing in each case was made in a standard plant-physiological solution and the growth of the straight, well-defined roots of Lupinus albus seedlings in the dark at a temperature of 20°C. for 24 hours was carefully measured. A comparison of these with control seedlings, grown under exactly the same conditions, was then made, and the effect of the washings, expressed as a phytotoxic index or percentage of growth in terms of the normal controls, was determined.

The average phytotoxic index obtained from stomach washings of 100 normal patients was found to be 86%. The lowest reading was 86%, while the highest was 100%. Stomach washings from cases of carcinoma showed no difference in toxicity as compared with normal subjects.

The experiments previously reported 1 have shown that rat embryos possess the potency to undergo a development of their tissues when implanted in strange surroundings. In order to test further the capacity of the embryonic parts for differentiation, transplantations of 8 and 9 day rat embryos have been made to the chorioallantoic membrane of the chick. This experiment was first performed by Hiraiwa 2 using rat embryos of a considerably older stage of development. He found that there was a considerable degree of self-differentiation in this form.

Up to the present about 200 such transplantations have been made; the eggs have been incubated from 7 to 9 days after transplantation (9 day hosts) and the grafts studied. In many cases the transplant causes only a minor reaction which is indicated by a slight thickening of the membrane on the site of the operation and a slight increase in the vascular field. This is the so-called membrane reaction. In some cases a distinct nodule of tissue is found which upon section shows degenerating embryonic tissue. In these cases there are no embryonic parts except isolated tissue fragments.

In the positive series, containing embryonic parts, there are two groups: (1) those in which there is rapidly differentiating tissue and (2) those in which the tissues have developed exceedingly well but have undergone subsequent degeneration. So far we have had the opportunity to study the sectional material from 6 of these cases. Of these only one shows tissues undergoing differentiation at a rapid rate with a negligible amount of degeneration; 4 cases show the mixed type in which some of the tissues are undergoing differentiation and some are degenerating, while the sixth case shows practically complete embryonic degeneration.

The value of plain roentgenograms of the abdomen without the administration of contrast substances in the diagnosis of ileus has been repeatedly emphasized by clinicians. Goehl, Lynch, Borman, and Wangensteen 1 found experimentally that roentgenologic evidence was of less value in the presence of strangulation than in simple obstruction. The roentgen diagnosis of ileus is based upon the fact that in obstruction there is an accumulation of gas and fluid proximal to the point of obstruction which can be visualized roentgenological. Whereas the presence of gas visualized in the roentgen plate is of diagnostic value, it is considered by many observers 2 that the finding of multiple fluid levels (demonstrated by obtaining the roentgenogram in such a way that the junction between gas above and fluid below may be visualized) is of greater diagnostic importance.

The present investigation was undertaken to compare the results obtained with roentgenograms taken in the horizontal position and those taken in the upright position, the latter technic demonstrating fluid level formation.

Ninety-four observations were made on 19 dogs. At each observation, 2 roentgenograms of the abdomen were obtained, one with the animal in the horizontal position, and the other in the upright position. The x-rays were made by means of a specially constructed radiographic animal board. 3

A simple obstruction of the jejunum and terminal ileum was produced in 3 and 7 animals, respectively. A “strangulated” obstruction of the jejunum, ileum and sigmoid flexure of the colon was produced in 2, 5, and 2 animals, respectively. Roentgenograms were taken at intervals after the obstruction, varying from 1 to 75 hours.

Fibrinogen content of plasma is not decreased but actually increased in acute experimental obstructive jaundice, probably due to irritative effect of bile on liver. Coagulation time remained above normal limits, but did not parallel the fibrinogen content of the plasma. Coagulation time is not a satisfactory index of a hemorrhagic tendency in the presence of jaundice. Diminution of fibrinogen content does not parallel the degree of liver destruction. The Wu method for determination of fibrinogen is not satisfactory when exact quantitative analysis of blood proteins is done. The Tyrosine equivalent for dogs was found to vary from 12.5 to 19.7. This was learned after determinations were made from 24 samples of blood. For comparative analysis as herein used, the Wu method serves the desired purpose.

It would appear from our observations that the cause of the hypomotility of the gastro-intestinal tract of rachitic rats is due to a lack of vitamin D and not to the unbalanced minerals of the diet.

Hill 1 demonstrated that in addition to the usual tremor frequency of 8 to 12 tremors per second, a tremor frequency of around 50 tremors per second could be recorded. He found that the tremors of higher frequency and lesser excursion were superimposed upon those of lower frequency and greater excursion. Travis and Hunter 2 called attention to the striking similarity between records of tremors and those of action currents and later recorded tremors of a rate as high as 200 per second. 3 The present paper reports the recording of tremors of a rate as high as 500 per second.

For the recording of the tremors we used an electrical current generator to activate, with or without the aid of amplification, a super-sensitive element of a Westinghouse oscillograph. All tremors were recorded from the extended forefinger of the firmly supported hand. Several healthy individuals served as subjects. By means of an electrical filter and amplifying system we were able to by-pass and amplify greatly only tremors of frequencies above 130 per second. When such recordings are compared with those obtained without the use of filter and amplifier some interesting relationships are revealed (Fig. 1). It is to be noted that each large tremor (in upper record) enduring approximately 0.1 sec. is really composed of a group of several oscillations (in lower record) occurring at a rate of about 200 per second. Further the tremor line between the volleys is not quiet. It presents both smaller oscillations of the same frequency as the larger waves in the volleys, and other generally still smaller oscillations of a frequency as high as 500 per second.

In a series of papers Dochez 1 and his associates have presented evidence for the virus etiology of the common cold and have described experiments on the cultivation of this virus in tissue media and its inoculation into human subjects and chimpanzees. The purpose of this investigation has been to cultivate the virus of common cold and during the period of the year when the incidence of common colds is low to conduct human inoculation experiments on volunteers not subject to isolation.

The strain of common cold virus employed in these experiments was derived from a patient, “A. R.,” suffering from a cold of more than usual severity. Nasal washings were obtained on March 26th, 1931, within the first 24 hours of the onset of the cold. Ten cc. of Tyrode solution were flushed through each nostril and expelled through the mouth. These washings were promptly passed through a Seitz filter and cysteine hydrochloride added to a concentration of 1:2000. The filtered washings were introduced in 1 cc. amounts into 9 cc. hashed chick embryo tissue culture medium containing cysteine hydrochloride in a concentration of 1:2000. The culture was incubated under vaseline seal at 37°C. for 4 days. A Seitz filtrate was prepared from this first culture generation and stored under vaseline seal at 4°C. Further tissue cultures and filtrates were prepared in series as described above, the periods of incubation being from 4 to 5 days and the intermediate periods of ice-box storage of the Seitz filtrate averaging 5 to 6 days. At the present time this cold virus is in the 31st generation and has been cultivated successfully outside the body for a period of 8 months.

Attempts to induce common colds by inoculation into human volunteer subjects were carried out as follows :

Lowering plasma proteins to below 3% and osmotic pressure of plasma colloids to below 10 mm. produces a tendency to edema formation. The edema is very slight and does not increase rapidly until XaC1 is given, when the urinary output falls suddenly and massive edema appears.

In a previous communication 1 a method was described for the detection and titration of immune bodies in poliomyelitis, with reference to the applicability of the test in the study of susceptible persons, carriers, therapeutic value of serums from normal adults, and in relation to prognosis and convalescence.

A total of 363 serums has been studied to date. These included 100 normal adults and 100 normal children (age groups 6 weeks to 19 years), 58 convalescent serums (monkey, adult human, and children), 76 normal animals (monkey, horse, goat, and sheep), and 29 immune animal serums (monkey, horse, goat and sheep).

The results have agreed with known facts. The specificity of the test has been demonstrated also by observations made during all stages of poliomyelitis in the human and in the monkey.

The progressive development of immune bodies could be shown to occur during convalescence, particularly in those cases where rapid and complete recovery ultimately took place. These antibodies could not be found in the blood serum of patients or animals with a poor outlook as to recovery. When the disease went on to a protracted convalescence with poor or slow restoration of muscular activity, these immune bodies were found to be weak or negligible in quantity.

The serums from infants and very young children did not show any protective properties whatsoever and the same was true for 34% of adults, thus agreeing with the known facts of susceptibility to poliomyelitis and the neutralizing power against the virus of serums from certain normal adult persons.

In a study of anthelmintics being carried out in this laboratory, it has been shown that certain resorcinols have marked anthelmintic properties, 1 hexylresorcinol having the most intense action of the normal alkyl resorcinols. Both hexylresorcinol and heptylresorcinol have been given in ascaris, hookworm, and trichuris infestations, hexylresorcinol being found to have the greatest efficiency. 2 The latter has now been given by us to over 2,000 cases controlled by the Stoll egg counting method, without any indication of toxic effects, and with an average efficiency of approximately 90% in ascaris, 85% in hookworm disease, and 55% in trichuriasis. 1 , 3 The lower resorcinols were found to be less active in vitro, resorcinol itself having relatively very little action. It was first shown by Johnson and Lane that the antiseptic action of resorcinol could be increased by the introduction of alkyl groups and that this antiseptic action increased with the length of the straight alkyl chain up through butyl resorcinol. 4 Leonard then showed that the antiseptic action of these alkyl resorcinols reached a peak with hexylresorcinol and that this member of the series had the least toxicity. 5 This idea of introducing alkyl groups into substances with known antiseptic action was recently taken up by Coulthard, Marshall and Pyman, who showed the variation of phenol coefficients in homologous series of phenols. 6 As with the resorcinols a similar increase in the phenol coefficient with an increase in molecular weight of the alkyl chain, the maximum being found in 5-n-amyl-o-cresol, was demonstrated.

1. Experiments 1 were carried out at 10°C.

2. Gelatin was placed in cresyl blue solution at pH 9 and at pH 5.5. Dye solutions were changed before alteration in the dye occurred. After 24 hours gelatin was removed, washed in distilled water, and melted in a test-tube. The concentration of the dye in the gelatin was determined colorimetrically. More dye was taken up by gelatin at pH 9 than at pH 5.5 due to greater concentration of negatively charged protein ions at pH 9 capable of combining with the dye cations.

3. The gelatin stained at pH 9 was placed in buffer solutions at pH 9 and at pH 5.5. After 12 hours the concentrations of the dye in the buffer solutions were determined colorimetrically. More dye passed out of the gelatin into the buffer solution at pH 5.5 than at pH 9. This is due to the decrease in the negatively charged protein ions at pH 5.5.

4. Experiments were repeated 10 times and without exception the above results were obtained.

5. These results are significant as indicating the following possibility. If the dye penetrates a living cell in a dissociated form from cresyl blue solution we may picture the mechanism as above described when the external protoplasmic surface and the vacuolar surface consist of protein or a substance behaving like it. No matter how readily the dye is taken up by the surface, it cannot penetrate into the interior of the cell unless it is capable of being given up at the inner side in such a manner as described above.

Georgia Cooper 1 and her coworkers, working with cultures from pneumonia patients at Harlem Hospital and Bellevue Hospital, segregated Type VII from the miscellaneous group. During the past 3 years 121 patients invaded with Type VII pneumococcus have been observed at Harlem Hospital. During this period 1407 patients were admitted to the adult Pneumonia Series. The incidence of Type VII was 7.5%. The mortality among 85 cases treated without serum was 25.9%.

Serum has been employed on 17 adult patients and 2 children. It was administered for the most part until agglutinins were demonstrable by the whole blood stained slide technique. The cases were chosen when serum was available by the chance of their alternate admission to the hospital. One patient, to whom serum was administered on what was thought to be the 23rd or 24th day of her disease, came in on the 20th day of her illness, with the history of a chill and pain in the side at the onset. She was obese, had an aortic insufficiency, and had had dypsnea on effort for 2 years. She died of exhaustion. All the other patients recovered. One of those who received serum and recovered, suffered from a bacteremia which was apparently increasing prior to serum administration.

The cases are insufficient in number to establish the curative value, but they furnish suggestive evidence that adequate doses of Type VII antipneumococcic serum may have therapeutic value in patients suffering from Type VII pneumococcus pneumonia. Observation of the cases led me to believe that it was beneficial.

The lots of serum, the method of production and refinement and reactions are shown in Table I:

Our observation of the presence of an abnormal amount of fat in the livers of rats fed a diet high in protein was first made in a series of rats in which we were attempting to determine the stages of development of chronic nephritis. In rats placed on Liver Diet I and killed from 2 to 3 months later, we found that we could tell from the presence of fatty infiltration of the liver which rats were on the diet and which were not; whereas, we were unable to detect any difference between the kidneys of the rats on the Control or the Liver I diet. This observation led to a search for the cause of the phenomenal difference in the livers of the rats fed the 2 diets.

The livers of rats fed Liver Diet I contain very large amounts of cholesterol and fat but less than the normal amount of lecithin. The

values for cholesterol have ranged from 2.14 to 5.51% and those for total fatty acids minus lecithin from 9.48 to 11.48%. The values for lecithin have varied from 2.11 to 2.37%.

When the liver residue remaining after the aqueous extraction of the pernicious anemia fraction replaces the dried, whole liver in the above diet, there is no excessive deposition of fat in the liver, (Liver Diet IV). Likewise, when dried, whole liver extracted with hot 95% alcohol is fed, fatty livers are not found, (Liver Diet VII).

All of these diets are high in fat and protein and contain very small amounts of carbohydrate. Liver Diets I and IV contain about the same large quantities of cholesterol and lecithin, while Liver Diet VII is practically free of these substances.