ETIOLOGY OF OROYA FEVER : I. CULTIVATION OF BARTONELLA BACILLIFORMIS.

A pure culture of a microorganism resembling in morphology and pathogenic action Bartonella bacilliformis has been obtained from blood taken during life from a case of Oroya fever which ended fatally. The blood taken at Lima into citrate solution and transported to New York at refrigerator temperature yielded positive cultures 28 days after its withdrawal from the patient. The strain of Bartonella bacilliformis thus isolated grows well on the semisolid leptospira medium, and also on slant agar containing animal blood. The initial growth is not readily recognizable to the naked eye, but the presence of the organisms can be determined by means of the dark-field microscope and by Giemsa and Gram staining methods. No growth has been obtained on the more ordinary culture media. The organism is an obligate aerobe, is Gram-negative, and under certain cultural conditions motile. All the forms which have been described as occurring in human red corpuscles may be found in the cultures, and in addition many granular and coarsely irregular forms have been met with. The inoculation of cultures of Bartonella bacilliformis into Macacus rhesus produces infection and gives rise to effects which differ with the mode of inoculation. The intravenous injection of the culture into young macaques induces a prolonged irregularly remittent fever. The organism can be cultivated from the blood over a long period, and it has been detected within the red corpuscles of the monkeys, reproducing the precise appearances observed in human cases of Oroya fever. The intradermal injection of the culture into the eyebrow of young macaques gives rise to nodular formations rich in new blood vessels and showing the bacilliform organism within the endothelial cells. From the experimentally induced nodules cultures of the organism are readily recovered.     

ETIOLOGY OF OROYA FEVER : XIII. CHEMOTHERAPY IN EXPERIMENTAL BARTONELLA BACILLIFORMIS INFECTION.

The therapeutic effect of several antiparasitic chemicals on experimental verruga peruana is described. The drugs were administered by intravenous injection according as the nodules (1) were already developed to an approximate maximum, or (2) were still in the active period of growth. The effect of the drugs was different under the two circumstances of their administration. When they were given after the maturity of the nodules they hastened the regressive process, but when given during active growth of the lesions no action whatever was detected. Bartonella bacilliformis in culture is acted upon injuriously by a number of the chemicals employed in the therapeutic tests, the most active being formalin and neutroflavine.     

ETIOLOGY OF OROYA FEVER : XI. COMPARISON OF BARTONELLA BACILLIFORMIS AND BARTONELLA MURIS. CULTIVATION OF BACTERIUM MURIUM,N. SP.

Bartonella muris appeared in the blood of all white rats, wild rats, Chinese hamster, and mice, from which the spleen was removed, but did not appear in that of splenectomized monkeys, rabbits, or guinea pigs. It has not been possible to transmit B. muris to normal rats, monkeys, rabbits, or guinea pigs, by intraperitoneal, intradermal, or intravenous injection of blood containing B. muris from splenectomized rats. In two instances an acute orchitis was induced in normal rats by injection directly into the testicle of blood or saline suspensions of the liver of splenectomized rats. The intracorpuscular elements occasionally identified as B. muris or as having appeared as a result of the inoculation. The acute orchitis of rats was transferable to normal rats in series. From the testicular tissue, as well as directly from the blood of a splenectomized rat, there was isolated in pure culture a microorganism which induced in the testicles of normal rats an acute orchitis such as resulted from inoculation of the blood or liver suspensions of splenectomized rats. While a few inclusions were found in the erythrocytes of some of the animals, their number was so small and their occurence so infrequent that they could not be definitely identified as B. muris. In morphological features the cultural forms of the microorganism isolated resemble B. muris. The organism found in the testicular tissues, however, is considerably coarser than B. muris and takes a deeper stain. Immunological study failed to settle the question of the relation between B. muris and the cultivated organism, which is provisionally called Bacterium murium. Bartonella muris, Bartonella bacilliformis, and Grahamella talpæ have characteristic individual morphological features.     

ETIOLOGY OF OROYA FEVER : IV. THE EFFECT OF INOCULATION OF ANTHROPOID APES WITH BARTONELLA BACILLIFORMIS.

The inoculation of a chimpanzee with cultures and a passage strain of Bartonella bacilliformis induced local reactions which, while definite and characteristic, progressed less rapidly and were much less striking than those in the control rhesus monkey. Bartonella bacilliformis was demonstrated in the blood corpuscles with difficulty, and the fever was slight compared with the high and persistent fever of the rhesus monkey. In both the swelling of the lymph glands was an early symptom and constantly present. Definite anemia developed in the chimpanzee only after accidental infection with Rocky Mountain spotted fever and may have been due to either one or both infections, though it disappeared when the blood had become negative by culture for Bartonella bacilliformis and the local lesions had disappeared. Incidentally, the chimpanzee was found in this one instance to be less susceptible to the spotted fever than Macacus rhesus and guinea pigs. In the ourang-utan, also, Bartonella bacilliformis induced a mild systemic and local infection. A rise of temperature occurred 10 days after inoculation) and fever continued for a week, though it was decidedly less severe than that in the control rhesus. The lesions induced by scarification were less definite than those which arose at the sites of intradermal inoculation. Bartonella bacilliformis was recovered from the blood on the 9th and on the 16th days after inoculation and from nodules excised on the 33rd and 53rd days. A few erythrocytes containing the organism were demonstrated in stained smears, but prolonged search was required to find them. The symptoms and lesions observed in the chimpanzee and ourangutan as a result of infection with Bartonella bacilliformis are far milder than those of rhesus monkeys and show less resemblance to human Oroya fever or verruga.     

ETIOLOGY OF OROYA FEVER : III. THE BEHAVIOR OF BARTONELLA BACILLIFORMIS IN MACACUS RHESUS.

The experiments reported here were carried on in the main with passage strains of Bartonella bacilliformis, and the results indicate that the virulence of the organism has been considerably enhanced by passage through susceptible animals. While the animals of the earlier experimental series showed no anemia, some of the present group manifested a definite reduction in the number of red cells and in hemoglobin, and in one instance (M. rhesus 25) anemia was of the extreme type so often associated with Oroya fever in man. The anemic condition appeared to be secondary in character, however, nucleated red cells being few in number. In this animal also Bartonella bacilliformis was readily demonstrated in the erythrocytes by means of stained smears, though the number of cells invaded by the parasites was by no means so great as in the human infection. In most instances of experimental Bartonella infection so far induced the demonstration of the parasites by ordinary routine examination of stained film preparations is possible only when the titer of the blood exceeds 1:1,000. Prolonged search of many slides has not been attempted, however. The number of microorganisms in the blood, as shown by culture tests of ascending dilutions, was in most instances highest (1:100,000 to 1:10,000,000) during the early period of the infection coincident usually with the period of highest fever, falling to a titer of 1:10 during the last half of the disease. In one of the fatally infected monkeys, however, the titer increased from 1:10 on the 4th day to 1:1,000,000 on the 24th day. The titer of the blood was equally great in Monkeys 5 and 6, although the former was inoculated locally, the other intravenously and intraperitoneally. The largest proportion of infected red cells was found in Monkey 25, while the blood titer, as shown by culture test, was highest in Monkey 7. The febrile reaction varied in the animals of this series from a severe continuous fever of 104–105°F., lasting 2 to 3 months, in one instance, with a remittence during the 3rd to 5th weeks, to the acute high fever (106°F.) of 1 day''s duration in the fatally infected monkey, No. 25. The more usual reaction, however, is an irregular course of moderate fever with one or more periods of high temperature (105°). Bartonella bacilliformis was constantly demonstrated, both microscopically and by culture tests, in the lymph glands of animals sacrificed 2 to 3 months after inoculation, and in two of three instances it was present also in the spleen, bone marrow, and heart blood. In the case of M. rhesus 6, which died 26 days after inoculation, the microorganism was obtained also in culture from the lymph glands, spleen, and heart blood taken at autopsy. In the other animal which died, a terminal bactelial infection, while not obscuring the effects of the Bartonella infection, made it impossible to isolate the parasite from either blood or tissues. The skin lesions, whether of the nodular type, induced by introduction of the virus intradermally or by application to the scarified skin, or of the miliary character occurring spontaneously as a result of systemic infection, always yielded cultures of Bartonella bacilliformis, and stained sections of such lesions revealed the parasites in large numbers in their characteristic situation in the endothelial cells. A chronic, systemic infection, in which the lymph glands are enlarged and Bartonella bacilliformis is present in the blood in high titer, may be induced by local inoculation, as shown in the case of M. rhesus 5. The local lesions induced in one instance by introduction of a passage strain, both intradermally and by scarification, attained within 2 months extraordinary size, the nodules arising at adjacent sites of inoculation on the right eyebrow having coalesced into a large pedunculated mass which overhung the eye. This type of reaction had not been observed hitherto in the course of the present study but has been described by earlier investigators as a result of the inoculation of monkeys with human verruga tissues. The striking fact brought out in the present study is the variety of responses to inoculation which animals of the same species may manifest. The clinical features of the infection may be typical of Oroya fever or may resemble those of verruga peruviana, and in M. rhesus 25 we have an instance of a type of infection in which the characteristic phenomena of both conditions are simultaneously present. Whether the appearance will resemble those of the one or the other condition appears to depend on the susceptibility of the individual as well as on the virulence of the organism. Moreover, it seems probable that different degrees of resistance to the invasion of the parasite on the part of the blood cells, internal organs, or skin of a given animal may determine the predominant clinical manifestations of the infection. The factor of variation in susceptibility of different individuals or different tissues of the same individual would account for the variety of types of human Bartonella infection.     

ETIOLOGY OF OROYA FEVER : XV. EFFECT OF IMMUNE SERUM ON THE COURSE OF BARTONELLA BACILLIFORMIS INFECTION IN MACACUS RHESUS

Experiments are reported on the effect upon the course of experimental verruga peruana in Macacus rhesus of the injection of (1) small quantities of rabbit immune serum simultaneously with living cultures, (2) one large dose of convalescent monkey serum 24 hours prior to inoculation, (3) a similar preliminary dose followed by three subsequent injections of the serum, (4) three large doses of convalescent serum, following the inoculation. The convalescent serum was found (1) to prevent the multiplication of Bartonella bacilliformis in the blood in most instances, and (2) to delay the development of the skin lesions for considerable periods, when given before inoculation. When the serum treatment was not begun until after the appearance of the skin lesions, it had no effect on the progress of the nodules, although the blood became free from Bartonella bacilliformis. Since the severe effects of verruga peruana (Carrion''s disease) are believed to be due to the multiplication of Bartonella bacilliformis within the blood, the injection of convalescent serum in cases of Carrion''s disease in man would appear to offer promise.     

ETIOLOGY OF OROYA FEVER : V. THE EXPERIMENTAL TRANSMISSION OF BARTONELLA BACILLIFORMIS BY TICKS (DERMACENTOR ANDERSONI).

Experiments are reported in which Bartonella bacilliformis was transmitted from infected to normal rhesus monkeys by the bite of the tick, Dermacentor andersoni. A long period of feeding, both on the infected animal and on the normal animal subjected to infection, was required in order to secure positive results. The infection transmitted by the ticks was mild, but definite, as shown by the recovery of Bartonella bacilliformis from the lymph nodes and blood.     

CONCERNING AGGLUTININS FOR TREPONEMA PALLIDUM

1. There is no demonstrable amount of agglutinin for Treponema pallidum (Noguchi) in normal human and normal rabbit serum in dilutions as low as 1:20. 2. Agglutinins for Treponema pallidum are readily produced in young rabbits by the administration of pure cultures of living spirochetes. 3. There is no appreciable amount of agglutinin for Treponema pallidum culture used in the sera of secondary and tertiary syphilis or in the cerebrospinal fluid of tertiary syphilis in dilutions of 1:20 to 1:640.     

ETIOLOGY OF OROYA FEVER : VII. THE RESPONSE OF THE SKIN OF MACACUS RHESUS AND ANTHROPOID APES TO INOCULATION WITH BARTONELLA BACILLIFORMIS.

Bartonella bacilliformis failed to induce lesions when merely rubbed on the surface of the intact skin of a chimpanzee, an ourang-utan, and numerous Macacus rhesus monkeys, although when applied to the scarified skin of the same animals it gave rise to extensive lesions. Application of infectious material to the scarified skin did not always induce verruga lesions, but intradermal inoculation almost invariably gave rise to nodule formation. The localization of Bartonella bacilliformis in the skin is not, in experimental animals, determined by mechanical factors, since scarification of the skin or intradermal injection of foreign substances in monkeys infected with Bartonella bacilliformis does not give rise to verruga formation. The degree of susceptibility of the skin tissues appears to be considerably diminished during the course of experimental infection with Bartonella bacilliformis. Inoculation of the scarified skin of infected animals gave uniformly negative results, and intradermal inoculation induced only a mild local reaction. In a few exceptional instances, however, of animals previously infected with the strain of Bartonella bacilliformis derived from a human verruga nodule, reinoculation with the same strain gave rise to unusually marked reactions. The evolution of the skin lesion induced in experimental animals by Bartonella bacilliformis may be divided into four stages, the period of incubation, the initial stage, the mature and vascular stage, and the regression. In the initial stage the lesion is a pure angioendothelioma, but in the stage of full development the histological picture is complicated by connective tissue proliferation and occasionally also by penetration of epidermis into the lesion. The demonstration of Bartonella bacilliformis in the endothelial cells distinguishes the lesion from others which simulate it. The cutaneous lesions known as verruga nodular, verruga mular, and verruga miliar have been reproduced in monkeys.     

ETIOLOGY OF OROYA FEVER : VI. PATHOLOGICAL CHANGES OBSERVED IN ANIMALS EXPERIMENTALLY INFECTED WITH BARTONELLA BACILLIFORMIS. THE DISTRIBUTION OF THE PARASITES IN THE TISSUES.

The pathological changes observed in the organs in Macacus rhesus monkeys which have succumbed to severe infection with Bartonella bacilliformis are similar to those found in human organs in persons dying of Oroya fever. The characteristic changes in the liver are the zonal necrosis of the cells around the hepatic veins, involving active macrophagocytosis of invading polymorphonuclear leucocytes in the necrotic areas, and a marked endothelial hyperplasia in the sinusoids or around the portal veins. In some instances there is fatty infiltration of hepatic cells. In the spleen persistent hyperplasia of the endothelial cells of the capillaries leads to the formation of minute foci of infarction, owing to occlusion of the lumina. The follicles are dispersed or reduced, and there is an active macrophagocytosis of cellular débris, polymorphonuclear leucocytes, and erythrocytes. In some specimens an increase in normoblasts is noted. Pigment is sometimes present. The lymphatic system shows general progressive endothelial hyperplasia, with active invasion of macrophages which contain polymorphonuclear leucocytes, erythrocytes, and greenish or dark pigments. In the bone marrow there is increased activity of macrophagocytes. Numerous normoblasts are found in some instances. In one monkey, sacrificed during the course of infection, small, verruga-like nodules were found in the lungs and spleen. Bartonella bacilliformis has been detected microscopically, though in small numbers, in all tissues showing histological changes. Parallel cultural determinations of the presence of Bartonella bacilliformis in the blood, liver, spleen, lymphatic glands, bone marrow, and local lesions established the relationship between the pathological conditions and the presence of the parasite. The organism seems to persist longest in the lymphatic glands. Cultural methods offer a simple and conclusive means for the determination of the presence or absence of the infecting organism.     

THE ETIOLOGY OF BARTONELLA MURIS ANEMIA OF THE ALBINO RAT : THE ISOLATION OF BARTONELLA MURIS

1. Bartonella muris has been isolated in pure culture on Noguchi''s leptospira medium from the blood of the splenectomized adult rat suffering with Bartonella muris anemia. 2. Bartonella muris is a small, actively motile, Gram-negative bacillus. It grows best on media containing blood and on Noguchi''s leptospira medium. The optimal temperature for growth is 20–25°C. It produces neither gas nor acid on media containing sugars. It does not hemolyze blood in artificial media. Viability of the cultures in leptospira media was maintained for 36 days. 3. With this culture a severe anemia was produced in rats weighing 30 gm., with the occasional appearance of Bartonella muris bodies on the red cells. The anemia occurred within 24 hours, and lasted for 3 to 5 days with recovery of the rat. Bartonella muris was recovered in pure culture from the blood of these animals. The blood of these rats was infectious for other 30 gm. rats. 4. 3 week old rabbits were infected with cultures of Bartonella muris, a severe anemia resulting after an incubation period of 48 hours. The organism was recovered from the blood on the 5th day after injection. Bartonella muris is non-pathogenic for adult rabbits. 5. A severe anemia was produced in young guinea pigs with cultures of Bartonella muris within 48 hours. The organism was recovered on the 2nd and 5th days after injection. Postmortem examination revealed changes in the organs similar to those found in splenectomized rats suffering with spontaneous Bartonella muris anemia. 6. The infection was reproduced in white mice. In one instance a severe anemia developed on the 5th and 6th days. The organism was recovered on the 6th day. 7. The anemia was produced in splenectomized adult rats of non-carrier stock. The organism was recovered from the blood stream of these rats. A marked leukocytosis was noted (65,000) at the peak of the anemia as is found in the spontaneous disease in infected splenectomized adult rats. 8. Serological tests have thus far failed to demonstrate the production of agglutinins, though complement-fixing antibodies are present in homologous sera in low dilutions.     

ETIOLOGY OF OROYA FEVER : X. COMPARATIVE STUDIES OF DIFFERENT STRAINS OF BARTONELLA BACILLIFORMIS,WITH SPECIAL REFERENCE TO THE RELATIONSHIP BETWEEN THE CLINICAL TYPES OF CARRION'S DISEASE AND THE VIRULENCE OF THE INFECTING ORGANISM.

Through the cooperation of Dr. Sebastian Lorente, Director of the National Department of Public Health of Peru, nine strains of Bartonella bacilliformis have been isolated, by means of the semisolid leptospira medium, from nine of twelve specimens of blood withdrawn from cases of verruga and forwarded from Peru under conditions of refrigeration. The cultural titer of the blood specimens immediately after their arrival (2 weeks after withdrawal) varied from 1:10 to 1:100,000. Blood from the severe anemic type of the disease, in which there was no eruption, had the highest titer. Blood agar slants yielded irregular results, but some strains grew well on these media. Morphologically the strains differed very little in fresh preparations examined by dark-ground illumination. In stained preparations some strains appeared coarser, others finer than the average. Special staining indicated that the flagella were characteristically unipolar and varied in number from one to four, some strains showing distinctly more wavy and heavier flagella than others. Young cultures grown on the surface of horse blood agar for 3 to 6 days show individuals with fairly sharp contours, short rods, often varying in thickness toward one or both ends, being intermingled with smaller oval or coccoid elements. Some strains show a predominance of bacillary, some of coccobacillary forms. It is not known whether these features are inherent or are due to conditions of growth, which, though identical, may react differently upon different strains. Definiteness in outline disappears with the age of the culture. More striking variations are found in the virulence of the different strains for the monkey (Macacus rhesus). Three of the nine strains isolated proved to be non-pathogenic for the monkeys. All three of these were derived from cases of benign verruga. The remaining six strains all gave rise to local lesions when intradermally inoculated and were recovered in culture from the blood of the animals. So far, severe anemia has not developed in any of the monkeys. It is significant that most of the severe cases yielded virulent strains, while some of the strains from benign verruga were non-pathogenic. It appears highly probable that the severe form of Carrion''s disease is, in general, caused by a virulent strain, while the benign forms are due to a strain of low virulence. On the other hand, a virulent strain may cause benign verruga in unusually resistant persons and a weak strain may give rise to severe blood infection in unduly susceptible individuals. The form of Carrion''s disease is probably determined primarily by the inherent virulence of the strain of Bartonella bacilliformis and is modified secondarily by individual predisposition in a given case. An interesting phenomenon brought out by the present investigation was the failure of the nine human blood specimens to induce local verruga in the same monkeys in which the corresponding cultures, inoculated simultaneously at separate sites, gave rise to typical lesions. Yet the original blood samples were shown by cultivation to have contained live bartonellas at the time they were inoculated, and blood culture revealed the presence of the microorganisms in the blood of monkeys which showed no other signs of infection after inoculation with the human blood. Whether this striking difference is merely a quantitative one or is due to some factor still unknown—such as, for example, a biological phase of the microorganism—has not been determined. The uniformly negative results of transmission experiments with blood by previous investigators is explained by an actual inability of the blood to induce skin lesions and the lack, until now, of a reliable method of detecting Bartonella bacilliformis in the monkeys'' blood. The strains isolated showed similar serologic properties, as tested by complement fixation.     

ON THE SPECIFICITY OF AGGLUTININS AND PRECIPITINS

The results of the partial saturation of precipitins with antigens related in derivation to the homologous one give no conclusive evidence of the regular existence in a single immune serum of multiple antibodies which act specifically on various chemical groups of the antigenic proteins. It seems possible to explain at least a part of the facts by the assumption that a single antibody will react to different degrees with several similar substances. By the partial absorption of hemagglutinins with heterologous blood, specific fractions were obtained. By such means one may readily differentiate the blood of related species, even when precipitins show but little difference. The peculiarities in specificity manifested by precipitinogens and agglutinogens suggest an essential difference in the chemical structures which determine the specificity of the two kinds of antigens.     

THE FORMATION OF AGGLUTININS WITHIN LYMPH NODES

Agglutinins are formed within the draining lymph nodes of mice, following intradermal injections of killed cultures of microorganisms.     

PHYSICOCHEMICAL CHARACTERIZATION OF MOUSE MYELOMA PROTEINS: DEMONSTRATION OF HETEROGENEITY FOR EACH MYELOMA GLOBULIN

Physicochemical characterization of mouse myeloma proteins revealed the individuality of each myeloma protein. When the myeloma proteins are considered collectively a wide range of individual properties were represented, including electrophoretic mobilities varying from the gamma to alpha region, hexose contents from 1 to 4 per cent, and ultracentrifugal components from 6.5 to 13 S. The 20 myeloma proteins could be divided into groups, the gamma type and the beta type myeloma globulins, on the basis of physicochemical, as well as immunoelectrophoretic, studies. Two gamma type myeloma proteins (5563, MPC-11) resembled normal gamma globulins, sedimenting as a single 6.5 S peak in the ultracentrifuge, and having a relatively low hexose content (1 per cent). Eighteen beta type mouse myeloma proteins differed from gamma myeloma proteins and, typically, were found on ultracentrifugal analysis to have multiple components with sedimentation coefficients of 6.5, 9, 11, and 13 S, having a higher hexose content (2 to 4 per cent) as well as distinctive chromatographic and starch gel electrophoretic properties. All of the mouse myeloma proteins were heterogeneous and heterogeneity of two types was observed. Polymer formation was responsible for the 9, 11, and/or 13 S components seen on ultracentrifugation of the beta type myeloma proteins. Starch gel electrophoresis revealed this type of heterogeneity as relatively widely separated myeloma protein components, presumably owing to the retardation effect of starch gel on the electrophoretic migration of the larger polymers. Starch gel electrophoresis revealed a different type of heterogeneity for the two gamma type myeloma proteins, each of these being shown to contain 5 or more components differing only in electrophoretic properties. The physicochemical characteristics of the γ-type and β-type myeloma proteins in the mouse indicated the close similarity of these proteins to the γ- and β-_2A-myeloma proteins in man.     

THE SOURCE OF AGGLUTININS IN THE MILK OF COWS

The foregoing experimental cases point to a distinct participation of the udder in the production of agglutinins when the gland is invaded by living or flooded by dead bacteria. The quarter injected reacts at first with a heavy influx of polynuclear leucocytes and later with an increase of agglutinins.     

STUDIES ON BARTONELLA MURIS ANEMIA : VII. THE PROTECTIVE ACTION OF COPPER AND IRON AGAINST BARTONELLA MURIS ANEMIA

The effect on Bartonella muris anemia of adding copper or iron or both to an adequate diet was studied. The addition to the diet of copper (0.1 mg. per rat per day), or iron (1 mg. per day), or both during a period of 2 days prior to splenectomy and 1 month subsequent thereto failed to protect adult albino rats against Bartonella muris anemia. The addition of copper to an adequate diet for a period of 10 days prior to splenectomy and 1 month subsequent thereto protected 75 per cent of the rats against the anemia. The addition of iron (1 mg. per rat per day) for a period of 10 days prior to and 1 month subsequent to splenectomy protected 50 per cent of the rats against this anemia. The addition of both copper and iron for a period of 10 days prior to and 1 month subsequent to splenectomy protected 75 per cent of these rats against Bartonella muris anemia. The addition of copper alone for a period of 2 months prior to and 1 month subsequent to splenectomy protected 63 per cent of the rats against Bartonella muris anemia. The addition of iron, or of both copper and iron during a period of 2 months prior to splenectomy and 1 month subsequent thereto protected about one-third of the rats against Bartonella murisanemia.     

THE VALUE OF MALLORY'S CONNECTIVE TISSUE STAIN FOR THE DEMONSTRATION OF VARIATION IN THYROID COLLOID

1. Mallory''s connective tissue stain used in connection with fixation of tissue by Zenker''s fluid or bichloride of mercury demonstrates the presence or absence of iodin in the thyroid colloid, as is evidenced by parallel chemical determinations, iodin feeding, and test-tube experiments. 2. Greatly increased activity of the heart is accompanied by a loss of thyro-iodin from the colloid of the thyroid. 3. This method, while apparently not adaptable to clinical use, may prove of value in further experimental work on the thyroid.