American Association of Anatomists. Sixty-ninth annual session; Marquette University School of Medicine Milwaukee Wisconsin April 4 5 6 1956. Officers abstracts demonstrations (pp. 449 У500)код для вставкиСкачать
ABSTRACTS OF PAPERS READ BY TITLE 449 406. An anatomical approach t o the problem of bronchial asthma. M. Wharton YOUKG, Howard University, College of Medicine. Bronchial asthma is a universal malady characterized by marked disturbance i n respiratory rhythm The etiological concept of spasm of the bronchus” antedates the microscopic revelation of the paucity of muscle in the bronchial wall. There is no definite muscular layer encircling the lumen of the bronchus and the bulk of cartilage which sustain the patency of this tube f a r exceeds the thin muscular network. Bronchoscopist Chevalier Jackson has never observed a “spasm of the bronchus. ” Breathing is controlled and regarded by a hypersensitive respiratory center located in the reticular formation in the hindbrain within which Magoun and Pitts ( ’39) have delineated an anatomically distinct inspiratory and expiratory division. I n asthma the derangement between inspiration and expiration could result from disorder between the two divisions of the respiratory center. The automaticity of respiration is revealed in sleep and anesthesia; the converse activity of inspiration and expiration suggests a sympathetic-parasympathetic control. The effreacy of adrenalin in asthmatic therapy may result from adrenergic influences which reestablish the normal interrelationship between the inspiratory and expiratory centers rather than to its presumed relaxation of visceral muscle spasm. Surgical removal of the carotid body has proved beneficial in chronic cases of asthma (Nakayama). The fact that asthmatic attacks may be initiated psychogenetically or by slight environmental changes further suggests its neurological origin. . DEMONSTRATIONS D $9. Quantitative data on lymphatic absorption of blood, yeast and tumor cells.’ Lane ALLEN and James MRACEK”, Department of Gross Anatomy, Medical College of Georgia. suspensions of cells were injeoted immediately upon the inferior surface of the diaphragm of adult rabbits. After a 10 minute interval the animals were exsanguinated, the thorax opened widely and lymph was obtained from nicked vessels on the superior surface of the diaphragm and analyzed. Under optimal conditions, as an average, for every 100 erythrocytes in the peritoneal cavity 85 erythrocytes appear in lymph, or 85% absorption. Leucocytes show 22.8% absorption, Yoshida r a t sarcoma cells show 19.1% absorption and yeast cells 16% absorption. Data, slides and photographs will be shown. - 1 Supported in part by Lederle. D 6 . A gold chloride-mercuric bromide impregnation.’ Rudolf ALTSCBUL, Department of Anatomy, University of Saskatchewan. Deparaffinized sections from tissues, fixed in 95% ethanol, are treated €or 4-6-12 hours in a saturated aqueous solution of mercuric bromide, containing 0.01% brown gold chloride. After rinsing, treatment with 2 4 % “hypo” and counterstaining with eosin, the sections are dehydrated and mounted. I n epithelial cells 450 AMERICAN ASSOCIATIOX O F ANATOMISTS of various types, in nerve cells, and in striated and smooth muscle, a black, finely granular deposit is found. I t is absent in endothelium, i n goblet cells, in the lining of pulmonary alveoli, in thyroid epithelium, in the collecting tubules of the kidney, and in lymph follicles. Fibroblasts are not showing it, but activated histiocytes are. Variations of intensity or lack of impregnation among elements of the same cell type seem t o depend on ceIIular activity prior t o fixation. Chick embryos are negative to the 5th day; but on the 6th day, liver, kidney and spinal ganglia can be impregnated. A similar change is seen in mouse fetuses between the 13th and 14th day. A reaction of the metal conipound with phospholipids has t o be considered. Although this is no final explanation, it is felt that these observations should be reported, because the lack of straight parallelism with other histochemical procedures, whether metal impregnations or not, suggests that this method is a new indicator of some metabolic processes. ~ 1 Supported by a grant from the Medical Division, National Research Council of Canada. D40. Evidence f r o m electron micrographs on the passage of material through pores of the nuclear membrane. Everett ANDERSON* and H. W. BEAMS, Department of Zoology, State University of Iowa. Electron micrographs of nurse cells in the ovary of Rhodnius prolixus reveal the nuclear membrane to contain pores of approximately 400 A i n diameter. Concentrated at certain regions near the nuclear membrane are masses of small graaules. Each granule measures about 200 A in diameter. Some micrographs reveal an apparent extension of nuclear material (including granules) through the pores into the cytoplasm where it is clustered in irregularly shaped bodies. 1 Supported by grant (B-301,C2)from the National Institutes of Health, U. S . Public Health Service. D 3 3 . d g e changes in nerve cells. Warren ANDREW, Department of Anatomy, The Bowmaa Gray School of Medicine, Wake Forest College.’ Photomicrographs, presented as color transparencies, illustrate the degenerative changes in the nucleus, cell body, and processes of nerve cells in old age, chiefly in human material. The general alterations, such as loss of Nissl substance, accumulation of pigment, increasing basophilia of the nucleus, and actual death and removal of cells are shown. The specific nature of alterations in particular groups of cells is emphasized for the Purkinje cells, cells of the inferior olive, and hypothalamus. The specificity of time of appearance and rate of progression of senile change in different parts of the brain, as presented in the conclusions of Oskar and CBcile Vogt, is confirmed and amplified. Illustrations are given of peculiar changes which apparently represent ‘ reaction” to senile change, a compensatory activity of nucleolus, nucleus, and cell body, as seen both in the author’s material and that of the Vogts. Preparation of this demonstration has been aided by the Gray Pharrnaccutical Company and the Josiah M a w . Jr., Foundation. ABSTRACTS O F DEMONSTRATIONS 451 D 2'. Skeletal changes correlated w i t h irradiation in radium and plutoniumintosicated dogs.' James S . AENOLD*, Radiobiology Laboratory, Department of Anatomy, Vniversity of Utah College of Meaicine. (Introduced by Thomas F. Dougherty) Dogs were given radium and plutonium intravenously in amounts calculated t o produce retention of 2-3 mc/kilo after one year. The skeletal changes involved massive death of both cortical and spongy bone with loss of osteocytes, fibrosis of endosteal surfaces, and later reconstruction and proliferation of metaplastic bone. Pathologic fractures occurred at a time following bone death but preceding extensive reconstruction. The bone marrow was all but destroyed in areas of spongy bone but later regenerated following fibrosis of bone surfaces. There was early and persistent hyperplasia of marrow in long bone shafts. I n spite of a considerable difference in the pattern of irradiation to bone between Ra and Pu, the morphologic pattern was quite similar, suggesting mechanisms other than direct irradiation involved in their pathogenesis. 1 Supported by A.E.C. Qrant dT' (11-1)-119. D J 4 . Stereoscopic color photographs of the heart. David L. BASBETT, Department of Anatomy, Stanford University, California. Normal internal structures o f the dissected heart are shown in three-dimensional color transparencies. D 8. Liver tumor cells in the anterior chamber of the eye. Sylvia H. BENSLES, Department of Anatomy, University of Toronto. A liver tumor, produced in a young white hybrid rat by feeding 4-dimethylaminoazobenzene, was found to be an adenocarcinoma. A suspension of cells from this tumor was injwted into the anterior chamber of the eye of another white hybrid control rat. The latter was sacrificed fourteen days later and the eye was prepared for microscopic study. Although there was considerable vascular and tissue reaction t o the tumor implant, the tumor cells grew well and there was very little evidence of necrosis. Where the tumor cells were free in the aqueous humor they were anaplastic; but where they came in contact with the iris they became differentiated so as to resemble intestinal epithelium, forming mucous glands and surface goblet and absorptive cells. Moreover, the tissue of the iris developed the structure of a lamina propria with papillary projeetions like villi, so that t h i s area resembled the mucous membrane of a diminutive duodenum. D I d . Sudan black staining of gross brain sections.' Harold BRODY* and John E. WIRTH", Department of Anatomy, University of Buffalo, School of Medicine. (Introduced by Oliver P. Jones) Sudan black B has been used in staining frozen sections of many tissues including nervous system. It has been found that it is particularly adaptable 452 AMERICAN ASSOCIATION O F ANATOMISTS for gross brain sections cut at any thickness. The differentiation between gray and white matter and the rapidity of the staining technique makes the procedure useful both in the neuroanatomy and neuropathology laboratories. Specimens will be shown which may be stored in formalin or embedded in plastic. 1 This investigation. was supported in part by research grant B-645 ( C ) from the National Institute of Neurological Diseaaea and Blindness of the National Instltutes of Health, Public Health Service. D 58. Hormonal dependence of transplanted adrenal cortical tumors of mice. Henry C. BROWNING, Department of Anatomy, University of Texas Dental Branch and Baylor Medical College, Houston. The intraocular transplantation technic was applied to four adrenal cortical tumors arising in castrate ce or C mice with the following results in autoIOgOUS hosts:a. One ce and two C tumors showed hormonal dependence. Suitability of the host, in terms of latency after transfer and actual growth rate, increased in the sequence; intact males, intact females, castrate females, castrate males. b. I n castrate males transplants commenced growth between the 30th and 60th day and filled the anterior chambers by the 120th day. In intact males the latency was approximately 120 days and the anterior chambers were not filled until the 180th day. c. Tumor growing i n intact hosts still showed, on retransplantation, increased latency in intact as compared with castrate hosts. d. Shortening of latency was only effective if castration was executed within 45 days of transfer. e. I n male castrates testosterone inhibited growth and caused some regression of growing transplants ; growth was resumed after cessation of administration. 1 Supported by a grant from ( C - 2 4 6 3 ) the National Cancer Institute. National Institutes of Health. D 52. Abnormalities produced in the chick embryo by radioactive phosphorus (Paa).IArthur 0. CHAPMAN and John 8. LATTA, Department of Anatomy, University of Nebraska, College of Medicine. Tissue necrosis in chick embryos resulting from ionizing irradiation has been previously reported (Chapman and Latta, Anat. Rec., 129: 292, ’52; Chapman, J. Morph., 95: 451-470, ’ 5 4 ) . A few embryos have developed regional malformations of the central nervous system, eyes, notochord and somites aa a result of Pn injected into the egg before starting incubation. Brain changes range from rosettes i n the wall t o severe distortions. I n one 5-day embryo, part of the forebrain wall projects inside-out through the unclosed anterior neuropore to form a large ‘‘encephalocele.” The eyes tend to be retarded and sometimes malformed. One eye i n the 5-day embryo mentioned above has a very small optic cup which failed to enclose the lens. In one ?-day embryo the optic CUPS show marked retardation and degeneration and the lenses failed to develop. ABSTRACTS O F DEMONSTRATIONS 453 The spinal cord also may show rosettes in the wall, have partial duplication, fail t o close dorsally, or be completely absent in some areas. Malformation or absence of the notochord or somites is seen in parts of some embryos. There is sometimes a regional correlation between spinal cord, notochord and somite abnormalities. Isupported by research grant No. B-534 (R) of the National Institute of Neurological Diseases and Blindness, United States Public Health Service. D 48. Neonatal differentiation of renal tubules in the mouse, studiea with the electron microscope.' Sam L. CLARK, Jr., Department of Anatomy, Washington University School of Medicine. Kidneys from fetal, neonatal, and adult mice were fixed in 1%osmium tetroxide in dichromate buffer at p H 7.2, embedded in methacrylate, and sectioned with glass knives. Sections 2~ thick were examined by phase contrast microscopy and after staining with periodic acid-Schiff technique, and sections approximately 200 A thick were observed by electron microscope. I n contrast to the proximal and distal convoluted tubules of the adult, those of full-term fetal mice do not possess rod-like mitochondria in parallel array, separated by invaginations of the basal plasma membrane. Instead, the mitochondria are round or oval and the basal plasma membrane is simple in contour. In addition, the proximal convoluted tubules of fetal mice are distinguished by variable, rudimentary brush borders, and the presence of electron-dense round bodies which stain with the periodic . micrographs will be acid-Schiff technique and reach a diameter of 3 ~ Electron shown to demonstrate the development of these tubules from the fetal to adult form during the first few days of life. 1 Supported by grant RQ-3784 USPHS. D 19. A method for implanting chronic electrodes in thc rat. B. Vaughn CRITCHLOW*, Department of Anatomy, University of California at Los Angeles and Veterans Administration Hospital, Long Beach, California. (Introduced by Charles H. Sawyer) A method developed by Green, Maxwell and Osborn for chronic implantation of electrodes in cats and rabbits (to be published) has been modified for the rat. The anesthetized animal is placed in a stereotaxic instrument, the skull is trephined, and a pattern of concentric, bipolar electrodes, which are attached to earrier tubes by a low-melting electronic cement, is lowered into a pre-determined position. The exposed portions of the electrodes and three small anchoring screws in the skull are then covered by a thin layer of dental cement. When the cement has hardened thoroughly the carrier tubes are removed by heating them slightly, a small electronic plug is soldered to the electrode leads, and a second layer of dental cement is poured around the solder joints and the base of the plug. This technique, which has proved quite satisfactory for electroencephalographic (EEG) studies on unanesthetized rats, affords an easy, stable and long-lasting connection to recording equipment ; and preparations with three sub-cortical and two cortical 454 AMERICAN ASSOCIATION OF ANATOMISTS electrodes have been maintained in good condition for as long as 5 months. The only artifacts seen thus f a r have been those associated with gross movements, and these show up in all EEG channels as easily recognizable, high-amplitude slow waves. Il 17. A method for reclaiming poorly impregnated silver nitrate nerve# preparations. J. David DECK*, Department of Anatomy, University of Virginia. (Introduced by C. C. Speidel) Sets of serial sections of larval urodele forelimbs were inadequately stained three years ago using a technique for the differentiation of nerves. These sections had been impregnated with silver nitrate, reduced in sodium bisulfite, toned with gold chloride, and fixed i n sodium thiosulfate. Due to the capriciousness of the technique, nerves were not selectively impregnated. Recently, sample sets of these serial sections have been reclaimed with very good results by bleaching with solutions of strong acids followed by reimpregnation. Cover glasses were removed from chosen slides and the sections were hydrated. A solution of acids (three parts cone. hydroch1oric:one part cone. nitric: 10 parts water) was prepared and the slides were immersed i n the acid mixture for a period of from one to 5 minutes, until the sections were bleached white. Sections were not destroyed or distorted, though care was necessary t o keep from washing them off the slides after the acid treatment. After prolonged washing, sections were reimpregnated, toned with gold chloride, and counterstained with eosin. Nerve fibers were differentiated quite as well as could be expected in an original silver nitrate impregnation. D d 6 . Some observations on neurosecretion in the rat. Jacob DE GROOT*, Department of Anatomy, School of Medicine, University of California a t Los Angeles. (Introduced by John D. Green) Female rats were subjected to various experimental procedures (dehydration by thirst or administratioil of hypertonic saline, administration of colchicine, pituitary stalk section, placement of electrolytic brain lesions) and their estrous cycles were studied. The amount and distribution of Gomori-positive material and its relation to nerve fibers were studied using the chrome-alum hematoxylin and Bodian methods, in these rats as well as in the normal rat, cat, dog and monkey. Material staining similarly to the neurosecretory substance in the supra-optic and paraventricular nuclei was found in the habenular nuclei, in ependyma and glia cells, and in macrophages near lesions. D 44. Electron microscope observations on several major cell types in bone marrow f r o m the human and the rat.' Quin B. DE MARSH and Jean KAUTZ*, Department of Anatomy, University of Washington. Human bone marrow was removed by sternal biopsy and fixed for two hours in 0.25% buffered osmic acid. Rat marrow was dissected out of the femur intact and similarly fixed. Electron microscope observations are compared and correlated with phase and polarization microscope observations. ABSTRACTS O F DEMONSTRATIONS 455 Some previously unreported phases in the life cycle of plasma cells will be pictured. These cells appear to have a “storage phase” during which the spaces within endoplasmic reticulum sacs are filled and swollen with a moderately dense, finely precipita€ing material. Except i n the case of plasma cells, endoplasmic reticulum is rarely found in mature blood cells. Clusters of Golgi membranes are often seen. Using the presence of cristae as a criterion, mitochondria can be clearly distinguished from specific granules in the granulocytes and in megakaryocytes. Details of the submicroscopic morphology of both megakaryocytes and platelets will be presented, as well as findings related to the production of platelets by megakaryocytes. Lymphoblasts, myeloblasts and monoblasts from leukemic patients will be shown. Marrow capillaries are often encountered, especially in the unbiopsied rat marrow. Possible morphological evidence for the way in which marrow cells may enter the circulation will be discussed. 1 Supported by a grant (C-133245 ) from the United States Public Health Service. D b 7 . The histology of the ovary of Thomomys, the western pocket gopher.’ Kenneth L. DUKE, Department of Anatomy, Duke University School of Medicine. The ovarian surface of Thornomys is covered by a germinal epithelium consisting of flattened cells. I n some ovaries, near the hilus, the cells may be cuboidal in shape. Epithelial invaginations into the cortex are uncommon. Numerous primary oocytes are located near the ovarian surface and are characterized by large, spherical nucleoli. Such oocytes measure 25 p in diameter. As the follicles grow in size, the most unique feature is the spectacular hypertrophy of the theca interna. Preovulatory follicles with a diameter of 8 0 0 p may be surrounded by a strip of theca interna measuring 500 p in thickness. This layer atrophies following ovulation. The cells of the theca interna are large epithelioid cells, and the layer is extremely vascular. There is a high rate of follicular degeneration. The theca interna of atretic follicles usually gives rise to interstitial cells. The degeneration of many intraovarian ova is accompanied by the appearance of large cytoplasmic vacuoles. Polar bodies are often formed in the ova of degenerating follicles, and the spindle for the second maturation division may develop. The ovarian stroma is well populated with argyrophilic fibers. Collagenous fibers are most apparent in the tunica albuginea of the hilar region and in the perivascular tissue of the medulla. Elastic fibers are confined to the vicinity of blood vessels, and the subperitoneal layer of the mesovarium. * Supported by a grant from the Duke University Research Council, D 9. Human liver carcinoma and vertebrate evolution? Hans ELIAS, Department of Anatomy, The Chicago Medical School. I f one studies the structure of many cases of primary carcinoma of the human liver, a chaotic picture seems to unfold. However, a student of comparative 456 AMERICAN ASSOCIATION O F ANATOMISTS embryology will find that every form of such tumors has its counterpart in the embryonic or adult liver of some vertebrate: muralia of many cells thick walls as in the liver of turtle embryos; globular masses as in snake embryo livers; trabeculae as in pig embryo livers; tubules as i n livers of embryos of sharks, alligators and song birds; duct-like structures as in chick embryo livers; spindle cell masses, as in marsupial, rat and primate embryo livers. Thus atavism manifests itself in the manifold varieties of hepatocellular tumors of man. This phenomenon provides a new piece of evidence for the theory of human evolution from lower vertebrate forms. 1 Supported by a grant from the U. 9. Public Health Service. DS. Rat and mouse whole bones grown as isogencuus subcutaneous implants? William J. L. FELTS, Departments of Anatomy, Tulane University and University of Minnesota. This demonstration will consist of line drawings and cleared specimens illustrating the following series. (1) Phalanges of 5-day rats, implanted i n littermates and allowed to remain for as long as 200 days. ( 2 ) Humeri of two-day mice, implanted in the female parent, remaining for 28 days. ( 3 ) Humeri of mice ranging i n age from three days prenatal t o 28 days postnatal, implanted in the parent for 28 days. ( 4 ) Humeri of two-day mice implanted for 4 weeks but held at constant room temperature (23-24°C.) for up to 72 hours before implantation. This material will show that immature long bones will grow and mature remarkably well as nonfunctioning implants, and maintain size and shape after major growth is completed. Data showing the effects of donor age upon subsequent behavior of the immediate implant will be presented. I n addition, it will be shown that the whole bone, even a t a relatively high storage temperature, retains a potential for growth for many hours (e.g., cartilage growth results in an appreciable length increase during the 28 day period even after 36 hours storage). However, an extended period of storage results in the orderly suppression of potentials for each of the several tissue regions. Currently under investigation are the question of induction phenomena i n implants and the effects of a wide range of temperatures and storage periods. * The latter part of this study has been aided by a grant from the Minnesota Chapter of the American Cancer Society. D 60. Cutaneous and transcutaneous respiration in man. Laurence R. FITZGERALD, Division of Anatomy, Medical Units, University of Tennessee. It has been shown by various workers that about 2% of the total oxygen taken up by the entire human body is taken in through the skin surface. Slightly more than 2% of the total carbon dioxide given off by the body passes out through this surface. Since the magnitude of the cutaneous respiration is less than the inherent error of measurement of the total respiration, it has justifiably been neglected in studies of over-all metabolism. Calculations will be presented which show that all or most of the epidermis of the skin can receive sufficient oxygen by diffusion through the surface to main- ABSTRACTS OF DEMONSTRATIONS 457 tain the observed metabolic rate. I n many regions of the body, oxygen can diffuse into the level of the capillaries of the dermis at a concentration higher than that in the blood. However, the small area of the capillaries in the skin prevents absorption of large amounts of oxygen through the skin into the blood vessels. Thus transcutaneous respiration probably can never be a major factor in total bodily economy in man. The role of cutaneous respiration in renewal of epidermal cells and in wound-healing seems to deserve further study. D 30. Abnormalities i n the pattern and relationships o f the major arterial trunks and aortic arch derivatives of fetal rats following maternal trypan blue injections? Marjorie H. FOX and Charles M. GOSS, Department of Anatomy, Louisiana State University School of Medicine. Approximately 30% of all fetuses recovered from dams injected with l m l of a 1% aqueous solution of trypan blue either 73 or 83 days after insemination exhibited some degree of transposition or rotation of the aorta and main pulmonary artery. All 4 major types of human congenital transposition complexes (overriding aorta, partial transposition, Taussig-Bing, complete transposition) were well represented. Complete transposition of the aorta was invariably accompanied by a counter-clockwise rotation of the aortic valve and alterations in the distribution pattern of the coronary arteries. Approximately one-half of the fetuses with aortic or pulmonary transposition had additional abnormalities in the pattern of the aortic arch derivatives. The tendency towards this concomitant aberration was more marked in the 83 day series. Over 1 0 abnormal patterns have been identified, but distally arising (retroesophageal) right subclavian artery, right aortic arch with mirror imaging of the normal pattern, and double aortic arch formed by various combinations of right and left 4th and 6th arches occurred most frequently. Supported by grant from the National Heart Institute. D 45. Electron microscopic study of normal human leukocytes? James A. FREEMAN"; Department of Anatomy, Louisiana State University School of Medicine. (Introduced by Frank N. Low) Normal leukocytes were recovered from human blood by a technic not utilizing anticoagulants or chelating agents, and fixed in buffered OsO,. The morphology of these cells is presented, with particular reference to ultrastructure i n mitochondria, specific granulations, nuclear membranes, and other organoids and inclusions. 1 Aided by grants from the Public Health Service (H-1663) and The Idly Research Laboratories. 2 Public Health Training Fellow (HTS-5111). D $3. Fused quartz rod demonstration of small blood vessels and circulating blood cells of A m p h h m a ? Fann EARDING*, Hilda DEBACKERI and Melvin H. KNISELY, Department of Anatomy, Medical College of South Carolina. Amphiumae have the largest red cells known to man, about 7 0 long. ~ White cells also are large. Relaxed or dilated capillaries are wide enough to permit 458 AMERICAN ASSOCIATION O F ANATOMISTS these large cells to pass undistorted. Capillaries may measure 100 i n diameter. Kidney glomeruli are enormous, measuring up to 3.5 mm i n diameter. L. Warner ('49) recorded in motion pictures the plugging of individual Amphiuma glomeruli with agglutinated blood cells. Thus, microphysiological and micropathological studies can be conducted at low magnifications. At any magnification, much more anatomical detail can be observed in Amphiumae than in other experimental animals. Therefore, many problems of physiology and pathology can be attacked easily i n Amphiumae. For instance, we need to know each and every anabolite necessary for nourishing the linings of small blood vessels sufficiently well so that they do not leak blood plasma proteins. We need to know the responses of small vessel linings to many kinds of specific toxins, so that we may use these responses to test for the possible presence of toxins in blood drawn from patients with various pathologic conditions and diseases. Amphiuma bladder mesentery is a n excellent area for experimental manipulation and study. Cannulation of individual capillaries is possible. A copy of a large bibliography on Amphiumae, and some sources of supply of these animals, will be given to those interested. Supported by USPHS Grant H-1683. D 49. A preliminary study of the electron microscopy of renal juxtaglomerular cells; correlation with light microscopy? Phyllis Merritt HARTROFT,, Department of Pathology, Washington University Medical School. (Introduced by W. Stanley Hartroft) I n an attempt t o elucidate further the nature of juxtaglomerular cells, a study using the electron microscope is currently underway. Renal tissue from normal, adrenalectomized, and salt-deficient rats was fixed in Dalton's ffuid (buffered osmic acid) and embedded in methacrylate. Thin sections were cut for examination with the electron microscope and thicker ones (under la) were prepared for phase microscopy. I n addition, paraffin sections of tissue from the same kidneys were stained by a special technique previously described. These sections and those prepared f o r phase microscopy served to correlate the appearance of juxtaglomerular cells as visualized with the light microscope with their appearance in electron micrographs. In paraffin sections, juxtaglomerular cells are most easily recognized by their granules. I n electron micrographs, these granules are very conspicuous by virtue of their osmophilia. They are spherical t o oval in shape, similar in appearance to granules seen in other secretory cells. Mitochondria can be readily distinguished as can the abundant ergastoplasm with its prominent ergastoplasmic granules. These features provide a sharp contrast between juxtaglomerular cells and adjacent cells in the arteriolar media and support the concept that they may be secretory in nature. Supported by a grant from the Nutrition Foundation. Acknowledgment is also made to the Departments of Anatomy and Radiology, Washington University Medical School, for facilities. ABSTRACTS O F DEMONSTRATIONS 459 0 5 5 . A human ovum with a n estimated ovulation age of about nine days.' Chester K. HEUSER, Department of Microscopic Anatomy, Medical College of Georgia. I n the uterus of a 34-year-old white woman removed surgically by Dr. Richard Torpin an intact ovum was found implanted in the crevice a t the junction of the posterior and anterior walls. The ovum is partly covered in by the endometrial epithelium. Reconstructions prepared from the serial sections reveal a symmetrical germ disk (0.05 X 0.09 X 0.132 mm) placed normally in a chorion measuring 0.36 X 0.58 X 0.59 mm. Size of chorionic cavity, 0.1 X 0.1 X 0.3 mm. The primary yolk sac is forming as is indicated by cells which have spread from the entodermal layer of the germ disk. Numerous small communicating lacunae are present in the thickened syncytium on the abembryonic pole of the ovum. A few lacunae communicate with surrounding endometrial vessels. Some of the lacunae contain small amounts of static blood. An unusual feature of the ovum - which may be related to the method of formation of the lacunae -is the presence of large ghostlike spheres or nuclei entrapped in the syncytial spaces. 'This investigation was supported (in part) by a research grant R.G.4222 from the National Institutes of Health, Public Health Service. D 35. Methods for studying regression of atheromata using intraocular transplants and implants? A. C. HIGGINBOTHAM, Department of Anatomy, Medical College of South Carolina. Atheromata contribute to the death of many persons today. Cholesterol is present in all atheromata, and its removal may be essential for their regression. Thus f a r no method has been developed for studying the regression of individual atheromata. Green ( '38), Turner ( '39), Markee ( '40), Martinovitch ( ' 5 5 ) , and Ramm ( ' 5 5 ) have successfully used anterior chamber transplantation for other purposes. Atheromata or large cholesterol crystals placed in the anterior eye chamber can be studied and their dimensions measured frequently. Experimental atherosclerosis can be developed easily in rabbits, but dogs are resistant. Homotransplants of two non-atheromatous and 4 atheromatous aortas were made into the anterior eye chambers of 6 young male rabbits. Two homotransplants of non-atheromatous aorta were made into young male dogs. Heterotransplants of atheromatous human aorta were made into the anterior eye chambers of two adult male rabbits. Large cholesterol crystals were implanted in one dog and 5 rabbits. Gross characteristics of transplants and implants can be visualized and photographed through the corneas of host animals. In histologic sections, unstained and following Oil Red 0 staining, birefringent substances, including cholesterol and its esters, can be identified and their distribution studied with the polarizing microscope. Other details of the transplants can be studied in variously stained sections. 1 Supported by USPHS Grant H-1020. 460 AMERICAN ASSOCIATION O F ANATOMISTS D 3 8 . T h e fine structure of normal and leultemic marrow cells.' A. F . ROWATSON* and E. A. McCULLOCH*, Departments of Anatomy and Medicine, University of Toronto. (Introduced by A. W. Ham) This exhibition consists of electron micrographs of sections of cells from bone marrow and peripheral blood, obtained from normal and leukemic patients. About 20 specimens of marrow and 4 of leukemic blood were examined. The fine structure o f cells from the lymphocytic, granulocytic, megakaryocytic, erythrocytic and plasma cell series is illustrated. I n some instances parallel examples of normal and leukemic cells are shown. I n addition the appearance of cells from marrow and peripheral blood after many months of continuous cultivation i n vitro is demonstrated. Aided by a grant from the hational Cancer Institute of Canada. D.20. Studies in dogs on absorption from the subarachnoid space and on the ultrastructure of the choroid plexus. Guy M. HUNT and E. Harold SHRYOCK, Department of Anatomy, College of Medical Evangelists. The rate o f absorption of Ringer '8 solution introduced a t graded pressures (from 100 to 600 mm of water) into the subarachnoid space at the cisterna magna was found t o be mathematically related to the pressure. Graphic analysis o f these data introduces the possibility of estimating rates of absorption at pressures less than those utilized in the experiments. A simple apparatus for measuring absorption rates while maintaining constant pressure levels will be demonstrated. Tissues for light microscopy were obtained after perfusing the anesthetized animals through both common carotid arteries with Ringer's solution followed by 10% formalin. The perfusate was injected under a pressure calculated to slightly exceed the animal's normal blood pressure. Sections were stained with hematoxylin and triosin, Heidenhain '8 hematoxylin, and Krichesky '8 modification of Mallory 's aniline blue collagen stain. Representative sections will be displayed on microscopes and by colored transparencies. Tissues for electron microscopy were removed promptly after the death of the animals, fixed in buffered osmium tetroxide and sectioned a t approximately 0.05 F . Prints of electron photomicrographs will be displayed. D99. Electron microscopy of growing oocytes of Fundulus? Norman E. KEMP and Margaret D. ALLEN*, Department of Zoology, University of Michigan. Small pieces of ovaries or single, mature eggs from specimens of Fundulus collected between June and August a t Woods Hole, Massachusetts, were fixed in 1% osmic acid in buffered artificial sea water, embedded in methacrylate and sectioned at .05 L./ with a Porter-Blum microtome. Young oocytes lacking yolk have numerous mitochondria, concentrated in the peripheral cytoplasm, and possess small granules of undetermined nature in the perinuclear cytoplasm. A few thick strands of highly osmiophilic material (eosinophilic with routine staining for light microscopy) may be present among the follicular cells. As growth continues, ABSTRACTS OF DEMONSTRATIONS 461 the zona radiata begins to differentiate a t the surface of the oocytes, accompanied by the appearance of lipid yolk and later of protein yolk and cortical alveoli i n the underlying cytoplasm. Follicular cells pull away from the oocyte, thus leaving spaces containing protoplasmic processes of the follicular cells. The thick osmiophilic strands among the follicular cells become abundant during early growth but later diminish in number. Within the differentiated zona radiata coiled fibers believed to be the distal ends of follicular cell processes extend between parallel bars, apparently the walls of cylinders, comprising a structural framework. Mature oocytes from which the chorion has been removed have microvilli at the surface. Cortical alveoli burst through the surface after fertilization. Supported by grants from the National Science Foundation ( 0 1 1 6 6 ) and the Michigan Memorial-Phoenix Project. D 59. Visualization under ultraviolet lighting of the inspiratory and exppiratory pathways through the dog's nose. J. Edward KING*, Department of Anatomy, Duke University. (Introduced by R. Frederick Becker) Gross specimens of parasagittally sectioned dog heads will be shown under ultraviolet light. The animals previously had inspired or exhaled an aerosol containing fluorescein. Inspiration occurred during quiet breathing or during a sniff. One-way passage of air through the nasal chambers was controlled by a special tracheal valve inserted by cannulization in the living animal and was also simulated in decapitated heads for comparison. Diagrams and photographs taken under ultraviolet light will also be shown. D 10. Radioautographs of young rat tissue after administration of C"-labelled bicarbonate, glucose and mannose. Y . KUMAMOTO*, Department of Anatomy, McGill University. (Introduced by C. P. Lehlond) One- and three-day-old rats were injected with C"-bicarbonate, -glucose or -mannose and sacrificed 3-4, 24, and 72 hours later. Tissue sections, prepared after fixation in alcohol-formalin, Orth, or Carnoy, were coated with Eastman Kodak NTB2 and 3 emulsions, exposed for 3 to 9 months, and processed by standard radioautographic procedure. A similar, widespread distribution of radiocarbon followed injection of the three labelled substances. The radioautographic reaction of soft tissues was maximal a t the earliest interval and decreased thereafter. This decrease was generally gradual, except in a few cases-chondrocytes and the mucus of the sublingual gland - where it occurred promptly. I n contrast, the radioautographic intensity observed in bone and dentin durieg the experimental period was permanent. The distribution of the isotope originating from the three substances studied differed in several aspects: both sugars, but not bicarbonate yielded an intense reaction in the goblet cells of the intestinal epithelium; whereas mannose but neither glucose nor bicarbonate elicited an intense reaction of enamel. It is 462 AMERICAN ASSOCIATION O F ANATOMISTS concluded from these results that mannose can transform into glucose or bicarbonate, and that glucose can transform into bicarbonate, but that transformations in the reverse direction do not occur to a sufficient extent t o be detectable radioautographically. D 22. Demonstration of spatial relationships of capillaries to neuronal elements in specific parta of cat nerz)ous syste7n.l Isabel LOCKARD*, J. BARHAM*, N. FORLIDAS* and R. MYERS*,Department of Anatomy, Medical College of South Carolina. (Introduced by J. A. Gavan) Many methods have been devised for demonstrating central nervous system capillary beds; few have demonstrated nerve cells simultaneously (The Circulation of the Brain and Spinal Cord, '38). Our demonstration material contains vessels injected by the Williams ('48) method, and cells stained with thionin, and permits measurements, comparisons and contrasts of specific and different parts of the nervous system. Krogh ('29) showed that each capillary supplies a cylindrical zone of tissue surrounding it. Flow through single opossum brain capillaries is necessary to maintain the surrounding neurones (Scharrer, '39). Equations describing the rates of supply of oxygen to parts of tissue cylinders have been developed by Ingram Bloch ('41, '43), G. W. Schmidt ('52, '53a, '53b), Opitz and Schneider ('50). Methods are available for the measurement of capillary densities (Craigie, '38; Chalkley, '43). Bed cells circulate freely in health but become agglutinated in many diseases, forming masses which resist passage through terminal arterioles, and can plug capillaries (Knisely et al., '47 ; Edward Bloch, '55). Reduced flow through capillaries, or other localized circulatory disturbances, may underlie many neurologic disorders. TO calculate oxygen supply of cells between capillaries in health and disease as a basis for contributing to our understanding of these phenomena, we need to know such information as: distance between capillaries, numbers per cubic millimeter, diameters, lengths, and relationships to neuronal elements. Supported by USPIlS Grant B-457 ( C ) . D 41. Electron Micrographs of protoplasmic double membrane systems.' Frank N. LOW, Department of Anatomy, Louisiana State University School of Medicine. Double membrane systems comparable to those of nuclear membranes, endoplasmic reticulum and mitochondrial cristae, but of lesser dimensions, are illustrated. They were observed in the striations and apical cell membranes of intestinal mucosa cells, golgi apparatus, single walls of cisternae of endoplasmic reticulum and in unidentified cytoplasm inclusions. The overall dimensions of these double membrane systems, which consist of two dense parallel membranes enclosing a lucid interspace, range from about 150 to 6 0 A or less. This implies a thickness of 20 A or less in the individual dense membranes in the finer complexes. 1 Aided by grants from the Public Health Service (H-1663) and The Lilly Research Laboratories. ABSTRACTS OF DEMONSTRATIONS 463 D 5 1 . A case of human cyolopia associated with rudimentary pinnae, absence of the right radius, polydactyly and syndactyly. E. W. LOWRANCE, M. D. OVERHOLSER, Stuart 0. LANDRY, Jr.* and F. E. DOENGESI, Department of Anatomy, University of Missouri. A human male premature (32 weeks prenatal, living 45 minutes following birth) 40 em in length and weighing 1485 gm presents the following apparent anomalies: classical cyclopia with a median single eyeball but with eyelids extending laterally; a soft, club-shaped proboscis, located above the eye, approximately 20mm long and 12mm in maximum diameter, with a single, offset, patent opening measuring approximately 1mm in diameter. The specimen also has very rudimentary right and left pinnae which are only slightly raised above the surface of the side of the head; right and left sixth digits present on the superior extremities ; syndactyly involving digits two, three and four on the right lower extremity, and digits three and four on the left lower extremity; the right wrist and hand deviating abruptly toward the thumb side. Roentgenograms show the absence of a right radius and the presence of five metacarpals on each side. However, the shaft of the left fifth metacarpal is thickened and its middle third bears a medially directed process with which the left sixth digit appears to articulate. An extended report will follow detailed dissection and study. D Z l . The relation of the cranial aura to the cavernous sinuses in the cow. C. P. MARTIN and William Henry BOYD, Department of Anatomy, McGill University. The cavernous sinuses are described as irregularly shaped, endothelial lined venous spaces between the inner and the outer laminae of the cranial dura at the sides of the body of the sphenoid bone. I n the cow, the cavernous sinuses are within the outer lamina of the cranial dura mater. The outer dura which forms the floor of the cavernous sinuses and the sella turcica, enclose the optic and the maxillary nervw in the side walls of the cavernous sinuses. The outer dura on the internal surface of the parietal bones, is continuous with the dura enclosing the nerves i n the side walls of the cavernous sinuses. The outer dura courses medially above the cavernous sinuses, and beneath the lamina of the inner dura. The inner and the outer laminae of the dura form the roof of the cavernous sinuses, and fuse about the stalk of the hypophysis in the formation of the diaphragma sella. The outer lamina of the dura encloses the hypophysis, forming its capsule and the medial walls of the cavernous sinuses. D 1 8 . The rat brain in stereotamic co-ordinates. Leo C . MASSOPUST, Jr., Laboratory of Neuroanatomical Sciences, National Institutes of Health. A new stereotaxic instrument and scaled co-ordinate maps of parasagittal and frontal sections of the rat brain to be used with it will be demonstrated. 464 AMERICAN ASSOCIATION O F ANATOMISTS Measurements were taken on 30 animals weighing 100 to 300gm. Due to the increase in brain size within this weight range, it was necessary to make separate co-ordinate maps for animals of 100, 200 and 300gm. Because of the large number of photographs for each weight group and the limitation of space, the demonstration will be limited to illustrations of the 200gm group. Coordinates are represented on uniformly enlarged photographs of frozen sections (100 p ) which were stained by the Sudan Black B technique. This method practically eliminates dehydration shrinkage. D5. S h i f t s in the lumbar plexus correlated w i t h the lumbarization of the last thoracic vertebra in mice. Wallace MCNUTT and Eddie DEAN*, Department of Anatomy, The University of Texas Medical Branch, Galveston. The lumbar plexus of 80 mice from strains SEA-se (vertebral formula 7 cervical, 13 thoracic, 6 lumbar) and SEC-dse (vertebral formula 7/14/6) were dissected. The pattern of each strain showed a constancy that was attributed t o the uniform genetic make-up. The caudal portion of the plexus was alike i n the two strains in that the obturator and femoral nerves were formed by the lst, 2nd, and 3rd lumbar nerves; the tibioperoneal by the 3rd, 4th, and 5th; and the pudendal by the 5th and 6th lumbar nerves. However, there was a strain difference in the cranial portion of the plexus, the subcostal and iliohypogastric arising from the last thoracic (12T) and the ilioinguinal from the 1st lumbar in the SEA-se mice and all three of these nerves from the last thoracic (13T) in the SEC-dse mice. Each of these strains has been bred brother by sister with forced heterozygosis for the short-ear gene, which has as one of its effects the suppression of the last pair of ribs without changing the number of presacral vertebrae. This has a secondary effect upon the pattern of the cranial portion of the lumbar plexus consisting of the spreading of the origins of the subcostal, iliohypogastric and ilioinguinal nerves ; besides, of course, the caudal shift by one lumber vertebra of the remaining nerves of the lumbar plexus. D 84. Occwrrence of nerve cells in the sciatic nerve of albino rats. George METZ* and Robert JUDICE*, Department of Anatomy, The University of Texas Medical Branch, Galveston. (Introduced by John C. Finerty) Sciatic nerves of albino rats were removed intact with their main branches and fixed in 10% formol saline solution. Some were sectioned longitudinally at 10 or 15I./ and stained with either Nissl stains or by various silver methods. I n addition t o the sectioned material, whole mounts of the entire nerve and its branches were prepared and stained with Cresyl echt Violet. Nerve cells were found in 42 of the 46 nerves studied. The number of cells was variable, ranging from 4 to 144 ganglion cells per nerve. The cells occurred singly or in groups along the entire length of the sciatic nerve, but the majority were located a t the bifurcation into tibia1 and common peroneal branches. Cell bodies were round t o oval, 25-45 p in diameter, and encapsulated by satellite cells. They showed the characteristic centrally located nucleus, prominent nucleolus and granular clumps of Nissl substance scattered throughout the cytoplasm. ABSTRACTS O F DEMONSTRATIONS 465 U 61. Interpretation of the electromyogram of graded voluntary movements in normal subjects.' Alice L. O'CONNELL* and 0. A. MORTENBEN, Department of Anatomy, University of Wisconsin. The conventional electromyogram of moderate or strong voluntary contraction records the characteristic interference pattern of action potentials. The number of active motor units during such a contraction is so great that the finer details of the relative activity in different parts of the muscle are obscured. I n this study an attempt was made to demonstrate such details during graded voluntary movements performed by normal human subjecta Action potentials were recorded by an 8 channel ink-writing electromyograph from 8 pairs of small surface electrodes arranged longitudinally along the tibialis anterior muscle. Intramuscular electrodes were used to supplement the records obtained from surface leads. Relative differences in activity were recorded from the several parts of the muscle. The superior portion consistently showed a lower threshold and a higher frequency. This pattern suggests a correlation with the internal structure of this muscle. The electromyogram also demonstrated the recruitment of additional motor units and the increase i n frequency of the same unit during increasing effort. The variation in distribution of action potentials, presumably from the same motor unit, in several channels may be evidence of the spatial distribution of the motor units. Differences in activity were most apparent during minimal contractions but also detectable during moderate effort. Maximum effort disclosed co-contraction of the tibialis anterior in the movement of eversion, but not i n plantar flexion. 1 Supported by a grant from the Wisconsin Alumni Research Foundation. D 50. Electron microscopic studv of absorption of particubte matter from the peritoneal cavity.' D. Louise ODOR, Department of Anatomy, University of Washington. Rats were injected intraperitoneally with a solution of colloidal mercuric sulfide or of thorotrast and were killed at intervals thereafter. Thin sections of mesentery and diaphragm were examined with the electron microscope. As early as 4 hour after the injection the administered particles adhered t o the surf aces of the microvilli and the plasma membrane of the mesothelium. At this and a t subsequent times the particles were localized within the mesothelial cytoplasm, confined either within clear vacuoles of varying sizes or aggregated in relatively dense bodies. No particulate matter was noted within the intercellular spaces. Some was found within the cytoplasm of macrophages of the subjacent connective tissue as early as f hour after the injection. Twelve hours later relatively few particles remained adherent to the mesothelial cell surf ace, but there was an increase in the number within both mesothelial cells and macrophages. The particles of mercuric sulfide in the mesothelial cytoplasm varied in size from about 50 to 220 A. More of the particulate matter was observed within the cyto- 466 AMERICAN ASSOCIATION O F ANATOMISTS plasm of the diaphragmatic than of the mesenteric mesothelium a t all time intervals studied. Many particles were located in macrophages in the connective tissue between the muscle fibers of the diaphragm, but none were situated within the sarcoplasm. -~ 1 Supported in part by grant RG-4296 from the Department of Health, Education and Welfare, and by the Life Insurance Medical Research Fund. D 5 4 . Apposition and fusion of placentae in the hamster.' Margaret Ward ORSINI, Department of Anatomy, University of Wisconsin. Apposition and actual fusion of adjacent placentae is not rare in the hamster. The degree of union varies. It has been found in animals bearing their first, second and third litters, and in individual horns containing as few as 5 young, and as many as 11. As many as three double gestation swellings have been found in one horn. The youngest found ( 9 days and 8 hours) appear grossly as abnormally large gestation swellings which, after clearing, show two embryos, each in its individual decidual cavity. By 12 days and 10 hours the individual placentae are distinguishable from the enveloping tissue. I n one horn of this age two placentae appear fused, in an adjacent swelling the placentae are only i n apposition. Both apposition and fusion increase in late gestation. Sectioned material from the 16th day, one preparturition and one midparturition, shows union of maternal tissue, apposition, and also of the fetal trophospongium, true fusion. The labyrinth is not involved so there is no mixing of the fetal circulations. All material indicates each placenta, despite union, still receives its individual maternal arterial blood supply. This intimacy of adjacent placentae appears t o be caused by nearness of the original implantation sites and subsequent crowding. High litter size favors this. 1 Supported by a grant from the National Science Foundation. D 67. Strains of mycotic parasites an red blood corpuscles f r o m hospital patients. James W. PAPEZ and B. Pearl PAPEZ, Columbus State Hospital, Ohio. Four strains of mycotic spores were found in red blood corpuscles (rbc) of hospital patients. Each strain began with single spore that entered rbc, dispersed in stroma into a colloidal stage, fluoresced in purple (indole) or rose, and organized into particulate globules. Under phase contrast, oil immersion microscopes, each strain and its stages were identified and watched for several days (3-21). Four strains are illustrated. Each strain ends in a definite form which we named rosette, beaded, mucoid and cystic. 1. Rosette strain, spore disperses, colloidal stage gives rise t o formative spots that grow protrusions from surface of rbc converting it to a rosette of 9 to 13 mycotic cells which separate. 2. Beaded strain, colloidal stage disperses as ring in periphery of rbc, purple fluorescence, globules form out of ring, project from surface as beads on a string. 3. Mucoid strain (Anat. Rec. V. 118, Suppl. P 403, 1954), colloidal stage is ring or dispersed, forms numerous particles, grows spines to outside, organisms reduce rbc t o mucoid sac. 4. Cystic ABSTRACTS OF DEMONSTRATIONS 467 strain, colloidal cyst forms blister inside of rbc which breaks open, cyst with sawteeth, organisms are globules on stems. I n contaminated blood (hemorrhages) a capsular strain grows a stout, pulsatile, globular filament which lays a gray capsule in remnant of rbc. Mycotic spores in sclerotic patches in walls of blood vessels are illustrated. D 28. Stereoscopic photographs and drawings f o r teaching anatomy and embryology. Anthony A. PEARSON, Luis A. TURNER* and William J. MIKKELSEN*, Department of Anatomy, University of Oregon Medical School. No abstract submitted. D 1 4 . Vital staining of nerve tissue with methylene blwe. E. H. POLLEY*, Department of Anatomy, Hahnemann Medical College. W. Angulo) (Introduced by A. The use of methylene blue as vital stain in neurohistological research has not enjoyed great popularity recently because of the great variability in the resulting preparations. We have succeeded in obtaining preparations that demonstrate the value of this technic despite its inherent variability. Animals were anesthetized and methylene blue was administered parenterally by a modification of the method of Feindel, Sinclair, and Weddell ('47). The animals were then sacrificed and tissues of the peripheral and central nervous system were removed for oxidation and subsequent fixation after the method of Bethe and Cajal. Paraffin and frozen sections as well as whole mount preparations will be demonstrated. D 16. A modified stain for degenerating terminals. Margaret M. POWERS*, Paul A. YOUNG* and George CLARK, Departments of Anatomy and Physiology, University of Buffalo Medical School. Silver stains are generally considered erratic and the stains for degenerating terminals, at least in our hands, have proved no exception. However such a stain was needed badly to complement the Nauta and Gygax stain for degenerating axons (Stain Tech., 2 9 : 91, 1954). On analysis of published methods there appeared to be an overwhelming number of variables involved. The procedures were varied as systematically as possible. On the basis of these experiments it was possible to develop a method which has given surprisingly consistent results. Test material has been mainly from cats with complete transection of the spinal cord. The changing picture of terminal degeneration, as seen in such preparations, will be shown after 3, 4 and 5 days postoperative survival. D 46. A n eZectronmicroscopic study of the Pncinian corpwscle. T. Andrew QUILLIAM, Department of Anatomy, University of California at Los Angeles and Veterans Administration Center, Los Angeles. The lamellae of the outer zone of the corpuscle are extremely thin yet truly cytoplasmic in nature, and they exhibit a radial assymetry about the central 468 AMERICAN ASSOCIATION O F ANATOMISTS longitudinal axis. They are arranged in two well-defined groups in the more superficial of which the lamellae are comparatively widely spaced, while i n the deeper group they lie closer together. Over the surfaces of the lamellae lie many circularly oriented collagen fibers. A distinct layer of thicker cells separates these outer lamellae from the inner core of the corpuscle. This latter consists of twin sets of cytoplasmic sheets bilaterally enfolding the terminal part of the central axon. The two halves of the core are separated from each other by a radial cleft. The axon, which is somewhat oval in cross section with its long axis coincident with that of the cleft, has a limiting membrane immediately deep t o which is a striking pallisade-like arrangement of mitochondria. No myelin is distinguished around the terminal axonal filament but, proximally, nearer the point of exit of the axon from the corpuscle a myelin sheath is acquired, and here a few centrally situated mitochondria are demonstrable. Vascular capillaries occur in considerable number near the myelinated segment of the axon but are not found further distally. D 47. Electron microscopic studies of kidney f r o m rats maintained on normal or low potassium diet dkring vital staining w i t h silver nitrate.' James F. REGER", Department of Anatomy, University of Colorado Medical Center. (Introduced by V. L. van Breemen) Electron microscopie studies were made of renal tissue from two groups of rats whieh were given 0.5% silver nitrate solution as drinking water for periods of 6-12 months. One group was maintained on normal diet plus silver nitrate; the second, on low potassium diet plus silver nitrate. Low potassium diet produced no differences in location or amount of silver deposition i n the kidney, though it did cause muscular atrophy. I n both groups of animals silver deposits were found as follows : sparsely distributed through perivascular and peritubular loose connective tissue ; rarely located within endothelial cells j precipitated in endothelial basement membranes ; precipitated between smooth muscle cells of arterioles ; most heavily precipitated in basement membranes of the glomerulus and convoluted tubules ; and sparsely precipitated in thin basement membranes of Henle's loop and collecting tubules. No silver was identified in urine collected from several animals of each group. Preliminary x-ray diffraction studies indicate that the silver is present i n the tissue as silver sulfide. It is possible that free sulfur or sulfhydryl groups in the basement membranes and ground substance precipitate the silver, the size of the precipitated granules varying with the amount of silver available and to a much less degree with the amount of sulfur available. 1 Supported by grants from the Muscular Dystrophy Associations of America, Incorporated, and the U. S. Public Health Service. D J 1 . T h e finer pulmonary vascular architecture in f e t a l and neonatal guinea pigs. S. R. M. REYNOLDS, Department of Anatomy, University of Illinois, College of Medicine. Thick (25 p ) sections of fetal guinea pig lung will be shown, demonstrating in the fetal and neonatal state ( a ) terminal arterioles; (b) convoluted arterial capillaries lying along the alveolar ducts; (c) the true capillary plexus that ABSTRACTS OF DEMONSTRATIONS 469 joins these convoluted arterial capillaries with each other; (d) venous drainage of convoluted arterial capillaries ; (e) elastic tissue about the arterial capillaries. Methods of preparation, sectioning and staining the tissues were described in abstracts for the meeting of the American Association of Anatomists in 1955 (Anat. Rec., 121 : 414, S.R.M. Reynolds: Pulmonary vascular changes and hemodynamic changes). D 3 6 . Heart Pain: Mechanisms and relief. Joseph T. ROBERTS, Veterans Ad- ministration Hospital and University of Buffalo School of Medicine. The exhibit shows work done on the role and changes in the vasa nervorum or blood vessels of the nerves, especially as affected by drugs or in experiments showing that the vasa nervorum of the left arm contract after ligation of a coronary artery. This supports our belief that cardiac referred pain is caused by ischemia in somatic nerves, due to a reflex vasospasm. This lowers the threshold of irritability for various sensory and motor changes in the nerves in the area of referral. This is thought to be a mechanism for persistent shoulder-hand pain, Dupuytren 's contracture and other sequels of cardiac infarction. Methods of revascularizing the ischemic heart are shown, e.g. the author 's method of arteriolizing the coronary sinus or increasing the abundance and size of the Thebesian vessels. The various anastamoses of the coronary system are demonstrated, and also the lymphatics of the heart muscle. D5.9. Weight changes associated wit71 pregnancy in the guinea pig.' James B. ROGERS and Richard C. TAYLOR", Departments of Anatomy and Pathology, University of Louisville School of Medicine. Weight changes during pregnancy, post-partum weights, litter weights and placental weights from 917 guinea pig pregnancies will be reported. The observations are made on guinea pigs from a colony which has been maintained for fifteen years under relatively constant conditions as to diet, temperature and care. Early i n pregnancy the weights were taken at weekly intervals. Late in pregnancy the animals were weighed daily, a t the same time of day each time and were handled by the same person. Post-partum weights were taken immediately after delivery. Each offspring was weighed individually soon after birth. Placentas were taken from the cage when ever possible. By selective breeding a strain of guinea pigs susceptible to a toxemia of pregnancy has been maintained. Toxemia of pregnancy characterized by sluggish behavior, convulsions and death, just before, during or immediately after delivery, occurs in 15% of pregnant female guinea pigs which have an inherited susceptibility to the disease. Another strain which does not develop the toxemia is maintained. Data will be presented in tabular form for three groups of pregnancies (1) nonsusceptible (2) susceptible to but not developing the toxemia and (3) susceptible animals which died from toxemia. Increased amount of amniotic fluid seems to be characteristic of toxemic animals. 1 Aided by a grant from the U. S. Public Health Service and by a grant to the University of Louisville from the Kentucky State Medical Research Commission. 470 AMERICAN ASSOCIATION O F ANATOMISTS D 2 5 . A schematic presentation of thalamic morphology and connections together with observations on details of thalamocortacal relationships.’ Glenn V. RUSSELL, Department of Anatomy, The University of Texas Medical Branch, Galveston. A schema is presented illustratiiig succinctly but in a simplified form a large part of the fundamental information on the morphology, connections, and organization of the dorsal thalamus. Evolved over the last several years by the author as an adjunct to teaching medical students the anatomy and connections of the dorsal thalamus, it has proved t o be a valuable teaching aid t o the lecturer and a worthwhile and instructive study guide and mnemonic device to the student. Elaborating upon the schema, recent observations of some details of thalaniic projections to the cerebral cortex of man are demonstrated. 1 Supported by USPHS-NINDB Grant B - 7 5 1 ( C ) . D 43. Electron microscopy of the epiphyseal apparatus. Bronnetta L. SCOTT*, Department of Anatomy, University of California a t Los Angeles and Veterans Administration Center, Los Angeles. (Introduced by Daniel C. Pease) Electron micrographs of undecalcified sections of the epiphyseal apparatus demonstrate some of the ultra-structural components and relationships inaccessible by previous methods. For this study the proximal end of the kitten humerus was used. I n the epiphyseal cartilage, the appearance of the endoplasmic reticlum indicates the approximate stage of cytomorphosis. The chondrocytes of the proliferating zone present a characteristic dehydrated appearance, and the endoplasmic reticulum is oriented into tightly-packed, undulating layers. Progressing toward the diaphysis as the cells become increasingly hydrated, the endoplasmic reticulum becomes more loosely disposed. The cartilagenous matrix consists of a network of fine fibrils without demonstrable periodicity. The capsular portion lacks these fibrils. Osteoblasts are separated from the calcified matrix by a layer of loose collagenous fibers. This layer is of variable thickness, reflecting the rate of deposition of the matrix, and its subsequent calcification. Calcification is essentially the same in both cartilage and bone. Small islands of crystals are deposited a t random and then coalesce. Individual crystals, at least in early stages, are less than 50 A wide and about ten times as long. The “brush border” of the osteoclast is an intricate infolding of the plasma membrane. I t s association with mineral resorption in cartilage and bone is suggested by the presence of crystals between its folds and by associated cytoplasmic vacuoles. D 4 . Some unusual perforations of Indian skulls. H. Alan SKINNER, Department of Anatomy, University of Western Ontario. Four Indian skulls were found in the same burial pit on an island in the St. Clair River. The 4 skulls show identical trepan openings in the same position in the vault of the skull about 4cm behind the coronal suture and crossing the ABSTRACTS OF DEMONSTRATIONS 471 median sagittal suture. The openings i n the skulls are i n the form of deep grooves and the inner margins of the openings are ellipsoidal in shape. They are unlike any other openings of the skull i n the author's experience and no reference to them has been discovered in the literature of trepanning. D1. The influence of Vesalius' Fabrica on Ruini's Anatomia d d Cavallo (1598). J. F. SMITHCORL%*,Department of Anatomy, Michigan State Universitg. (Introduced by Miriam Scott Lucas) Carlo Ruini 's magnificent Anatomia deZ cuvuZZo is the first comprehensive anatomy of a single animal. Attempts have been made to discredit the Ruini plates as a theft from the work of da Vinci, but da Vinci never did more than the surface anatomy of the horse. Rather, the superb craftsmanship of the unknown creator of the Ruini plates appears to be directly related to that of Vesalius' Fabrica. A curious, but hardly credible suggestion i n a posthumous edition of Ruini, credits the plates t o Titian (died 1576), although Titian did visit Bologna while Ruini resided there. Inasmuch as many of the Vesalius plates are the work of students of Titian, it seems logical that other students or followers of the Titian school may have been responsible for the Ruini drawings being done in the Veealius tradition. The number of similarities between the two works is more than coincidental; the posing of subjects, the rendering of muscle masses, the stylized representation of organs and systems, and the landscape backgrounds all point to this. With nothing in animal anatomy as a precedent, Ruini's work required the artistic tradition established by the Fabrica of Vesalius. D 13. A method for fixing guinea p i g cochleae under "physiological conditionsJ' and preparing ribbons of sections following double embedding? Richard H. SWIGART, Arthur L. JlLJERS* and James B. ROGERS, Departments of Anatomy and Otorhinolaryngology, University of Louisville School of Medicine. During studies on age changes in various organs of senile guinea pigs it seemed advisable to correlate cytological changes with tested deafness. Standard methods for preparing cochleae were first tested using young adult guinea pigs from an inbred strain. These methods did not meet our requirements, for good cytological preservation was not consistently attained, and serial sections of cellodion embedded specimens were cumbersome to prepare. The method presented here was modified after that of Koenig et al (Stain Tech., ZU: 13-22) f o r brain. After perfusion a t 37°C. with 0.9% Naa-xcacia solution followed by Heidenhain's Suss' '-acacia solution, cochleae were dissected and immersed in the latter for 24 Iiours, decalcified in 570 trichloracetic acid i n 10% aqueous formalin, washed in 9570 alcohol for 12-24 hours, dehydrated in 100% alcohol for 1 hour, in a 1 : l ether-alcohol mixture for 1 hour, placed i n 276, 4%, and 6% parlodion for 2, 3 and 4 days successively, and embedded i n 6% parlodion hardened with chloroform vapor for 6-8 hours. Blocks were trimmed, cleared in benzene and infiltrated i n three changes of 52"-54" 472 AMERICAN ASSOCIATION O F ANATOMISTS paraffin for one day each and embedded. Ribbons of 1 0 p sections could bc efficiently cut on a rotary microtome. Charts, pictures of the constant temperature perfusion apparatus and other features of the method, and photomicrographs of the results attained will be presented. 1 Supported by a grant from the American Otologieal Society and by a grant to the University of Louisville from the Kentucky State Medical Research Commission. D 6 2 . Injected vessels in &eared tissues. P. F. SWINDLE*, Department of Physiology, Marquette University School of Medicine. (Introduced by Walter Zeit) The gross and microscopic specimens are from a collection of many which were injected and cleared over a period of 32 years in studies of comparative physiology and anatomy. The most common injection media were red cinnabar and India ink. The cinnabar was used quite extensively because of its radiopacity, because it shows up as white in the infrared print (if reflected light is used in exposing the plate), and also because it can be urged into the very small arterioles and arteriovenous anastomoses without driving it into the capillaries in non-pathologic tissues. The clearing was done by using a Spalteholtz method which leaves the tissues preserved in methyl salicylate. The animals were obtained mainly from the Washington Park Zoo in Milwaukee, Wisconsin. D 5 6 . Variations in depth o f implantation o f the human ovum correlated with placental anomalies, spontaneous abortion and premature separation. Richard TORPIN, Department of Obstetrics and Gynecology, Medical College of Georgia. See abstract of paper from platform No. 179. D 3 2 . Conduction tissue of anomalous human hearts.' R. C. TRUEX and J. A. KAIN*, Department of Anatomy, Hahnemann Medical College. This study was based on celloidin serial sections of nine human hearts ( 2 days t o 3 years of age) with high interventricular septal defects. Histologic appearences Qf the conductive elements in certain hearts, as well as some variations in the atrial cardiac muscle fibers will be demonstrated. The relationship of the septal defect to the common bundle and its left and right bundle branches are to be illustrated. 1 Supported by U. S. Public Health Qrant H - l 5 9 1 ( C ) . D 46. Electron microscopic studies of lung tissue from guinea pigs and pre,mature infants.' V. L. van BREEMEN and H. B. NEUSTEINr2, Departments of Anatomy and Pathology, University of Colorado Medical Center. Electron microscopic studies were made of normal and pathological lung tissue from guinea pigs and premature human infants. Observations of normal lung ABSTRACTS OF DEMONSTRATIONS 473 corroborated Lowe ’s observations on alveolar epithelium. Attenuated epithelial sheets overlay the capillaries, separated from endothelial cells by basement membranes. Epithelium apparently covered septa1 surfaces except where macrophages rested within the septum and protruded into the alveolar space. Macrophages (alveolar cells) occurred itlso as apparently free cells. Pulmonary hyaline membrane disease occurs a s the only pathological finding in about 25% of premature infant deaths. Lung tissue was obtained from premature infants who died in this hospital. For comparative study hyaline membrane disease was produced in guinea pigs by exposure for 72 hours to 98% oxygen atmosphere with 95% humidity. Electron microscopic study indicated that the hyaline membranes in the guinea pig lungs were essentially the same a s those in the infant lungs. Hyaline membranes occurred in alveolar space overlying the epithelium. Finely granular material (probably plasma proteins) was found next to the capillaries in the alveolar space. Fibrils extended from this material into a mass of fibrils and debris, identified as the hyaline membrane. I n our interpretation the fibrils are fibrin and the hyaline membrane is essentially a fibrin clot enclosing some cellular and other debris. Supported in part by a research grant from the National Institutes of Health, Public Health Service. Maytag Fellow of the American Csiicer Society. D 7. Tissue culture studies of the rat thymus.’ Mina Lee VERNON”, Department of Anatomy and The Tissue Culture Laboratory, University of Oklahoma School of Medicine. (Introduced by Kenneth M. Richter) Roller tube cultures of the r a t thymus were studied i n a living state, and after fixation and staining “in toto” by a modified Giemsa method. Time-lapse cinephotomicrographic data was collected on some of the cultures. Comparative studies were made on routine histologic preparations of the uncultured thymus. These studies revealed firstly, that many of the several cell types distinguishable in ordinary histologic preparations of the r a t thymus can also be found in cultures. Secondly, most of these cell types actually grow and reproduce in tissue culture including fibroblasts, stellate shaped reticulum forming cells, endothelial cells, and epithelioid cells. Thirdly, certain cells of thymic explants undergo such extreme pleomorphism that a t times free cells (thymocytes9) change into epithelioid cells, and, conversely, epithelioid cells change into free cells (thymocytes?). This has been observed only by means of cinephotomicrography. Fourthly, histological differentiation of special structures occurs in thymic tissue cultures including ( a ) a reticulum formed by stellate shaped cells; ( b ) Hassall’s corpuscles with central differential staining suggestive of hyalinization ; (c) capillaries many of which were patent proximally and contained fragments which, on the basis of their differential ietaining, were suggestive of blood cells; and (d) conspicuous epithelioid sheets of undetermined origin. Basis of thesis submitted to the Qraduste Faculty of the University of Oklahoma in partial fulfillment of requirements for the degree of Master of Medical Science. 474 A M E R I C A N ASSOCIATION O F A N A T O M I S T S D 15, A Bielchowski-Marchi technique f o r parafin sections. Gin0 DI VIRGILIOI, Monroe GREG0R.Y" and Joseph HOLLENBERG*, Department of Anatomy, Howard University. (Introduced by W. Montague Cobb) The finely myelinated fibers which are a source of equivocation with the Marchi technique show up well with a silver method, i n which sections are mounted in the usual manner and passed through mordants, oxidizing and reducing agents; toned with gold (Auer and Di Virgilio, '53) and intensified with a Bielchowski formula (Nauta and Gygax, '51). The treatment of the tissue with the various chemicals inhibits silver deposition on the normal tissue. Silver will be deposited on the degenerating axons. The end result is an extended Marchi picture. Demonstrations are provided from the brains of cats, rats, rabbits and mice subjected to various types of lesions and from pathological human material from the Armed Forces Institute of Pathology, Washington, D. C. The sections are from serially cut brain tissue, selected at regular intervals, and stained for both normal and degenerated fibers. Corresponding series, selected a t the same intervals were stained for the degenerating axons only. A limited number of specimen slides is available on request. D 37. Akimesia, rigidity, tremor and associated phenomena i n reserpinized monkeys and chimpanzees. William F. WINDLE, Jan CAMMERMEYER, Jane T. JORALEMON", John 0. SMART*, Earl FERINGA" and Michael McQUILLEN", Laboratory of Neuroanatomical Sciences, National I n stitutes of Health. Reserpine in single doses of 2.5-3.0 mg/kg orally, intravenously or subcutaneously in Af rican green monkeys and chimpanzees produced phenomena resembling those i n human parkinsonism. These were recognizable within 30 minutes and became maximal in 2-4 hours, full recovery requiring 24-48 hours. All animals showed in varying degrees: (1) muscular rigidity of the "cogwheel" type, better in the chimp than the monkey; ( 2 ) akinesia, torpor, catalepsy and lethargy; ( 3 ) tremor which varied inversely with degree of lethargy and disappeared during sleep (resting 3/sec. tremor, disappeared on voluntary movement); (4) sialorrhea and some increased waxiness of facial skin; ( 5 ) changes in mood and ( 6 ) general appearance including fixed facial expression, poverty of bodily movements, marked kyphosis with head bent forward, and elbows and hands flexed. Phases of akinesia often alternbted with brief spontaneous activity, changes to successive voluntary movements being impaired. Grasp and other reflexes were present. Reserpine administered to monkeys daily for 4 months in doses of 0.2-0.4 mg/kg subcutaneously induced hypokinesia, slight rigidity and marked tremors, but no vegetative phenomena. The maximum effect occurred a t the third or fourth hour after the drug was given. D 11. Early stages of intraepidermal melanoblasts in A-egro fetuses. Arnold A. ZIMMERMANN and S. W. BECKER, Jr.", Departments of Anatomy and Dermatology, University of Illinois. Split-skin preparations treated with Masson's ammoniated silver nitrate reveal immature forms as well as fully differentiated dendritic cells in surface views. ABSTRACTS O F DEMONSTRATIONS 475 Such propigment cells appear first in the 11th week and rapidly form extensive intraepidermal distribution patterns. At 12 weeks they are present in all skin areas of the body, including palm and sole. Cell counts on such preparations are more reliable than those made on sectioned material. The technique essentially illustrates the maturation process of the propigment cells. The “mongolian spot” of a 4.8 month fetus, consisting of dermal melanoblasts, is also demonstrated. MOTION PICTURES M P 12. Collateral ventilation in the lung of dog.’ A, W. ANGULO, Vincent KOWNACKI* and Edmund HESSERT, Jr.*, Department of Anatomy, Hahnemann Medical College. This Kodachrome film summarizes the results obtained in a series of in vitro experiments in which a colloidal suspension of carbon particles ( 2 7 mp in diameter) was injected into a single secondary brounchus at pressures that ranged within physiological limits. I n each preparation the injection mass was observed to fill first the pulmonary segment directly attached t o the injection apparatus. The injection mass was then seen to appear in adjacent pulmonary segments. These results clearly indicate that there is ample communication between the segments of each lobe. Therefore, the bronchopulmonary segments are not discrete anatomical or physiological units in the lung of dog. ~ Supported by U. S. Public Health Grant H-1366Ca. * Supplied by Columbian Carbon C!ompany through the courtesy of Doctor George S. Samuelsen. M P 6. Platelets in the periphe:ral circulation of the hamster (Melsocricetcs auratus).’ Herbert J. BERMAN*, George P. FULTON and Hrenton R. LUTZ, Department of Biology, Boston University. Platelets were cinephotomicrogrrzphed in civo in eddies, columns of plasma, and in plasma spaces in vessels with flowing blood. I n wityo, filamentous and dendritic processes, lysis, adhesiveness, and agglutination of platelets were recorded with phase microscopy. Freshly drawn platelets appeared disc-shaped in surf ace view and lenticular in side view. Later they became filamentous and dendritic. Granules appeared to extend into the filamentous processes. Lysed platelets appeared pale, smaller, and less granular with some loss of content. They were observed adhering to glass. The agglutinated and nonagglutinated platelets in a 24-hour slide preparation were lysed. Platelets observed in living blood vessels were also disc-like on surface view and lenticular on side view. Filamentous and dendritic processes were not observed in v i v a Platelets were photographed plugging a hole made in a blood vessel wall with a microrod. Adhesion of platelets followed 476 AMERICAN ASSOCIATION O F ANATOMISTS mechanical, electrical, or chemical injury of a vessel wall. Some of the coagulation factors such as commercial thrombin and invasive thromboplastin (Russell’s viper venom), caused platelet thrombo-embolism. ISupported by grant H-902 ( e 4 ) , National Heart Institute, PHS, and the Department of the Army, Office of the Surgeon General. M P 5 . Color tebvision of microcircidation. Edward H. BLOCH, Louise WARNER, Murray C. BROWN* and John W. CHRISTENSEN*, Department of Anatomy, Western Reserve University; Department of Anatomy, Georgetown University ; The Clinical Center, National Institutes of Health and Columbia Broadcasting System Laboratories. The film depicts a pilot exploring some possibilities of electronic image processing by color television. Frogs were anaesthetized, laparotomized and their mesentery or liver quartz-rod transilluminated. The tissue was observed with a stereobinocular microscope ( x 32-150), an area selected and a CBS color television camera swung over the ocular. Subsequently the images were observed and recorded from a monitor. The images were studied and recorded first at normal color balance, light intensity and Kelvin temperature and then the following adjustments were made : light intensity reduced, contrast varied for each color, colors removed, ‘ ‘crispening” circuits added t o vary apparent contrast and phase shifts produced between camera color wheel and the monitor and recorder color wheels. The images produced by this field sequential system had good resolution and the color compared favorably with standard recording methods. I n general, surface details were better and the images recordable at lower light intensities. Since it was possible t o balance the colors of the image, variations i n Kelvin temperature was not important. When the colors of the image were modified individual cellular components were made either more or less prominent than normal. Thus with this method it is possible to rapidly vary contrast by removing or adding colors without affecting the tissue. MP 10. Reduction and substitzition experiments on the peripheral nerves of respiration. Victor CASTLEBERRY*, T. H. BARNETT* and H. A. HOLTMAN*, Department of Anatomy, The University of Texas Medical Branch, Galveston. (Introduced by Donald Duncan) A film illustrating tolerance of the dog to bilateral phrenectomy and section of 8 pairs of intercostal nerves, and a series of cats t o show effects of various substitutions for the phrenic nerves. One cat illustrates the effects of vagophrenic anastomosis ; namely, a well preserved and powerful diaphragm but inadequate due to lack of effective natural impulses. Other cats display the adequacy of one phrenic nerve alone, the effectiveness of the levatores costarum, and bilateral substitutions of the phrenic nerves by the nerves to the infrahyoid muscles. ABSTRACTS O F DEMONSTRATIONS 477 M P 11. An experimental study 01%the passage of air through the nose. Seymour FRIEDBERG*, Department of Anatomy, Duke University. (Introduced by Kenneth Duke). No abstract submitted. M P 8. Neurosonic surgery.' William J. FRY*, Frank J. FRY* and John W. BARNARD, Bioacoustics Laboratory, University of Illinois. The multiple, focussed beam, ultrasonic method for producing selective accurately localized reproducible changes in the central nervous system is illustrated in this motion picture. Surgical preparation of the subject (monkey), the technique of irradiation and histologically pipepared tissue sections are shown. The operation of the instrumentation is demonstrated by producing a lesion in the fiber tracts of the internal capsule. This technique is in current use in studies of brain structure and function. A lesion of practically any desired size (as small a s 1 mm in diameter) and shape can be produced a t almost any specified position in the central nervous system without disturbance of intervening tissue and without disruption of blood vessels even within the site of the lesion. I n addition, it is possible by proper choice of dosage to irreversibly affect fiber tracts without destroying surrounding or neighboring nuclei which receive the same dosage of acoustic radiation. The stained tissue sections shown in the motion picture illustrate the production of ultrasonic lesions of a variety of sizes and shapes in both white and gray matter a t various depths in the brain. 'The support in this research field of the Physiology Branch of the Office of Naval Research is acknowledged. M P 3 . Alkaline tolerance of the isolated embryonic rat heart.l E. K. HALL, Department of Anatomy, University of Louisville School of Medicine. The effects of alkalosis were studied on isolated hearts of ll$-day r a t embryos in Ringer made alkaline with NaOH. I n the pH range 8.5-9.49 (30 cases), the hearts accelerated during the first 4 minutes (from 158 to 177/minute), then decelerated slowly during the rest of the 60-minute experimental period (to 132), then accelerated slightly in the terminal control solution. I n the p H range 9.5-10.5 (30 cases), the same sequence of events occurred, except that changes were more marked. The rate in the terminal control solution attained the initial control rate. I n the p H range 110.6-11.5, there was immediate acceleration in 27 of 34 cases, then rapid deceleration and arrest within several minutes; upon immediate return to normal Ringer, 25 hearts began to beat again, some within a few minutes. Results with NH,OH were similar (80 cases), except that deceleration and arrest, with subsequent recovery in control solutions, occurred a t lower p H levels (9.2-10.7). The alkaline tolerance of the embryonic r a t heart is evidently greater than is generally stated for adult mammalian myocardium. 1 Supported by Grants-in-Aid from the -4mericau Cancer Society, upon recommendation of the Committee on Growth of the National Research Counci!, from the Kentucky Siate, Medical Research Commission, and from the National Heart Institute of the National Institutes of Health. Public Health Service. 478 AMERICAN ASSOCIATION O F ANATOMISTS M P 4 . The effect of poliomyelitis virw on luman brain cells grown in tissue culture.' Mary J. HOGUE, Robert McALLISTER", Arthur E. GREENEX and Lewis L. CORIELL', Departments of Anatomy and Pediatrics, School of Medicine, University of Pennsylvania. A large neuron, a Purkinje cell, in a 94-day-old culture of cerebellum from a 127mm crown rump human fetus has been photographed for 4 hours and 20 minutes. The tips of the cell processes are active and there is streaming of cytoplasmic granules in the cell body, but little locomotion is seen. This feature is followed by pictures of a group of three cells from a 108-day-old culture of the cerebellum of a 127mm human fetus. The culture has been inoculated with Photographing began 28 poliomyelitis virus, type I, Mahoney strain, titer minutes after the cuIture was inoculated with the virus and continued for 5 hours and 40 minutes. The pictures show the contraction of the cell processes and the withdrawal of these processes into the cell body. During this activity, the cells are pulled about by the powerful contraction of their processes. They become masses of granules. There is no recovery from this reaction to the polio virus. Supported by a grant from the Foundation of Infantile Paralysis. MP8. The ingu.ina1 region and scrotal contents.' Joseph E. MARKEE, Department of Anatomy, Duke University School of Medicine. This 16 mm color motion picture with optical sound illustrates the anatomy of the inguinal region and canal and the scrotal contents. Emphysis is placed on the relationships which are pertinent to femoral and inguinal herniation. The morphological details and the concepts are illustrated partly by models, drawings and animation and partly by the demonstration of dissections. P a r t of the film deals with inguinal ligament and the relationship of the structures which pass through the lacuna1 musculorum and vasorum. By means of animation, another part of the film illustrates the development of the inguinal canal and the continuity of the coverings of the testicle with the layers which form the abdominal wall. Another animation sequence deals with the additional layers which are formed during the various types of inguinal herniation. A dissection of the inguinal canal in the female is demonstrated. The final sequence demonstrates a dissection of the scrotum and its contents. Aided by a grant from the National Foundation for Infantile Paralysis. MPl. Effects of narcosis and hypotherrnia upon resistance to asphyxia in newborn guinea pigs.' A Kodachrome moving picture. James A. MILLER, Jr. and Faith S. MILLER*, Department of Anatomy, Emory University. Earlier experiments have demonstrated that hypothermia is effective in protecting newborn guinea pigs from asphyxia (Miller and Miller, '54). Since narcosis reduces motor activity, blocks shivering, reduces muscle tone, particularly at low temperatures, and depresses basal metabolism, experiments were performed to test the effects of narcosis upon asphyxia1 resistance a t body temperatures between 40°C. and 14°C. Two mg/cc/100 gm nembutal (sodium pentobarbital) ABSTRACTS O F DEMONSTRATIONS 479 in water was injected intraperitoneally into day-old or younger guinea pigs. Littermate controls received water only. Temperatures were adjusted by radiant heat or immersion to the neck in ice water. Asphyxia was produced by exposure in a bell jar to 95% N, 5% C02. Results: Pentobarbital narcosis prolongs asphyxia1 survival a t all temperatures tested, but is most effective a t temperatures below 20°C. Narcotized animals can recover completely from exposures which are lethal to non-narcotized littermates a t the same temperature. With cooling superimposed upon narcosis most animals recover from two times the lethal exposure. As employed here nembutal narcosis has the protective action of a 10°C. depression of body temperature. An analysis of the data suggests that the benefits of narcosis in asphyxia result chiefly from its effect upon cellular metabolisim. The results are demonstrated in a Kodachrome moving picture. + Aided by grants from the National :Institutes of Health and the Emory Research Committee. M P 7. T h e H e L a cell -its activities as seen w i t h phase microscopy.' C. M. POMERAT and C. G. LEFEBER", Tissue Culture Laboratory, Department of Anatomy, University of Texas Medical Branch, Galveston. This is the first of a series of films based on living human cells being prepared as medical school teaching aids. The phenomena of cellular outgrowth, nuclear rotation, normal and abnormal mitoses, pinocytosis, intercellular filopodial activity, and mitochondria1 movements are shown with the use of phase contrast, time-lapse cinematography. Aided by grant from the Association of American Medical Colleges. M P 9. Peripheral and central faoial nerve paralysis. Othmar C. SOLNITZKY, Department of Anatomy, Georgetown University. This 16mm silent kodachrome teaching film shows the anatomy of the facial nerve by drawings and diagrams in relation to facial paralysis. Patients with various lesions and deficits are shown. I n peripheral facial paralysis, both the temporofacial and cervicofacial divisions are affected with homolateral flaccid paralysis of one side of the face. Unilateral peripheral facial paralysis is shown in a patient following a n attack of bulbar polio. A case of bilateral peripheral facial paralysis, resulting from CNS syphilis is also shown. Central facial paralysis is of two types. The first type of central facial paralysis is characterized by loss of emotional facial expression (amimia) i n the lower half of the face on the contralateral side with retention of voluntary facial expression. This type is associated with lesions of the thalamus, basal ganglia or tegmental region of the upper brainstem. Iit will be demonstrated by diagrams. I n the second type, there is loss of voluntary facial expression in the lower half of the face on the contralateral slide, with retention of emotional facial expression. This type results from involvement of the voluntary motor pathway and is demonstrated by two patients with hemiplegia. 480 AMERICAN ASSOCIATION O F ANATOMISTS MP 19. The termination f o r t h e bile ducts. Juliaii A. STERLING*, Department of Surgery, Graduate School of Medicine, University of Pennsylvania. (Introduced by Oscar V. Batson) This motion picture film demonstrates untraumatized specimens of the termination for the bile ducts. Photography of cleared specimens, the ducts having been injected with pigmented gelatin-iodide, indicates the relationships of the ducts, the papillary sphincter and the ‘common channel. ” The characteristics of the duct terminations are examined in the normal, in choledocholithiasis and in pancreatitis. Forty per cent of specimens were found to have separate orifices for bile and pancreatic ducts. The other sixty percent were evaluated further with regard to the significance of the common channel. An ampulla was found present in only 5 % . I n these interductal reflux is possible. I n other instances the presence of a common channel does not signify the existence of interductal reflux. The author indicates that the terminations for the bile and pancreatic duct is not through an ‘ ampulla, ” but through a ‘papilla. ” ABSTRACTS O F DEMONSTRATIONS 481 TISSUE CULTURE ASSOCIATION ABSTRACTS TC 9. Yucopolysaccharide production b y normal human tendon cells in mass tissue culture.' C. Andrew L. BASSETT and Karl MEYER ", Departments of Orthopedic Surgery and Medicine, and Histochemistry Research Laboratory, Columbia University College of Physicians and Surgeons. Cells arising from plasma iinbedded explants of normal human toe extensor tendon, obtained at operation, grew readily through the clot periphery onto glass. From D 3.5 Carrel flasks, in which the central explant and plasma clot had been excised, T-60 cultures were established, with cells directly on a glass substrate. The nutrient consisted of 20% chick EE-50, 40% human placental cord serum, and 40% Earle's saline with antibiotics. Medium removed a t bi-weekly fluid changes was subjected to m u c h clot test, pooled, frozen, and mucopolysaccharide content determined. The mucin clot test was positive during the first 3 months of cell propagation. However, it rapidly became negative during the 4th month and remained negative f o r the next 6 months that the strain was maintained. Certain growth and morphologic alterations occurred concomitant with the change in the mucin clot test. Medium collected during the first 3 months showed a total mucopolysaccharide content about 5 times the amount accounted for by chick embryo extract in the medium. After 5 months in vitro, the cells produced less than 3 the total mucopolysaccharide of earlier 3 month samples. Acetone powder residues, containing chiefly protein and nualeic acid, remained unchanged in amount during the entire observation period. Hyaluronic acid was isolated only from the first 3 month collections. The study indicates that these tendon cells in vitro are capable of producing mucopolysaccharides, but in decreasing amounts with the passage of time. 1 Supported in part by U. S. Public Health Service Grant A 817 and a grant from the Masonic Foundation for Medical Research and Human Welfare. TC40. Gonads of W mice cultured in vitro. E. BORGHESE, Department of Anatomy, University of Pavia, Italy. The relationship between anemia and gonad sterility has been studied in mouse embryos of the W strain, by means of tissue culture. The gonads used were from embryos a t the 12th day of gestation. These embryos were obtained from crossing W+ X W+. At this age neither anemia nor gonad sterility are recognizable. After cultivation for 4 days, four different structures were found: fertile testes, sterile testes, fertile ovaries, sterile ovaries. Of the total number of cultures, the ratio of fertile gonads to the sterile ones appears to be 33:s. This number is not significantly different from the 3 : l ratio of fertile-normal embryos to sterile-anemic ones on the 16th day of gestation. Sixteen days (12 4) is the total age of the cultured gonads. The + 482 AMERICAN ASSOCIATION OF ANATOMISTS - Xa is 0.6585, with P 0. 45. I n each of seven embryos, the right and left gonad behaved the same. The probability of getting this result by chance was calculated to 0.4970. Thus, the development of a gonad in culture as fertile or sterile depends upon its genotype and not upon environmental conditions. It can be concluded that gonads taken from embryos before the appearance of anemia, continue to develop in culture into sterile or fertile gonads aecording to their genotype. The gene acts directly on gonads and not through the anemia. TC 39. The behavior of nuclear and nucleolar sizes in tissne cultures of different growth intensity. Otto M. BUCHER, Department of Histology and Embryology, University of Lausanne, Switzerland. Frequency curves of nuclear and nucleolar sizes, obtained from hanging-drop cultures of fibroblasts and osteoblasts, correspond to normal distribution (for the technique see Internat. Review of Cytology 111, 69-111, '54). Moreover, a linear correlation exists between the nuclear and nucleolar volume. I n order to characterize mathematically the quantitative relationship between the nuclei and the nucleoli we calculate a quotient E / N by dividing the nuclear size (K) by the nucleolar size (N). I f the intensity of growth of tissue cultures is influenced experimentally, the nucleoli react to a greater extent than the nuclei, which can be explained by the participation of the former in protein formation. As the nucleolar size represents the denominator i n the nucleo-nucleolar quotient, the latter decreases, if the tissue growth is augmented and vice-versa. Therefore, the variation curves of the quotients become displaced respectively to the left and t o the right. This conception, can be easily proven since it is not difficult t o regulate the growth rate of the cultures, for instance, by changing the conditions (temperature or composition of the medium, see Zschr. Anat., 118: 150-164 and 531-542, '54-'55) as well as by administrating appropriate chemical substances (see Aeta. anat., 25: 45-52, '55 and Zschr. mikrosk.-anat. Forsch., '56 in press). TC 68. Some factors affecting the growth promoting properties of serum in tissue culture.' Relda CAILLEAU*, Cancer Research Institute University of California Medical Center, San Francisco. (Introduced by Morgan Harris) Cord, human, monkey, horse, ox, sheep, hog, rabbit, chick and r a t sera were tested as growth media for HeLa, strain L, and mouse liver epithelial cells. The medium consisted of 40% serum, 40% medium 199 and 20% Tyrode's solution. Three or more subcultures were carried out before a medium was considered to be growth promoting. The effects of sex, age and heating (30 minutes at 56°C.) were studied with certain sera. Sera which showed good growth promoting qualities when unheated were usually adversely affected by heat. Sera which proved toxic when unheated were generally 483 ABSTRACTS OF DEMONSTRATIONS less toxic after exposure to heat. Toxic sera often caused the cells t o clump or become extremely granular. No universal relationship could be established between sex, age or f a t content of the serum and it growth promoting activity. ~~~ ~- Supported by a grant from the U. 8. Public Health Service, National Institutes of Health, to Dr. P. L. Kirk, Department of Bioclhemistry, University of California, Berkeley. I T C 3 3 . Growth of carrot root tissue i n the presenca of coconut milk.’ Samuel M. CAPLINI, Department of Biology, University of Rochester, and Eastman Kodak Company, Rochester, N. Y . (Introduced by M. S. PARSHLEY) A cylindrical explant of secondary phloem, 1.8 mm in diameter, 1mm high and weighing 2.5mg, was placed on 100 ml of White’s medium containing 0.8% agar and 10% coconut milk. I t was photographed in color during a period of 38 days a t the rate of one frame every 64 minutes, the culture exposcd t o a few seconds of light for each picture. Growth began as a uniform, progressive swelling over the visible surface, like rising dough, which soon became somewhat nodular; growth proceeded, nevertheless, in a steady manner. To prevent condensation of moisture on the vertical viewing window a piece of flexible heating tape was placed above it against the container. After 50 days the cultiw weighed 2.63 gm, making a relative increase of 1,050 times (as compared with 2-4 times in the absence of coconut milk). According to Blackman’s (Ann. Bot., 93: 353. 1919) compound interest formula for growth, this represents an average relative growth rate of 15.9% per day for the 50-day period, comparable in relative rate t o the normal vegetative growth of an intact plant. Other (unpublished) work has shown that explants from 46 different carrots in the presence of indole3-acetic acid averaged in 14 days less than 25q.b of the final weight attained in medium containing coconut milk. Isupported by n research grant, No. C-755 (CS), from the National Cancer Institute, Public Health Service. P C l l . Studies on the growth of mammalian cells in agitated fluid media. William R. CHERRY and Robert N. HULL, Lilly Research Laboratories, Indianapolis, Indiana. The growth of cell suspensions in flasks incubated on a rotary Brunswick shaker has been described by Earle et al. This accomplishment has been successfully duplicated in our laboratory using cell strains other than those employed by Earle and with some modification in the procedure. I n addition to the duplication of the shaker flask cultures a second technic employing a suspended magnetic stirrer bar f o r agitation has been developed. Results obtained by this method compared favorably with those achieved with the shaker cultures grown in our laboratory. The technics of both of these methods will be described as well as the variations in medium, speed of rotation and size of cell inoculum required for each cell type studied. The relative efficiency of the fluid culture to conventional flasks in terms of cell populations obtainable will be discussed. 484 AMERICAN ASSOCIATION O F ANATOMISTS TC 34. Functional and histological observations on islet tissue grown ‘ i n vitro’. Robert E. COALSON”, Department of Anatomy and The Tissue Culture Laboratory, University of Oklahoma School of Medicine. (Introduced by Kenneth M. Richter) Explants of histologically undifferentiated 9-10 day chick embryo pancreata and of fetal C3H and C57 brown mice pancreata were grown in culture by roller tube and hanging-drop methods for periods ranging up to 58 days. Histologic studies of chick pancreatic cultures revealed (1) little or no evidence of acinar tissue development or differentiation, but ( 2 ) a differentiation of tinctorially identifiable alpha and beta islet cells. Limited histologic preparations of cultivated mouse pancreas showed no differentiation of either acinar tissue or tinctorially identifiable islet cells. Assays for ‘insulin activity’ (Randle’s rat diaphragm technique, ’54) were made a t specific intervals on the supernates of mouse pancreatic and liver (control) cultures during 58 days of cultivation. The assays demonstrated clearly that the supernates from the pancreatic cultures from the 17th day of cultivation on possessed a high degree of insulin activity. ‘Insulin activity’ assays and expressed as milligram of glucose utilized by 100mg of rat diaphragm per unit of time showed that pancreatic supernates effected an average glucose utilization of 2.987 mg whereas control liver supernates effected an average glucose utilization of 1.960 mg. This data comprises the first positive demonstration of the production of insulin or an insulin-like substance by pancreatic tissue grown in culture. 1 Part of study submitted to the Graduate Faculty of the University of Oklalioma in partial fulfillment of the requirements for the degree of Doctor of Philosophy. TC20. A comparison of the Nissl substance of living and fixed spinal ganglion cells. Arline D. DEITCH” and Margaret R. MURRAY, Departments of Surgery and Anatomy, College of Physicians and Surgeons, Columbia University. Tissue culture preparations of chick embryo spinal ganglia were studied by phase-contrast microscopy and ultraviolet photography. I n a medium of plasma, chick embryo extract and Parker’s 858, cultures were maintained i n good health up to 5 weeks; within a week neurones less than 10 p thick could be found without cellular over- or under-lay. Such cells, in various stages of chromatolysis and regenerative maturation, were photographed living, fixed with osmium tetroxide followed by formalin, and rephotographed under phase-contrast, then stained with azure A or cresyl violet and again rephotographed. The phase-contrast image was thus related to the stained Nissl picture. I n living neurones there are mediumdark relatively homogeneous cytoplasmic areas in the form of larger flattened plates or smaller flakes. These occupy the same positions as the fixed, stained Nissl substance. The granular and fibrillar elements of the cytoplasm are sharply delimited from the Nissl masses. The latter appear practically unchanged after fixation; dehydration, however, produces some shrinkage and distortion. It is nevertheless possible to homologize at least the larger masses with the same Nissl areas after staining. Ultraviolet photographs taken by Dr. Montrose J. Moses ABSTRACTS O F DEMONSTRATIONS 485 at 262 mp show Nissl bodies in living cells to be discrete darkly absorbing cytoplasmic bodies remaining unchanged after formalin fixation. These observations support the concept that the cla.ssica1 Nissl bodies represent essentially similar structures pre-existing in the living state. 1 Postdoctoral Fellow of the National Institute of Neurological Diseases and Blindness, National Institutes of Health, U. 9. Public Health Service. T C 3 . Recent results from the growth of cells of long-term strains in shaken fluid suspension cultures. Wiltcin R. EARLE, J a y C . BRYANT and Edward L. SCHILLIKG, National Cancer Institute, Bethesda, Maryland. No abstract was submitted. TC 16. T h e growth of cells on a transparent gel of reconstituted rat-tail collagan. Robert L. EHRMANN* and George 0. GEY, Division of Cellular Pathology, Department of Surgery, Johns Hopkins University, Baltimore, Maryland. With the knowledge that collagen is a universal component of connective tissues, serving as a kind of solid substratum and support for cells in the organism, an effort has been made to reduplicate some aspects of this natural system i n tissue cultures. By dializing an acetic acid solution of rat-tail collagen against distilled water, a clear gel is formed. Slices of this gel were placed in roller tubes, equilibrated with culture medium, and tested as a substratum for the growth of 28 cell strains, of which 23 were of human origin. Besides these, a strain of human kidney fibroblasts was grown on collagen which had been jelled by exposure of the acetic acid solution to ammonia vapor. Collagen gel proved superior to the glass wall of roller tubes as a surface on which t o cultivate these strains. On collagen gel, colony outgrowth wa,3 frequently more extensive, and almost always occurred without the retractions so commonly seen on glass. Even after continual exposure, the gel showed no eivdence of lysis by the cells. TC 2. Nutritional studies on certain long-term cell strains. Virginia J. EVANS, Benton B. WESTFALL, Ka,therine K. SANFORD, Naomi M. HAWKINS" and William T. McQUILKIN, National Cancer Institute, Bethesda, Maryland. No abstract was submitted. TC.99. Toxicity of blood serum of schizophrenics in tissue culture. II.I Sergery FEDOROFF* and Anthony F. MESZAROS* ', Department of Anatomy, University of Saskatchewan and Saskatchewan Hospital, North Battleford, Canada. (Introduced by Rudolf Altschul) Continuing the work reported last year on the tosicity of blood serum of schizophrenics in tissue culture (Anat. Rec., 1 8 2 : 394, 1955), it was found that although there was no correlation between the degree of serum toxicity and the clinical condition of the schizophrenic patients, the toxicity was higher in female than in male patients, and generally less in patients of older age groups. The 8erums of a few normal individuals who were under conditions of stress a t the time blood was obtained from them, were also highly toxic in tissue cultures. 486 AMERICAN ASSOCIATION O F ANATOMISTS Toxic serum can be made non-toxic by heating it a t 56°C. for 35 minutes. Blood serum which was toxic to strain L cells was never toxic to strain HeLa cells, despite the fact that both cell strains had been grown i n the same medium for 6 months prior to the tests. The cerebrospinal fluid of schizophrenics is also toxic to strain L cells. Preliminary results indicate an even greater difference in the toxicity of cerebrospinal fluid of schizophrenics and of non-schizophrenics than in the toxicity of the blood serums of the two groups. 1 Supported by a National Health grant. Saskatchewan Committee on Schizophrenia Research. TC r. Exfoliative cytology as n tool in tissue culture. Robert H. FENNELL, Jr.”, Department of Pathology, University of Pittsburgh School of Medicine. (Introduced by Joseph Leighton) Contact smears from gelfoam particle tissue cultures were fixed and stained using Papanicolaou ’s technique. The examiner had no previous experience with tissue culture preparations and applied criteria used i n clinical exfoliative cytology. By these criteria, smears were classified as ‘‘malignant’ ’ or “benign. ” The cells examined included 4 derived from normal tissues (Chang’s conjunctiva, Earle’s skin, Henle ’s intestinal epithelium and Leighton’s D-189) and three derived from cancers (Gey’s HeLa, Eagle’s K B and Osgood’s J-96). The cytological criteria ordinarily used t o classify a smear as malignant were found in smears from several of the “normal” cell lines as well as from the “tumor” cells’ lines. The results of this exploratory study indicate that the techniques of exfoliative cytology can contribute to the evaluations of tissue cultures. The alcohol-ether mixture is a good fixative and the stain, a modified hematoxylin-eosin method is excellent, especially for nuclear details. Identification of cells of a certain striin and detection of even minor alterations in cells should be possible, but to answer the question on morphology alone of whether a given cell or group of cells is “malignant” may not be possible in some cases where cells from many different sources are considered. TC $0. Tissze culture studies in rheumatic fever.’ Lloyd FLORIO*, Gertrud WEISS* and Gladys Kinsman LEWIS* ’, Department of Preventive Medicine and Public Health, University of Colorado School of Medicine. (Introduced by C . M. Pomerat) Among the more commonly accepted theories of causation of rheumatic fever, is one that it is due t o a delayed hypersensitivity of the tuberculin type. Since measurement of this type of sensitivity has been reported in tissue culture, rheumatic and non-rheumatic tissues have been compared in their responses t o crude streptococcal filtrates and t o disintegrated organisms derived from strains presumed to have caused the disease, both in the presence of pooled serum from normal individuals as well as in serum from rheumatic fever cases. Migration was observed after one day and spindle shaped transformation of certain white blood cells was measured about one week after explantation in both rheumatic and non-rheumatic bloods. The effect on migration after phagocytosis of dead ABSTRACTS OF DEMONSTRATIONS 487 streptococci by rheumatic andl non-rheumatic white blood cells was likewise observed. Fibroblasts were compared from skin and heart auricular tips of rheumatic and non-rheumatic individuals and from an occasional rheumatic nodule grown in various concentrations of those same materials. Using similar tissue culture techniques, we also re-examined the concepts involved in the delayed type of tuberculin hypersensitivity using tissues from tuberculous and non-tuberculous humans and guinea pigs. Our results in these experiments were not nearly as definitive as many of the reports in the literature. We were not seeking a statistical answer, but, a n “all or none phenomenon” that would clearly distinguish a rheumatic from a non-rheumatic individual. We failed to find it. 1 Supported by a. b. c. d. grants from National Heart Council United States Public Health Service. The Delta Delta Delta Alliance of Denver, Colorado. The Mary Ruby Murphy Fund. The Barth Foundation. TC 14. Use of manometric method in nutritional studies of strain HeLa.’ George E. GIFFORD” ”, Hugh N. ROBERTSON* ’ and Jerome T. SYVERTON, Department of Bacteriology and Immunology, University of Minnesota, Minneapolis. (Introduccd by Thurlo B. Thomas) The respiration of growing HeLa cells was measured with a standard Warburg apparatus. The oxygen consumption rate was proportional to cell number, while the rate of change of oxygen consumption rate was directly proportional to rate of cellular multiplication and inversely proportional to inoculum size. Maximum populations for growth were determined. Addition of human serum to growth medium in amounts greater than 40% did not increase HeLa cell respiration. Yeast extract, 0.1 %, added to media containing 40% serum stimulated respiration significantly. Cells in 10% human serum with 0.1% yeast extract or in 40% serum without yeast extract respired @t equal rates. The amount of glucose commonly added to media, 100 mg%, limited respiration after 49 hours when approximately 1 million cells per milliliter were employed. For short term maintenance of a coiistant rate of cellular respiration 10% chicken serum in Hanks ’ balance salt solution was adequate. Respiration accelerated slightly with 20% chicken serum, and was increasingly depressed in the absence of serum. With 10% serum, semi-synthetic and synthetic media were not found superior to Hanks’ solution for cell maintenance. Phosphite 0.02 M, phosphate 0.02 M and tris (hydroxymethyl) aminomethane 0.01 M buffers were not toxic f o r HeLa cells. 1 Supported by a grant from the National Foundation for Infantile Paralysis, Inc. T C Q 6 . T h e mitotic activity in vitro of rat kidney tubular epithelium and preliminary @dings on the effects of milateral nephrectomy. And& D. GLINOS’ and Allan M. CAMPBELL* ”, Growth Physiology Laboratory, Division of Surgery, Walter Reed Army Institute of Research, Washington 12, D. C. (Introduced by Duncan C. Hetherington) The tube slip method was used in developing a short-term culture test for this study. Cortex, outer and inner medulla were cultured separately. Inner 488 AMERICAN ASSOCIATION OF ANATOMISTS medulla yielded pure epithelial outgrowth and was therefore used in all subsequent work. Mitotic and total cell counts were performed on fixed and stained slips a t the end of desired culture periods. It was found that following the establishment of the epithelial membranes, the mitotic index decreases rapidly between the fifth and seventh day. Five adult male rats were subjected to a unilateral nephrectomy, and cultures were prepared from the removed kidney. The animals were sacrificed at 1, 2, and 7 days after the operation, and cultures were prepared from the remaining kidney. All cultures were fixed on the fifth day of incubation. The ratios of the mitotic indices of the cultures from the remaining kidney t o those of the cultures from the removed kidney were as follows: First day 1.6, second day 2.3, seventh day 1.7. The significance of these preliminary results with regard to the study of the mechanism controlling kidney tubular epithelium regeneration will be discussed. TC 3.2. Morphological and cytochemical observations of normal and leukemic blood cells in tisszse culture. Milton N. GOLDSTEIN* and Terence McCORDepartment of Biology, Roswell Park Memorial Institute, MICK* Buffalo, N. P. (Introduced by S. Culver WILLIAMS) O, Peripheral blood from normal and leukemic individuals and bone marrow samples from leukemic patients were collected in citrated vials. After centrifugation, fragments of the buffy coat and bone marrow were explanted i n D-35 Carrel flasks on the surface of perforated cellophane and in plasma clots formed on cellophane. The cultures were maintained at 36°C. in a medium consisting of two parts Gey’s solution and one part pooled human sera. The cells i n buffy coat and bone marrow cultures from patients with myeloblastic, lymphoblastic and monocytic leukemias developed giant cells which resembled those seen in normal blood cultures. The formation of giant cells on cellophane from leukemic leucocytes took longer to develop, but resembled those giant cells seen in preparations of normal blood cultures. There was an increase in polysaccharide and acid phosphatase, which was also seen i n normal blood cultures. Many cells in cultures of normal as well as leukemic Ieucocytes developed a fibroblast-like appearance i n plasma clots, and showed the same cytochemical staining reactions as the giant cells. The potentiality of leukemic cells t o differentiate like the normal monocyte in vitro suggests the possibility of a maturation defect in some leukemias, TC 41. Tissue1 culture studies of fish melanomas. Sylvia S. GREENBERG M. J. KOPAC and Myron GORDON Department of Biology, Graduate O, School of Arts and Science, New York University. The 4 expressions of spontaneous pigment cell tumors of hybrid xiphophorin fishes (typical melanomas, melanomas with pigmentless zones, amelanotic melanomas of albino hybrids, and amelanotic melanomas with melanotic zones) were grown in tissue culture. The medium contained human ascitic fluid, chick embryo extract, fowl plasma, and Holtfreter’s solution. Growth was faster and more luxurious with ascitic fluid than with carp serum, the usual component of fish culture media. ABSTRACTS O F DEMONSTRATIONS 489 The pigmentless zones of melanomas are of two diverse types. Growth of cells from fibrous, white zones is slow, with only a sparse proliferation of small spindle cells. Disintegrating pigment cells are present in the esplants. Such zones within the melanoma, indicate a cessation of pigment cell growth. I n contrast, the pigmentless, soft, edematous, and translucent zones of- some melanomas grow rapidly, with nielanocytes as the preponderant cell type. The cells are not well differentiated, as manifested by a sparsity or complete lack of pigmented granules, and inability to maintain the typical dendritic shape. Rapid growth, or a n increase in number of immature pigment cells can therefor account for pigmentless zones in some melanomas. I n cultures of amelanotic melanomas, the inelanocyte with colorless granules is the preponderant cell type. The pigmented zones contain mature, non-dividing cells with brown granules. Cytochemical studies on the cultures indicate that variations in pigment patterns of xiphophorin pigment cell tumors are expressions of a single tumor, and cannot be considered as 4 distinct tumor typeg. 1 Damon Runyon Cancer Research Fellow. TC 2.9. Acid ?nzicopolysaccharide’s produced in tissue culture.’ Henry GROSSFELD*, Karl MEYER’ ’, and Gabriel C. GODMAN*, Histochemistry Research Laboratory and the Department of Medicine, Columbia University, College of Physicians and Surgeons, New York. (Introduced by Wm. R. Duryee) Mass cultures from explants of human foetal skin, and bone, of embryonic bovine bone and rat subcutaneous tissue were grown in Petri or Petroff dishes. They were fed biweekly with a Holution of 20-250/0 EE, 10% serum and 60-650/0 beef amniotic fluid. The pooled “used” medium, and the harvested cell mass were submitted to analysis for mucopolysaccharides. From human skin cultures a mixed polysaccharide was obtained, approximately 3 of which was hyaluronic acid and about & chondroitin sulfate C. The fraction containing the latter was “undersulfated,” possibly owing t o the production in vitro of insufficiently sulfated chondroitin. Cells from human bone produced almost equal amounts of hyaluronic acid (26.0 mg/100 mg acetone dry material) and chondroitin sulfate (31.9 mg/100 mg acetone dried material). Bovine bone cells also produced appreciable quantities of mucopolysaccharide, including a sulfated fraction. 705 ml of supernatant from cultures of r a t subcutaneous fibroblasts yielded 101 mg of a mixed polysaccharide (14.1 mg/100 ml) containing equal amounts of chondrosamine and glucosamine. From its sulfate content and from its optical rotation, this material is believed t o be a mixture of hyaluronic acid and chondroitin sulfate C, which fractions are in process of being isolated. The content of mucopolysaccharide in EE could in no case account for more than 140 of that recovered and was considered negligible. 1 Supported in part by grants A-21 end A-817 of the U. S. Public Health Service. 490 AMERICAN ASSOCIATION O F ANATOMISTS T C W . The virus spectra of several new cell strains. Janet (Lavelie) HERBERG" and Robert N. EIULL", Lilly Research Laboratories, Indianapolis, Indiana. (Introduced by Duncan C. Hetherington) The ability of established cell strains of mouse, rat, calf, monkey and human origin t o support the growth of a group of viruses has been studied. The group included polio-virus, mumps, measles, herpes simplex, A. P. C. vaccinia, influenza, lymphocytic choriomeningitis, lymphogranuloma venereum, MM virus of mice and others. The problems encountered in the adaptation of some of the viruses to growth in several of the cell strains will be presented. Other problems occurred in the preparation of certain cell strains for virus studies and will be discussed. The latter difficulties primarily involved the elimination of virus inhibitors from the complex medium used to sustain rapid proliferation of the cell strain. The significant or characteristic cytopathogenic effects produced by some viruses in a particular cell strain have been compared. Also differences in the C. P. E. of a given virus has been noted in several cell strains. The relative sensitivity of various cell strains to a given virus has been observed as well as the capacity of the cell strains to produce the virus. Preliminary observations on the growth of viruses in suspended cell cultures have been made. TC 12. T h e adaptation and maintenance of mammalian cells to continuous growth in tissue culture. Robert N. HULL, William R. CHERRY and Irving S. JOHNSON, Lilly Research Laboratories, Indianapolis, Indiana. During the past 5 years a number of cell straiirs have been isolated and maintained in our laboratories. Several variations of the plasma clot technic have been used t o initiate growth of cells on glass as well as the direct preparation of cell suspension from animal tissues by the method of trypsin digestion. These technics as well as procedures used for maintenance and subculture of various cell strains will be discussed. Cell strains started in our laboratory include two normal, and one malignant strains of mouse origin, one malignant rat cell, four normal monkey cells of embryonic torso, adult kidney and testicular origin, one of normal human embryonic origin, and one from calf embryonic tissue. Our experience in,the isolation and handling of other cell strains not included in the list above will be presented. These include cell strains which we developed but which were lost by accident as well as strains from other laboratories. T C 1 8 . Depletion of some of t k e serum proteins in tissue culture mediunc. H. Naim KENT* and George 0. GEY, Division for Cellular Pathology, Department of Surgery, The Johns Hopkins University School of Medicine. O Experiments have shown that significant variations occur in the protein composition of the media user1 to cultivate cells. The cells tested were from the rat normal strain 14p; the rat tumorous strains T-72 and T-333; and the human tumorous strain A. Fi. from Gey and Gey stock. Microchemical analyses have demonstrated a considerable decrease of the protein concentration levels during in vitro growth. Electrophoretic studies show that only the alpha and beta globulins reveal a significant decrease in concentration. The strains listed ABSTRACTS O F DEMONSTRATIONS 491 above produce varying degrees of depletion. The analytical results confirm the depletion seen in the electrophoretic patterns obtained by zone and conventional electrophoresis. The question of the mode of incorporation of these two protein fractions by the cells will be discussed. I n direct tests of the utilization of euglobulins and pseudoglobulins isolated from human placental cord serum there was evidence of a depletion of these added globulins. This was also confirmed by direct chemical analysis. The question of whether this depletion is due to an intracellular funcion rather than extracellular protein degradation will be discussed. T C 13. Growth characteristics of two mouse fibroblast strains in stationary and in shaken cultures.' Robert J. KUCIILER and Donald J. MERCHANT, Department of Bacteriology, University of Michigan, Ann Arbor, Michigan. Two stable strains of mouse fibroblasts (Strain L and Strain LLCMI) were grown both in stationary and in shaken cultures and growth curves were plotted from the results of quantitative measurements of the cell populations. The growth curves for each strain were similar in both stationary and in shaken cultures. When either stationary or shaken cultures were started with cells taken from the stationary growth phase the lag phase lasted for 48 to 96 hours. Starting cultures with cells from the log phase of growth shortened the lag phase to 24 hours or less. Taking advantage of this fact it was possible to develop a growth and feeding cycle for Elhaken cultures which approached a steady state. Under these conditions no appreciable p H shift occurred during the 4-5 day growth cycle and no aeration was required. A continuous large yield of cells was obtained. Supported by U.S.P.H.S. grant no. (32539 ( c ) M & I. T C B 7 . A method of cultivating the normal adult mammary gland of the mouse.' E. Y. LASFARGTJES*, Departments of Microbiology and Surgery, Columbia University, Go llege of Physicians and Surgeons, New York. (Introduced by M. ChBvremont) A collagenase which can hydrolyze 25mg of collagen in 18 to 24 hours at the concentration of 0.02mg/ml has been used to separate ducts and acini of the mammary gland from the adipose tissue surrounding them. The glands from C. 57 mice in an early stage of pregnancy are minced in a collagenase solution 10 times as concentrated (0.2 mg/ml), then agitated for two hours a t 37" C. After 10 minutes centrifugation a t 1200 rpm most of the f a t can be discarded. The sedimented cells are washed twice in plain Simrns' solution and resuspended in the culture medium. One drop of this tissue suspension is spread on clotted plasma on a Maximow cover slip and 1 m l is introduced into each roller tube prepared. The slide preparations are washed and refed every 3 to 4 days; the medium in the roller tubes is renewed twice a week. The epithelial sheets, many of them in pure form, have spread widely by the 4th day of cultivation. 492 AMERICAN ASSOCIATION O F ANATOMISTS Human placental serum seems t o be a fundamental element. A 50% dilution of this serum in Simm’s solution supports the growth of the mammary epithelium for periods over one month. I n roller tubes, a nutritive fluid composed of Parker’s 858 supplemented with 25% of human placental serum still supports the growth of the epithelial cells after 5 weeks. Isupported by research grant C-2520 from the National Cancer Institute, U. S . Public Health Service. TC 6. Some histologic featurt-s of human “normal” and “cancer” cell strains in sponge matrix tissue culture. Joseph LEIGHTON, I r a KLINE, Morris BELKIN O , Frances LEGALLAIS and Henry C. ORR, Laboratory of Chemical Pharmacology, National Cancer Institute, Bethesda, Maryland. Sponge matrix cultures of three cell strains that arose from human tumor tissue - Gey ’s HeLa, Eagle’s KB and Osgood ’s J-96 -were examined in histologic sections and found to be morphologically indistinguishable from each other: Sections of D-189, a strain that arose in vitro as a morphologic “malignant transformation” from normal connective tissue, had the same appearance a6 the three strains originally from cancer tissue. All four had malignant features, i.e. absence of architectural patterns indicating tissue of origin, hyperchromatism, high nuclear, cytoplasmic ratio, prominent and often multiple nucleoli, and many mitoses including numerous tripolar and other abnormal forms. Two strains of “normal” human cells - Henle’s Intestine 407 and Chang’s conjunctiva - were indistinguishable on histologic examination from the four strains mentioned above. The morphologic transformation in vitro of normal human cell into cells resembling those from cancer does not necessarily imply that they are equivalent in any other quality. The similarity may have resulted from the selection of variants capable of rapid growth. Cell strains arising from many kinds of tissues, fed on the same kinds of media and selected through many subculture generations for rapid growth may eventually possess many biologic properties in common, including microscopic structure. Variations in the ability of these strains to invade and replace histologically normal tissues are being studied. T C 2 4 . Some problems involved in the culture of cells in a perfusion chamber.’ P. J. LEINFELDER” and B. Shannon DANES”, Department of Ophthalmology and Physiology, State University of Iowa, College of Medicine, Iowa City. (Introduced by Robert Chambers) I n the development of a perfusion chamber for the study of the metabolic and morphological activities of cells grown in culture, it was necessary to set up a control environment in which the physiologic factors that effect growth and maintenance of cells can be experimentally established. The stainless steel chamber that permits continuous perfusion with nutrient fluid is a modification of the Pomerat (’51), Christiansen (’53)’ and Buchsbaum (’54) perfusion chambers. The unit is composed of twin chambers so that simultaneously the activities of sister cultures under control and experimental ABSTRACTS O F DEMONSTRATIONS 493 conditions can be determined. The perfusion fluid (4 parts horse serum, 4 parts Txrode's and two parts embryonic chick extract), is gassed during delivery to the chamber with 5% carbon dioxide and 95% air. Cultures used were primary 7-day embryonic chick heart explants set up in hanging-drop preparations. After 12 hours sister cultures were transferred to the twin perfusion chambers. I n order to simulate flow of tissue fluid continuous perfusion was used. Perfusion rates from 7 p 1 per hour to 1600pl per hour were used. A definite curve of activity based on migration and mitoses was obtained. The experimental data indicates that under these controlled conditions flow of approximately 1 5 0 ~ 1per hour is optimum for the maintenance and growth of the chick heart fibroblast. 1 Supported by a grant from the Iowa Division of the American Cancer Society. TC31. Effects of antibodies on cells i n tissue culture. Charles E. LUMSDEN", Maida Vale Hospital, London, W. 9, England. (Introduced by William F. Windle) A study has been made on the eff ects, upon cells in tissue culture, of heterologous non-immune and immune sera, and of homologous agglutinating sera. This communication deals chiefly with the morphological changes, the phenomenon of resistance, and - in the case of human tissue elements- the presence in the cells of haemagglutinogens. The mechanism of these effects has been compared in mammalian erythrocytes, nucleated erythrocytes (fowl), and in mammalian fibroblasts, mesenchyme and nerve fibres. Though the various stages of the antibody effect succeed each other extremely rapidly, this begins primarily in the ground substance of the cytoplasm, and is followed by mitochondria1 fragmentation, and then by nuclear shrinkage associated with - or due to -passage of nuclear substances into the cytoplasm. Complement is essential to the process: in its absence, fibroblasts can fix antibody and grow unhampered but, even after washing out of the serum, the antibody effect occurs a t once if complement is added. Compared with red cells, living tissue cells have a relatively enormous carrying poxer for antibody before succumbing to its effects. TC 38. Radiosulphur intake b y mucopolysaccharides of embryonic and oultured connective tissues. R. E. MANCINI ",E. 8. LUSTIG and C. NfifiEZ O, Instituto de Oncologia and A. H. ROFFO, Comisidn Naeional de la Energia Atbmica, Buenos Aires, Argentina. Using autoradiographic techniques employing stripping film" of high resolving power, the following studies were undertaken: (1) Intake pathway of S3Gin adult, embryo and cultured connective tissue cells; (2) Nature of the substance responsible for the intake by submitting sections to specific chemical extractions or incubation with connective tissues enzymes. Pele film was applied directly t o hanging-drop cultures in a liquid medium. Six microcuries of radioactive substance proved to be a minimum useful dose to obtain satisfactory images without disturbing culture growth. First evidences of sulphur intake were given by cells of the central zone from the third hour 494 A M E R I C A N ASSOCIATION O F A N A T O M I S T S of culturing with progressive increase up t o 24 hours. I n the migration zone, the 5% appeared to be concentrated selectively in the perinuclear cytoplasm of the cells. Intake increased until it reached a peak at 48 hours at which time the sulphur diffused into all the cytoplasm even into the prolongations of the fibroblasts. From the third hour on, as intracrllular intake increased, an extracellular arrangement of sulphur can be observed in the neighborhoood of the cells and later on in the intercellular spaces, as observed in chick embryo mesenchyme. Images are more evident and intense near the explant where the cellular population is denser. Autoradiographs were negative in the controls using a non-cellular medium. A partial removal of S35from fibroblasts can be obtained by previous treatment with normal hydrochloric acid or with saturated barium hydroxide or by hyaluronidase. Collagenase and elastase, which are specific enzymes for collagen and elastic fibers respectively did not act upon the uptake of the radioisotope. T C 4 . Tissue culture) as a biochemical tool. Joseph F. MORGAN and Helen J. MORTON, Laboratory of Hygiene, Department of National Health and Welfare, Ottawa, Canada. Freshly explanted chick embryonic heart tissues have been used t o study cell nutrition and metabolism. The effect of variations in the synthetic media and culture survival has been correlated with changes in these media during the cultivation period. A specific requirement for L-cystine and a supplementary requirement for either L-or D-methionine were found. Paper chromatography was used to follow changes in the amino acid content of the synthetic medial during tissue cultivation. The observed changes were related to the effect of each individual amino acid on culture survival, employing a nutritional depletion technique. Total protein determinations showed a progressive decrease during cultivation i n synthetic media but the addition of certain natural extracts caused a net protein increase. Synthetic media published by various workers were compared, using one standardized type of culture, and the efficacy of these media was related t o specific components. The advantages and disadvantages of fresh explants and established cells ' strains are discussed in relation to the above experiments. !CC 5. Altered animal cell strains f o r quantitative cultlire work.' Raymond C. PARKER, LaRoy N. CASTOR ' * and Ernest A. McCULLOCH ConO, naught Medical Research Laboratories and Department of Therapeutics, Faculty of Medicine, University of Toronto. Over the past several years, there have been numerous instances i n our laboratory of the development of strains of altered cells that cannot readily be identified with any cell type existing i n the animal body. These altered cells are characterized mainly by their ability to multiply with great rapidity to yield uniform populations of free-living cells that can be centrifuged, washed and subcultured by quantitative procedures. Sometimes, these altered cells appear during sudden bursts of activity i n old cultures undergoing marked cellular 495 ABSTRACTS O F DEMONSTRATIONS degeneration. At other times, healthy cultures become altered either i n part or in their entirety, Sometimes, the alterations take place in tissues only recently explanted from the body; a t other times, the ce1l:s affected have been cultivated for months or years. The cells of some of these altered strains live and multiply while attached to the glass walls of the culture containers. Cells of other strains, smaller in size, have a tendency to live and multiply in suspension, even in stationary cultures. Strains of altered cells of one type or the other have been obtained from bone marrow, kidney, liver and peripheral blood, from several species, and from various long-established strains of cells from other sources. A description will be given of the development of the altered strains and of efforts that are being made t o determine their nature. Supported in part by grants from the National Cancer Institute of Canada. a Bellow of the American Cancer Society. Present addreams : Johnson Foundation for Medical Physics, University of Pennsylvania. TPC 37. Netabolic changes in chick embryonic heart and liver during adaptation H.E.R.T. t o in vitro conditions. John PAUL and Enid S. PEARSON O, Tissue Culture Unit, Biochemistry Department, Glasgow, Scotland. Glasgow University, Immediately after explantation 15-day chick embryonic liver had a very high oxygen uptake, associated with almost compleie utilization of lactic acid in the medium and production of hexose in addition to that originally present. On the other hand heart tissue had vitrtually no oxygen uptake but rapidly utilized glucose with production of pyruvate and lactate. One day later both tissues had a low oxygen consumption of approximately the same order and both utilized glucose with formation of pyruvate and lactate. By €he second day these changes were exaggerated and liver often had a lower oxygen consumption than heart. Immediately after feeding, at this stage, liver oxygen consumption increased while heart oxygen consumption tended t o decrease. Both used glucose and produced pyruvate and lactate. By the third day the picture had reverted to one similar to that a t the second day. I n both tissues there was an initial decrease in DNA followed in a day or two by an increase, the decrease being more prolonged i n the case of liver. incorporation took place into lipid phosphorus, acid-soluble phosphorus, Active P32 RNA and DNA a t all stages in both tissues. The explants were also analysed for total lipid, carbohydrate and protein and these results along with the histochemical picture will be reported in full elsewhere. T C d l . T h e behavior of chick spinal ganglia explanted in a chemically defined medium.’ Edith R. PETERSON*, Department of Surgery, College of Physicians and Surgeons, Columbia University, New York. (Introduced by Margaret R. Murray) While Parker’s synthetic medium 858 will maintain growth in Earle’s L-stain indefinitely, it will not support organized growth of chick spinal ganglia. It is 496 AMERICAN ASSOCIATION OF ANATOMISTS inadequate for this tissue even though supplemented with chicken plasma, which is necessary t o provide a proper physical substrate. I n addition to the plasma, 6 biological components have beeen tested as supplements to the synthetic medium. Only with the addition of embryo extract has complete differentiation, including myelinization, occurred. This and the action of serum and plasma will be discussed in detail in relation to supporting cells m d to neuronal Nissl pattern, mitochondria, fibrous sheath and myelin. I n inadequate media a reduced percentage of cells survive but with abnormal form and behavior. I n these ganglion explants one is dealing with a mixed population of cells whose regeneration, reconstitution, and progressive differentiation must be maintained in delicate balance for several weeks, and in which survival and growth alone are not sufficient for orderly development. 1 Supported in part by research grant B-858 from the National Institute of Neurological Diseases and Blindness, U. S. Public Health Service. l'C42. Further studies on connective tissue mechanics in vitro. Hans H. P F E I F FER, Laborat. f. Polarization Microscopy, Bremen I, Germany. The experiments tended to bring out a standardization of the culture medium to elude deviations of the results. Objects studied were reticular connective tissue of axolotl and perichondrial fibroblasts of chicken embryos. A mixture of plasma and chicken embryo extract was suspended on a stocking woof of colourless Dralon stained with HARRIS' hematoxylin.' Just before clotting a fragment of tissue was transferred into the medium, and the whole preparation was placed in a moist chamber, and incubated. A quick emigration of fibroblasts began around the explant, and repeated passages were possible. The movement of the fibroblasts is determined by tactile excitement starting from the Dralon net. The cells do not grow out as isolated elements, but in a reticular formation. Sporadic elements one finds, a t best, near the margin of the preparation. Step by step, the cultures stop as a veil, the gaps of the Dralon net. Using Y. GERENDAS' apparatus the Dralon woof, in this state, was fixed in a stretched position. Measurements of double refraction, by means of a micra compensator, prove that the cells stretch themselves while growing and utilizing the most suitable fibers of their Dralon medium. Always the main directions of growth correspond to midpoints of the distances of the connections of Dralon net. I f now the fibrillar stroma of the cells was separated by digestion with alkaline pancreatine, unbranched fibrils of leptonic thickness came across each other and formed fibers and bundles undigestible by trypsin, swelling in acetic acid, and showing double refraction like collagen. Some fibroblasts stretched between Dralon fibers are, no doubt, in a state of increased tension and show dark parallel striations indicating intracellular fibrils. They give evidence of the fact that their arrangement corresponds (just like hematocytes repopulating a trypsin digested omentum membrane [PFEIFFER, Anat. Rec., 121, n. 2, 19553) to the tensile trajectories within the culture medium. Other cells, verisimilar, not exposed t o any tractions, find their way, during the growth, down into the medium. '1 have to thank Doctor Harms (Farbenfabriken Bayer, Leverkusen) for yielding up this and other stains for histochemical purposes. ABSTRACTS OF DEMONSTRATIONS 497 TC36. A sample holding vessel permitting the p H measurement of onn/fourth cc samples w i t h ordinary-sized electrodas.‘ Kenneth M. RICHTER, Department of Anatomy and The Tissue Culture Laboratory, University of Oklahoma School of Medicine. It would seem that one of the principles followed in the design and fabrication of electrodes has been ‘to make the electrodes fit the size of the sample.’ It is possible, by utilizing a different approach, namely one of “making the sample fit the electrode,” to extend downward the present useful range of conimercially available electrodes. This approach literally is a matter only o f fabricating a sample holding vessel in which the sample chamber proper redeets closely the size and shape of the tips of the electrodes available. For electrodes measuring 7/16” by 54” the following type of vessel su&m for samples down to ace. It consists simply of a block of plexiglass (1” x 13’’ J( 2 ” 1 in which two parallel but closely spaced 3” diameter wells are drilled afii3 interconnected a t their bottoms in U-tube fashion by a 1/16” o r Q” channel. The electrodes when inserted into the arms of the U-shaped chamber displace the sample upwards and effectively increase sample/electrode contact. Hundreds o f check measurements attest to the reliability of determinations made with this sort of vessel. It has been used routinely in this laboratory for several years and has been especially useful in measuring the p H of roller tube supernates ( 2 cc and less). 1 Supported by grantein-aid from the Helen Hay Whitney Foundation. TC35. Studies on the individual and joint e r e c t s of ATP, glutathione and histamine-HC1 on the fibroblast growth of chick heart in culture.’ Kenneth M. RICHTER, B. R. RITCHESON” and .J. MURRAY”, Department of Anatomy and the Tissue Culture Laboratory, University of Oklahoma School of Medicine. Experiments involving planimetric measurements and microscopic study of 1845 cultures bearing on the individual and joint effects of ATP, glutathione and histamine-HC1 at concentrations of 100 pg/cc of nutrient on the growth and cytomorphology of chick heart tissue in roller tube cultivation were made. ATP and glutathione, when used singly or t,ogether, had no statistically significant effect on growth during 48 hours of experimental treatment. Histamine, given alone, had a statistically significant and marked (46%) growth inhibitory effect. The growth inhibiting action of histamine was completely nullified when histamine was administered in combination with either glutathione or ATP. The combined administration of ATP, glutathione and histamine produced a marked (223%) and statistically significant growth stimulating effect. No specific correlations were evident between the p H changes observed in the nutrient media and the effects of the above compounds on growth. Phase contrast studies and study of the cultures after staining by a modified Giemsa revealed no significant cytomorphic alterations from the normal controls relative to the mitochondrial, golgi, nucleolar or nuclear components. 1 Supported by grants-in-aid from the Helen Hay Whitney Foundation. 498 A M E R I C A N ASSOCIATION O F A N A T O M I S T S T C 1. Immunologic and growth properties of low and high sarcoma-producing lines of cells derived in vitro f r o m a single adult mouse cell. Katherine K. SANFORD, Gwendolyn L. HOBBS and Mary C. FIORAMONTI, National Cancer Institute, Bethesda, Maryland. No abstract submitted. TC 19. Pineal tumor giant cell.' Machteld E. SANO, Department of Pathology, Newton and Morristown Memorial Hospitals and Department of Tissue Culture, Temple University Medical School. The giant cell of a human pineal tumor was studied in tissue culture by cinematography. Rhythmic phases of nuclear and cytoplasmic activity were observed. Interpretation of these phenomena from a cytophysiologic standpoint have been made and will be discussed. 1 Supported in part by the Chase Research Foundation and the F. E. M. Sano Fund. TC 15. T h e influence of elevated temperatures on the growth and morphology of H e L a cultures.' Oleg S . SELAWRY" and Terence McCORMICK* Department of Biology, Roswell Park Memorial Institute, Buffalo, N. Y . (Introduced by Machteld E. Sano) O O, HeLa cells were cultured in D/35 Carrel1 flasks in a fluid medium composed s f one-third calf serum and two-thirds medium 199 a t 36" C. The cells were subjected to heat treatment (waterbath) in order to determine the upper level of their biokinetic temperature and their morphological changes under such treatment. 100% of a cell population show irreversible damage after exposure to 42 f 0.1"C. for 10-12 hours, to 43°C. for three hours, to 44°C. for two hours and to 45°C. for 55 minutes as determined by further observation of the culture for 25 to 30 days. Surviving cells after shorter exposure time are less heat susceptible, especially in the range o f 42°C. After intermittent treatments i n the course of 8 months they became adapted to 42°C. for more than 100 hours, t o 45°C. for up to 75 minutes. The influence of elevated temperatures on the mitotic cycle and on cytoplasm and nucleus of the interpliases will be described. 1 Supported by the L-S-W Qrant. T C 8 . T h e Detroit lines of human epithelium-like cells: observations on morphology and biological behavior.' Cyril S. STULBERG, Lawrence BERMAN ' and Frank H. RUDDLE, The Child Research Center of Miehigan and the Department of Pathology, Wayne University College of Medicine, Detroit. Previous communications have described a stable strain of human epitheliumlike cells (Detroit-6) derive2 from a tissue culture of bone marrow. Further studies have resulted in the development of seven additional lines with similar niorphologic and growth characteristics. Three of the strains (Detroit-6, 32, -34) came from cultures of sternal marrow of patients with carcinomatosis. Two strains (Detroit-30a, -56a) were obtained from the carcinomatous ascites of two patients, and one strain (Detroit-116a) from the pleural effusion of a ABSTRACTS OF DEMONSTRATIONS 499 patient with lymphosareoma. Two strains (Detroit-52, -98) developed from the sternal marrow of individuals with no known malignancies. I n the bone marrow cultures the epithelium-like cells first appeared a s isolated plaques of polygonal cells on the 50th to 65th day of continuous culture. Each marrow culture passed through myeloid, monocytoid, and sometimes fibroblastic phases before epithelium-like cells were observed. Epithelium-like cells occurring in the original ascitic and pleural fluids gradually increased in number during one to six weeks. Once epithelium-like cells appeared, they were mechanically separated from the parent culture or primary cultures were trypsinized, and after subculture, other morphologic cell types disappeared. Comparative studies on morphology, growth behavior, transplantation t o heterologus hosts, sponge culture, and virus susceptibilities, illustrating similarities and differences in these cell lines, will be presented. Supported by a grant (E859)from the National Microbiological Institute, and by 8 grant (C-2149) from the National Institutes of Health, Public Health Service. TC 17. The amino acid requirements of Auman uterine fibroblasts, strain U 12.' H. E. SWIM* and R. 1. PARKER* Department of Microbiology, Western Reserve University. (Introduced by Ivor Cornman) O, The results of pre,vious studies indicate t h a t chronic toxicity of conventional cell culture media is partially responsible for the failure of normal (unaltered) human fibroblasts to proliferate after 4 to 8 months of serial cultivation. I n an attempt to circumvent some of these difficulties thc amino acid requirements of uterine fibroblasts have been investigated. Strain U12 in contrast to other human fibroblasts studied in this laboratory, became altered in vitro, as indicated by changes in morphology of the cells and in their loss of susceptibility to infection with poliomyelitis virus. Strain U12 was selected for study because it probably represents the closest practical parallel available at the present time to a n unaltered human fibroblast. The method employed was based on studies of Fischer employing a defined medium supplemented with dialysed horse serum and dialysed embryo extract. The cells degenerated rapidly when each of the following was omitted from the medium: arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine. The concentration of each amino acid and glutamine required for maximal growth under the experimental conditions has. been established. The quantitative aspects of these studies will be presented and discussed in relation to the continuous serial propagation of unaltered human fibroblasts. 1 Supported by a grant from The National Foundation for Infantile Paralysis. T C I O . Some quantitative studies on the growth o f Strain L (Earle) mouse cells in serum-free nutrient solutions. Charity WAYMOUTH, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. The growth of strain L cells in media containing no serum or tissue extracts has been studied with a simple quantitative method using a microhematocrit (Waymouth, '56, in press). The effects on cell proliferation of omitting or varying the concentration of certain components of the medium have been examined and new information on nutrients essential f o r cell proliferation will be presented. 500 AMERICAN ASSOCIATION O F ANATOMISTS TC25. Air-ion concentration and the growth of cells in vitro? John L. WORDEN*, Department of Biology, St. Bonaventure University and James R. THOMPSON* ', Inst. f o r Cellular Research, University of Nebraska. (Introduced by Tobie Mullerj Recent investigations have demonstrated the effects of air-ion concentration and polarity on various physiological values i n intact mammals. Statistically significant changes have been noted in carbon dioxide capacity of plasma, p H of whole blood, nerve regeneration time, adrenal secretory activity and others. Apparatus for the generation and dissemination of ions of determined polarity has been adapted to tissue culture procedures so that cells may be propagated in liquid medium with an overlay of 5% CO, in air with (1) normal ion content, ( 2 ) increased negative ion content, ( 3 ) increased positive ion content. Experimentation has shown that an excess of ions of one polarity is essential for physiological effects. I n conditions 2, 3, above, ions of opposite polarity are a t near zero level. Cultures of pure strain L cells (Earle's) were employed. Replicate planting8 and cell enumerations mere made according t o techniques of Earle, Evans, Sanford et al. Determinations were run in sets of 36 replicates, 9 used to determine initial counts and uniformity of planting, and 9 placed in each of three incubators at 37.5"C. for 72 hours. Atmospheric conditions were normal in one incubator, elevated negative ion concentration i n the second, elevated positive ion concentration in the third. Statistically significant differences in rate of proliferation occurred -reproduction was accelerated under conditions of negative ionization and decelerated under conditions of positive ionization. The nature of the effects is compatible with that observed in intact animals. 1 Supported by a grant from the Wesix Research Foundation, San Francisco, California.