Ink Particles Research Articles

MORPHOLOGICAL STUDIES OF THE NYMPHAEACEAE. V. DOES NELUMBO HAVE VESSELS?

The examination of the primary xylem of roots of Nelumbo has revealed the presence of vessel elements with intra‐face, transversely‐rowed to obliquely‐rowed bordered pitted side walls and scalariformly perforated end walls. Whole mounts of macerated xylem, serial sections of roots, and cells through which a water suspension of India ink particles under slight or no pressure was passed, were the means of investigation. It is our opinion that this discovery does not support theories which derive monocotyledons from the Nymphaeaceae. A discussion leading to this conclusion is presented.

Wachstum und Ausbreitung des Ehrlich-Asziteskarzinoms der Maus im Hühnerembryo

Growth and spread of Ehrlich ascites carcinoma of the mouse in the chick embryo were studied after injection of different amounts of tumor cells into chorioallantoic veins on the 11th day of incubation. Inoculation of 1×10<sup>3</sup>-1x10<sup>5</sup> tumor cells did not result in tumor growth. After injection of 1 × 10<sup>6</sup> tumor cells, metastases were found in heart, liver, mesonephros, brain, and chorioallantois; nevertheless most embryos hatched out. The dose of 1 × 10<sup>7</sup> tumor cells extended the formation of metastases to lung and metanephros; most embryos, however, died before hatching. The difference of localization of metastases is thought to be connected with the different stages of organogenesis on the 11th day of incubation. Four hours after injection, tumor cells were observed within the blood vessels of all organs examined. The same localization was ascertained for india ink particles. Twenty hours after injection tumor cells were found within the parenchyma of all organs, and none could be detected in blood vessels by 48 h. It is assumed that these findings are an expression of active locomotion displayed by the tumor cells. Almost always groups of tumor cells within the organs were surrounded or infiltrated by pseudoeosinophilic granulocytes. After the 14th day lymphocytes were occasionally present in the vicinity of metastases. However, no clear-cut indication of an anti-neoplastic function of these leukocytes could be acquired.

Studies on the internal defense mechanisms of sponges: II. Phagocytosis and elimination of India ink and carmine particles by certain parenchymal cells of Terpios zeteki

Specimens of the sponge Terpios zeteki were challenged with 1:10 sea-water dilutions of India ink and carmine particles, and examined in histological sections at 1, 6, 12, 24, 48, and 96 hr post-injection. It was determined that foreign particles injected into the mesoglea formed extracellular clumps by the 1st hr. These clumps were subsequently resolved with the concurrent phagocytosis of the particles by archaeocytes and less frequently by collencytes. Ink- and carmine-laden sponge cells moved through the mesoglea and entered excurrent canals by migration between the lining pinacocytes. In this manner, the sponges became cleared of foreign particles. Carmine particles were completely eliminated by the 24th hr and ink particles by the 96th hr when maintained at 20°C in sea water with a salinity of 35 ‰ Differential counts of the five types of free parenchymal cells did not reveal any significant increase in the number of cells during the course of the experiment although dividing archaeocytes and collencytes were observed.

THE FEEDING AND SWIMMING OF CONCHOECIA (CRUSTACEA, OSTRACODA)

1. The swimming and feeding of living Conchoecia is described. In general, the observations reported support conclusions arrived at by Iles from anatomical studies.2. Freshly caught animals swam constantly, except during feeding. Apparent running over the bottom was achieved by beating of the antennal exopods, as in swimming, and was not the result of any action by the more leglike appendages.3. The food consisted almost entirely of dead crustaceans. Formed masses of detritus were also eaten. Living Crustacea were generally ignored, although one attack on a possibly moribund specimen was observed.4. There was much shifting and rotation of the food by the mandibular palps, the exopods of the maxillae and first trunk limbs, and the caudal furca. This manipulation served to press different parts of the food against the mandibular gnathobases. Loss of food was prevented by the sticky secretion of the marginal carapace glands.5. The dorsal condyle of the mandible seemed to be more loosely articulated than would appear from the description by Iles.6. There was no significant filtration of particles from the current produced by the beating of the vibratory plates.7. Swirls caused by the more leglike appendages brought in particles which became trapped in the sticky secretion of the marginal carapace glands. Resulting boluses of Sepia ink particles were pushed to the mouth and swallowed. However, there are reasons to doubt the significance of this mechanism in the natural habitat.

Microcirculatory dynamics in the normal and inflamed pancreas

When an India ink solution was infused into the main pancreatic duct of the dog, the particles were shown to pass between the cells of the acinar unit without apparent ductal rupture. The ink accumulated in a well defined space immediately adjacent to the basilar aspect of the acinus. This periacinar space appeared to communicate directly with terminal ramifications of the local lymphatic circulation. When the lymphatic system was patent, an ink solution introduced into the periacinar space was removed by this route, and the ink particles were deposited in the lung. When the lymphatic system was obstructed by ligation of the thoracic duct, much of the ink remained in the periacinar spaces; however, there was also a considerable concentration of the particulate material in the liver. The latter suggested that the periacinar spaces also communicated directly with the local capillary (portal) circulation. During acute pancreatitis the periacinar spaces became acutely distended, and congestion and stasis were present in both the lymphatic and capillary components of the circulation. Ultimately, erythrocytes filled the periacinar spaces, suggesting the communications between the capillaries and periacinar spaces became pervious to both the fluid and cellular components of the blood. The latter may in part explain the development of hematochylia during acute experimental pancreatitis. The existence of a periacinar space which communicated with both the lymphatic and the capillary circulation of the pancreas was supported by the distribution of ink when it was infused directly into the local lymphatic, venous, or arterial systems during acute pancreatitis.

Fine structure of experimentally produced subsynovial carbon granuloma.

RECENT electron microscopic investigations of synovial joints have been concerned with the phagocytic nature of synovial membrane cells1,2. In order to investigate this phenomenon further, 1 c.c. of colloidal carbon in the form of Indian ink (particles 200–2000 A in diameter) was injected into the knee joints of rabbits. 104 days later the animals were killed, and large granulomata were evident in the subsynovial tissues. This report is concerned with the structure of these carbon granulomata.

The formation of lymph in the ovary

The lymphatics of the ovaries of pregnant and non-pregnant ewes were outlined either by direct or retrograde injection of indian ink or Berlin blue and the distribution of these vessels within the ovary was determined. In active ovaries the mature follicles and corpora lutea contained a profuse network of lymphatics. In inactive ovaries the lymphatics were very small and poorly developed. Lymph was collected from the ovaries of conscious ewes for periods of several days. The flow of lymph from ovaries with corpora lutea averaged 4.15 ml./h; a maximum rate of flow of 14.9 ml./h was recorded in one ewe. The protein concentration of ovarian lymph was 73 % of the plasma concentration. When 131 I labelled albumin was injected intravenously into ewes it entered the ovarian lymph very rapidly and the specific activities of albumin in the plasma and lymph equilibrated within 10 to 20 min of injection. The structure of the ovarian blood capillaries provided an explanation for the very high rate of lymph flow and protein leakage in the ovary. The endothelium of the blood capillaries was discontinuous with gaps up to 1 to 2 μm in diameter through which red cells, indian ink particles and ferritin passed into the interstitial spaces. Where the basement membrane of the capillaries was deficient over gaps the surfaces of the luteal cells came into direct contact with the circulating plasma, and occasionally cytoplasmic extensions from the luteal cells projected into the lumen of the blood capillaries. The lymphatics which were associated with many of the blood capillaries had open intercellular junctions and material could enter these vessels readily from the interstitial spaces. The blood capillaries in the ovarian stroma and those around immature follicles appeared less permeable than the capillaries of the corpus luteum. The association between the development of the corpus and an increase in lymph production in the ovary suggests the possibility that the changes in capillary permeability may be related to the synthesis and secretion of steroid hormones.

THE PHAGOCYTIC NATURE OF SCHIFF-POSITIVE INTERSTITIAL CELLS IN THE RAT TESTIS.

Recent studies (Clegg & Macmillan, 1965) on the response of the Sertoli cells to vital dyes or particulate matter introduced directly into the seminiferous tubules led to the incidental observation that these substances were taken up and stored by certain interstitial cells if an accidental 'overspill' into the intertubular tissues occurred. The cells were studied further by injecting 0·25 ml. of a 1% suspension of Indian ink (Pelikan) in 0·9% NaCl solution directly beneath the tunica albuginea of one or both testes of adult albino rats. Animals were killed at intervals up to 14 days after the injection; equatorial sections of the testes were subjected to the PAS procedure and counterstained with Ehrlich's haematoxylin; a few sections from each testis were exposed to diastase digestion before staining. Within 48 hr. of injection the ink particles had been cleared completely from the intertubular spaces and lay within the cytoplasm of interstitial

THE FLOCCULATION OF SUSPENDED MATTER BY PARAMECIUM CAUDATUM.

SUMMARY: Paramecium caudatum has been shown to cause the flocculation of Indian ink particles. Flocculation has been demonstrated to be due to: (a) the secretion of a substance ‘P’, a carbohydrate whose monosaccharide constituents are glucose and arabinose, (b) the ingestion of particles by the cell which are then bound together by a sticky mucoprotein. Substance ‘P’ is adsorbed on to Indian ink particles present, which results in a change of their surface charge and the aggregation into floccules. Substance ‘P’ has been labelled with [14C] glucose which then appeared in the floccules.

Further observations on mechanism of hypoglycorrhachia in experimental meningitis.

When dogs with aseptic meningitis were infected with pneumococci intrathecally, a profound drop in CSF glucose occurred which was not observed in control animals given pneumococci without antecedent production of aseptic meningitis, or in animals with aseptic meningitis in absence of superimposed bacterial infection. Hypoglycorrhachia also occurred when heat-killed pneumococci, India ink particles and bacterial endotoxin were administered to animals with aseptic meningitis. These data suggest that the fall in CSF glucose is dependent upon presence of leukocytes engaged in active phagocytosis.

Development of Lipemia in Rats Following Intravenous Injection of India Ink.

In the course of studies of the role of the liver in destruction of clearing factor, Higgins’ India Ink was injected into rats. It was observed that blood samples withdrawn one to 2 hours after injection were lactescsent. This lactescence was associated with an increase in level of total fatty acids. The lipemia occurred in fasted rats thus indicating that the India Ink was either stimulating an increase in rate of mobilization of fat from the depots or blocking the exit of the mobilized fat from the blood stream. Since the reticuloendothelial system is readily saturated with the India Ink particles, the lipemia could have resulted from inhibition of the normal function of these cells. It has been reported (1) that the reticuloendothelial system functions in uptake of cholesterol from the blood. The evidence (2,3) is against these cells playing any major role in uptake of chylomicirons. The following experiments were carried out in an attempt to determine the mechanisrn by which India Ink induced lipemia. Methods and materials. Total fatty acids. The lipids were saponified by heating in presence of alkali and alcohol. The alcohol was removed and the fatty acids were freed by addition of acid in excess. The fatty acids were transferred to a solvent and, after evaporation of the solvent, were titrated by standard alkali. The details were as follows: 0.2 ml of plasma was added to 5 ml of alcoholethyl ether mixture (3: 1 v/v) and heated to boiling in a water bath. The mixture was then cooled and filtered into a graduated test tube. The residue was reextracted, filtered

The absorption of particles by the lymphatics of the diaphragm.

The absorption of particles from the peritoneal cavity by the terminal lymphatics, or lacunes, of the rabbit's diaphragm has been studied with the light and electron microscopes. Absorption occurs predominantly through those parts of the peritoneal surface of the diaphragm which overlie the lymphatic lacunes. These parts are referred to as the lacunar roofs.The mesothelial cells of the roofs have features which distinguish them from mesothelial cells elsewhere on the peritoneal surface of the diaphragm. The cells are more closely set, stain more darkly, and separate from each other more readily, particularly at the base of the intercellular junctions. They are supported by a lattice of coarse and fine fibres. In the meshes of this lattice mesothelial and lymphatic endothelial cells are separated only by the basement membrane of the mesothelium which in places may be incomplete.Erythrocytes pass through the roof into the lumen of the lacune through gaps which are formed by separation of the mesothelial cells and of the endothelial cells at the intercellular junctions. Particles of India ink and colloidal particles of thorium dioxide and saccharated iron oxide enter the intercellular spaces of the mesothelium and spread freely within the meshes of the fibre lattice. These particles appear to pass through the mesothelium by a predominantly extracellular pathway and probably enter the lymphatic lumen through temporary channels which are formed by separation of endothelial cells at the intercellular junctions.Absorbed colloidal particles accumulate in the cytoplasm of mesothelial and lymphatic endothelial cells in the roofs and a proportion of absorbed material may be transported intracellularly through these two layers in cytoplasmic vesicles. Lymphatic endothelial cells at other sites in the diaphragm can take up colloidal particles from the lumen of the lymphatics.

COUNTING PROCEDURE FOR PHASE CONTRAST MICROSCOPY

The bacterial culture to be investigated is mixed at 45 C with an equal amount of liquid culture medium containing 1 to 2 per cent agar and a small amount of India ink. About 5,L of this mixture is rapidly spread on a slide kept at 45 C. A cover slip is immediately placed on top of the film and the preparation is left to cool. Within a few minutes, a solid, bacteria-containing layer, 10 to 20 ,u thick, is formed between slide and cover slip. The preparation is studied under the microscope using an oil immersion objective. The bacteria in a field of view are counted by focusing gradually from the slide to the cover slip. The thickness of the agar layer is measured using the scale of the fine adjustment of the microscope. This can be done with a precision of about 0.5 ,u by focusing on some India ink particles immediately below the cover slip and immediately above the slide. The position of the slide on the stage is then changed and new areas of the agar layer are investigated in the manner just described. The diameter of the field of view is measured with a stage micrometer, and the average number of bacteria per ml of the original bacterial suspension is calculated. In an experiment of this kind, performed with a Leitz Ortholux microscope, 14 fields, containing together 243 bacteria (Proteus vulgaris), were investigated. The thickness of the agar layer varied between 7 and 24 ,u. From the data obtained it was calculated that the original bacterial suspension contained (1.92 + 0.39) X 108 bacteria (99 per cent fiducial limits). Counts performed with a conventional counting chamber having a depth of 0.1 mm gave the figure (2.19 0.30) X 108. The procedure described above would be especially useful for investigations concerning material that is not easily stained and thus can be most suitably studied with phase contrast microscopy, e. g., L-forms and pleuropneumonialike organisms. It is the author's experience that this method gives more reproducible results than counts performed with a conventional counting chamber having a depth of only 0.01 mm.

The ultrastructure of mouse lung: the alveolar macrophage.

Free alveolar macrophages of normal mouse lung have been studied in the electron microscope. The tissue was obtained from several young adult white mice. One other animal was instilled intranasally with diluted India ink 1(1/2) hours prior to the removal of the lung. Thin sections of the osmium-fixed, methacrylate-embedded tissue were examined either in an RCA EMU 2 electron microscope or in a Siemens and Halske Elmiskop I b. A few thick sections obtained from the same embeddings were stained for iron. The normal alveolar macrophages, which are usually in contact with the alveolar epithelium, were found to contain a variety of inclusion bodies, along with the usual cytoplasmic components like mitochondria, endoplasmic reticulum, and Palade granules. Another typical component of the cytoplasm of these cells which appears as small ( approximately 6 mmicro) very dense granules of composite fine structure is interpreted as ferritin. It is assumed that this ferritin is formed from red blood cells ingested by the alveolar macrophages. The macrophages in the alveoli were found to phagocytize intranasally instilled India ink particles. Such cells, with engulfed India ink particles, were often of more rounded form and the particles were frequently seen lying inside membrane-bound vacuoles or vesicles of the cytoplasm. The membrane of a few vesicles containing India ink particles was seen as the invaginated portion of the cell plasma membrane, and in one instance these same vesicles were seemingly interconnected with a rough surfaced cisterna of the endoplasmic reticulum. The process of phagocytosis is recognized as related to the "normal" process of pinocytosis.

STUDIES ON THE EFFECTS OF PHAGOCYTIC STIMULATION ON MICROBIAL DISEASE: I. ACTION OF SOME DERIVATIVES OF THE BICYCLO[0.3.5]DECAPENTAENE SKELETON ON ENDOTHELIAL CELLS OF SKIN VESSELS

If the depilated skin of white mice is rubbed with solutions of histamine or certain derivatives of bicyclo[0.3.5]decapentaene, simultaneous intravenous injection of a suspension of India ink leads to an intracellular accumulation of ink particles inside the endothelial cells of small vessels at the site rubbed. This phenomenon of phagocytosis is induced in cells which have normally no phagocytic capacity and can be inhibited by antihistaminics. Solutions of these derivatives are quickly absorbed and are thought to liberate sufficient quantities of histamine to induce phagocytic activity of the endothelial cells. Some derivatives of the bicyclo[0.3.5]decapentaene skeleton have been tested for similar action and a quantitative difference was observed. These observations confirm those of Jancsó who states that histamine stimulates the endothelial cells of small vessels to phagocytic function. The phenomena described seem to have a certain importance because if the hypothesis is correct, a high number of normally inactive cells may be placed at the disposal of the natural cellular defense mechanism of the organism by means of a physiological stimulus.

The role of stereocilia in removing India ink particles from the lumen of the rat epididymis.

The role of stereocilia in removing India ink particles from the lumen of the rat epididymis.

Composition of mitochondrial preparations in relation to intracellular localization of azoprotein in the mouse.

Conclusions and SummaryThese studies substantiate previous reports that a relatively large proportion of injected labeled protein is found in the mitochondrial fraction of homogenized liver(8). Failure to demonstrate in this fraction the azoprotein which was added to the whole liver homogenate indicates that an in vivo process is involved and that simple adsorption of the azoprotein by mitochondria does not occur. Results of microscopic study of wafers, made by centrifuging mitochondrial fractions, indicate that the intracytoplasmic granules of azoprotein seen in sections from injected animals(1-3,5) are found predominantly in such mitochondrial fractions. However, the facts that injected India ink particles occur in a similar proportion in such fractions, that the azoprotein granules do not take various mitochondrial stains, and that other impurities can be demonstrated in such preparations, indicate that such mitochondrial fractions do not consist only of mitochondria. This conclusion has also been expr...

The fate of india ink injected intracardially into the oyster, ostrea virginica gmelin.

The responses of the oyster to an intracardial injection of a sea water suspension of india ink were followed grossly and microscopically. The ink suspensions agglomerated readily and produced emboli which virtually occluded the arterial vessels of viscera, mantle and adductor muscle. Subsequent events, with considerable overlapping, were in sequence : (a) phagocytosis of the injected ink particles by mobile phagocytes, (b) distribution of the ink in the phagocytic amebocytes to all parts of the organism with concomitant resolution of the emboli and (c) eventual elimination of the ink from the organism by the migration of ink-laden phagocytes through the epithelial layers of the alimentary tract, digestive diverticula, palps, mantle, heart and pericardium into lumina from which they were voided. The epithelia of gonaducts, nephridia and shell-forming mantle were not routes of migration. A close relationship is noted between the role of the phagocytes in the normal digestive process and in the "defense" reaction to such a foreign body as india ink. The possible significance of the responses noted with respect to unexplained mortalities of the oyster is considered.

The influence of testicular extracts upon the fragility of red blood cells and upon the dispersion of indian ink particles in the dermis

The influence of testicular extracts upon the fragility of red blood cells and upon the dispersion of indian ink particles in the dermis

THE INFLUENCE OF TESTICLE EXTRACT ON THE INTRADERMAL SPREAD OF INJECTED FLUIDS AND PARTICLES

The experiments in this paper show that testicle extract causes India ink particles and those of Prussian blue to spread much more extensively through the intercellular spaces than similar suspensions made with Ringer's solution. Methylene blue inoculated intravenously localizes more extensively in areas previously injected with testicle extracts than in control areas receiving injections of tissue extracts without enhancing power. Kidney extracts have this property to a less degree, whereas spleen extracts and blood serum are devoid of it. The spreading power of extracts is destroyed by heating at 60 degrees C. for 30 minutes, as is also the power to enhance infections. The precise mode of action of the Reynals factor is not known, but the results of the experiments here presented suggest that it may depend at least in part on the property whereby testicle extract increases the spread of injected material and alters the permeability of tissue cells. It is not inconceivable that changes in permeability facilitate the passage of vaccine virus through the endothelial cells of the blood and lymph vessels, and lead to the generalized vaccinia which is of frequent occurrence in the reported results (20). It has been shown that fluids and suspensions of inert particles are spread by the extract.B. tetanus and B. coli exotoxins and trypsin were not enhanced at all in their action despite the fact that they were spread through a more extensive area in the tissues. Viruses, on the other hand, are markedly influenced and in this respect resemble bacteria, not toxins and enzymes. It appears probable that a definite capacity for multiplication on the part of an injected substance is required if its pathogenic effects are to be enhanced. It may be concluded tentatively that the enhancing power of the testicle extract may depend on that property which not only spreads the injected material through a larger area but renders the tissue cells more easily penetrable by the agents.