Frog Blood Research Articles

ADDITIONAL MECHANISMS FOR THE ORIGIN OF BUBBLES IN ANIMALS DECOMPRESSED TO SIMULATED ALTITUDES

1. A heavy ingestion of frothy emulsified fat by rats and bullfrogs does not increase susceptibility to bubble formation when the animals are decompressed 2 to 72 hours later. This indicates that gaseous films (bubble nuclei) initially present do not pass across the intestinal wall with the digested fat, and also that high fat content per se in the lymph and blood does not increase susceptibility to bubble formation. 2. Liquid caprylic acid injected into veins of bullfrogs crystallizes when the frogs are cooled. The crystallization causes bubbles to form without muscular activity on subsequent decompression. Cooling normal bullfrogs to 1-2 degrees C. fails, however, to crystallize any substances occurring naturally in the animals that might act in a similar manner. 3. When bullfrogs are cooled (e.g. to -5 degrees to -10 degrees C.) until ice forms in the blood vessels, and are then warmed and decompressed, bubbles form in the absence of exercise. Crystallization of water in the body thus forms nuclei or even small bubbles that persist. If only one foot is frozen, bubbles originate in the frozen foot. In some cases visible bubbles were observed in thawed feet at sea level (i.e. without decompression). When frog's blood is partly frozen in test tubes or in tied off sections of veins, bubbles will appear on decompression in the absence of mechanical agitation. The practical relation of this phenomenon to flight at high altitude should not be overlooked. 4. Fracturing a leg bone (tibia or femur) in a frog induces bubble formation on subsequent decompression. Bubble nuclei, which persist for (1/2) to 1 hour, are probably formed as a result of the intense mechanical disturbance when the bone snaps. Fracturing of bone is considerably more effective than crushing muscles for producing bubbles in frogs.

Copper Peroxidase Reaction of Frog's Red Cells: A Morphological Demonstration of Close Relation between Per=oxidase and Hemoglobin

By making the most of the smear peroxidase method of Sato and Sekiya and especially of the counting chamber peroxidase method of Sato and Shoji, I have made a study of frog's blood and come to the following conclusions. (1) Frog's red cells show different or rather all possible stages of peroxidase picture in the cytoplasma and in the nucleus, especially in the counting chamber method. (2) There is a possibility that these peroxidase-positive gran-ules may be equal to the acidophil substance, which will be manifested by the Al, ay-Giemsa stain. (3) The localization and the degree of development of these granules in each red cell are different according to the state of differ-entiation of red cells. (4) Thus, it is probable that these granules are closely related to the formation of hemoglobin in red cells.

Effects of the Commonly Used Anticoagulants on the Ultramicroscopic Appearance of Frog's Plasma

A series of investigations are in progress in this laboratory dealing with the changes brought about in the blood plasma by the injection of drugs and also by various pathological conditions.Since some method of preventing the coagulation of the blood plasma has to be employed, it became essential to study the effects of the commonly used anticoagulants on the appearance of plasma under the ultramicroscope. In previous communications1, 2 oxalated rabbit's plasma had been used.Samples of frog's blood (Rana catasbiana) were collected into oiled tubes without the use of any anticoagulant, centrifuged, the plasma pipetted off and diluted 1:5 with specially prepared, particle free, 0.75% physiological saline solution, and examined under the ultramicroscope. Similar samples were received into heparin (1/3 mgm. per cc.), potassium oxalate (2 mgm. per cc.) and sodium citrate (2.5 mgm. per cc.). The blood was also made non-coagulable by injection of heparin into the dorsal lymph sac 45 minutes previous to bleeding...

Blood Pressure and Blood Protein Determinations in the Frog.

In a previous note, the authors called attention to the low protein content of frog's blood. The low colloidal pressure of the blood is an important factor in determining the production of urine. The blood pressure of the frog is not nearly as high as in mammals, but if filtration is the basis of glomerular activity, there should be enough pressure to overcome the osmotic pressure of the colloids of the frog's blood. It seemed desirable, therefore, to make some actual blood pressure determinations and determinations of the protein content of the blood on the same animal. In order to make blood pressure determinations on the intact animal, the following apparatus was devised. A modified apparatus such as is used for determining arterial blood pressure in man was used. Small rubber cuffs were constructed from small rubber balloons, varying in size from one-half to one inch wide, and 3 to 5 inches long. A rubber tube was cemented into the open end of the balloon and by means of it the balloon was connected to a mercury manometer, an outlet valve, a Woolf bottle to act as a buffer, and a rubber atomizer bulb. The frog was placed in a suitably sized box with its right leg protruding through a small opening. The rubber cuff was then wrapped around the thigh or leg, the web of the foot placed under the low power of the microscope and as large an arteriole as could be found was brought into focus. A pulsating stream could be observed. The pressure in the cuff recorded by the mercury manometer was raised until pulsations in the arteriole could no longer be seen. The air was then gradually let out of the system and when pulsations first were observed, the mercury manometer was read for systolic pressure. As the pressure continued to fall slowly, it was noticed that between systolic peaks of pressure, the blood stream slowed to complete stoppage and sometimes to a reverse flow. When this reverse flow disappeared and the stoppage between systoles began to disappear, the diastolic pressure was taken. The readings obtained in this manner have been checked up with a mercury manometer attached directly to an artery in the abdominal cavity and with a Hurthle manometer attached in the same manner, and the readings were found to be within the limit of error.

THE SPINDLE‐CELLS IN RELATION TO COAGULATION OF FROG'S BLOOD

1. The thrombocytes of frog's blood, which alter so quickly on contact with glass or other water‐wettable surfaces, undergo no deformation and cytolysis as long as the blood is kept on solid paraffin.2. While the erythrocytes and the leucocytes of frog's blood can by means of the centrifuge be separated from the plasma, the thrombocytes, which have a density identical with that of the plasma, cannot be thus removed. A specimen of plasma obtained by simple use of the centrifuge clots on coming in contact with glass.3. From the centrifuged plasma of the frog the thrombocytes can be removed by passage through a charcoal filter. Plasma thus freed of thrombocytes readily clots on addition of tissue extract (thrombokinase), but will not spontaneously clot on contact with glass. In this respect it resembles hydrocele fluid, which is platelet‐free.4. Spontaneous coagulation of centrifuged frog's plasma is due wholly to disintegration of the contained thrombocytes. This disintegration in its turn is due to contact with a special kind of surface. As a result of the cell disintegration thrombin is liberated.5. Bird's blood, or centrifuged bird's plasma, has less tendency to undergo spontaneous coagulation on contact with glass. In this respect bird's blood differs both from amphibian and from mammalian blood.6. Tissue extract or thrombokinase prepared from the frog's ventricle is all but impotent to induce coagulation of cell‐free plasma. In this regard the tissue of the ventricle differs markedly from the connective tissue, liver, muscle, etc., of the frog.7. Contact cytolysis of frog's thrombocytes is due to a purely physical cause, and does not depend upon absorption by the cells of any soluble material derived from the effective water‐wettable surface. The better an adequate surface of insoluble material is chemically purified and freed of grease, the more rapidly does it induce cytolysis of the thrombocytes.8. Thrombokinase or tissue extract plays no part in the production of thrombin during simple spontaneous coagulation of the blood. In this regard the theory of Morawitz as to the precursor of fibrin‐ferment requires amendment.9. The word “prothrombin” (or “thrombogen”) might well be replaced by the phrase “unruptured thrombocytes,” for it would seem to be these that constitute prothrombin.10. In any representation of the chain of phenomena involved in spontaneous blood coagulation place must be given to an indispensable physical process, as a result of which the thrombocytes are induced to yield thrombin.The expenses of this research were defrayed in part by a grant from the James Cooper Fund of M'Gill University for Research in Experimental Medicine.

Protein content of frog's plasma

In the Journal of Experimental Pathology of October, 1921, Hill and McQueen made some observations on capillary pressure of the frog's kidney, which they believe indicate that filtration is not possible. They found capillary pressure of about 10 millimeters, while the pressure in the arterioles was from 25 to 30 millimeters. A pressure of 10 millimeters might still be large enough to produce filtration if the colloid content of the frog's blood was low enough. We have accordingly made some determinations on the protein content of the frog's plasma. This was done by drawing blood from the heart of frogs into tubes containing finely divided potassium oxalates. The blood was then centrifuged and the plasma obtained. Kjeldahl determinations were done on the plasma and reckoning the total nitrogen found as all protein, and using the factor 6.25, the protein of the frog's blood is between 0.6 and 0.8 per cent.—approximately 10 per cent. of that of mammals. With this small colloidal content it is evident that ve...

Scientific Serials

The Quarterly Journal of Microscopical Science for January, 1882, contains: H. Marshall Ward, B.A., report on the morphology of the fungus of the coffee disease of Ceylon (Hemileia vastatrix, Brk. ard Br.), plates 1, 2, and 3. This fungus probably belongs to the Uredines; still some structures, such as the curious spore-bearirg head and the long-necked haustoria are opposed to this alliance. The history of the adult fungus from the uredospore, and the formation of the teleutospores, are described and figured.—Dr. F. M. Balfour, on the nature of the organ in adult Teleosteans and Ganoids, which is usually regarded as the head-kidney or pronephros. It would seem probable that, though found in the larvæ or embryos of almost all the lcthyopsida, except the Elasmobranchii, this is always a purely larval organ, which never constitutes an active part of the excretory system in the adult forms —Dr. K. Mitsukuri (Japan), on the development of the supra-renal bodies in mammalia (plate 4).—Pat. Geddes, observations on the resting stage of Chlamydomyxa labyrinthuloides, Archer (plate 5), some very characteristic figures of the resting stage of this strange protean form are given.—J. T. Cunningham, a review of recent researches on Karyokinesis and cell division (plate 6).—Dr. Reuben T. Harvey, a note on the organ of Jacobson.—Prof. E. Ray Lankester, on Drepanidium ranarum, the cell-parasite of the frog's blood and spleen (Gaule's Würmschen). This very interesting memoir is illustrated with several woodcut illustrations.—G. F. Dowdeswell, M.A., on the micro-organisms which occur in Septicæmia (plate 7).—Prof. Bayley Balfour, Pringsheim's researches on chlorophyll, translated and condensed (plates 8 and 9).

Phenomena of Coagulation in Frog's Blood*

I WAS endeavouring in the autumn of last year, at Prof. Sanderson's instigation, to demonstrate upon the frog some of Briicke's fundamental experiments on the coagulation of the blood, which he performed on the tortoise; I was surprised at the apparent failure of some of them. For instance, having tied a glass tube into the animal's aorta and allowed it to fill with blood, I expected that which was in the tube speedily to coagulate, that which remained in the heart to continue liquid for a considerable time. But no such contrast was observable, both portions of blood remained perfectly fluid for an indefinite time. I say apparently, for, in fact, on subsequently turning out the blood, a slight film of coagulated fibrin was observable attached to the walls of the tube. Of course the corpuscles being the heavier gravitate to the bottom, and the blood thus becomes divided into two portions, a clear fluid above and a mass of red corpuscles below, with a thin filmy stratum of white again on the surface of the latter.