Gordon H. Scott

Distribution of Mineral Ash in Striated Muscle Cells

This report is a brief account of one phase of a study of the distribution of inorganic salts, in various tissues and cell types, as revealed by the technique of microincineration. The method of incinerating thin sections of tissue without disturbing the topography of the mineral components was suggested by Liesegang, 1 but its advantages were never realized until the researches of Policard. 2 Until recently the technique has been largely a gross histologic one because of inherent difficulties in making observations with the higher powers of the microscope. However, it was discovered by the writer 3 , 4 , 5 that the use of the dark field condenser of Zeiss removed to a large extent these difficulties and rendered it possible to follow through even the changes of the fixed minerals of the nucleus during mitosis. It is necessary to use a fixative which neither removes nor increases the inorganic elements of the tissue. Absolute alcohol is best adapted for this purpose, but gives poor fixation even under the most favorable circumstances. It was found after some experimentation that a mixture of 9 parts of absolute alcohol to 1 part of neutral formalin (Will Corporation) gives rather good cytologic detail and yet, for all practical purposes, does not change the original inorganic constitution of the tissue. After fixation for 24 hours the tissue is passed through several changes of absolute alcohol to complete the dehydration. The specimens are then cleared in xylol, embedded in paraffin and cut serially at 4 microns. Alternate sections are mounted according to the usual technique and stained with haematoxylin and eosin. The intervening sections of the series are mounted on glass slides with liquid petrolatum as a medium in which to spread the tissue.

Mineral Distribution in Some Nerve Cells and Fibers.

It is known from examination of many types of tissue that Mg and Ca, as revealed by the electron microscope, is located in areas which show white ash following microincineration. In nerve tissue certain difficulties have hampered a direct study of Ca and Mg by means of the electron microscope. Some of the findings in incinerated sections of frog sciatic and sympathetic ganglia are believed to be of significance although we have not been able to identify the salts as clearly as is desirable. When sections of frozen and dehydrated (Scott and Packer 1 ) frog sciatic are carefully incinerated and examined by dark field (Scott 2 ) the large myelinated fibers at the periphery of the nerve leave residues of white ash probably consisting largely of Ca and Mg. The ash is clearly the remains of the myelin sheath as it corresponds almost exactly with stained preparations of the same nerve taken a few levels either above or below. The point to emphasize, however, is that there is no visible residue of any sort in the tissue spaces surrounding the nerve fibers. In sharp contrast to plentiful mineral in the nerve fibers and little if any in the tissue space is the picture obtained when sympathetic ganglia are incinerated following the same treatment. The sympathetic ganglion cells are recognizable by their residue. Nuclear, nucleolar and Nissl substance ash is dense and of the variety associated with the presence of Ca and Mg. There is as a general rule a wide band, varying from an eighth to a sixth of the cell diameter, of dense white ash concentrated at the periphery of the cell. The tissue spaces immediately about the ganglion cells are filled with mineral residue not unlike, in quality and quantity, that seen in the neurones.

A New Development in Histospectrography.

Histospectrography, as developed by Policard, 1 and by Gerlach and Gerlach, 2 is a method of examining the elements in tissues which consists of passing a high frequency spark through a predetermined area in a section of tissue and by means of the spectrograph analyzing the rays emitted. The spectrograms will contain the lines characteristic of the elements encountered by the spark in passing through the tissue. It has been pointed out by Policard 1 and Gerlach and Gerlach 2 that one of the greatest difficulties encountered is the selection of electrodes; not only do the characteristic lines of the major element of the electrodes appear on the spectrum, but also those of even small impurities in the metal. In the course of our experiments with the technique, a means was devised whereby the purity of the electrodes is rendered immaterial, and the choice of basic metal almost so. In fact, for all practical purposes, our spectrograms contain lines characteristic only of the tissue. The tissue support assembly differs entirely from that employed by other investigators. Policard supports tissue sections for analysis on a metal plate, which forms the lower electrode of the spark gap, carried by a special mechanical stage to permit the selection of the area to be sparked while it is under observation with a microscope. The upper electrode is a metal point immediately above the tissue. Gerlach and Gerlachs apparatus differs from Policards in that a glass plate is interposed between the specimen and the base plate. With both procedures the possible area of localization is of the order of 1 mm. in diameter and lines are photographed from one or both electrodes.

An Electron Microscope Study of Calcium and Magnesium in Smooth Muscle.

Recent investigations of electrolytes in Thyone muscle by Steinbach 1 have led him to the conclusion that Ca is present in cells in lower concentration than that of the surrounding medium. Since these results are at variance with our own in a number of respects we have studied again the localization of Ca and Mg in mammalian and frog smooth muscle by the electron microscope as described by Scott and Packer. 2 Naturally, the results obtained cannot be compared directly with those of Steinbach as the same form was not used. Furthermore, our interest lay in determining the localization in smooth muscle cells which had been preserved as nearly as possible in their normal physiological state. Thin strips of smooth muscle were taken from the duodenum and stomach wall of cats and frogs. These pieces were frozen in liquid air while still actively contracting from the mechanical stimulation of the manipulation. The frozen tissues were dehydrated in vacuo at -63°C and infiltrated in water-free paraffin without breaking the vacuum. This procedure precludes any salt shift during dehydration and the addition of water from outside sources at any time and thus maintains the spatial relations of the element in question. Thin sections (8μ) of muscle were prepared and examined by heating on a surfaced cathode in the electron microscope. The technic of preparation of tissues for examination and the method of localizing Ca and Mg are believed to preserve the exact topical cellular relationships. Unfortunately, it is impossible by the method used to differentiate Ca and Mg, since both activate the cathode surface. Quantities of these elements in less than 1 × 10-12 g are easily detected. In no instance was either Ca or Mg found outside the cell wall.

Quantitative Estimation of Ash after Microincineration.

One of the things to be desired in the study of microincinerated sections is an accurate means of estimating the relative quantities of inorganic salts in various cells. Such a method would permit a quantitative comparison of tissues taken from animals subjected to experimental procedures with similar normal cells. Furthermore it would be possible to study such processes as normal growth and differentiation in a more exact manner than the microincineration technique has permitted. A quantitative method also has many advantages in the examination of pathologic tissues and comparable normal ones. The technique for quantitative photography of incinerated sections devised by Schultz-Brauns 1 has many points to commend it; but it is, in the final analysis, a procedure which depends on the visual judgment of the observer. It seemed advisable, therefore, to develop a method which would be free from this objection. The nature and distribution of the inorganic salts in cells and tissues has been described.2, 3 The appearance of the ashed preparations in darkfield illumination is that of hoar frost on a blackened surface. With this means of illumination only the ash of the section is revealed by light reflected from the surfaces of the individual particles of mineral. It is assumed, therefore, as the particles are approximately the same size and nature, that the more mineral present in the microscopic field the greater the quantity of light reflected into the eye of the observer. This assumption has been checked, in as far as possible, by examining colloidal solutions at different dilutions with the darkfield microscope with the result that given a constant source of light the intensity of the beam emerging from the ocular is roughly proportional to the number of particles in the microscopic field.

Cesium in the Mammalian Retina.

Fox and Ramage 1 and Sheldon and Ramage 2 reported that they were unable to identify, by spectrographic methods, cesium in the tissues of animals of widely divergent genera. Since it is well recognized that different methods of spectra excitation favor the identification of some elements it occurred to us that by use of the interrupted are method (McMillen and Scott 3 ) we might get the identifying lines. Fox and Ramage and Sheldon and Ramage used flame excited spectra in their studies. An examination of several hundred tissue and fluid samples from various mammals by our method failed to give any positive results. Through the kindness of Dr. Douglas Coles of The Veterinary Research Laboratories, Onderstepoort, Transvaal, South Africa, we obtained several ox eyes. In the course of routine spectrographic analysis of these specimens for barium, cesium was found in the retina. This led us to study spectrographically the retinae of some 60 eyes obtained from a local abattoir. Of this group 19 were from pigs, 17 from oxen and 24 were from sheep. Presumably the animals came from a rather wide though illy defined area of the Middle West. No more definite information could be obtained on this point. By carefully controlling the method used it was possible for us to exclude contamination from other sources. The eyes were slit and fixed in cesium-free formaldehyde as it was found much easier to separate the retinal layers in the hardened material. The instruments used in manipulating the tissue did not add cesium to samples of other tissues and consequently it is not at all likely that they did so in this case. The tissues to be examined were allowed to digest in a small amount of cesium-free HNO3 in chemically clean vessels.

Spectrographic Analyses of Human Spinal Fluid.

In connection with other studies on inorganic salt and metal distribution in cells and tissues we have had occasion to examine spectrographically a series of samples of spinal fluids taken from the usual hospital and clinic population. Our attention was directed toward a search for elements that are not usually recorded as being normal constituents of human spinal fluid. These are Pb, Al, Ba, Sr, B, Sn. Samples of spinal fluid were generously provided for us by Drs, A. F. Hartman and W. B. Wendell. The method used routinely in our examinations was as follows: 2 cc. of spinal fluid were placed on a carefully cleaned glass plate and evaporated to dryness at 100°C. The residue was scraped together, placed on a pure carbon electrode, wetted slightly with a small drop of the original fluid and dried. The loaded carbon was placed in front of the slit of a Bausch and Lomb Medium Quartz Spectrograph and the salt ignited by means of an intermittent arc. About 100 flashes of the arc covering a total time interval of 80 seconds sufficed to produce good pictures of lines throughout the ultra-violet spectrum. The intermittent arc is formed by making and breaking electrical contact between 2 vertical electrodes. The upper one has a motor-driven piston-like motion while the lower one is fixed and capped with the sample. In our search for Al we used 22 samples of spinal fluid. The line used to detect the presence of aluminum was the 3082.16 Å line and as a reference line we employed the conveniently placed 3096.92 Å line of Mg. Those Al lines of greater intensity than that of the Mg were called “strong” and those of much less intensity were designated as “weak”.

Nuclear Inclusions Suggestive of Virus Action in Salivary Glands of the Monkey, Cebus fatuellus L.∗:

Evidence is fast accumulating that we must recognize a special group of salivary gland viruses. All of them have been discovered by chance. They are so benign that attention was not directed to them by distinctive clinical symptoms. What attracted notice was the extraordinary hypertrophy of certain acinous, or duct, cells accompanied by the formation in their nuclei of inclusions resembling those caused by viruses. The first inclusion-laden cells were reported under the heading of “protozoan-like bodies” in the parotids of 2 infants by Ribbert 1 and in the submaxillary glands of guinea pigs by Jackson. 2 Credit is due to Goodpasture and Talbot 3 for recognizing the close resemblance between the bodies in humans and guinea pigs and for pointing out the similarity of both to the intranuclear inclusions described by Tyzzer 4 in varicella. Lipschütz 5 then rediscovered the intranuclear inclusions in herpes admirably described and illustrated by Kopytowski, 6 and emphasized the great importance of these bodies in “inclusion diseases” in general. But Kuttner and Cole 7 and Kuttner 8 led in the demonstration that the inclusions in guinea pigs are actually caused by a virus. Investigators, while examining the salivary glands of other animals, have been on the lookout for nuclear inclusions with the result that they have been reported in rats, 9 moles, 10 , 11 mice 12 and hamsters. 13 Finally Kuttner and Wang 13 have proved that the intranuclear inclusions in hamsters, mice and wild rats are caused by a virus which is very similar to the submaxillary gland virus of guinea pigs.

Post-mortem Changes in Mineral Salt Distribution in Nerve Cells.

An analysis, by microincineration, of the distribution of inorganic salts in anterior horn cells following temporary vascular occlusion of the spinal cord (Tureen 1 brought forward the necessity of examining the post-mortem changes in these elements in similar tissues. It seemed to be especially advisable also because of recent reports on the ash distribution in human nerve cells in various pathological conditions. Tissues were removed from etherized and bled cats at intervals ranging from 5 minutes to 27 hours. One series of animals was permitted to remain at room temperature; a second series of animals was kept in the ice box at 60° F. for similar periods. Sectioning and incineration were carried out as suggested by Scott. 2 Alternate sections of the series were stained with hematoxylin and eosin as controls. The incinerated sections were studied by dark field illumination. The findings will be related briefly in two parts, the first of which is the appearance of incinerated anterior horn cells after immediate fixation. The results are in general in accord with those for similar types of material described by Scott 3 and by Patton. 4 , 5 The first consideration is to establish a “normal” picture—a task admittedly difficult since there is considerable variation in the appearance of the anterior horn cells even under optimum conditions. In general the ash residue of the well-fixed anterior horn cell is uniformly distributed throughout the cytoplasm. This mineral is in small deposits approximately 1 to 2 microns in diameter. It is in this cytoplasmic ash that the greatest variation occurs. In some cases, for example, the remains of the Nissl substance are clearly discernible, in others not. It is as yet impossible to assign this variation to a definite physiological state of the cell.

On the Classification of Cells According to Their Inorganic Structure in vitro.20

One of the greatest difficulties in carrying out any problem in vitro is that of establishing a morphological criterion by which the various cell elements composing the growth can readily be classified. When dealing with pure culture strains of “Fibroblasts” and epithelial cells it is a comparatively simple matter to distinguish between the 2 cell types, as they not only exhibit distinct differences in their morphology, but also in their mode of growth. But when attempting to distinguish these 2 different types of cells in a mixed growth colony it becomes more difficult, as the epithelial elements composing the advancing free edge of the new growth invariably isolate themselves from the epithelial sheet and migrate into the culture medium as separate units, assuming a contour similar to fibroblasts (see Fig. 1). But far greater difficulties confront the investigator when he has to identify cells of the mesenchyme type, such as osteoblasts, chondrioblasts, and heart fibroblasts which, although possessing varying functional activities, exhibit similar morphological values. Parker and Fischer, 1 realizing this difficulty, successfully demonstrated that mesenchyme elements in tissue cultures, which would be designated morphologically as “Fibroblasts”, are found to possess different inherent growth potencies when cultivated under similar conditions in vitro. On these grounds they rightly contended that “the physiological properties of the cell should constitute the first claims to any definition.” Recently Horning 2 found that tissue culture cells which exhibit the same morphological characteristics and behavior under normal conditions in vitro, differ, however, in their reactions to similar pathological conditions, as the rate of cytolysis was in all cases found to be dependent upon the inherent growth energy of the given strain.