Arthur T. Henrici

A Microscopic Study of Pulps from Infected Teeth

It is generally considered that so long as the pulp is protected by an intact wall of dentin, it is free from infection; that only when the pulp chamber becomes exposed by caries or trauma does the pulp tissue become invaded by microorganisms, and that then it usually undergoes suppuration or gangrenous inflammation; and that the pain which occurs previous to actual exposure of the pulp is due to hyperemia, caused either by temperature changes or by lactic acid absorbed through the dentinal tubules. This conception of the process of natural revitalization finds general expression in the text-books on dental pathology. We believe, on the contrary, that the pulp is frequently invaded by microorganisms long before it has been exposed by caries or, indeed, in the absence of caries, by organisms which have passed into the pulp from the gingival region; and that such invading organisms give rise in the pulp to localized areas of inflammation which are not necessarily suppurative or gangrenous in nature, but rather granulomatous and progressive, leading to fibrosis or other changes. When suppuration and gangrene finally occur, it is in a tissue which has

Experimental Trichophytid in Guinea Pigs.

Summary Guinea pigs which had recovered from infection with Trichophyton mentagrophytes reacted to intraperitoneal injections of live spores, cell sap, or polysaccharide by generalized erythema of the skin followed by desquamation. This condition is considered identical with trichophytid in human cases. No organisms could be found in the allergic lesions. These experiments strongly support the theory that trichophytid is an allergic response of the skin to substances in solution distributed to it by the blood.

A statistical study of the form and growth of Bacterium coli.

In previous studies 1 of the form and growth of bacteria, the length of the cells has been taken as a measurement of size because apparently the width did not vary nearly so much, and because the width could not be determined accurately with the technique used. In the present study which has had for its purpose the establishment of the normal variations in size and form of Bacterium coli when grown on standard beef extract agar, a somewhat different technique was used. Photomicrographs were made from slides prepared by the negative staining method of Benians, and the photographic negatives were again projected so that the final magnification was 30,000 diameters. The projected image was traced on paper and from these tracings the length and area were determined. The growth curve was obtained by counting the cells by a technique which has been previously published. 2 The results are shown in the accompanying graph. The diagrams of the cells have been traced from composite photographs of the tracings of the projected images and indicate roughly the average size and form of the cells at the various time periods. The rate of multiplication is indicated by the curve of the logarithm of the number of cells. Assuming that the cells are symmetrical about the axis, the area of the projected image divided by the length squared may serve as an index of variation in form, just as the weight-length index has been used for higher organisms.

Influence of age of parent culture on size of cells of Bacillus megatherium.

In a previous communication 1 I have pointed out that during the early hours of a culture the cells of Bacillus megatherium become greatly increased in size and then rapidly decrease; that the increase in size is accompanied by increased variability, the frequency curves showing skewness and a tendency to bimodality; and that the degree of duration of the increase in size is greater with smaller seedings. These observations were made on cultures inoculated from a culture 12 hours old, i. e., one which had already reached the maximum of growth. In the present study I have observed the variations in size in cultures transplanted from a parent culture after 2, 4, 6 and 8 hours of growth, i. e., during the period when the cells were increasing and decreasing in size. In the accompanying graphs the solid lines indicate the average length of the cells, the broken lines the coefficient of variation. The abscissæ indicate the age of the cultures in hours, the ordinates to the left length of cells in microns, those to the right coefficients of variation. The curves for the parent culture are repeated in the graphs for the various subcultures up to the time of transplanting. When transplanted during the period of increasing size, at 2 hours, or at the time of maximum size, at 4 hours, the organisms continued to increase in size. It is noteworthy that the greatest decrease in size in these subcultures occurred between the 5th and 6th hours, as was also the case with the parent culture. The entire curves for these two subcultures, including the time spent in the parent culture, bear the same relationship to the parent culture as do curves obtained from cultures with small inoculums to cultures more heavily seeded; there is a greater increase in size over a longer period of time.

Immunologic Studies of Actinomycetes, with Special Reference to the Acid-Fast Species

The serums of six out of eight actinomycotic cattle fixed complement with four different antigens prepared from Actinomycetes isolated aërobically from lesions in cattle, and not with antigens prepared from saprophytic strains. The complement fixation test may be of diagnostic value in this disease. The serum of a rabbit immunized against A. bovis fixed complement with antigens prepared from A. bovis and A. maduræ, but not with antigens prepared from the acid-fast varieties A. asteroides and A. gypsoides, or from Mycobacterium tuberculosis. The serums of rabbits immunized against A. gypsoides fixed complement equally with antigens prepared from A. gypsoides and A. asteroides, and in lower dilutions with M. tuberculosis, but not at all with A. bovis and A. maduræ. The serums of rabbits immunized against M. tuberculosis fixed complement with the homologous antigen, and in lower dilutions, with antigens prepared from the acid-fast Actinomycetes, A. asteroides and A. gypsoides, but not with A. bovis and A. maduræ. It would seem, therefore, that the acid-fast Actinomycetes are more closely related to the acid-fast bacteria than to the non acid-fast Actinomycetes. A. gypsoides does not secrete a soluble toxine, but forms a very protent endotoxine. By careful vaccination there can be produced in rabbits an active protective immunity. The serum of such rabbits injected into guinea pigs gives the latter partial protection.

The rate of spore formation in bacteria.

The rate of spore formation in Bacillus megatherium is shown in Fig. 1, in which the logarithms of the number of vegetative cells and the logarithms of the number of spores are plotted. It will be noted that spore formation is initiated at the end of the active growth period. I have previously shown that the rate of variation in the size of the cells of Bacillus megatherium is dependent in part on the size of the inoculum, 1 in part on the concentration of nutrients in the medium. 2 I have also observed the influence of these factors on the rate of spore germination and spore formation in Bacillus cohaerens. Four sets of cultures were inoculated, two of normal strength agar, and two of quarter strength agar. One set of each kind was seeded with a very heavy emulsion, the other with the same suspension diluted 1-50. The culture used for seeding was an old one, composed almost entirely of spores. The curves indicate the percentage of free spores among 200 cells counted at each time period. The time intervals are plotted on a more extended scale during the period of spore germination than during the period of spore formation. Although the beginning of germination was noted in some spores after an hour, no increase in vegetative cells was demonstrable until the third hour. It is noteworthy that germination proceeded more rapidly in the lightly inoculated culture than in the heavily seeded one; and commenced more rapidly in the dilute medium than in the full strength agar. The process slowed up more rapidly in the former, and spores never completely disappeared in the heavily seeded dilute medium.

Differential counting of living and dead cells of bacteria

By means of the procedure here described it is possible to determine with considerable accuracy the number of bacterial cells in a suspension and at the same time to determine the size and form of the cells and to differentiate the living and dead cells. The method makes use of the principle of the Breed and Brew 1 method of counting bacteria in milk, and the negative staining method of Benians. 2 A measured quantity of bacterial suspension is mixed thoroughly with an equal quantity of 2 per cent. aqueous Congo red solution; the mixture is allowed to stand ten minutes. After again shaking the mixture, 0.01 c.c. is removed by means of a capillary pipette of that capacity and discharged on to a clean slide which has been clamped to the table over a piece of white paper on which a 2 cm. square has been ruled. By means of a stiff wire the drop of liquid is spread as evenly as possible over this area. After it has become thoroughly dry the slide is immersed a moment in a 1 per cent. solution of hydrochloric acid in 95 per cent. alcohol. This turns the dye blue and also fixes the film. If covered with a layer of cedar oil the slides will keep indefinitely, but if exposed to the air they fade considerably. Cells which were alive at the time of staining are unstained and appear as white spots on a blue ground. While the cells themselves may shrink considerably after fixation and drying, a number of comparative measurements have shown that the clear space in the film faithfully reproduces the size and form of the living wet cells. With favorable material flagella may be demonstrated by this stain.

A statistical study of the form and growth of a spore-bearing bacillus:

The rate of growth of Bacillus megatherium has been measured by direct counting of the cells, using a haemocytometer; and the average length of the cells has been determined by measurements made at the same time. In agar cultures inoculated from a 12-hour agar culture (which has nearly reached the maximum of growth but has not yet formed spores) it was found that the cells began to increase in size during the lag phase and reached a maximum length, about six times that of the inoculated cells, shortly after the beginning of the maximum growth phase, then rapidly becoming shorter. During the period of increase in length, frequency curves showed a tendency towards bimodality, indicating that possibly a process of selection of rapidly growing cells may occur during the lag phase, as has been suggested by some investigators. Two series of broth cultures inoculated from a 7-hour agar culture (during the period of maximum growth) showed no lag phase; nevertheless an increase in the size of the cells was observed beginning two hours after inoculation. Two series of broth cultures inoculated from a 7-hour agar culture (during the period of maximum growth) showed no lag phase; nevertheless an increase in the size of the cells was observed beginning two hours after inoculation. The cells did not become so large as did those on agar, and the variation was not so great, bimodality being present in but one of the frequency curves. One series was inoculated with 10 times as many bacteria as the other, and the series receiving the lesser amount of inoculum showed a slightly greater increase in the size of the cells over a slightly longer period of time.

A statistical study of the form and growth of a diphtheroid bacillus

In a previous communication 1 I described changes in the size of the cells of Bacillus megatherium during the growth of a culture, and variability in size, particularly the bimodality of the frequency curves in the early stages of growth. Further observations of this organism, varying the number and age of the cells in the inoculum showed that these changes are constant but vary in degree, the increase in size, and variation in size, and the tendency to bimodality being greater with smaller and older seedings; and that, while these changes take place during the vegative phase of the culture, there is apparently no actual correlation between the variations in size and the rate of cell division. It is noteworty that the coefficient of variation increases and decreases with the size of the cells. I have made a similar study of a chromogenic diptheroid bacillus isolated from lake water. It is larger than most members of this group of bacteria, but like the rest of the group decreases in size during the vegetative stages of growth and increases during the resting period; the curve for size is therefore just the reverse of that for B. megatherium. The decrease in size began after a latent period and during the logarithmic growth phase. As the cells decreased in size the frequency curves became more symmetrical and the variation decreased; as the size increased again the curves became more extended and showed skewness. There was no tendency to bimodality the single mode gradually shifting. The coefficient of variation decreased with the actual decrease in size, i.e., during the period of miost rapid cell divisions. Therefore the coefficient of variation cannot be used as an index of rate of reproduction with organisms such as these, where the entire population is subject to fluctuations in size independent of the growth of the individuals from youth to maturity.

Influence of concentration of nutrients on size of cells of Bacillus megatherium.

A young spore-free culture of Bacillus megatherium was inoculated on slants of standard beef extract agar, and on slants containing the same proportion of agar, but with one-half, one-fourth, one-eighth, and one-sixteenth as much of the nutrient ingredients, beef extract and peptone. The average length of the cells was determined at hourly intervals for eight hours. The results are shown in the accompanying graph. The curves for eighth-strength and sixteenth-strength agar were very similar to that for the quarter-strength, and have been omitted for the sake of clearness. At the beginning of growth protoplasm is synthesized more rapidly than cell division occurs, and the cells become increasingly larger until a critical point is reached, when the reverse conditions obtain and the cells decrease to their original size. I have previously pointed out that this critical point is reached earlier, the maximum size attained being smaller, when the culture is heavily seeded than when small inoculums are used; 1 and that it may be postponed, the cells reaching a larger size, if a portion of the culture is transferred to a new medium before attaining their maximum length. 2 It would appear then that the size of the cells is determined, in part at least, by the density of the population, i. e., the concentration of bacterial substance. But that this is not the sole factor is indicated by the experiment here reported; for there was much less growth in the mediums of lower concentration, yet the maximum length was smaller, and was reached earlier. Reducing the concentration of the nutrients has the same effect on the size-curve as increasing the concentration of bacterial substance, and the form of the curve is therefore determined by the proportion between these two factors.