A NEW- OR DELTA-TYPE STREPTOCOCCUS

Transcription

1 A NEW- OR DELTA-TYPE STREPTOCOCCUS CARRIE KIRK BRYANT Prom the Department of Microbiology and Bacteriopathology oj the Thomas W. Evans Museum and Dental Institute School of Dentistry, University of Pennsylvania, Philadelphia Received for publication June 27, 1924 INTRODUCTION In making some routine studies of streptococci isolated from root canals of pulpless teeth at the Evans Institute, I have found a type which can not be classified as any one of the four outlined by Brown (1919). The following description taken from Brown's work, will give the general characteristics of these four types. Type a... at the end of approximately 48 hours incubation... To the eye the deep colonies appeared as small biconvex greenish colonies.... surrounded by zones in which the blood corpuscles were distinctly greenish in color. Under the low power of the microscope.... the greenish zone was less apparent, but in the outer portion of the zone there was possibly a slight hemolysis After refrigeration for forty-eight hours.... Examined under the low power of the microscope the deep colonies were seen to be surrounded by what may be called "double zones".... composed of a distinct inner collection of... greenish nonhemolyzed corpuscles next to the colony and an outer more or less hemolyzed ring.... Apparently the greenish discoloration of the corpuscles was accompanied by a fixation against hemolysis for (1) it preceded hemolysis, (2) the hemolytic substance must have passed out from the colony through the collection of corpuscles, and (3) it must have been present in greater concentration among these corpuscles next to the colony than farther out where hemolysis did occur.. As it has been noted that the zone of greenish corpuscles was formed during incubation and that hemolysis took place during refrigeration, 53

2 54 CARRIE KIRK BRYANT the blood agar plate was replaced into the incubator for another period of forty-eight hours. There appeared outside the hemolyzed zone another ring of greenish corpuscles.... Still another exposure to the temperature of the refrigerator resulted in the formation of a second ring of partial hemolysis.... Apparently the alternate throwing off of rings of fixed corpuscles and of hemolysis in response to alternate incubation and refrigeration is limited only by the exhaustion of the medium and the accumulation of metabolic products which inhibit further growth and activity. Type j3. This type of appearance in blood agar may be described as a colony surrounded by a perfectly clear, colorless zone of hemolysis. *... There is no trace of discoloration and when viewed microscopically no corpuscles are seen in the medium surrounding the colony. The colony itself is grayish by transmitted or reflected light.... after refrigeration for forty-eight hours they showed no change.... nor did they change during a second period of incubation and refrigeration. The difference in behavior of the two types in respect to refrigeration is so great as to suggest different hemolytic substances or processes. Type a'.... It can scarcely be called anything but a hemolytic streptococcus for there are distinct hemolyzed zones and no apparent discoloration... colonies of the alpha prime type appear somewhat hazy or poorly defined within the hemolyzed zones. Under the microscope... the reason for this haziness is apparent; a few corpuscles are seen to remain throughout the zone but are most numerous next to the colony. After forty-eight hours incubation the plate.... was refrigerated for forty-eight hours. The zones were noticeably enlarged.... Placed into the incubator for another period of forty-eight hours the zones became still larger and rather diffuse but no multiple zones of concentric rings were formed. Type y. By the gamma type is meant the growth of the streptococcus colonies within and on the blood agar plate without the production of any perceptible hemolysis or discoloration of the surrounding medium during incubation or refrigeration. ORIGINAL OBSERVATIONS Grown in 5 per cent blood agar plates, using defibrinated horse blood, the surface colonies of the type under consideration produce no visible change in the surrounding medium. The deep

3 NEW- OR DELTA-TYPE STREPTOCOCCUS colonies after forty-eight hours incubation appear macroscopically, irregularly shaped, olive green in color and about 0.75 mm. in diameter. Each is surrounded by a pale green zone slightly narrower than the diameter of the colony. The illdefined outer margin of this green zone is not surrounded by a clear colorless zone, but merges directly into the unchanged blood agar beyond. This appearance is unchanged by forty-eight FIG. 1. PLATE COLONIES OF ORGANism GROWN IN 5 PER' CENT BLOOD AGAR,) DRAWN AFTER FORTY-EIGHT HOURS INCUBATION AND FORTY-EiG;HT HOURS IN ICE Box SUBSEQUENTLY There were approximately 175 colonies on the plate. Naked eye study seen by transmitted light andx ea. 8 diam. hours subsequent subjection to refrigeration (fig. 1). Under the, low power of the microscope in the zone immediately surrounding the colony the blood cells are seen to have lost all their pink color and to appear as minute pale grey-brown dots or when seen! in clumps, as slightly darker grey-brown masses. There is no apparent decrease in their number, and they lie on an absolutely clear colorless back ground. The outer margin of this zone is very ill-defined and merges gradually into the unchanged medium where the agar is tinted a distinct pink from the laked hemoglobin 55

4 ~~~~~~* ';';;.....h...'*. 60~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 56 CARRIE KIRK BRYANT and the blood cells have retained their normal tone-the clumps especially being seen as definitely red masses (fig. 2). The colonies resemble Brown's alpha type in that they are immediately surrounded by a zone, greenish to the naked eye, in which the blood cells still persist but without their normal color. But they differ from the alpha type in that refrigeration fails to produce an outer zone of hemolysis. They differ FIG. 2. COLONY TAKEN FRom FIo.1AS SEEN THROUGH Low POWER OF THE MICROSCOPE, SHOWING COLONY &ND ZOE IMMEDIATELY SURROUNDING- IT, WITH SINGLE AND SMALL CLUMPS OF ERYTHROCYTES, GREY-BROWN IN COLOR, IN A CLEAR COLO'RLEss BACKGROUND Beyond this and merging into it are to be seen the erythrocytes as they appear throughout the rest of the plate, single ones as brownish or red-brown, and clumps always red, lying in a pale pink backgro.und. X ca. 30 actual size of colony. from, both the alpha prime and the beta types in that there 'is no decrease in the number of blood cells in the zone surrounding the colony. That the action of these delta-type streptococci on blood -agar is characteristic, and is not due to some environmental peculiarity, is proven by the fact that they have been found present in plates showing typical colonies of the alph'a type in mixed cub-

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6 58 CARRIE KRKI BRYANT ture. When isolated and replated in pure culture they have retained their characteristic action in blood agar plates through repeated generations. The morphological study was made from the water of condensation of an agar slant which had been incubated for twentyfour hours after inoculation with a typical deep colony picked from a blood plate. The surface growth on agar was frail, effused and filmy. The organism appeared as a Gram-positive micrococcus about 0.75 micron in diameter, in short chains varying from four to about fifteen elements and in irregular clumps. By reference to chart 1 it will be seen that of the four strains isolated, all acidified and coagulated milk, all were bile insoluble, and none fermented inulin, raffinose, or sucrose. They varied in their proteolytic action on milk and gelatin. We seem then, in these organisms, to be dealing with true streptococci which are of a type until now undescribed as to their behavior in blood agar plates. REFERENCE BRowN, JAmms HowARD 1919 The use of blood agar for the study of streptococci. Monographs of The Rockefeller Institute for Medical Research, January 21, No. 9.