mixing the inoculated medium is poured into a Petri dish and

Transcription

1 APPEARANCE OF DOUBLE-ZONE BETA HEMOLYTIC STREPTOCOCCI IN BLOOD AGAR J. HOWARD BROWN Department of Pathology and Bacteriology, Johns Hopkins University, Baltimore, Maryland Received for publication January 28, 1937 In 1913 Smith and Brown (1915) isolated from the milk of four cows in a herd where mastitis was prevalent, four strains of streptococci with the hemolytic properties to be described. In the primary blood agar plate the deep colony of one strain was surrounded by "a clear inner circle (of hemolysis) three millimeters in diameter and a partly cleared outer zone, up to ten millimeters in total diameter." Later study and repeated replating in blood agar has shown that these four strains are alike in all respects. One of them, designated Streptococcus A-18 (Cow) B, has been described and photographed by Brown (1919). Apparently similar strains were isolated from horses by Jones (1919) who also published a photograph of one of them. Since then, many strains producing similar appearances in blood agar have been isolated from bovine and human sources. A brief description has been published (Brown, 1934). The method for demonstrating the double-zones is as follows. To a tube of infusion agar, melted and cooled to 45 to 50'C., is added about 1 cc. of defibrinated blood and a loop of suitably diluted culture. The dilution of the culture should be such that there may be fewer than 100 colonies in the plate. After thorough mixing the inoculated medium is poured into a Petri dish and allowed to harden. The plates are incubated at 370C. and then refrigerated. During the first 24 hours of incubation deep colonies with small zones of hemolysis appear and become larger as incubation is continued. Usually the zones of hemolysis have a rather poorly defined periphery but they will be found to be 35

2 36 J. HOWARD BROWN clear immediately next to the colony and are therefore of the beta type. To determine this, the colony should be viewed in optical section under the 16 mm. objective of the microscope (figs. 1, la, 2, 2a). When the plate is refrigerated there appears a broad outer zone of partial hemolysis, resulting in the so-called "double-zone" (figs. 3, 3a, 3b). It is to be noted, however, that between the inner clear zone and the outer partly hemolyzed zone is a fairly distinct ring or zone of unhemolysed blood corpuscles. Throughout the zones there is no greenish or brownish discoloration such as appears in alpha zones. For comparison there are shown in figures 4, 4a, 5 and 5a, photographs of streptococci producing alpha and single beta zones in blood agar plates similarly incubated and refrigerated. Although streptococci producing the above described appearance in blood agar were often isolated from mixed milk and the milk of cows with mastitis between the years 1915 and 1928, it was not until December of the latter year that a strain producing a similar appearance was obtained from a human source. Since then, over 100 strains have been isolated from human subjects during life and post-mortem. During life, strains were isolated from the throat, vagina, urine, a gangrenous leg and the blood stream. Many of those from the vagina and the throat were not associated with pathological conditions in these localities. Strains were isolated post-mortem from the heart blood, lung, ventricular fluid, peritoneal fluid and an abscess of the neck, often associated with other bacteria. Various factors influence the readiness with which the doublezone appears, the degree of hemolysis in the outer zone, and the density of the intermediate zone of unhemolysed corpuscles. If the plates are incubated only 24 hours and then refrigerated, the colonies and the double-zones are smaller than when they are incubated 48 hours before refrigeration. Usually the outer zones do not appear until the plates have been refrigerated, although they are likely to be manifest if the plates are left at room temperature after incubation and rarely may appear during the latter part of the period of incubation. Double-zones are not produced equally well in media made up

3 FIG. 1. A double-zone beta hemolytic streptococcus in a blood agar plate incubated 24 hours; natural size. Fig. la: Deep colony marked in figure 1; magnified 37 diameters. FIG. 2. The same plate as shown in figure 1 incubated 48 hours. Fig. 2a. The same deep colony as shown in figure la. FIG. 3. The same plate as shown in figures 1 and 2 refrigerated overnight. Fig. 3a. The same colony as shown in figures la and 2a; magnified 8 diameters. Fig. 3b. The same colony; magnified 37 diameters. 37

4 _4 FIG. 4. An alpha streptococcus in a blood agar plate incubated 48 hours and refrigerated overnight; natural size. Fig. 4a. Deep colony marked in figure 4; magnified 37 diameters. FIG. 5. A single-zone beta hemolytic streptococcus in a blood agar plate incubated 48 hours and refrigerated overnight; natural size. Fig. 5a. Deep colony marked in figure 5; magnified 37 diameters. FIG. 6. A double-zone beta hemolytic streptococcus in blood agar plate incubated 48 hours, with colony removed and placed onto the surface of the neighboring medium; natural size. FIG. 7. The same plate as shown in figure 6 refrigerated overnight, natural size. 38 L

5 DOUBLE-ZONE BETA HEMOLYTIC STREPTOCOCCI with various kinds of blood. A few bovine strains produce the zones most readily in horse blood but others, and most human strains, do not form double-zones in this blood. In the media used, rabbit blood served best for most strains, and was used routinely for this work unless otherwise stated. In a series of representative bovine and human strains human blood gave about the same results as rabbit blood; beef and sheep blood were less satisfactory. With representative strains, pork infusion, veal infusion and beef infusion agars have been tried and each agar has been made with Wilson P, Witte, Bacto, Fairchild, Proteose and Neopeptone. In all of these media and with either horse or rabbit blood all of the strains formed beta zones of varying size, but in certain of the media some of the strains failed to form double zones or some of the zones were atypical. A frequently encountered atypical double-zone was one which lacked the intermediate zone of unhemolysed corpuscles. In all three infusions typical double-zones were obtained most frequently when rabbit blood and Witte, Neo-, or Wilson P peptone were used.' Rarely a strain failed to form double-zones in one of these media but formed them in another. The reaction of the agar used was about ph 7.6. In extract agar made with Wilson P peptone all of a few representative strains formed double-zones with rabbit blood but not with horse or beef blood. Although more or less atypical double-zones appeared in various agars at ph 6.5, none appeared at ph 6.0. Reactions more alkaline than ph 7.0 were optimal for typical double-zone formation. Eight representative human and bovine strains were plated in duplicate in rabbit-blood veal infusion Witte peptone agar and incubated in Brown (1921, 1922) anaerobic jars. After incubation for 48 hours, one jar was opened and the plates were examined. All showed characteristic poorly defined single beta 1 It is regretted that the results depended upon the use of proprietary products of incompletely known source, method of manufacture and composition. It is hoped that an investigation of peptones, now in progress, may help to remedy this situation. 39

6 40 J. HOWARD BROWN zones of hemolysis similar to those produced by the same strains under aerobic conditions. The plates from this anaerobic jar were then refrigerated aerobically and within a few hours showed double-zones. The other anaerobic jar containing a duplicate set of plates was refrigerated without being opened after incubation for 48 hours. The double-zones formed in this jar were like those in the plates from the other jar refrigerated aerobically. It may be said, therefore, that these streptococci form doublezones after proper incubation and refrigeration under either aerobic or anaerobic conditions. At present it is impossible to offer an adequate explanation of the double-zone phenomenon although certain observations may be suggestive. The alternate zones of hemolysis and unhemolysed corpuscles suggest Liesegang rings and this interpretation has been offered by Kortenhaus (1929) for somewhat similar zones formed by staphylococci and with respect to the alpha zones formed by pneumococci and alpha streptococci in blood agar. In an interesting series of papers Idzerda and van Everdingen (1932) have also indicated that these appearances in blood agar may be due to physico-chemical causes. Using hydrogen peroxide, mercury, or an electric current as hemotoxic agents they were able to produce zones of alternate rings of hemolysed and unhemolysed blood cells in sterile blood agar plates. The problem, however, is not as simple as it may seem. A comparison of the hemolytic action of double-zone strains with that of the well known pathogenic hemolytic streptococci, such as Streptococcus pyogenes which may cause scarlet fever and septic sore throat, may be useful. After incubation of blood agar plates for 48 hours the double-zone strains form relatively small poorly defined zones of hemolysis; Streptococcus pyogenes forms larger, clearer, better defined zones. Broth cultures of double-zone streptococci vary in their ability to lake 5-per-cent suspensions of rabbit blood cells within 2 hours at 370C., whereas cultures of Streptococcus pyogenes produce complete making. Apparently Streptococcus pyogenes is more hemolytic than the double-zone streptococci and yet when 48-hour blood agar plates

7 DOUBLE-ZONE BETA HEMOLYTIC STREPTOCOCCI 41 of Streptococcus pyogenes are refrigerated, little or no visible change occurs; double-zones do not appear. The comparison suggests (a) that different hemolytic substances are produced by these two groups of streptococci, or (b) that another factor or substance is involved. When incubated blood agar plates of double-zone streptococci are placed into the refrigerator the double-zones may appear in from 30 minutes to several hours. On one occasion no doublezones were present after refrigeration for 3 hours and were fully developed 30 minutes later. These observations suggest that during the period of incubation some substance has diffused outward from the colony and affected the fragility of the blood corpuscles in the surrounding medium and that making of these corpuscles is brought about by exposure to lower temperature. Confirmation of this theory was obtained by removing by means of a cork-borer one of the colonies from an incubated blood agar plate and depositing the removed colony onto the surface of the neighboring medium (fig. 6). The plate was then refrigerated (fig. 7). The outer zone of partial hemolysis appeared not about the colony but about the area from which the colony had been removed. An adequate explanation of the cause of the zone of unhemolysed blood corpuscles appearing between the inner and outer zones of hemolysis cannot be given. The more or less discolored rings or zones of corpuscles which appear in alpha zones of streptococci are thought by Hagan (1925), Leifson (1932) and others to be the result of methemoglobinization of corpuscles by hydrogen peroxide, but the unhemolysed corpuscles in the zones of double-zone beta streptococci are usually not discolored. That the phenomenon is not, at least quantitatively, the same as that appears with alpha zones is indicated by the following experiments. A blood agar plate was streaked with a culture of alpha streptococcus and immediately afterward this streak was crossed by similar streaks of beta streptococci. After 24 hours incubation the plate appeared as in figure 8. Streak I is of the alpha streptococcus and is greenish by transmitted light but without

8 FIG. 8. A blood agar plate streaked with an alpha streptococcus (I) and then cross-streaked with single-zone beta streptococci (Ha and Hila), incubated 24 hours; natural size. FIG. 9. The same plate as shown in figure 8 after again cross-streaking with single-zone beta hemolytic streptococci (JIb and HlIb) and incubating for another 24 hours; natural size. FIG. 10. A blood agar plate streaked with a double-zone beta hemolytic streptococcus (1) and then cross-streaked with a single-zone beta hemolytic streptococcus (2), incubated 24 hours; natural size. 42

9 DOUBLE-ZONE BETA HEMOLYTIC STREPTOCOCCI hemolysis. Streaks Ha and IIIa are of beta streptococci and show wide zones of hemolysis without discoloration. The hemolysis is not interrupted at the crossing of Streak I by Streaks Ha and IIIa. At the crossing, however, edges of the hemolysed streaks are indented where the corpuscles under Streak I had become "fixed" before the hemolysis had advanced that far. At this time Streak I was again crossed by streaks of the two hemolytic streptococci and the plate again incubated. After reincubation for 24 hours the result was as shown in figure 9. It is seen that in crossing Streak I, the hemolysis of Streaks IIb and IIIb is interrupted, the corpuscles underlying the growth of the alpha streptococcus having been fixed by it and rendered resistant to hemolysis by the beta streptococci (Brown, 1919). A similar experiment was conducted with a double-zone beta streptococcus in place of the alpha streptococcus (figs. 10 and 11). It was found that the unhemolysed corpuscles beneath the streak of the double-zone streptococcus were not rendered resistant to hemolysis by the beta streptococcus. In view of the fairly common occurrence of double-zone beta hemolytic streptococci their lack of recognition may be due to several reasons. (1) It is necessary that blood agar pour plates be inoculated since double-zones often appear only about the deep colonies. (2) There must be a smooth even distribution of the blood in the agar and not too many colonies, preferably less than 100. (3) Plates should be incubated 48 hours at 370C. and then refrigerated overnight since the double-zone usually appears only after refrigeration. (4) The kind and amount of blood used are important. Routinely we have used defibrinated rabbit blood and this has been satisfactory although some bovine strains produce double-zones more readily when horse blood is used. Horse FIG. 11. The same plate as shown in figure 10 after again cross-streaking with the single-zone beta hemolytic streptococcus (3) and incubating for another 24 hours; natural size. FIG. 12. A double-zone beta hemolytic streptococcus in a blood agar plate incubated 48 hours and then refrigerated; natural size. Photographed in March FIG. 13. The same strain of streptococcus as shown in figure 12. Blood agar plate incubated 48 hours and then refrigerated; natural size. Photographed in April

10 44 J. HOWARD BROWN blood is not satisfactory for many strains of human origin. The amount of blood in the agar should be between 5 and 10 per cent or about 1 cc. of blood to 12 cc. of agar which, when poured into a 10-cm. Petri dish makes a layer of medium about 2 mm. thick. (5) The agar used has been a meat infusion agar with a reaction of about ph 7.6. Pork, beef and veal infusions have been tried as have also various peptones. Veal infusion with Witte peptone has given the most uniformly satisfactory results. Other combinations give good results with certain strains but may fail to allow the production of double-zones with others. Further experiment is necessary to determine whether extract agar may be used with equally satisfactory results. That others may have observed the double-zones here described is indicated by the following quotations. "The streptococcus on the plates which I show you this evening was isolated by Dr. Baumann from the blood of a case of phlebitis and paxaphlebitis of the leg. On the blood agar plates it was noticed that after 24 hours the organism was surrounded by a clear area. Surrounding this clear area the blood on the plate was darker in color than on the rest of the plate. After forty-eight hours a second clear ring appeared outside of this area of darkened blood. In one observation the process extended so that there were three clear concentric rings about the streptococcus colony." (E. Libman, 1905). Because of the lack of technical details it is impossible to decide whether the appearance described was alpha or beta. This is rendered more doubtful by a similar description of a pneumococcus colony by the same author in which be says "next to the colony was a clear ring, surrounding this was a ring of somewhat darkened blood, outside of which there was again a clear ring." (E. Libman, 1905). We know that the pneumococcus forms an alpha zone, often with considerable hemolysis. We must reemphasize that unless the deep colony in blood agar is studied under the microscope it is unsafe to make a diagnosis of alpha or beta zone. Taylor and Wright (1930) describe certain strains of hemolytic streptococci isolated from the human vagina which apparently formed double beta zones in blood agar plates left at room tem-

11 DOUBLE-ZONE BETA HEMOLYTIC STREPTOCOCCI perature or refrigerated after incubation. "In horse blood agar little or nothing happens except a slight lightening in colour, barely amounting to more than a haziness, at the periphery of the haemolysed zone. In rabbit-blood agar a similar zone develops much more commonly, at first as a wide belt, rather light in colour, surrounding the haemolysed zone next to the colony. Later, this belt tends to differentiate into an outer haemolysed zone and an inner usually quite narrow unhaemolysed part. Further incubation for 48 hours leads simply to greater emphasis of the outer zone of haemolysis and sometimes to disappearance of the intermediate unhaemolysed zone... There is one strain described by Brown and illustrated in Figures 3-6, Plate 4, of his monograph illustrating a change not unlike what is here iescribed-the formation on horse blood agar of a certain discolouration beyond the haemolysed area." This description ages with our observations of the double-zone beta streptococci of which the one referred to by Taylor and Wright was one of the first that we isolated. The reference to "a certain discolouration beyond the haemolysed area" is an error. We were careful to state on page 30 of the monograph "There is no discoloration," meaning by this that the remaining corpuscles were red, not greenish or brown. Stableforth (1932). states that all of his strains of Group I, type b were somewhat peculiar in regard to hemolysis. "After 24 or 48 hours incubation a large part of the colonies were surrounded by a narrow but clear zone of hemolysis outside which was produced a greenish ring similar to that found immediately around the colony of an alpha strain. Outside this ring of fixed cells was another in which hemolysis was again complete. This appearance does not exactly correspond to Brown's description of an alpha prime (a,) nor in fact to any of Brown's categories. There was, moreover, another character which precluded the placing of these amongst the alpha (a,) group, viz., the marked lysin production in fluid media. It should be added that in strains of no other type was this strong double-ringed tendency observed." It seems quite likely that Stableforth observed the double-zone beta appearance although the blood which he used 45

12 46 J. HOWARD BROWN (ox-blood) has in our hands not been favorable for this purpose. The greenish discoloration which he mentions may have been due to the medium used. We have noticed it when sheep blood was used and it is favored by some brands of peptone. He did not recognize the 'similarity of the appearance which he describes to the illustrations published by the author in 1919 (plate 4, figs. 3, 4, 5 and 6). Hare and Colebrook (1934) describe strains of streptococci from bovine mastitis and from the human vagina which doubtless belong to the group of double-zone beta hemolytic streptococci. They used horse-blood agar and state that "the zone of hemolysis is only complete in the immediate neighbourhood of the colony and outside this is a hazy zone consisting of islands of unhemolysed cells in a hemolysed matrix. Brown (1919) figures this appearance in his monograph (plate 5, figs. 1, 2, la and 2a)." In defibrinated horse blood the corpuscles normally are agglutinated. The "islands of unhemolysed cells" noted by Hare and Colebrook are doubtless due to the fact that the larger aggregates of cells are less readily hemolysed than others. Continuing, "With some strains from the human vagina and from the cow's udder this partially hemolysed zone may be quite wide and sharply demarcated from the inner zone of complete hemolysis." These were probably what we call double-zones similar to those illustrated in plate 4, figures 3, 4, 5 and 6 of the monograph (Brown, 1919). As stated above, some strains which produce double-zones in rabbit blood agar may not do so in horse-blood agar. This has been found to be true of the strain illustrated in Plate 5 (Brown, 1919) and referred to by Hare and Colebrook. Apparently the ability of these strains of streptococci to form double-zones is not a transient character, but is quite stable. We have not noted its disappearance from any of the more than 150 strains which we have in cultivation. The first strains were isolated by Smith and Brown (1915) in In figure 12 is shown a blood agar plate of one of these strains photographed in In figure 13 is shown a photograph of the same strain plated in Both show double-zones. Minor differences in the appearance of the zones in the two photographs may be due

13 DOUBLE-ZONE BETA HEMOLYTIC STREPTOCOCCI 47 to unrecognized variations in the media, but are partly due to differences in the methods of photography. SUMMARY The streptococci of a certain group of beta-hemolytic streptococci form double zones in blood agar plates refrigerated after incubation. This characteristic persists after long periods of cultivation of these strains. These streptococci are from human and animal sources. In a forthcoming publication the cultural characteristics of double-zone beta hemolytic streptococci will be described with some reference to their serological grouping, source and pathogenic significance. REFERENCES BROWN, J. H The use of blood agar for the study of streptococci. Monograph No. 9, Rockefeller Institute for Medical Research. BROWN, J. H Double-zone beta hemolytic streptococci. Jour. Bact., 27, 84. BROWN, J. H An improved anaerobe jar. Jour. Exp. Med., 33, 677. BROWN, J. H Modification of an improved anaerobe jar. Jour. Exp. Med., 35, 467. HAGAN, W. A The green coloration by certain streptococci on blood agar. Jour. Inf. Dis., 37, 1. HARE, R. AND COLEBROOK, L The biochemical reactions of hemolytic streptococci from the vagina of febrile and afebrile parturient women. Jour. Path. and Bacteriol, 39, 429. IDZERDA, J. AND VAN EVERDINGEN, W. A. G Zur Kenntnis der Aenderungen, die in Blutnahrboden durch Streptokokken, Typus a (Brown) verursacht werden. I. Zentralblatt f. Bakt., Abt. I, Orig., 123, 401. II. Ueber die Wirkung von sterilen Agarfiltraten auf kleine Erythrozytenmengen. Zentralblatt f. Bakt., Abt. I, Orig., 124, 78. III. Ueber die Wirkung von sterilen Bouillonfiltraten und von geringen H202-Konzentrationen auf kleine Erythrozytenmengen. Zentralblatt f. Bakt. Abt. I, Orig., 124, 185. IDZERDA, J. AND VAN EVERDINGEN, W. A. G Sur le changement de la resistance globulaire comme la cause de la formation des zones dans la g6lose au sang. I. Par le peroxyde d'hydrogene. Arch. N6erlandaises de Physiol. de 1'Homme et des Animaux, 17, 134. IDZERDA, J. AND VAN EVERDINGEN, W. A. G Experimentelle Untersuchungen uber die mittels H202 und Hg in Blutagarplatten erhaltenen Strukturen. Biochem. Ztschr., 249, 381. IDZERDA, J. AND VAN EVERDINGEN, W. A. G Experimentelle Untersuchungen uber die bei Stromdurchgang vermittelst Gold- und Platinelektroden in Blut-Agar erhaltenen Strukturen. II. Biochem. Ztschr., 254, 88.

14 48 J. HOWARD BROWN JoNEs, F. S The streptococci of equines. Jour. Exp. Med., 30, 159. KORTENHAUS, F Zonenzyklohfunolyse auf Blutagar bei Staphylokokken sowie bei Viridans-Streptokokken und Pneumokokken. Zentralblatt f. Bakt. Abt. I, Orig., 113, 499. LEIFsoN, E Types of bacteria on blood and chocolate agar and the immediate cause of these types. Jour. Bact., 24, 473. LIBMAN, E A note on a peculiar action of a streptococcus on blood plates. Proc. N. Y. Path. Soc., 5,59. LIBMAN, E A pneumococcus producing a peculiar form of hemolysis. Proc. N. Y. Path. Soc., 54,168. STABLEFORTH, A. W Studies on bovine mastitis. VII. The serological characters of mastitis streptococci. Jour. Comp. Path. and Therap., 45, 185. TAYLOR, J. AND WRIGHT, H. D The nature and sources of infection in puerperal sepsis. Jour. Obst. and Gyn. British Empire, 37, 213. VAN EVERDINGEN, W. A. G. AND IDZERDA, J Experimentelle Untersuchungen uber die bei Stromdurchgang vermittelst Gold- und Platinelektroden in Blut-Agar erhaltenen Strukturen. I. Biochem. Ztschr., 254, 59. Downloaded from on June 14, 2018 by guest