THE ISOLATION AND IDENTIFICATION OF BETA- HEMOLYTIC STREPTOCOCCI

THE ISOLATION AND IDENTIFICATION OF BETA- HEMOLYTIC STREPTOCOCCI AN EVALUATION OF THE USE OF OUTDATED HUMAN BLOOD-BANK BLOOD BERKLEY H. JOHNSON, M.S., STANLEY C. BRAUNSTEIN, M.S., AND CHARLES KASPER Division of Bacteriology, Naval Medical Research Unit No. 4, Administrative Command, U. S. Naval Training Center, Great Lakes, Illinois Bacteriologists generally agree that human blood should not be used in the preparation of mediums for the primary isolation of beta-hemolytic streptococci because of the possibility of streptococcal inhibitory substances being present in some specimens of blood. Rothbard 3 demonstrated that fresh human blood that contains complement, viable leukocytes, and type-specific antibody is bacteriostatic for homologous types of streptococci. Furthermore, it is not without reason to speculate that some human blood may contain sufficient antistreptolysin "0" to preclude the detection of beta-hemolytic strains that produce streptolysin "0" but little or no streptolysin "S." In this laboratory, a reduction in hemolysis and streptococcal growth has been observed when fresh blood from a patient with rheumatic fever was used in a 10 per cent concentration in a blood agar plate that was seeded with a virulent, matt-strain of type 19 streptococcus. The study described in this paper was designed to detect differences in (1) the amount of growth, (2) the appearance of colonies, and (3) the hemolytic characteristics of representative strains of group A, C, and G beta-hemolytic streptococci when outdated human blood (from the blood bank of a hospital) and sheep blood were used, the latter serving as a reference blood for the routine isolation of beta-hemolytic streptococci. MATERIALS AND METHODS The 4 cultures of streptococci used in these studies were strains that were maintained in the lyophilized state for varying periods of time, and the organisms were identified, previously and subsequently, as group A type 19, group A type 17, group C, and group G strains. Both of the group A strains were in the matt-phase of growth. The type 19 strain was selected for study because this type is, and has been, epidemic among Navy recruits at Great Lakes Naval Training Center. Because the recruits comprise the greater number of donors for the blood bank, there is some possibility that this blood contains type-specific Received, July 3, 1957; accepted for publication September 4. Mr. Johnson and Mr. Braunstein are Supervisory Bacteriologists (Medical), and Mr. Kasper is Research Technician. From Project Report NM 52 06 04.1.2, the Bureau of Medicine and Surgery, Navy Department, Washington 25, D. C. The opinions expressed herein are those of the authors and can not be construed as reflecting the views of the Navy Department or the Naval Service at large. The use of commercially available products does not imply endorsement of these products or preference to other similar products on the market. 587

588 JOHNSON ET AL. Vol. 28 inhibitory substances against type 19 streptococci, as well as antistreptolysin "0." On the other hand, a type 17 strain was selected to represent a streptococcal strain which has not been isolated as an epidemic strain at Great Lakes Naval Training Station since World War II, so that, if any inhibition were observed, the role of type-specificity could be studied. Group C and G strains were included in order to determine whether or not nongroup A inhibitory factors might also be present in the blood of some donors, thereby leading to interference in the recognition or isolation of such, not infrequently isolated streptococcal strains. All beta-hemolytic streptococci used in this study were originally isolated from Navy recruits in conjunction with epidemiologic studies that were performed during a period of several years. After several passages of the lyophilized strains in broth and on plates of blood agar, the organisms were incubated in Todd- Hewitt broth at 37 C. for 18 hours, and serial dilutions were prepared in order to obtain a plate count in the range of 30 to 300 colonies. This was accomplished as follows: 0.2 ml. of the culture grown in 10 ml. of broth for a period of 18 hours was transferred into 99 ml. of sterile water; this represents a primary dilution of 1:496. Five additional dilutions of 10-fold intervals were made in 20-ml. volumes, and 1 ml. of each of the last 4 dilutions was plated in order to determine the streptococcal count. Duplicate plates were poured for each of the dilutions, and the streptococcal colonies were counted, by means of a Quebec colon}'' counter, after incubation for 24 hr. at 37 C. Streak plates were prepared from the nondiluted culture, or the lower dilutions, in order to observe any differences in colonial or hemolytic characteristics, as manifested with the 2 species of blood. The basic agar medium consisted of 1 part Todd-Hewitt broth and 2 parts of a blood agar base (Difco), with the ph adjusted to 7.8. Blood was added to the autoclaved agar base (previously cooled to 45 C.) in order to obtain a 10 per cent concentration in earlier experiments, and, in later experiments, a 5 per cent concentration. Defibrinated sheep blood was used within 5 days after bleeding the animal. Human blood was used within an average of 3 to 5 days after the 21-day expiration date. The human blood was obtained from the blood bank of the U. S. Naval Hospital at Great Lakes Naval Training Station, and it contained 120 ml. of acid-citrate-dextrose (ACD) solution (formula B of the National Institutes of Health). All of the specimens of blood collected throughout the months of February, March, April, May, and June, 1956, were sterile. The specimens were selected during this period in order to maximize the possibility of obtaining blood most likely to contain appreciable amounts of antistreptolysin "O" or growth-inhibitory substances, inasmuch as streptococci are most prevalent in the area at this time. Titers of antistreptolysin "O" were determined with the plasma of the human blood, with commercially prepared streptolysin "O" reagent (Difco) and by a method that was previously described. 2 KESULTS During the 4-month period of this study, 30 samples of outdated human bloodbank blood were studied for the presence of inhibitory substances against betahemolytic streptococci. A pictorial representation of a typical experiment, in-

Dec. 1957 STREPTOCOCCI IN BANK BLOOD 589 dicating the growth and hemolysis of the 4. test strains of streptococci on human and sheep blood (using streak-plate and pour-plate methods), is reproduced in Figures 1 to 8. It may be noted in Figures 1 to 4 that the colonies and surface hemolysis are virtually identical on both species of blood. Similarly, no differences in colony counts, or in submerged hemolysis, are grossly evident in Figures 5 to 8. A summary of the counts of streptococcal colonies in aliquots from 30 pints of blood-bank blood, and from 13 samples of sheep blood (performed concomitantly as controls), is illustrated in Figure 9. The average plate counts indicated in this figure were obtained by means of averaging the colony counts from a selected FIGS. 1-4 FIG. 1 (upper left). Growth of type A17 streptococci on streak plates with 5 per cent human blood (la) or 5 per cent sheep blood (16). FIG. 2 (upper right). Growth of type All) streptococci on streak plates with 5 per cent human blood (2a) or 5 per cent sheep blood (26). FIG. 3 (lower left). Growth of Group C streptococci on streak plates with 5 per cent human blood (3a) or 5 per cent sheep blood (36). FIG. 4 (lower right). Growth of Group G streptococci on streak plates with 5 per cent human blood (4a) or 5 per cent sheep blood (46).

590 JOHNSON ET AL. Vol. 28 inoculum-dilution that yielded counts in the range of 30 to 300 colonies per plate on the agar medium with sheep blood. This same dilution of inoculum was used to compare the colony counts for all blood-bank blood that was tested on a single day. Any daily variation in inoculum was compensated, therefore, and an equitable comparison of all analyses performed throughout the period of study was thereby permitted. There were no differences in colony counts for any 1 of the 4 test strains of streptococci with either of the 2 species of blood (Fig. 9). Although there seemed to be no inhibitory substances in the human blood at a maximal concentration of 10 per cent, it was thought pertinent to determine whether or not the donors used in this study had any evidence of a recent streptococcal infection prior to their donation of blood to the blood bank. Determinations of antistreptolysin "O" antibody were performed with all 30 specimens of blood, and the distribution of titers is indicated in Table 1. Only 3 (10 per cent) of the donors had relatively increased titers of antistreptolysin "O" (250 units), or could be regarded as having had a recent streptococcal infection. On the other hand, there was no significant difference in the colony counts or the hemolytic and colonial characteristics of the streptococci cultured on these 3 bloods, as compared with the other 27 specimens of blood. DISCUSSION Recently Neussle and co-workers 1 reported an increase in recovery of betahemolytic streptococci among 2 series of hospitalized persons when sheep blood was substituted for human blood in the procedure of primary isolation. In addition, cultures from the throats of another series of 20 patients, plated simultaneously on both species of blood, similarly yielded fewer isolations of streptococci on human blood. The authors suggest that antistreptolysin, or unknown inhibiting substances present in human blood, may have resulted in the lower recovery rates. There is some question, however, as to whether or not the apparently lower rate of isolation of streptococci on human blood agar was actually the result of the type of blood, or a lower incidence of carrier-rates for betahemolytic streptococci in the Fall months, when human blood was used, than in the Winter months, when sheep blood was substituted. In our study, the experimental approach was designed to control or limit the variables, as much as possible, only to the species of blood used in the tests. Thus, it was possible to detect even small differences, not only between human and sheep blood, but also differences within both of the 2 species of blood that was used throughout the period of study. As illustrated in the figures, there seemed to be no differences in the amount of growth, the morphologic appearance of the streptococcal colonies, or the hemolytic characteristics of the 2 group A and 2 nongroup A strains on agar plates that contained sheep or human blood. Theoretically, inasmuch as (1) the bacteriostatic action of human blood against beta-hemolytic streptococci results from antibodies in the presence of viable phagocytes, active complement, and other labile substances, and (2) these factors are active only in fresh blood, one would not expect any inhibitory action by these substances in the agar plates that contained outdated human blood.

. o v C Q c o O 0 o 0 0 a o o o o O O 0/ \U O OD C!<& 8 ^7 oc V ^O ; - " \n - > O ^. -> ^ o V\ o d Or n ^' 7a 7b 8 a 8 b FIGS. 5-8 FIG. 5 (upper left). Growth of type A17 streptococci on pour plates with 5 per cent human blood (5a) or 5 per cent sheep blood (56). FIG. 6 (upper right). Growth of type A19 streptococci on pour plates with 5 per cent human blood (6a) or 5 per cent sheep blood (66). FIG. 7 (lower left). Growth of Group C streptococci on pour plates with 5 per cent human blood (7a) and 5 per cent sheep blood (76). FIG. S (lower right). Growth of Group G streptococci on pour plates with 5 per cent human blood (8a) or 5 per cent sheep blood (S6). STREPTOCOCCAL INOCULUM SHEEP BLOOD HUMAN BLOOD A 19 100 80 60 40 20 20 40 60 80 100 AVERAGE POUR PLATE COUNTS FIG. 9. Average counts of colonies of streptococci in agar mediums with human or sheep blood (pour-plate technic). 591

592 JOHNSON ET AL. Vol. 28 TABLE 1 DISTRIBUTION OF TITERS OF ANTISTREPTOLYSIN "0" IN 30 SPECIMENS OF HUMAN BLOOD- BANK BLOOD Number of Specimens of Blood 2 4 2 11 7 0 1 3* Titers of Antistreptolysin "0' units 0 to 25 26 to 50 51 to 75 76 to 100 J01 to 125 126 to 150 151 to 175 176 to 200 (ASO) * Each of the 3 specimens contained 250 units. Furthermore, from the data presented, it was demonstrated that, in spite of the fact that 3 of the 30 donors (10 per cent) probably had a recent streptococcal infection (as evidenced by an elevated titer of 250 units of antistreptolysin "O"), there was no apparent interference in detecting beta-hemolysis on these specimens of blood. From this study, therefore, there was no experimental evidence to support the supposition that interfering substances, either known or unknown, are present in outdated human blood-bank blood that would preclude the use of such blood in the primary isolation of beta-hemolytic streptococci. One should bear in mind, however, that human blood does not contain the inhibiting substance against Hemophilus hemolyticus, as does sheep blood. Therefore, Gram stains should be made with colonies that resemble those of beta-hemolytic streptococci isolated on human blood. SUMMARY AND CONCLUSION A comparative evaluation of aliquots from 30 pints of outdated human bloodbank blood and sheep blood, using the pour-plate technic, revealed no detectable differences in the amount of growth, the colonial characteristics, or the hemolytic properties of the 2 group A and 2 nongroup A strains of streptococci observed in this study. It is concluded that the use of outdated human blood-bank blood is suitable for the preparation of blood agar to be used for the isolation and identification of beta-hemolytic streptococci, provided Gram stains of suspected colonies are examined in order to exclude the possibility of Hemophilus hemolyticus. SUMMARIO IN INTERLINGUA Un evalutation comparative de aliquotes ab 30 pintas de supervetulate sanguine human ab un banca de sanguine e de sanguine ovin, sub le conditiones del technica a plattas coperite per effusion, revelava nulle detegibile differentias in le quantitate de crescentia, le characteristicas colonial, o le proprietates hemolytic

Dec. 1957 STREPTOCOCCI IN BANK BLOOD 593 del 2 racias de giuppo A e del 2 racias nonde gruppo A de streptococcos observate in iste studio. Es concludite que le uso de supervetulate sanguine human ab bancas de sanguine es legitime in le preparation de agar a sanguine destinate al empleo in le isolation e identification de streptococcos hemolytic beta, providite que suspecte colonias es examinate sub coloration per le Gram pro excluder le possibilitate de Hemophilus hemolyticus. Acknowledgment. The authors wish to acknowledge the assistance of Paul F. Frank, Director, Division of Bacteriology, in preparation of the manuscript; Robert L. Woolridge, Director, Division of Immunology, and Wilber F. Smith, HMC USN, for the performance of the antistreptolysin "0" titers; Walter T. Stille, Statistician, for aid in the preparation of the statistical data; and Wayne C. O'Neill, HM1 USN, for the photographic prints. REFERENCES 1. NEUSSLE, W. F., WRIGHT, A. E., AND JONES, P. R.: Comparison of human and sheep blood agar in detecting streptococcus. U. S. Armed Forces M. J., 6: 320-323, 1955. 2. ROBINSON, J. J., CRAWFORD, Y. E., AND Roiioi/r, D. M.: The determination of antistreptolysin "0." Am. J. Clin. Path., 22: 237-246, 1952. 3. ROTHBARD, SIDNEY. Bacteriostatic effect of human sera on group A streptococci. J. Exper. Med., 82: 92-132, 1945.