Evaluation of Three Commercially Available Test Products

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1980, p /80/ /07$02.00/0 Vol. 11, No. 3 Evaluation of Three Commercially Available Test Products for Serogrouping Beta-Hemolytic Streptococci M. SLIFKIN* AND GAIL R. POUCHET-MELVIN Department of Laboratory Medicine, Section of Microbiology, Allegheny General Hospital, Pittsburgh, Pennsylvania Three beta-streptococci serogrouping kits, Phadebact, SeroSTAT, and Streptex, were evaluated as to their sensitivity, accuracy, and suitability as methods for serogrouping streptococci in a clinical microbiology laboratory. The majority of the primary isolates examined by the various methods associated with each of the three kits were correctly identified. The Streptex direct mixed-culture procedure was more often associated with the observation of cross-reactivity than with the direct procedures of the other two kits which did not employ mixed growth cultures. Furthermore, the Streptex kit was associated with more falsenegative responses than those determined by the other two kits under evaluation. These results appeared to be due to the relatively poor sensitivity of the Streptex grouping reagents. The Streptex test procedures required more labor than the other kit procedures, requiring a 1-h enzymatic extraction step for the release of the group antigens. The SeroSTAT kit provided only a direct procedure and, thus, is limited in its application. The Phadebact procedures were the most versatile by providing not only a direct and a 24-h grouping procedure, but also by including a 4-h method that may be employed as required by the clinical microbiologist. The coagglutination method was previously shown to be an accurate, rapid, and simple method (1, 2, 6, 10, 12, 13, 16, 17) for the serogrouping of beta-hemolytic streptococci. This method employs, in part, Cowan I staphylococci that are coated with rabbit gamma globulin specific for a streptococcal group. The adsorbed antibody has its Fc portion attached to the staphylococcal protein A and its antigen-combining Fab parts directed outward (4). Kits for serogrouping various beta-hemolytic streptococci by coagglutination are commercially available (Phadebact Streptococcus Test kit, Pharmacia Diagnostics). Recently, it was shown that latex particles sensitized with rabbit gamma globulin specific for various streptococci could be employed in the serogrouping of these bacteria (14). At present, two commercially available latex agglutination kits for serogrouping these bacteria are available (SeroSTAT, Scott Laboratories, Inc.; Streptex, Weilcome Research Laboratories) for use in the clinical microbiology laboratory. Materials for various procedures including direct, nonmixed, direct mixed, and 4-h and 2-h cultures, as well as for mixed cultures, are included in the respective coagglutination and latex agglutination kits. Accordingly, the purpose of this investigation was to evaluate the serogrouping reagents from these three commercially available kits as to their 249 sensitivity, accuracy, and suitability as methods for serogrouping streptococci in a clinical laboratory. MATERIALS AND METHODS Organisms. The cultures of bacteria were obtained from clinical specimens and stock cultures. Stock cultures of serogrouped beta-hemolytic streptococci were maintained in Trypticase (BBL Microbiology Systems) soy broth containing 15% (vol/vol) glycerol at -70 C (18). Clinical specimens received on transport swabs (Culturette, Marion Scientific Corp.) and the stock cultures were streaked on Columbia sheep blood agar plates (BBL) to obtain isolated organisms. The plates were incubated at 35 C under anaerobic conditions in GasPak (BBL) units for 12 to 18 h. The plates were then observed for the presence of betahemolytic streptococci with the application of Gram staining and the determination of hemolytic and catalase activity (7, 8, 20). Isolated colonies of beta-hemolytic streptococci from each clinical primary plate were selected and employed for the direct-plate coagglutination method with the Phadebact, Streptex, and SeroSTAT reagents. However, approximately 40% of the beta-hemolytic streptococci employed for the direct-plate test with the Streptex reagents were obtained as mixed cultures by streaking a loop across the path of the streptococci. One isolated colony from each clinical primary plate was selected and inoculated into 2 ml of Todd-Hewitt broth (BBL) for use in the respective 4- (9, 16) and 24-h Phadebact and 24-h Streptex methods.

2 250 SLIFKIN AND POUCHET-MELVIN Similarly, isolates were inoculated into 40 ml of Todd- Hewitt broth manufactured by BBL and Difco Laboratories and were examined for cross-reactivity by the various serogrouping reagents. Primary isolates (327) of beta-hemolytic streptococci were derived from the following locations (number of isolates in parentheses): upper (143) and lower (77) respiratory tract, blood (26), wounds (70), urine (59), and the umbilicus (2). These 12- to 18-h primary isolates were serogrouped by all three kits with their associated procedures when possible. The results were compared with those obtained by the autoclave extraction method. Grouping of streptococci by coagglutination. The Phadebact Streptococcus Test kit containing reagents for serogrouping groups A, B, C, and G streptococci was employed following the manufacturer's protocol for the direct-plate, 4-, and 24-h procedures. At least five isolated colonies from each clinical primary plate were used when applying the direct-plate procedure (17). Grouping of streptococci by latex agglutination. The SeroSTAT Streptococcus Test was employed following the method as described in the kit. This method employed only isolated colonies from an isolation medium and thus is classified in this investigation as a direct-plate method. The Streptex protocols presented in the kit permit a 24-h test and a direct-plate method that permits the use of either a pure culture of isolated colonies of beta-hemolytic streptococci or a mixed culture obtained by one or more passes of a loop across the surface of a primary plate containing beta-hemolytic streptococci. After 1 h, an enzyme extraction step employing pronase was used with the direct and 24-h Streptex methods. Lancefield grouping. Extracts were prepared for the Lancefield grouping by the autoclave extraction method (15). Commercial antisera (Burroughs Wellcome Co.) for groups A, B, C, D, F, and G were employed in the performance of the precipitin test in capillary tubes (19). All the grouping methods were performed blind, relative to each other. TABLE 1. Group Several approaches were utilized to determine the sensitivity of each kit in the ability to sero-react with beta-hemolytic streptococci. Strains representing group A streptococci were washed twice in sterile HA buffer (Difco) and suspended in 1.0 ml of HA buffer. The suspensions were diluted in twofold steps and tested for coagglutination or agglutination with the serogroup A reagent from each of the three kit systems. The strength of reactivity was observed as well as the time in seconds required for the response to occur. Plate counts of each dilution of bacteria were prepared with a calibrated 0.01-ml loop. In other experiments, various numbers of group A colonies were picked with an inoculating needle to determine the minimal number of colonies required for a serogrouping response. RESULTS Accuracy of reagents. The comparative results of the three serogrouping procedures are shown in Table 1. The direct-plate Phadebact procedure, when compared to the precipitin method, permitted the correct identification of 86 primary beta-hemolytic isolates from the 87 isolation plates (98.9%). A minimum of five colonies was required for testing with each of the four Phadebact reagents. The 4- and 24-h Phadebact readings yielded results that corresponded in all cases (100% correct) to those obtained by the Lancefield method. The direct-plate SeroSTAT method yielded results that correctly identified 166 of 167 (99.4%) isolates, compared to the reference method. However, due to the lack of a 24-h method, not all of the isolates tested by the other two kits could be identified. The direct-plate Streptex method was associated with the misidentification of 15 beta-hemolytic streptococcal isolates (95.4% correct). Comparison of three test products and associated methods for the serogrouping of beta-hemolytic streptococci from primary isolation plates Phadebact procedures SeroSTAT procedure Streptex procedures J. CLIN. MICROBIOL. Lancefield Direct 4-h 24-h (direct) Direct' 24-h pro- cedureb No. No. No. No. No. No. (no. No. cor- No. cor- No. cor- No. cor- No. cor- No. cor- grouptested rectly tested rectly tested rectly tested rectly tested rectly tested rectly ed) grouped grouped grouped grouped grouped grouped A B C D NTe NT NT NT NT NT NT NT F NT NT NT NT NT NT NT NT G Non grouped Isolated colonies or mixed cultures containing beta-hemolytic colonies were employed. Rantz-Randal autoclave extraction method. NT, Not tested; kit did not provide reagent to serogroup D or group F streptococci.

3 VOL. 11, 1980 These misidentifications were due, in part, to the lack of responsiveness of five group A isolates, and false-positive reactivity with the group C serogrouping reagent. The latter cross-reactivity was due to the presence of Streptococcus pneumoniae and other bacteria collected on the loop with the mixed-culture procedure, as will be discussed in the section on cross-reactivity. Furthermore, two group A isolates gave equivocal agglutination responses with the group C reagent. The seven misidentified group C isolates and one group F isolate did not react with their respective serogrouping reagents. Another group F isolate yielded a cross-reactive response with the group C reagent with the mixed-culture method, whereas another isolate did not agglutinate with the specific reagent. Two nongroupable strains also yielded cross-reactive agglutination with the group C reagent with the direct mixed-culture method. Two misidentifications occurred with the 24-h Streptex method (99.5% correct). These were due to the cross-reactivity of a group C isolate with group A reagent and the false-negative response of a group F isolate with the specific serogrouping reagent. Sensitivity of reagents. Approximately 40,000 to 60,000 colony-forming units combined with both Phadebact and SeroSTAT reagents to cause a minimal response, whereas 80,000 to 100,000 colony-forming units resulted in a strong serogrouping response. On the other hand, the minimal number of colony-forming units to cause a latex agglutination response with the Streptex group A reagent was 80,000 colonyforming units (Table 2). Both the Phadebact and SeroSTAT group A reagents reacted with five colonies to form a weak response with 1 min of observation. The application of 10 or more colonies with either of these two reagents, however, resulted in stronger reactions within 30 s or less. The enzyme-extraction procedure of the Streptex method required 10 colonies for a minimal latex agglutination reaction, whereas 20 or more colonies yielded relatively stronger agglutination responses (Table 3). A third experiment was employed to further pursue the sensitivity of the three kit reagents for the serogrouping of group A and group C streptococci. The polyvalent control antigen from a Streptex kit was diluted in twofold steps, and one drop of each dilution was mixed, respectively, with one drop of group A and group C reagent from each of the three streptococcal serogrouping test kits. Both the Phadebact and SeroSTAT reagents responded similarly by producing their coagglutination or agglutination responses with their group A reagents through a 1:64 dilution of polyvalent antigen. Furthermore, EVALUATION OF SEROGROUPING KITS 251 TABLE 2. Minimal number of colony-forming units required for coagglutination and agglutination of group A streptococci with Phadebact, SeroSTAT, and Streptex reagents Number of col- Response with group A reagenta ony-forming units Phadebact STrAT Streptex 1, , , , , , a +, Weak response; -, no response; ++, moderate response; +++, strong response. TABLE 3. Minimal number ofgroup A streptococci colonies required for coagglutination and agglutination with three commercially available serogrouping kitsa No. colonies Phadebact SeroSTAT Streptex 3 _b _ _ a Twelve-hour colonies grown on sheep blood agar at 350C. b-, No response; +, weak response; ++, moderate response; +++, strong response. the periods of time for the reactions to occur through the various dilutions of polyvalent antigen were quite similar. The Phadebact group C reagent also produced observable coagglutination responses through a 1:64 dilution, whereas the SeroSTAT group C reagent yielded slightly slower responses, especially at the higher dilutions of the polyvalent antigen. In contrast to the latter two reagents, the response time of agglutination with the group A and group C Streptex reagents was considerably slower. Also, the greatest dilution at which detectable agglutination was evident occurred at a 1:8 dilution of the polyvalent antigen. Twenty colonies each of four group A streptococci stock strains derived from the American Type Culture Collection and three clinical isolates of group A streptococci were serogrouped by each of the three products to determine the response time with the group A reagents for each of the three kits. The time for serogrouping response by both the Phadebact and SeroSTAT reagents for these seven strains of group A streptococci was similar and occurred between 6 and

4 252 SLIFKIN AND POUCHET-MELVIN 10 s. The response time with the Streptex reagents, employing the enzyme extract from each of these bacterial strains, was consistently prolonged. The latter agglutination reactions occurred between 16 and 21 s. Cross-reactivity. The potential for crossreactivity of the serogrouping reagents from each of the three kits was determined for 13 species of bacteria including five members of the Enterobacteriaceae, Pseudomonas aeruginosa, Staphylococcus aureus, and five species of viridans streptococci. The Phadebact group C reagent cross-reacted with all of the strains of Klebsiella pneumoniae and S. pneumoniae examined. The reaction time for the coagglutination response with S. pneumoniae was quite rapid in contrast to that observed for K. pneumoniae. The cross-reactivity responses for the Streptex reagents were similar to those of the Phadebact reagents except that the group A reagent also produced agglutination with all the test strains of K. pneumoniae. Cross agglutination with the SeroSTAT reagents occurred with K. pneumoniae and S. aureus with the group A, B, C, and G reagents at just under 40 s of observation. The group C reagents from the SeroSTAT Kit reacted with all the S. pneumoniae strains (Table 4). The group A reagents of the Phadebact and SeroSTAT kits were shown to cross-react with drops of Todd-Hewitt broth manufactured by Difco but not with the BBL product (Table 5). Since the manufacturer of Streptex stated in the protocol of the kit that mixed growth may be employed for the serogrouping of beta-streptococci from a culture plate, the possibility of TABLE 4. Cross-reactivity of commercial streptococcus agglutination grouping reagents with various bacteria Cross-reactive reagents Organism Phade- SeroSTAT Streptex bact Ca A B C G A C K. pneumoniae +C (10)b S. aureus (10) S. pneumoniae (10) All others (53)d a Phadebact reagents A, B, and G and Streptex B, D, F, and G did not react with any of the bacteria tested. b Number of strains tested. c +, Weak response; -, no response. d Other bacteria tested: Citrobacter diversus (5), Enterobacter cloacae (10), Escherichia coli (10), P. aeruginosa (10), Serratia liquefaciens (10), viridans streptococci (8). TABLE 5. Cross-reactivity response of Phadebact, SeroSTAT, and Streptex group A reagents with BBL and Difco Todd-Hewitt broth Phade- Sero- Strep- Todd-Hewitt Todd-ewittbroth bact STAT tex BBL (lot no. EIDHRS) -a BBL (lot no. EIDHRT) Difco (lot no ) + + Difco (lot no ) + + a -, No response; +, weak response. cross-reactivity problems with other bacteria with this procedure was examined. Sweeps were taken from 10 primary plates containing 5 to 200 beta-hemolytic colonies. The agglutination with the direct mixed-culture method from eight of the plates containing group A streptococci was somewhat ambiguous since a response was observed with both the group A and group C Streptex reagents. Furthermore, the time of the agglutination response was only slightly faster with the group A reagent than that with the group C reagent for four of these plates and essentially the same with the other four plates. Another plate containing 80 beta-hemolytic colonies of group A streptococci did not respond with the group A reagent when approximately 20 colonies were collected with a sweep of the loop and extracted for the serogrouping procedure. A sweep of colonies from two primary plates containing only two and five colonies of group A streptococci, respectively, yielded only false-positive group C responses. On subculture, these organisms were correctly serogrouped by the Streptex group A reagent (Table 6). DISCUSSION J. CLIN. MICROBIOL. Numerous investigations have shown that coagglutination testing, particularly with the Phadebact Streptococcus Test, affords the clinical microbiologist a rapid and reliable method to serogroup beta-hemolytic streptococci (10, 16, 17). The present investigation confirms the diagnostic potential of this method and shows the capabilities of the relatively new SeroSTAT and Streptex latex agglutination tests for the serogrouping of beta-hemolytic streptococci. This work substantiates our previous report (7) which showed that the Phadebact test system permits the serogrouping of colonies taken directly from the surface of a primary isolation plate when at least five beta-hemolytic streptococci are present. These results are in contrast to a recent report stating essentially that no direct-plate method exists for the Phadebact Streptococcus Test (13). Likewise, as previously reported (17), when sufficient numbers of these colonies are not available for this direct proce-

5 VOL. 11, 1980 TABLE 6. Cross-reactivity of response with Streptex reagents using mixed-culture plate procedure with bacteria from primary throat culture plates No. of Plate byta beta-he- Streptex re- Final identificano. molytic colonies sponsea tionb on plate 1 5 C(10) Beta-hemolysis due to S. pneumoniae 2 30 A(12), C(10) Group A 3 2 C(10) Group A 4 80 c Group A A(15), C(25) GroupA A(11), C(15) Group A A(10), C(20) GroupA A(18), C(18) Group A A(20), C(19) Group A A(8), C(15) Group A a Time of agglutination response (seconds) in parentheses. Rantz-Randal extraction. e, No reactivity with any serogrouping reagent. dure or when the possibility exists of picking colonies of other species of bacteria with the loop, either the 4- or 24-h Phadebact procedure may be utilized. Furthermore, an earlier investigation has shown the coagglutination procedure to be applicable to serogrouping streptococci directly on the colonies growing on primary plates (6). There is only one procedure for the Sero- STAT kit and this one, like that of the Phadebact direct method, employs colonies from a primary plate or from a pure culture obtained by subculture. Furthermore, its sensitivity and specificity response with a minimum of five colonies was similar to that observed with the direct-plate Phadebact procedure. In contrast to these two kits, the Streptex procedure required a greater number of bacterial colonies with its required extraction step to visualize its agglutination response. Not all primary isolation plates may contain the minimal number of colonies required for the seroidentification of streptococci with each of the respective kits. This dependence upon minimal numbers of isolated streptococcal colonies may present some limitations in the application of these kits. In these instances, the 4-h broth-culture procedure may be employed with the Phadebact reagents, or a subculture with a blood agar plate may be used to seroidentify the isolate. The major discrepancies that were detected with the Streptex kit appear to be related to the use of the mixed-culture method. This method appears to be associated with cross-reactivity between the Streptex group A and group C EVALUATION OF SEROGROUPING KITS 253 reagents and other bacteria collected in the sweep across an isolation plate. Furthermore, the relatively slower reactivity of its serogrouping reagents in conjunction with their cross-reactive potential resulted in the misidentifications or equivocal serogrouping responses observed in this investigation. Agglutination responses occurring from 1 to 2 min with Streptex were recently reported (K. C. Gross, M. P. Houghton, and L. B. Senterfit, Abstr. Annu. Meet. Am. Soc. Microbiol. 1979, C170, p. 338). The coagglutination of all the strains of S. pneumoniae tested in this investigation with the group C reagents from each of the three serogrouping kits is consistent with the fmdings of other investigators (3, 10, 11, 14). The crossreactivity of this bacteria with the Phadebact and SeroSTAT serogrouping reagents would not cause any difficulties due to the use of only isolated colonies with these two methods. This is in contrast to the Streptex kit (i.e., 4.6% misidentification) which permits the application of mixed as well as pure cultures of beta-streptococci by direct serogrouping. When employing any of these kits in the clinical laboratory, it is imperative to screen out pneumococci through careful examination of the morphology and hemolytic activity of the bacterial colonies. This protocol will reduce the cross-reactive potential for error with all these kits regarding the pneumococci (Table 4). This potential was minimal with the Phadebact and SeroSTAT serogrouping reagents because the manufacturer suggests the use of isolated colonies. In contrast, the Streptex kit permits the application of both mixed and pure culture of beta-streptococci by direct serogrouping. Certainly, the lower sensitivity of the Streptex serogrouping reagents was evident in this investigation. When compared to the Phadebact and SeroSTAT procedures, the Streptex procedures required extra labor, particularly a 1-h extraction step before using the latex agglutination reagents. Furthermore, the Streptex extraction method, as borne out in this investigation, may not yield enough group antigen for a serogrouping response and, thus, was frequently associated with false-negative responses more than with either the Phadebact or SeroSTAT kit. This was the case when fewer than 20 colonies were used for an extraction. A possible explanation for these negative responses with the Streptex kit may be associated with the vagueness of the explanation of its package insert concerning the density of the bacterial suspension required for the pronase extraction. Extraction enzymes such as Pronase B have been shown to be effective for the preparation of group-specific streptococcal antigens (5). However, this procedure re-

6 254 SLIFKIN AND POUCHET-MELVIN quires the use of chemically clean glassware and double-distilled water for an unimpeded extraction (21). Thus, although the Streptex test system, unlike the Phadebact and SeroSTAT systems, permits serogrouping group D and group F betastreptococci, it is recommended that alternative tests be additionally employed if the beta-hemolytic streptococci are other than group A, B, C, or G. Namely, group D beta-streptococci may be identified with the use of bile-esculin agar (8). At present, except for their relatively small colony size, no biochemical nor physiological test exists for the identification of group F betastreptococci (7, 8). Therefore, the precipitation method employing antisera to group F carbohydrate should be utilized if desired by the laboratory. Due to experience in the recognition of the morphology and type of beta-hemolysis of the streptococcal colonies as well as their source, particularly with the group A and group B isolates, we often initially employed the grouping reagents for these beta-hemolytic streptococci with certain isolates. Furthermore, these two serogroups were isolated in greater numbers than the other serogroups. Accordingly, it was considered a disadvantage, in contrast to the Phadebact and SeroSTAT kits, not to have separate commercial serogrouping Streptex reagents available. One SeroSTAT dispenser of latex grouping reagent became blocked with latex particles so that release of reagent was impeded. We solved this problem by opening the orifice with a pin. Loss of reactivity of one vial of group A Streptex reagent for group A streptococci was observed after 6 months of storage at 10 C. Neither of these difficulties was noted for the Phadebact kit, although slight leakage from the dispensing vials occasionally occurred. We solved this problem by lightly tapping the vial on the countertop to remove reagent from the cap. As with the SeroSTAT kit, the Phadebact kit does not require an enzymatic procedure for extraction of the group antigens. The Phadebact procedures are more versatile than those associated with the other two kits by providing not only a direct-plate and 24-h protocol, but also a 4-h method that may be employed as required by the clinical microbiologist. The cross-reacting nature of Todd-Hewitt broth manufactured by Difco was observed in this investigation. Thus, it became evidence that the 4- and 24-h Phadebact procedures should be employed only with the BBL product as recommended by the manufacturers. In summary, all three kits evaluated in this J. CLIN. MICROBIOL. investigation permitted the correct serogrouping of the majority of the clinical isolates of betastreptococci examined. In contrast to the directplate procedure, the Streptex kit offers both a direct-plate procedure on isolated as well as on mixed growth cultures. However, the Streptex direct-plate procedure on mixed cultures was more often associated with cross-reactivity. Furthermore, the Streptex test was associated with more false-negative responses than that observed with the Phadebact and SeroSTAT reagents. It was concluded that the Phadebact Streptococcus Test was the most proficient through the availability of its three procedures of serogrouping that include direct, 4-h, and 24- h, procedures. LITERATURE CITED 1. Arvilommi, H Grouping of beta-hemolytic streptococci by using coagglutination, precipitation or bacitracin sensitivity. Acta Pathol. Microbiol. Scand. Sect. B 84: Arvilommi, H., 0. Uurasmaa, and A. Nurkkala Rapid identification of group A, B, C, and G betahaemolytic streptococci by a modification of the coagglutination technique. Comparison of results obtained by co-agglutination, fluorescent antibody test, counterimmunoelectrophoresis, and precipitin technique. Acta Pathol. Microbiol. Scand. Sect. B 86: Austrian, R., C. Buettger, and M. Dale Problems in the classification and pathogenic role of alpha and nonhemolytic streptococci of the human respiratory tract, p In L. W. Wannamaker and J. M. Matsen (ed.), Streptococci and streptococcal disease. Academic Press Inc., New York. 4. Christensen, P., G. Kahlmeter, S. Jonsson, and G. Kronvall New method for the serological grouping of streptococci with specific antibodies adsorbed to protein A-containing staphylococci. Infect. Immun. 7: Ederer, G. M., M. M. Herrmann, R. Bruce, J. M. Matsen, and S. S. Chapman Rapid extraction method with Pronase B for grouping beta-hemolytic streptococci. Appl. Microbiol. 23: Edwards, E. A., and G. L Larson New method of grouping beta-hemolytic streptococci directly on sheep blood agar plates by coagglutination of specifically sensitized protein A-containing staphylococci. Appl. Microbiol. 28: Facklam, R. R Streptococci, p In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 8. Facklam, R. R., J. F. Padula, L. G. Thacker, E. C. Wortham, and B. J. Sconyers Presumptive identification of group A, B, and D streptococci. Appl. Microbiol. 27: Farrell, B., and I. Amirak Agglutination grouping of streptococci. (Letter to the editor.) Lancet ii: Finch, R. G., and I. Phillips Serological grouping of streptococci by a slide coagglutination method. J. Clin. Pathol. 30: Hahn, G., and L. Nyberg Identification of streptococcal groups A, B, C, and G by slide co-agglutination of antibody-sensitized protein A-containing staphylococci. J. Clin. Microbiol. 4: Kirkegaard, M. K., and C. R. 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7 VOL. 11, 1980 streptococci. J. Clin. Microbiol. 6: Lrm, D. V., R. D. Smith, and S. Day Evaluation of an improved rapid coagglutination method for the serological grouping of beta-hemolytic streptococci. Can. J. Microbiol. 25: Lue, Y. A., I. P. Howitt, and P. D. Ellner Rapid grouping of beta-hemolytic streptococci by latex agglutination. J. Clin. Microbiol. 8: Rantz, L. A., and E. Randall Use of autoclaved extracts of haemolytic streptococci for serological grouping. Stanford Med. Bull. 13: Rosner, R Laboratory evaluation of a rapid fourhour serological grouping of groups A, B, C, and G betastreptococci by the Phadebact Streptococcus Test. J. Clin. Microbiol. 6: Slifkin, M., C. Engwall, and G. R. Pouchet Direct-plate serological grouping of beta-hemolytic streptococci from primary isolation plates with the EVALUATION OF SEROGROUPING KITS 255 Phadebact Streptococcus Test. J. Clin. Microbiol. 7: Slifkin, M., and Pouchet, G. R Rapid carbohydrate fermentation test for confirmation of the pathogenic Neisseria using a Ba(OH)2 indicator. J. Clin. Microbiol. 5: Swift, H. F., A. T. Wilson, and R. C. Lancefield Typing group A hemolytic streptococci by M precipitin reactions in capillary tubes. J. Exp. Med. 78: Washington, J. A., II, W. J. Martin, and A. G. Karlson Identification of bacteria, p In J. A. Washington II (ed.), Laboratory procedures in clinical microbiology. Little, Brown & Co., Boston. 21. Watson, B. K., R. C. Moellering, Jr., and L. J. Kunz Identification of streptococci: use of lysozyme and Streptomyces albus filtrate in the preparation of extracts for Lancefield grouping. J. Clin. Microbiol. 1: