Identification of streptococci: use of lysozyme and Streptomyces albus filtrate in the preparation of extracts for Lancefield grouping

Self Cite

A total of 256 beta-hemolytic streptococcal isolates were subjected to serological and physiological tests to identify those which could be classified as Streptococcus milleri. S. mileri accounted for 75% of 70 group C isolates, 15% of 69 group G isolates, 75% of 16 nongroupable Isolates, and 100% of 20 group F isolates examined. No S. mileri isolates were encountered among the 90 group A streptococci studied. Of the 95 beta-hemolytic S. miller isolates examined, 81% were recovered from respiratory specimens.

Section: Methodsmentioning

confidence: 99%

Occurrence of Streptococcus milleri among beta-hemolytic streptococci isolated from clinical specimens

Ruoff¹,

Kunz²,

Ferraro³

1985

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“…quires the use of chemically clean glassware and double-distilled water for an unimpeded extraction (21).…”

Section: Slifkin and Pouchet-melvinmentioning

confidence: 99%

Evaluation of three commercially available test products for serogrouping beta-hemolytic streptococci

Slifkin¹,

Pouchet-Melvin²

1980

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.

“…Relatively large numbers of beta-hemolytic streptococci and high temperatures are required for extraction of pronase (3) and Streptomyces albus lysozyme (2,6,10). Heavy metals can inactivate pronase, and chemically clean glassware and double-distilled water must be used in its preparation.…”

mentioning

confidence: 99%

“…Heavy metals can inactivate pronase, and chemically clean glassware and double-distilled water must be used in its preparation. Pronase is not effective in the extraction of group D streptococci (4,5,10). Group D streptococci may be extracted with mutanolysin only after a 4-h incubation (1).…”

mentioning

confidence: 99%

Serogrouping single colonies of beta-hemolytic streptococci with achromopeptidase extraction

Slifkin¹,

Cumbie²

1987

Achromopeptidase (TBL-1; Wako Chemicals, USA, Inc., Dallas, Tex.) prepared as a 2,000-U/ml solution will extract the serogroup antigens from single colonies of groups A, B, C, F, and G streptococci in 1 min at room temperature. This enzyme extraction is not effective for the serogrouping of all group D streptococcus species. Achromopeptidase extracts can be used with latex or coagglutination reagents. Achromopeptidase is a bacteriolytic enzyme derived from Achromobacter lyticus (7, 8). This enzyme has been reported to lyse a relatively wide spectrum of bacteria including Micrococcus, Bacillus, Sarcina, Staphylococcus, Streptococcus, and Clostridium spp., as well as some gramnegative bacteria (Bacteriolytic Enzyme for Biochemical Use: TBL-1, Wako Chemicals, USA, Inc., Dallas, Tex.). Achromopeptidase has been used for the preparation of bacterial enzymes, DNA, and RNA (7, 8) and for the preparation of protoplasts and spheroplasts (8). The enzyme is now commercially available as a crude product (TBL-1) from Wako Chemicals, USA, Inc. This study demonstrates that achromopeptidase may be used to rapidly extract the specific antigens of groups A, B, C, F, and G streptococci from single colonies grown on sheep blood agar plates. The streptococci used in this investigation were obtained from stock cultures maintained in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) and 10% glycerol (vol/vol) at-70°C. The serogroups included groups A (15 strains), B (22 strains), C (15 strains), D (11 strains), F (8 strains), and G (10 strains). All of the bacteria used in this investigation were streaked on Columbia sheep blood agar (Scott Laboratories, Inc., Fiskeville, R.I.) and incubated at 35°C in ambient air for 12 to 18 h. Gram staining and the catalase test were done on all of the cultures to presumptively identify them as beta-hemolytic streptococci. The serogroups of the strains grown on the blood agar were confirmed by nitrous acid extraction of single colonies (9) and serogrouping with Streptex reagents (Wellcome Diagnostics, Research Triangle Park, N.C.). One colony of each strain of streptococcus was placed in 0.20 ml of 0.05 M Tris hydrochloride buffer solution (pH 8.0) containing 50 to 2,000 U of achromopeptidase per ml. The bacterial suspensions were incubated for 1 to 60 min at 37°C. In other experiments, incubations were done at room temperature. Serogrouping of the respective extracts of streptococci was done with Streptex latex reagents (Burroughs Wellcome Co., Research Triangle Park, N.C.) and the Phadebact coaggfutination reagents (Pharmacia Diagnostics, Piscataway, N.J.). A drop of each extract was placed on a glass microscope slide and mixed with a drop of the respective serodiagnostic reagent. The reactants were mixed by hand for 1 min to determine the agglutination or coagglutination response of each extract.