Rapid Extraction Method with Pronase B for Grouping Beta-Hemolytic Streptococci

The enzyme, Pronase B, was used to extract the C carbohydrates of betahemolytic streptococci for serological grouping. Reference strains of streptococci, groups A through N, were accurately grouped by using this extraction method. Of the beta-hemolytic streptococci isolated from patients' cultures, 1,197 were classified as either group A or not group A by this method. There was 100% correlation with the reference Lancefield method. In contrast, false reactions occurred with the presumptive bacitracin disc method in 4.1% of the group A and 12.7% of the non-group A cultures. The Pronase method proved simple, accurate, and readily adaptable to the clinical laboratory routine.     

Serological Grouping of Hemolytic Streptococci by Counter-Immunoelectrophoresis

The counter-immunoelectrophoresis (CIE) method of grouping streptococci was more sensitive than the capillary precipitin method. The precipitate was easier to read, and the test was simple to perform and required fewer reagents. An autoclave-Pronase-B extraction procedure is described and was found superior to either acid, Pronase-B, or the autoclave extraction procedures for recovering groupable polysaccharide from different strains of streptococcal groups A, B, C, D, F, and E. Data obtained from over 400 strains indicate that the sensitivity of the CIE and a more efficient extraction of groupable polysaccharide provides a simple method for rapid diagnosis of streptococcal infections.     

New Method of Grouping Beta-Hemolytic Streptococci Directly on Sheep Blood Agar Plates by Coagglutination of Specifically Sensitized Protein A-Containing Staphylococci

A technique is described that allows the grouping of beta-hemolytic streptococci directly upon the primary colony. This was accomplished by applying a small drop of specifically sensitized protein A-containing Staphylococcus aureus over a colony of streptococci, rocking the plate to allow mixing of the particles with the soluble group-specific polysaccharide, which in the case of beta-hemolytic streptocicci was produced in abundance during colony formation, and observing for agglutination of the sensitized particles. Such a simple test for group A beta-hemolytic streptococci should allow accurate identification of group A streptococci in small laboratories, such as in clinics or physicians' offices, as well as in the larger public health and private laboratories.     

Simple Procedure for Production by Group C Streptococci of Phage-Associated Lysin Active Against Group A Streptococci

Phage-associated lysin of high potency was prepared by growing the host group C streptococcal strain 26RP66 in a semisynthetic medium. The lysin was stabilized by adding dithiothreitol and neutralized ethylenediaminetetraacetic acid (EDTA) to facilitate further concentration and partial purification. The lysin remained active when stored at -65 C for 1 year. Lysin was active against all strains of group A streptococci tested and was more active against living cells than heat-killed cells. The procedure outlined is practicable for most bacteriological research laboratories and does not require column purification or other complex biochemical procedures. It should be useful to any laboratory which requires small amounts of lysin to produce L-forms and protoplasts or to release streptococcal antigens.     

Comparative Grouping of Mastitis Streptococci by Means of Co-Agglutination and Precipitation

Two hundred bovine mastitis streptococcal strains belonging to groups B, C, E, G and L were tested comparatively by means of the co-agglutination and precipitation technique. Identical results were obtained with the two tests in 191, or 95.5 %, of the strains. Six strains, which could not be grouped by co-agglutination, proved to belong to group B when grouped by precipitation. On the other hand, one strain which proved to belong to group G and two strains to group L when grouped by co-agglutination, could not be grouped by precipitation. Some cross-reactivity was observed between groups A and C, B and G, B and L. Only a few L strains showed marked cross-reactivity which was not easily distinguished from specific reactions. However, the cross-reactivity ought to be eliminated by dilution or adsorption. Using the precipitation test as a supplementary method, the easy and rapid co-agglutination test was found to be a suitable procedure for routine grouping of mastitis streptococci.     

Grouping of Lactic Streptococci by Gel Electrophoresis of Soluble Cell Extracts

Soluble cell proteins obtained from 35 strains of lactic streptococci were examined by gel electrophoresis. A mathematical analysis of the densitometer scans of the gels enabled strains to be grouped according to their overall similarity. Strains which were known to be variants of the same parent strain fell into the same group, supporting the validity of the method. It is suggested that strains which are alike according to their gel electrophoretic patterns when grown under standard conditions have an overall phenotypic similarity and that this indicates a similarity in genotype. The relevance of this to selection of strains of lactic streptococci for cheesemaking is discussed.     

Serological Relationships of Type I Antigens of Group B Streptococci

Some of the complex antigenic relationships of type I group B streptococci from various clinical sources were defined by means of immunodiffusion, absorption, and precipitin tests. Three predominant types are described: Ia, Ib, and Ii. Methods for preparing antisera for differentiating type I strains are presented.     

Simplified Fluorescent-Antibody Staining Method for Primary Plate Isolates of Group A Streptococci

Beta-hemolytic streptococci were identified from primary plates by using a simplified fluorescent-antibody staining method. There was 100% correlation between this method and standard precipitin grouping.     

Extraction of Cell-Wall Polysaccharide Antigen from Streptococci.

Slade, Hutton D. (Northwestern University Medical School, Chicago, Ill., and Max-Planck Institut für Immunbiologie, Freiburg, Germany). Extraction of cell-wall polysaccharide antigen from streptococci. J. Bacteriol. 90:667-672. 1965.-The carbohydrate grouping antigens in the cell walls of streptococci belonging to groups A, E, G, L, and T were extracted with 5% trichloroacetic acid at 90 C. The antigens were removed also from dry whole cells by extraction with trichloroacetic acid followed by treatment with phenol-water. Details of the methods are presented. The antigens obtained by use of either of these procedures were suitable for studies on immunological specificity and chemical structure. Quantitative enzymatic and chemical analyses of two group E antigens and one group T preparation showed the presence of l-rhamnose (22 to 44%), d-glucose (7 to 22%), d-galactose (T antigen only, 26%), glucosamine (2 to 16%), and galactosamine (T antigen only, 3%). In addition, analyses of A and G antigen preparations are presented. The protein and phosphate content of the A and E antigens were about 1% each. Quantitative precipitin curves of these antigens are presented.     

Simplified Gas Chromatographic Procedure for Identification of Bacterial Metabolic Products

A rapid and simple procedure is described for analysis of fermentation products from anaerobic bacteria grown in glucose broth. A 1-ml sample of the culture is drained through cation-exchange resin in a Pasteur pipette. The effluent fluid is directly analyzed isothermally in a gas chromatograph for volatile fatty acids (C(2) to C(6)) as well as for lactic, pyruvic, and succinic acids. This procedure is considered to be suitable for routine use in clinical bacteriology.     

Simplified Procedure for Preparation of Specific Antibodies to Gamma Globulins

Antibodies were prepared to rhesus monkey, rabbit, dog, and hamster gamma globulins which had been purified by agar-block electrophoresis. Immunoelectrophoretic analysis demonstrated that these antisera were specific for gamma globulin components in whole serum. The use of these antisera as secondary serological reagents was examined.     

A Simplified Procedure for Preparation of Undecalcified Human Bone Sections

A new type of apparatus for sectioning samples of hard, undecalcified bone is described. Slices of fresh or archeological human bone 4-5 mm thick are dehydrated and then embedded in epoxy resin. The apparatus used to prepare sections from the resulting blocks consists of a low-speed rim-type diamond cut-off wheel and a slowly advancing table carrying the specimen held in a rotating mount. Sections may be cut at a thickness of 80 μm ± 1%. After cleaning in an ultrasonic bath, these can be mounted on slides for quantitative microscopic examination with transmitted light. Grinding and polishing are not necessary. The results obtained are illustrated.     

A Simplified Procedure for Isolation of Golgi Apparatus from Rat Liver

A simplified method is described for isolation of Golgi apparatus from rat liver. The procedure provides high yields of intact, enzymatically active preparations relatively free from contamination by other cell components. When large volume, swinging bucket rotors are employed, it is possible to process livers from 10–12 rats in a single run of less than 2 hours.     

Simplified Procedure for the Isolation of Intact Chloroplasts from Chlamydomonas reinhardtii

A simple procedure that yields highly purified intact chloroplasts from Chlamydomonas reinhardtii is described. This procedure involves breakage of cell wall-deficient cells by passing them through a narrow bore syringe needle. The intact chloroplasts are then purified from the crude homogenate by differential centrifugation and Percoll gradient centrifugation. This procedure generates relatively high yields of chloroplasts capable of CO₂ fixation. These chloroplasts were characterized by electron microscopy, marker enzyme analysis, and ferricyanide exclusion. Transmission electron microscopy indicates that these chloroplasts retain their pyrenoids and eyespots. Scanning electron microscopy confirms that the characteristic cup shape of C. reinhardtii chloroplasts persists in vitro. This rapid, inexpensive procedure produces chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts.     

Procedure for Drying Leptospiral Antibody on Sand and Sugar for Serological Studies in Leptospirosis

A simple technique is described for drying sera on washed, dry sand or ordinary sugar cubes for the sero-diagnosis of leptospirosis. The results are shown to be very similar to those obtained with fluid sera or sera dried on filter paper discs. Sera adsorbed on sand or absorbed in sugar eliminated some of the problems associated with sera dried on paper. This method is suitable for use in the field and is expected to be of value in epizootiological studies where contamination and chemical denaturation of fluid serum samples held without refrigeration is a problem.     

Enrichment Procedure for Use with the Membrane Filter for the Isolation and Enumeration of Fecal Streptococci in Water

By the use of PYC enrichment medium, the recovery of fecal streptococci from river water has been increased more than twofold over that of M-Enterococcus Agar. Of the isolates tested, 94.6% could be classified as enterococci or enterococcus biotypes. This method seems to yield a larger number of strains which would not normally be revealed. Serological typing of atypical streptococcus strains isolated indicates that the majority of these “biotypes” can be placed in the enterococcus group.