A method is described for the preparation of protoplasts of Streptococcus mutans BHT. The muralytic enzyme mutanolysin was prepared free of contaminating proteinases and shown to completely dissolve cell walls of this strain. Whole cells were converted to stabilizable protoplasts by using the enzyme in an isotonic medium containing 40% raffinose. Experiments using [3H]thymidine and [I4C]leucine as cytoplasmic pool markers revealed only minimal (10%) leakage during a 1-h incubation. Examination by electron microscopy revealed the apparent absence of structural cell wall on the enlarged spherical bodies. Quantitative chemical analyses of membranes prepared by lysing protoplasts demonstrated only very small amounts of rhamnose and trace amounts of galactose. These sugars are the principal components of the BHT cell wall polysaccharide. Also, there were only small amounts of peptidoglycan components (e.g., N-acetylglucosamine) in the purified membranes obtained by this method.

Thirty strains of streptococci were tested for lysis with lysozyme, and 29 of these could be lysed by the following method: (i) suspension of the cells to a Klett reading of 200 units (no. 42 filter) in 0.01 M tris(hydroxymethyl)aminomethane buffer, pH 8.2, after washing twice with the buffer; (ii) addition of lysozyme to a final concentration of 250 ug/ml with incubation for 60 min at 37 C; (iii) addition of sodium lauryl sulfate (SLS) to a final concentration of 0.2% and incubation up to an additional 15 min at 37 C. Significant lysis was obtained only after the addition of SLS. (Strains of groups A, E, and G were treated with trypsin at a concentration of 200 Ag/ml for 2 hr at 37 C before exposure to lysozyme.) These parameters for optimal MATERIALS AND METHODS Bacteria.

Hydroxy-interlayered vermiculite (HIV) is a ubiquitous phyllosilicate in the <0.05-mm fraction of sandy soils on the U.S. southeastern coastal plain. Extensive areas of soils with abundant HIV (i.e., peninsular Florida) have no detectable mica; yet the coarseness, platy habit, and nonexchangeable K associated with HIV grains suggest a mica precursor. The objectives of this study were: (1) to probe for mica zones (1.0-nm) within HIV grains, using high-resolution transmission electron microscopy (HRTEM), and (2) to determine intragrain elemental distributions via electron microprobe analysis (EMA). HIV grains from a Quartzipsamment medium-silt fraction, which contained no detectable mica by X-ray diffraction (XRD), were concentrated via high-density liquid separation. EMA transects and X-ray dot maps showed zonation or trends of K depletion near edges of some grains, with K2O contents ranging from trace levels to >40 g kg-1. Elemental oxide data indicated a dioctahedral phyllosilicate structure, with some octahedral substitution of Fe and Mg for Al. Intermittent 1.0-nm lattice-fringe images obtained by HRTEM supported the presence of mica zones within grains. There were no detectable 1.4-nm fringes, despite the dominance of a 1.4-nm XRD peak, indicating the instability of the HIV specimen under the electron beam. Results support a transformational link between mica and HIV in these soils. Rapid incursion and polymerization of Al following loss of K from mica may limit the extent of the vermiculite intermediate. The latter idea is consistent with the paucity of vermiculite in Florida soils. Traces of occluded mica may be the last remnants of the precursor grain. A sand-sized mica precursor would likely have weathered in place during the period when colloidal components such as kaolinite illuviated to deeper zones. Thus, the transformation product (HIV) would comprise a significant proportion of the <0.05-mm fraction persisting in sandy eluvial horizons.

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Mutans streptococci (MS) representing eight different serotypes were tested for their ability to coaggregate in vitro with oral actinomyces and other streptococcal species. Of the mutans streptococci tested, only strains of S. cricetus (formerly S. mutans serotype a) displayed pronounced coaggregations and only with certain strains of actinomyces. S. cricetus coaggregated, by lactose nonreversible mechanisms, with serotype 4 Actinomyces naeslundii WVU963 and WVU924 and with serotype 2 Actinomyces odontolyticus WVU758. The first pair was disaggregated by protein denaturants (e.g., sodium dodecyl sulfate and urea) and EDTA. This coaggregation was inhibited when the streptococcal, but not the actinomyces, partner was pretreated with either heat or protease, suggesting the presence of a protein mediator on only the streptococcal cell surface. The S. cricetus-A. odontolyticus coaggregation appeared to involve protein components on each cell, as shown by the lack of coaggregation after pretreatment of either cell type with heat or proteases. This coaggregation was also reversed by sodium dodecyl sulfate and urea, as well as by sodium deoxycholate, but not by EDTA. The data indicate that different mechanisms may be involved in each of these coaggregations. * Corresponding author. nism) in gnotobiotic rats (27). In many of the studies, however, MS were included in the battery of coaggregation test organisms but either no intergeneric coaggregations were observed with MS (5,27,29,39) or the reactions were weak and not studied further (11,13,31).Since MS, along with Streptococcus sanguiis and Streptococcus mtzitis, are considered to be early colonizers of tooth KH2PO4. and Tween 80 (26) and supplemented with 0.1% 2695 on August 3, 2020 by guest http://iai.asm.org/ Downloaded from

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We undertook a morphological and immunochemical comparison of purified outer membrane antigens from oral and nonoral isolates of Bacteroides asaccharolyticus. Electron micrographs of thin sections of whole bacteria revealed a compact, electron-dense capsule external to the outer membrane of oral strains. A loose, web-like material was noted on the surface of several nonoral strains that was distinct from the dense capsule seen on oral strains. Polyacrylamide gel electrophoresis showed distinct differences in the protein band pattern between oral and nonoral isolates; sugar composition was similar with a few exceptions. An indirect fluorescent-antibody test utilizing antiserum to a purified capsular antigen from a single oral strain cross-reacted with all of numerous oral and nonoral strains of B. asaccharolyticus, thereby demonstrating a shared antigen that is species specific for B. asaccharolyticus. However, antibodies to an oral strain-derived capsular antigen were detectable by enzyme immunoassay only in serum from rabbits immunized with oral strains. Thus, definite morphological and immunochemical differences were found between oral and nonoral isolates of B. asaccharolyticus.

A method is described for lysing cariogenic and noncariogenic streptococci with lysozyme. It suggests that the lysozyme may affect oral populations of streptococci.