Use of self-assembled monolayers of different wettabilities to study surface selection and primary adhesion processes of green algal (Enteromorpha) zoospores.

We investigated surface selection and adhesion of motile zoospores of a green, macrofouling alga (Enteromorpha) to self-assembled monolayers (SAMs) having a range of wettabilities. The SAMs were formed from alkyl thiols terminated with methyl (CH(3)) or hydroxyl (OH) groups or mixtures of CH(3)- and OH-terminated alkyl thiols and were characterized by measuring the advancing contact angles and by X-ray photoelectron spectroscopy. There was a positive correlation between the number of spores that attached to the SAMs and increasing contact angle (hydrophobicity). Moreover, the sizes of the spore groups (adjacent spores touching) were larger on the hydrophobic SAMs. Video microscopy of a patterned arrangement of SAMs showed that more zoospores were engaged in swimming and "searching" above the hydrophobic sectors than above the hydrophilic sectors, suggesting that the cells were able to "sense" that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of microorganisms to substrata having different wettabilities.

Colloidal gold immunolabeling of immunoglobulin-binding sites and beta antigen in group B streptococci.

We have characterized the immunoglobulin A (IgA)-Fc-binding properties and beta-antigen expression of several strains of group B streptococci by using ultrastructural immunocytochemistry. Colloidal gold-labeled tracers were used with intact and sectioned bacteria in order to gain information regarding the location and distribution of cell surface and cytoplasmic IgA-Fc-binding molecules and beta antigen. Colloidal gold (5- or 15-nm particles) was conjugated to IgA to characterize IgA-binding properties and to IgG to test for IgG binding. Rabbit anti-beta antiserum was reacted with the bacteria and then with protein G labeled with 15-nm colloidal gold particles. A double-labeling technique was used for simultaneous localization of IgA-Fc- and anti-beta-antibody-binding properties on sectioned bacteria. The data corroborated previous results which indicated that (i) IgA-Fc-binding and IgA-Fc-nonbinding forms of beta antigen can be secreted by strains which do not express beta antigen on the cell surfaces (HG806, VC75); (ii) differences in levels of expression of beta antigen and/or IgA-Fc-binding proteins can be detected among various group B isolates; (iii) group B streptococci do not express human IgG-Fc-binding proteins; and (iv) not all forms of beta antigen are capable of binding human IgA.

Intergeneric bacterial coaggregations involving mutans streptococci and oral actinomyces.

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.

Evaluation of anti-inflammatory agents using a model of granuloma formation evoked by divinyl copolymer beads in the mouse lung.

We have developed and quantitated a reproducible standardized granulomatous inflammatory reaction using divinyl copolymer beads. Approximately 10000 gas sterilized beads (43-53 micron in diameter) are injected into the tail veins of mice and embolize to the lungs where they evoke granuloma formation which is maximal at 48 h. The anti-inflammatory effects of both steroidal and nonsteroidal agents, namely, bacterial levan, hydrocortisone acetate, polyanetholsulfonate, indomethacin, acetylsalicylic acid, ellagic acid, and aminophylline were determined by comparing granuloma size in treated animals with those in untreated controls. Granulomas in paraffin sections were traced on the ground glass screen of a light microscope and the area of each granuloma measured with a digitizer-computer programmed to prepare histograms and merge data from replicate experiments. Of the agents tested, the greatest reductions in granuloma size occurred after treatment with bacterial levan (71%), hydrocortisone (70%), polyanetholsulfonate (58%), and indomethacin (55%).

Assessment of tissue reactions to biomaterials: a model system using the foreign body response in the mouse lung to intravenously injected divinyl copolymer beads.

We have developed a reproducible model of granuloma formation in the mouse lung using a narrow size range (45-53 micron) of divinyl benzene copolymer beads as a standard test material. Approximately 10,000 beads are given by intravenous injection into the tail veins of mice whence they embolize to the lung and incite granuloma formation. This delivery system eliminates the superimposed inflammatory reaction due to trauma that occurs when materials are directly implanted at the test site by surgical incision. Granuloma size was quantitated at intervals from 3 h to 6 weeks by tracing mid-bead granuloma areas on the ground glass screen of a light microscope at a known magnification and measuring the areas with a digitizer interfaced with a microcomputer programmed to prepare histograms and to merge data from replicate experiments. At 3 h only a few polymorphonuclear leukocytes could be observed adhering to the beads. At 48 h, the time of maximum granuloma size (mean area = 7501 micron2), the granulomas consisted of both polymorphonuclear and mononuclear leukocytes. After 6 weeks, the granulomas were smaller, composed predominantly of mononuclear leukocytes and had a mean area of 2893 micron2. This model system allows the analysis of a large number of measurements, is reproducible, and provides a useful method for comparing the hosts' inflammatory response to a variety of potential biomaterials, as well as for determining the effectiveness of antiinflammatory drugs.

Mutanolysin-induced spheroplasts of Streptococcus mutants are true protoplasts.

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 [14C]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.

Immunochemical differences between oral and nonoral strains of Bacteroides asaccharolyticus.

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.

Quantitation of particles in the freeze-fractured nuclear membrane after renal ischemia.

Changes in the number and sizes of membrane-associated particles have been quantitated in the protoplasmic (P) and exoplasmic (E) fracture faces of the outer membrane of nuclei isolated from the inner cortex following renal ischemia and reflow in the rat. No changes were observed in the inner nuclear membrane. After 20-min ischemia, the number of particles in both fracture faces decreased. With reflow, the total number of particles decreased after both 20- and 60-min ischemia. The partition coefficient (Kp = CPF/CEF) increased from 10 to 11 and 17 at 20- and 60-min ischemia then fell below control values to a Kp of 7 after 120 min. After reflow, Kp steadily decreased except after 20-min ischemia followed by 240-min reflow when Kp began to rise. The sizes of particles were predominantly 60 A in the P face of control outer membranes but became larger after ischemia. After 20- and 60-min ischemia with reflow, the size distribution became more normal. The shifts in particle numbers and sizes seem to indicate modifications within the membrane resulting from ischemia.

Ultrastructural, physiological, and cytochemical characterization of cores in group D streptococci.

Cores are large, rod-shaped structures that have been found almost exclusively in group D streptococci, measure 0.1 to 0.16 mum in diameter, and extend the width or length of cells. This study has shown that cores are produced in the cells at a reproducible point in early stationary growth after extensive mesosomal formation and after the pH has dropped below 6.5. When cells containing cores were introduced into a fresh medium with a pH above 6.5, the structures disappeared within 5 min. The structures were not found in young, logarithmically growing cells but formed in these cells upon autolysis or treatment with penicillin. Cores that were forming or disintegrating appeared to have a lamellar substructure. When chloramphenicol was added to the medium before the culture reached stationary phase, no cores were found in the cells. Cytochemical studies indicated that cores contain protein and are not composed of cell wall material or other polysaccharides that contain 1,2-glycol groups.

Plasma membrane changes in freeze-fractured rat kidney cortex following renal ischemia.

Ischemia was produced in the inner cortex of the rat kidney by clamping the pedicle (artery, vein, and ureter) and severing collateral connections. After 30 minutes of ischemia, a slight aggregation of membrane-associated particles was observed in the freeze-fractured plasma membranes. The aggregation was progressive after 60 and 120 minutes of ischemia. These changes were reversible after 15 and 240 minutes of reflow of blood following 30 or 60 minutes of ischemia. The changes were irreversible after 120 minutes of ischemia. The cells were vacuolated after ischemic periods of 30 minutes or longer and after 120 minutes of ischemia the tissue was severely damaged and aggregation of membrane-associated particles was evident in the vacuolar membranes. No changes in the tissue or plasma membrane were observed after 5 or 20 minutes of ischemia.

Tridimensional ultrastructure of freeze-fractured rat kidney cortex (studied in stereo).

The tridimensional ultrastructure of the inner cortex of rat kidney has been studied by observing freeze-fractured tissue in stereo. The complex structure of the tubules, fenestrated capillaries and glomeruli is more readily observed through the irregular fracturing of the tissue that occurs in this technique. The ultrastructure of the brush border and interdigitating membranes of the proximal tubules, the structure of the fenestrated endothelial membranes of the capillaries and the ribbon-like appearance of the filtration space between the foot processes of the glomeruli were especially well depicted in stereo images of freeze-fractured renal tissue.

Lysis of grouped and ungrouped streptococci by lysozyme.

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 mug/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 mug/ml for 2 hr at 37 C before exposure to lysozyme.) These parameters for optimal lysis of streptococci by lysozyme were established by testing the group D Streptococcus faecalis strain 31 which lyses readily with lysozyme and the group H strain Challis which is less susceptible to the action of the enzyme. Viability of S. faecalis decreased 96% after 3 min of exposure to 250 mug of lysozyme per ml, whereas the more resistant strain Challis retained 27% of the initial viability after the same period. After 60 min, there was almost total loss of viability in each case. Variations of three methods of lysing streptococci with lysozyme were compared with respect to the decrease in turbidity and the release of protein and deoxyribonucleic acid (DNA) effected by each variation. The method presented in this paper allowed the greatest release of these cytoplasmic constituents from S. faecalis and strain Challis. Transformation experiments using DNA obtained from strain Challis (streptomycinresistant) by this method showed that the DNA released is biologically active.