Electron probe microanalysis (EPMA) measurement of thin-film thickness in the nanometre range.

The thickness of thin films of platinum and nickel on fused silica and silicon substrates has been determined by EPMA using the commercial software STRATAGEM for calculation of film thickness. Film thickness ranged in the order 10 nm. An attempt was made to estimate the confidence range of the method by comparison with results from other methods of analysis. The data show that in addition to the uncertainty of the spectral intensity measurement and the complicated fitting routine, systematic deviation caused by the underlying model should be added. The scattering in the results from other methods does not enable specification of a range of uncertainty, but deviations from the real thickness are estimated to be less than 20%.

[Effects of serums and plasmas of various species on a protease of Staphylococcus aureus. Human alpha2-macroglobulin as an inhibitor (author's transl)]. / Wirkungen von Seren und Plasmen verschiedener Spezies auf eine Protease von Staphylococcus aureus. Humanes alpha2-Makroglobulin als Inhibitor.

Serums and plasmas from various animals and man inhibited 85-100% of the activity of a purified protease from Staphylococcus aureus, strain M 135 (table 1). The inhibitory effects of corresponding serums and plasmas were approximately equal. The inhibition in human serum was caused by alpha2-macroglobulin. On the other hand, alpha1-antitrypsin proved to be ineffective (table 2). The complex alpha2-macroglobulin-protease could be separated by vertical polyacrylamide discelectrophoresis. Immunelectrophoretic analysis revealed no changes by the staphylococcal protease neither on alpha2-macroglobulin, nor on other human serum proteins. Only fibrinogen was split into at least 2 components (fig. 1).

[Purification of protease from staphylococcus aureus (author's transl)]. / Reinigung einer Protease von Staphylococcus aureus.

380 (80%) of 475 Staphylococcus aureus cultures isolated from humans, cattle and dogs were proteolytically active either on casein or gelatin or both (table 1). Protease-activity could also be demonstrated in experimental body-cavities of rabbits (fig. 1). The enzyme-activity was estimated with azocasein. Protease from S. aureus, M 135 precipitated from the culture supernatant with ammonium sulfate at 65% saturation (table 2). It was purified by 2 filtrations on Ultrogel AcA 44 (fig. 2,3) and subsequent isoelectric focusing between pH 3.5-7.0 (fig. 4). The purified protease yielded only 1 line in the SDS-polyacrylamidegel-electrophoresis, in the gelatin-polyacrylamidegel-electrophoresis and in the double immuno-diffusion test (fig. 5). Its isoelectric point was at pH 4.6, and its highest proteolytic activity between pH 7.5-8.3. The molecular weight was estimated by SDS-polyacrylamidegel-electrophoresis to be near 29.000. The protease-activity was completely inhibited in the presence of EDTA, partially inhibited by Cu2+ and Zn2+ and increased by Mn2+ (table 3).

[Demonstration and partial purification of staphylokinase (author's transl)]. / Bestimmung und partielle Reinigung von Staphylokinase

In these studies the fibrinogen plate medium proved to be not sufficiently specific for the demonstration of staphylokinase (SAK). For this purpose we modified the azocasein-test by the addition of plasminogen. Protease, on the other hand, we measured without plasminogen. This enabled us to differentiate between the 2 enzymes. After precipitation with ZnCl2 from the culture supernatant of Staphylococcus aureus strain, V8, SAK could be prepurified by filtration on ultrogel AcA 44 (tab. 1, fig 1). A more than 100-fold increase in specific SAK-activity (in comparison to that in the culture supernatant) to 34.722 units/mg protein was achieved after 2 X isoelectric focusing between pH 3.5-10.0 and refocusing between pH 5.0-7.0 (fig 2). The partially purified SAK was free of protease, coagulase, beta- and delta-hemolysins. DNase, lipase and phosphatases, but it contained minor amounts of alpha-hemolysin. It revealed only 1 band in the SDS-polyacrylamide-gel-electro-phoresis and 1 precipitin-line in the double immunodiffusion test with an antiserum against the SAK preparation after ultrogelfiltration.

[Influence of "clumping factor" from Staphylococcus aureus on phagocytosis (author's transl)]. / Einfluss des "Clumping Factors" von Staphylococcus aureus auf die Phagozytose

In phagocytosis experiments the pathogenic significance of the "clumping factor" (CF) of Staphylococcus aureus was studied. CF-positive staphylococci were engulfed considerably less by leukocytes from rabbits and cattle than CF-negative staphylococci. Equally, the engulfment of staphylococcal skeletons (SSk) coupled with partially purified CF was significantly less than that of the SSk. The SSk had been prepared by extraction of staphylocci with formic acid. By this method the SSk were freed of all substances associated with staphyloccal virulence. Before coupling with CF the SSk were activated by cyanogen bromide. These in vitro-observations could be confirmed by the corresponding phagocytosis studies in experimental body cavities of rabbits. Thus, CF apparently interfered with the engulfment of the staphylococci by the leukocytes.

[Effects of staphylococcal leucocidin in mice (author's transl)]. / Wirkungen von Staphylokokken-Leukozidin bei Mäusen

After intraperitoneal injections of mice with paraffin-oil and sodium caseinate the numbers of macrophages and granulocytes increased respectively as well in their peritoneal exsudate as in their blood. On the other hand, cyclophosphamide markedly decreased the numbers of leucocytes. All mice were more susceptibel to infections with leucocidin (PVL-R)-positive staphylococci than to those with leucodidin-negative staphylococci of comparable hemolytic properties. Generally, mice with increased numbers of leucocytes were relatively more resistant to the staphylococcal infections. Concentrated PVL-R-leucocidin injected intraperitoneally into mice caused a temporary decrease in the number of leucocytes, especially granulocytes and their phagocytic activity.