None of the procedures currently used for producing cell-free filtrates provided preparations containing all of the extracellular products that the culture was able to produce. Even the membrane-filter technique failed to yield such preparations unless the cultures were grown on dialysis membranes laid over Brain Liver Heart Agar plates at 37 C for 48 h in an atmosphere containing 10% CO2 in air and the growth from 25 such membranes was harvested in 12.5 ml of phosphate-buffered saline. These preparations contained detectable quantities of coagulase, phosphatase, DNAse, RNAse, lipase, esterase, egg yolk factor, lysozyme, gelatinase, staphylokinase, and caseinase and were able to hemolyze human, horse, rabbit, and sheep erythrocytes. Broth and semisolid cultures, however, yielded more units of beta hemolysin than did the membrane cultures.
Diethylstilbestrol (DS) has been shown to be active against staphylococci and other gram-positive bacteiia but not against gram-negative microorganisms. The present study extends these findings. Standardized suspensions of '4C-labeled Staphylococcus aureus serotypes III and IV and Shigella flexneri were prepared and exposed to pharmacological concentrations of DS (1 to 20 ,ung/ml) under diverse environmental conditions; the cells were removed by membrane filtration and the presence of radioactive substances in release to the supernatant fraction was followed by standard radioisotopic techniques. Controls were exposed similarly to the hormone vehicle alone (buffer containing 2% ethyl alcohol). DS at bactericidal concentrations above 6 ,g/ml caused significant leakage of cellular radioactivity of S. aureus labeled with 'IC-glucose and 14C-glutamic acid within 1 to 4 hr after exposure to DS. Maximum leakage of radioactivity occurred under anaerobic conditions at 37 C. Absorption studies of "C-labeled DS indicated that the affinity of S. flexneri for DS is markedly less than that of S. aureus. This might be one reason was added and the cells were incubated for 1 hr at 37 C on a rotary shaker. The 14C-labeled cells were har-474
Diethylstilbestrol (DS) has been shown to be active against staphylococci and other gram-positive bacteria but not against gram-negative microorganisms. The present study extends these findings. Standardized suspensions of
14
C-labeled
Staphylococcus aureus
serotypes III and IV and
Shigella flexneri
were prepared and exposed to pharmacological concentrations of DS (1 to 20 μg/ml) under diverse environmental conditions; the cells were removed by membrane filtration and the presence of radioactive substances in release to the supernatant fraction was followed by standard radioisotopic techniques. Controls were exposed similarly to the hormone vehicle alone (buffer containing 2% ethyl alcohol). DS at bactericidal concentrations above 6 μg/ml caused significant leakage of cellular radioactivity of
S. aureus
labeled with
14
C-glucose and
14
C-glutamic acid within 1 to 4 hr after exposure to DS. Maximum leakage of radioactivity occurred under anaerobic conditions at 37 C. Absorption studies of
14
C-labeled DS indicated that the affinity of
S. flexneri
for DS is markedly less than that of
S. aureus
. This might be one reason for the resistance of gram-negative bacteria to DS.
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