Color change of pH indicators in broth medium is commonly used to quantify growth of ureaplasmas. These organisms differ from other members of the Mollicutes by their abiity to hydrolyze urea to C02 and NH3. This study describes a method which continuously monitors color change in ureaplasmal broth cultures. Using this technique we found: (i) there was a pH-dependent absorbance at 554 nm in ureaplasmal broth medium containing phenol red, (ii) a sigmoidalshaped color changing curve (absorbance at 554 nm versus time) was produced by metabolizing organisms whereas a linear curve was generated by antibioticinhibited ureaplasmas, and (iii) the minimum cell density which elicited a growthinhibited color change was 1.25 x 104 colony-forming units per ml. Others have shown that apparently dead ureaplasmas can cause a color change in broth media. This color change is probably due to the presence of an active urease. This study graphically and quantitatively assesses growth-inhibited color change.
A competitive inhibition enzyme immunoassay for the detection of Streptococcus pyogenes directly from throat specimens or from solid bacteriological medium is described. Group A-specific polysaccharide adsorbed onto treated polystyrene beads, in conjunction with rabbit antibody to S. pyogenes, was used to determine the presence of the polysaccharide antigen. Inhibition values in excess of 65% were observed with 104 or more CFU of S. pyogenes per test. An inhibition of 25% was demonstrated with as few as 103 CFU per test. Heterologous microorganisms tested at 106 CFU per test reacted at levels of inhibition less than 25%. Two types of bacterial transport medium and swabs of different fiber compositions did not alter the assay performance. Accurate identification of S. pyogenes was achieved by testing single colonies picked directly from blood agar plates which had been incubated for 18 to 24 h. In addition, the assay was performed on throat specimens from children and adults having pharyngitis. A single-swab, blind study was conducted in which enzyme immunoassay reactivity was compared with results of blood agar culture and bacitracin sensitivity. When there were discordant results, serological identification was used as the confirmatory test. At an optimal cutoff value of 40% inhibition, sensitivity and specificity by enzyme immunoassay were 97.0% and 97.9%, respectively, as compared with confirmed culture results. The assay has an incubation time of 3 h and is a sensitive and specific method for the detection of S. pyogenes antigen.
Three streptococcal pyrogenic exotoxins (SPEs), designated as SPE A, B, and C, have been purified and characterized. Routine purification of the SPEs includes the technique of isoelectric focusing. An earlier study showed that the removal of commercial carrier ampholytes (Ampholines) from SPE was difficult. The physicochemical properties of SPE C were previously reported; however, the SPE C preparation used in those experiments was contaminated with Ampholines. As an alternative to Ampholines, we used simple buffers to generate the isoelectric focusing pH gradient and used this SPE C in a comparative study to evaluate the effects of Ampholine contamination on the biological and biochemical properties of this toxin. We found that Ampholine contamination overestimates protein concentration; consequently, the biological activity of SPE C was actually greater than reported. The most serious effect of Ampholines in SPE C was on amino acid analysis. The presence of Ampholines causes an apparent increase in neutral amino acids and a decrease in basic amino acids.
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