Detection of Neisseria gonorrhoeae by ultraviolet illumination of NADH generated by an immobilized antibody-gonococcal enzyme complex.

Frozen, cervical swabs were placed in a lysis buffer containing an immobilized antibody to the gonococcal enzyme, 1,2-propanediol oxidoreductase. The immobilized antibody--enzyme complex that formed was active after the addition of substrate (1,2-propanediol and NAD) and this activity could be detected by visual inspection of NADH fluorescence under ultraviolet illumination.

Rapid estimation of biochemical oxygen demand.

The feasibility of using columnar reactors containing immobilized microorganisms for the rapid estimation of BOD was demonstrated in this study. Dilutions of three types of industrial effluents were tested by the BOD5 test and by this experimental system. A high degree of correlation (r = 0.98) was observed between results of the two tests. The mean standard error of estimation of the experimental system was 11%.

Enzymatic detection of Neisseria gonorrhoeae.

In a study using a non-serological enzymatic approach for the detection of Neisseria gonorrhoeae in cervical and urethral swabs, the technique was shown to be technically feasible. The enzyme, 1, 2-propanediol oxidoreductase, was used as a presumptive diagnostic marker for N gonorrhoeae. Enzymatic activity was measured with a fluorometer. Two assay procedures were performed: (a) enzyme detection (two-tube and three-tube assays) requiring 60 minutes; and (b) enzyme inhibition (EI) (90-minute and modified 20-minute assays). Sensitivities of the two-tube, three-tube, and the 90-minute EI assays with male urethral specimens from a high-prevalence population were 80%, 84%, and 91% respectively. The specificities of these assays in a low-prevalence male population were not determined. Sensitivity of the 90-minute EI assay in a high-prevalence female group was 77% and specificity in a low-prevalence female group was 75%. The modified EI assay was tested only in a low-prevalence female group and had 87% specificity. Although the specificity of the assays needs improvement, several advantages--including early case detection, rapid availability of results, detection of current active infections, and the possibility of automation--are intrinsic in this enzymatic approach.

The isolation and characterization of a 1,2-propanediol oxidoreductase from Neisseria gonorrhoeae.

An enzyme which oxidizes 1,2-propanediol in the presence of NAD+ has been purified from lysates of Neisseria gonorrhoeae. The enzyme was activated by monovalent cations, had a pH optimum between 9 and 10, and showed a substrate specificity unlike any known alcohol or glycerol dehydrogenase. The enzyme had an apparent Km of 17 mM for 1,2-propanediol and 0 . 37 mM for NAD+. When chromatographed on a Sephadex G-150 column, the enzyme eluted as a single peak in the molecular weight region of a bovine serum albumin marker. An antibody to the purified enzyme was prepared in goats. When antiserum was reacted with the enzyme in immunodiffusion experiments, a single precipitin band was detected. When the enzyme was mixed with an excess of antibody and then reacted with substrate, enzyme activity was completely inhibited.

The occurrence of 1,2-propanediol oxidoreductase in micro-organisms and its use as a possible diagnostic marker for Neisseria gonorrhoeae.

The cervical microbial flora of 25 females and stock cultures of various micro-organisms which may be present in the human female cervix were examined using a fluorimetric assay for 1,2-propanediol oxidoreductase. Results indicated that only members of the genera Neisseria and Acinetobacter possess appreciable activities of the enzyme, whose physiological function is not yet known. The activity of this enzyme in N. gonorrhoeae appeared to be significantly higher than the activities observed in mot of the other Neisseria species and in the Acinetobacter species. These results indicated that it may be possible to utilize this enzyme as a presumptive diagnostic marker for N. gonorrhoeae in cervical secretions. 1,2-Propanediol oxidoreductase may also be of taxonomic significance for the classification of various bacterial species.

Applications of fluorophore-containing microbial growth media.

Media containing the fluorogenic compound 8-anilino-1-naphthalene sulfonic acid may be used to discriminate between gram-positive and gram-negative bacteria and to differentiate between various species of bacteria. Fluorescent light emitted from colonies of gram-negative bacteria on 8-anilino-1-naphthalene sulfonic acid-containing agar was visually more intense than that on gram-positive bacteria. The emitted light from the gram-negative bacteria differed in wave-lengths from that of light emitted by colonies of gram-positive bacteria. The fluorescent intensity of colonies on complete 8-anilino-1-napthalene sulfonic acid agar supplemented with 1% of single substrates varied depending on the bacterial species, thus allowing the development of profiles used to identify 12 different species.

Constricted tube system for presumptive identification and differentiation of group D streptococci.

A single, constricted tube containing two differential media to identify and differentiate group D streptococci was developed. Test results with a limited number of group D streptococcal isolates were in complete agreement with results of conventional procedures.