Antitoxin-in-Membrane and Antitoxin-in-Well Assays for Detection of Toxigenic Corynebacterium diphtheriae

The Elek culture plate precipitin test is routinely used for the detection of exotoxin from toxigenic strains of Corynebacterium diphtheriae. Recently, the World Health Organization standardized this test to ensure accuracy, reliability, and reproducibility. In this study, we further modified the standard Elek test by using the antitoxin-in-membrane (AIM) and antitoxin-in-well (AIW) approaches. In the AIM tests, each strain was stabbed and streaked backwards and away from a point approximately 7 mm from the edge of a sterile cellulose acetate-cellulose nitrate filter membrane disk (pore size, 0.45 μm; diameter, 25 mm) containing 25 IU of diphtheria antitoxin. For AIW tests, a central well (diameter, 5 mm) containing 9 μl of antitoxin (4.5 IU) was surrounded by eight equidistant stab-streaks of each strain placed 10 mm from the well. In both methods, precipitin bands of identity typically were noted after 24- and 48-h incubations at 37°C. Both toxigenic and weak toxigenic strains gave clear and reproducible results. Compared with the standard Elek test, the AIM and AIW tests each use 50% less medium and 75 and 87% less antitoxin, respectively. AIM has the potential to test up to 14 isolates and AIW has the potential to test up to 24 isolates on the same plate. Furthermore, clearer positives were noted with weak toxigenic strains. In a blinded test of 209 verified C. diphtheriae isolates, a 99.5% agreement with the standard Elek test was obtained overall. Both modifications conserve reagents and medium, permit the simultaneous testing of a larger number of strains, and may be particularly suitable for reference laboratories or hospitals involved in diphtheria epidemic settings.

Substrate surface influences upon germination of Colletotrichum truncatum conidia

Substrates influenced germination of conidia of the mycoherbicide, Colletotrichum truncatum. Very few (< 10%) conidia germinated when incubated while suspended in water or when incubated on or in partially liquefied agar. Many (> 70%) were induced to germinate when incubated on firm agar. The percentage of germinated conidia increased as agar firmness increased. Not all solid substrates equally influenced germination. Over 50% of the conidia germinated on chromatography paper, cellulose acetate filter, or on hard (plastic cover slip) substrates. In contrast, germination was relatively poor on cellulose nitrate filter and glass cover slips. Some natural substrates of dissimilar texture (wood, live plant leaf) produced good germination. Lower O2 levels or limited gas exchange for submerged conidia did not prevent germination because many conidia germinated while submerged on or between firm substrates. Subjecting conidia to motion during incubation between glass cover slips favored germ tube production rather than appressoria formation. Evidence suggests a positive germination response of C. truncatum conidia to solid substrates that occurs prior to the well-documented thigmotropic response of germ tubes of germinated conidia. Key words: Colletotrichum truncatum, conidia germination, thigmotropism, substrate.

The selective eosinophil chemotactic activity of histamine.

Histamine diphosphate was shown to selectively attract human eosinophils from mixed granulocyte populations when over 20% eosinophils were used in a modified Boyden chamber chemotactic assay system. This effect of histamine is abolished by incubation with diamine oxidase (histaminase) and was generated by decarboxylation of L-histidine. A linear dose dependent increase in eosinophil migration was observed between 3 X 10(-7) M and 1.25 X 10(-6) M, while higher concentrations of histamine inhibited the migration of eosinophils. The attractant activity of histamine was not inhibited by H-1 or H-2 receptor antagonists, however, the inhibition of migration observed at higher histamine concentrations was reversed by metiamine, an H-2 receptor antagonist. The effects of histamine upon eosinophil migration were demonstrable using three different assays: (a) counting cells that had traversed 5-mum pore, 12-mum thick polycarbonate filters, (b) counting cells that had migrated various distances into a 3-mum pore, 145-mum cellulose nitrate filters, or (c) measuring the number of cells that had traversed an upper polycarbonate filter and migrated into a lower cellulose nitrate filter using 15Cr-labeled cells. The ability of histamine to enhance eosinophil migration was shown to be dependent upon the presence of a concentration gradient; histamine did not cause a dose-dependent increase in random motility. Furthermore, preincubation of the eosinophils with histamine deactivate the cells to further stimulation by histamine or by C5a. It is concluded that in low doses histamine is a chemoattractant for human eosinophils, while in higher doses histamine inhibits eosinophil migration. These observations may relate to the influx and localization of eosinophils in immediate hypersensitivity reactions.