Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood.

Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5-10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.

Techniques for clearing ovules for studies of megagametophyte and early postfertilization development in Rhododendron.

Using ovule clearing, more than 33,600 ovules of Rhododendron nuttallii T. W. Booth (Ericaceae) were examined for megagametophyte and early postfertilization stages at daily intervals from anthesis until 3 weeks after pollination. Pretreatments with amyloglucosidase to digest integumentary and gametophyte starch and Stockwell's bleach to remove tannins from the integumentary epidermis were necessary. Ovules were cleared by a combination or modifications of Herr's four-and-a-half or Stelly's hemalum-methyl salicylate techniques and were observed using differential interference contrast optics. The method proved suitable for large-scale quantitative studies of ovule development and fertilization. A general protocol is suggested as a starting point for ovule clearing studies.

Post-pollination callose development in ovules of Rhododendron and Ledum (Ericaceae): zygote special wall.

In Rhododendron spp. and Ledum groenlandicum a callose wall is laid down around the zygote in the first 2 days after fertilization. The periodic acid/Schiff-positive, aniline blue-fluorescence-positive callosic wall is initiated adjacent to the degenerating synergid, extends to cover the entire zygote surface, and remains visible during the initiation of embryogeny as the zygote elongates before the first proembryonal division. Unfertilized ovules show eventual callose deposition in the ovule wall cells during senescence in undeveloped abscising pistils, but show no development of callose within the embryo sac. Possible roles of a zygote special callose wall are discussed.