Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

The dorsoventral polarity of unequally cleaving spiralian embryos becomes established at an early stage. The factors determining the position of the dorsoventral axis are still unknown. We present data showing that the sperm entry point (SEP) in both normal development and under experimental conditions determines the position of the first cleavage furrow in Dreissena embryos. The position of the spindles at second cleavage is directed by the site of fertilisation also, and the large, dorsal D quadrant of the 4-cell stage always forms opposite the SEP. The spiral chirality at third cleavage seems to be independent of both the fertilisation point and the arrangement of the quadrants. Dextral and sinistral third cleavages are found in a single egg batch, but sinistral cleavages prevail. We postulate that two factors coordinate the proper positioning of the dorsoventral axis. The sperm entry point as an epigenetic factor determines the dorsal side of the embryo. But since the dorsoventral axis forms oblique to the first cleavage furrow, this first decision is still ambiguous, and a second decision is required that, due to the alternative chirality of spiral cleavage, finally sets up the dorsoventral axis.

A centrifugal method for extracting apoplastic sap from roots of lupin (Lupinus angustifolius) and pea (Pisum sativum) plants, and a method to analyse malic dehydrogenase in the sap using capillary electrophoresis, are described. Osmolality of apoplastic sap was relatively constant at relative centrifugal forces (RCFs) between 600 and 3000 g for lupin, and between 600 and 4000 g for pea. Electropherograms of a marker enzyme (malic dehydrogenase) and other components in apoplastic and symplastic saps revealed that contamination occurred at 7000 g. It is suggested that apoplastic sap expelled from plant roots at RCF between 600 and 3000 g is free from symplastic contamination, and is regarded as being of apoplastic origin. The proposed method was used to measure apoplastic pH changes in the plant roots in response to external pH, ammonium, nitrate and vanadate.