Purity Of Strains Research Articles

Combination of Phenotypic Tests as a Screening Approach for the Differentiation of Cryptic Species Candida albicans and Candida dubliniensis

Background: Candida albicans, an important pathogen for humans and animals, shares phenotypic features with Candida dubliniensis, leading to misidentification. Thus, the goal of this study was to apply a combination of phenotypic tests for the differentiation of these cryptic species. Methods and findings: Thirty-seven azole-resistant C. albicans from animals and 03 C. dubliniensis from humans were included in this study. Purity of strains was evaluated on CHROMagar Candida™, on which both species present green colonies. Then, phenotypic characterization was performed based on the growth pattern on sunflower seed agar, where C. albicans presents smooth colonies and C. dubliniensis presents rough colonies, and esterase production on Tween 80™ agar, which is positive for C. albicans, forming an opacification zone, and negative for C. dubliniensis. Molecular differentiation was performed with primers CALF/CALR for the amplification of ITS1/ITS2 regions of rDNA, yielding an amplicon of 100 bp for C. albicans, but not for C. dubliniensis. Of the 37 C. albicans, 35 showed green colonies on CHROMagar Candida Medium™, 36 presented smooth colonies on sunflower seed agar, while 34 showed an opacification zone on Tween 80™ agar. PCR yielded 100-bp-amplicons for all 37 C. albicans strains, confirming their identification. Control strains of C. dubliniensis showed the expected phenotypic features and amplicons were not obtained for the specific PCR reaction. Conclusions: The phenotypic methods used were not absolutely effective, however, the combined observation of smooth colonies on sunflower seed agar with an opacification zone on Tween 80™ agar leads to a reliable presumptive phenotypic identification of C. albicans.

DNA amplification fingerprinting as a tool for checking genetic purity of strains in the cyanobacterial inoculum

The electrophoretic patterns for 17 different cyanobacterial cultures derived from 6 different decamer primers were analysed to provide diagnostic fingerprints for each culture and their genetic distances based on RAPD markers.The primer OPB 09 produced a maximum of 24 amplified products. The primers OPB 09, OPG 04 and OPAH 02 generated markers specific for Nostoc cultures. Westiellopsis was found to be distinct from other cyanobacterial cultures in the RAPD profile obtained with the primer OPAH 02. The primer OPF 03 generated specific markers for Tolypothrix tenuis. Fischerella cultures could be identified with the primers OPB 09, OPAG 03 and OPF 05. The study revealed that these RAPD markers could be further used to identify and establish the genetic purity of the strains in the cyanobacterial inoculum. There was a similarity of 60–90% within Westiellopsis cultures. Nostoc cultures shared 50–80% similarity with Westiellopsis cultures. Anabaenacultures were similar to Westiellopsiscultures by 60–70. The markers produced for each culture were also applied to phylogenetic analysis to infer genetic relatedness in this group of prokaryotes. The dendrogram analysis clearly revealed that free-living cyanobacterial cultures are closely related to each other and are distinct from the symbiotic forms.

Myelin-associated glycoprotein and complementary axonal ligands, gangliosides, mediate axon stability in the CNS and PNS: Neuropathology and behavioral deficits in single- and double-null mice

Complementary interacting molecules on myelin and axons are required for long-term axon–myelin stability. Their disruption results in axon degeneration, contributing to the pathogenesis of demyelinating diseases. Myelin-associated glycoprotein (MAG), a minor constituent of central and peripheral nervous system myelin, is a member of the Siglec family of sialic acid-binding lectins and binds to gangliosides GD1a and GT1b, prominent molecules on the axon surface. Mice lacking the ganglioside biosynthetic gene Galgt1 fail to express complex gangliosides, including GD1a and GT1b. In the current studies, CNS and PNS histopathology and behavior of Mag-null, Galgt1-null, and double-null mice were compared on the same mouse strain background. When back-crossed to >99% C57BL/6 strain purity, Mag-null mice demonstrated marked CNS, as well as PNS, axon degeneration, in contrast to prior findings using mice of mixed strain background. On the same background, Mag- and Galgt1-null mice exhibited quantitatively and qualitatively similar CNS and PNS axon degeneration and nearly identical decreases in axon diameter and neurofilament spacing. Double-null mice had qualitatively similar changes. Consistent with these findings, Mag- and Galgt1-null mice had similar motor behavioral deficits, with double-null mice only modestly more impaired. Despite their motor deficits, Mag- and Galgt1-null mice demonstrated hyperactivity, with spontaneous locomotor activity significantly above that of wild type mice. These data demonstrate that MAG and complex gangliosides contribute to axon stability in both the CNS and PNS. Similar neuropathological and behavioral deficits in Galgt1-, Mag-, and double-null mice support the hypothesis that MAG binding to gangliosides contributes to long-term axon–myelin stability.