Loading...
2 results
Search Results
Now showing 1 - 2 of 2
- The use of DRAQ5 to monitor intracellular DNA in Escherichia coli by flow cytometryPublication . Silva, Filomena; Lourenço, Olga; Vaz, Cidália Pina; Rodrigues, Acácio Gonçalves; Queiroz, João; Domingues, F.C.Flow cytometry provides a rapid and high-content multiparameter analysis of individual microorganisms within a population. In the past years, several fluorescent stains were developed in order to monitor DNA content distribution and cell-cycle phases, mainly in eukaryotic cells. Recently, due to its low detection limits, several of these fluorescent stains were also applied to prokaryotic cells. In this study, the ability of a novel far-red fluorescent stain DRAQ5 in assessing intracellular DNA content distribution in Escherichia coli DH5alpha was evaluated. The results showed that a DRAQ5-labelled live E. coli suspension can be obtained by incubation of 1 x 10(6) cells/mL with 5 microM DRAQ5 in PBS buffer supplemented with EDTA (pH = 7.4) during 30 min at 37 degrees C. Flow cytometric analysis of fixed E. coli cells revealed that ethanol should be used in detriment of glutaraldehyde for DRAQ5 labelling. After the analysis of RNase and DNase digested samples, DRAQ5 was proven to be a specific DNA labelling stain. The present study demonstrates that the use of DRAQ5 as a DNA-labelling stain provides an easy assessment of intracellular DNA content and cell-cycle phases in gram-negative bacteria such as E. coli.
- Anti-Candida activity of a chitosan hydrogel: mechanism of action and cytotoxicity profilePublication . Oliveira, Ana Palmeira de; Ribeiro, MP.; Oliveira, Rita Manuela Palmeira de; Gaspar, Carlos; Oliveira, Sofia Costa de; Correia, I.J.; Vaz, Cidália Pina; Oliveira, José António Martinez Souto de; Queiroz, João; Rodrigues, AcácioCandida spp. are common causative agents of mucocutaneous infections. New therapeutic antifungal drugs are needed to treat chronic disease as these are frequently clinically resistant to azols. Chitosan, among other possible vehicles for active compounds, shows an added value as it appears to have intrinsic antimicrobial properties. The aim of the present study was to evaluate the anti-Candida activity of a medium-molecular-weight chitosan hydrogel (CH), to clarify its possible mechanism of action and to evaluate its cytotoxicity on human fibroblasts. CH antifungal activity was assessed according to CLSI reference M27-A3 protocol; its mechanism of action was investigated by flow cytometry, and its cytotoxicity was studied by MTT assay. CH demonstrated a full inhibition of C. tropicalis, C. krusei, C. guilliermondii and C. parapsilosis growth while impairing C. albicans and C. glabrata viability. Flow cytometry tests showed that CH acts by inducing primary lesion of the cytoplasmic membrane. However, CH showed no cytotoxic effect upon human fibroblasts cells. Resistant strains will require new therapeutic approaches. Chitosan being a good carrier and having itself anti-Candida activity seems to be a promising vehicle to be used for the treatment of mucocutaneous candidosis.