Instruments and supplies

A new device Inoclic™ based on classical platinum wand-type inoculator system was studied for inoculation of a wide range of culture media. Inoclic™ device appears a convenient and valid alternative to agar diffusion or broth dilution to determine susceptibility of aerobic pathogens.

New device INOCLIC™ for inoculum preparation and standardization, and new rapid medium MHR-SIR™ were tested for Staphylococcus aureus A.S.T. Sixty five different clinical strains of S. aureus, including fifteen Methicillin-Resistant S. aureus (MRSA) strains, were tested using two different methods for inoculum standardization: Inoclic device (i2a, Montpellier, France) versus standard 0.5 McFarland suspension according to CLSI guidelines. And two different culture media: classical Mueller-Hinton medium (MH, i2a, Montpellier, France) versus fast Mueller-Hinton medium (MHR-SIR™, i2a, Montpellier, France).

We here show that hypoxia induces a marked shift of the CSC fraction towards EMT leading to altered cell morphology, an increased proportion of CD44high/ESAlow cells, patterns of gene expression typical of EMT, and enhanced sphere-forming ability. The size of EMT fractions returned to control levels in normoxia indicating a reversible process. Surprisingly, however, even under normoxic conditions a fraction of EMT CSCs was present and maintained high levels of HIF-1a, apparently due to actions of cytokines such as TNFa. Functionally, this EMT CSC fraction showed decreased mitochondrial mass and membrane potential, consumed far less oxygen per cell, and produced markedly reduced levels of reactive oxygen species (ROS). These differences in the patterns of oxygen metabolism of sub-fractions of tumour cells provide an explanation for the general therapeutic resistance of CSCs and for the even greater resistance of EMT CSCs. They also identify potential mechanisms for manipulation of CSCs.

The increased sensitivity of molecular assays means that a greatly reduced initial sample volume may still be useful in pathogen detection. Therefore, investigations were undertaken to assess the suitability of a swab transport system rather than faeces for the diagnosis of viral gastrointestinal disease. The use of swab based systems reduces the amount of sample required and can negate the need to perform pre-extraction procedures that may be time consuming, costly and involve hazardous chemicals.