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We describe the protocol for an inexpensive and nondestructive optical reflectance assay for the measurement of biofilm formation. Reflectance data are obtained using an Ocean Optics (Dunedin, Florida) USB 2000 spectrometer with a polychromatic light source. A fiber optic cable is used both for illumination and collection, and Ocean Optics OOIBase32 Platinum software is used for preliminary processing of the data. Differences in reflectance data collected at times ranging from 2 to 24 h distinguish between cell attachment and volume growth for two strains of Enterococci. Confocal scanning laser microscopy imaging is used to confirm these results. Phase contrast microscopy images are also obtained in conjunction with reflectance measurements for several different biofilm specimens. The experiments consider biofilm formation on glass and polystyrene substrata, but the method can be used for many other abiotic substrata of interest, both opaque and nonopaque.
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Colcemid (0.4 microgram/ml) induced DNA synthesis in quiescent chick embryo fibroblasts, and increased the amount of F-actin. Cytochalasin D, which disorganized actin filaments, inhibited the stimulation of DNA synthesis by colcemid. These results suggest that actin filaments are involved in the sequence of events leading from the depolymerization of microtubules to the initiation of DNA synthesis. They also demonstrate that depolymerization of microtubules can influence the amount of F-actin in the cell.
Atomic force microscope-based phase imaging in air is capable of elucidating variations in material properties such as adhesion, friction, and viscoelasticity. However, the interpretation of phase images of specimens in a fluid environment requires clarification. In this report, we systematically analyzed atomic force microscope-derived phase images of mica, glass, and collagen under the same conditions as used for living cells at various tapping forces; the resulting data provide critical information for the interpretation of phase images of living cells. The peripheral regions of COS-1 cells consistently show a more negative phase shift than the glass substrate in phase images at set-point amplitude: free amplitude (Asp/A0) = 0.6-0.8. In addition, at all Asp/A0 values suitable for phase imaging, tapping frequency appears to be high enough to ensure that phase shifts are governed primarily by stiffness. Consequently, phase imaging is capable of high resolution studies of the cellular surface by detecting localized variations in stiffness. We demonstrate that phase imaging of a bifurcating fiber in COS-1 cell cytoplasm is readily capable of a lateral resolution of approximately 30 nm.
The phase separation and dewetting of thin films of blends of deuterated polystyrene (dPS) and poly(p-methylstyrene) (PpMS) were investigated during annealing. The surface morphology, obtained from atomic force microscopy and phase measurement interference microscopy, the density profile, determined by X-ray reflectivity in the region of total external reflection, and the surface composition obtained from static secondary ion mass spectroscopy, are reported. This system is only weakly incompatible. The interaction of the components with substrate and air during phase separation leads to a bilayer formation with a broad polymer-polymer interface. PpMS segregates to the air interface. The bilayer structure is unstable and defines the starting point for the dewetting of PpMS on top of the dPS layer. In the final dewetting state a homogeneous layer of dPS on top of the substrate is covered with an ultrathin layer of PpMS as well as with quite thick mesoscopic drops of PpMS.
A successful attempt has been made to identify microridges on squamous cells in routine smears using phase contrast microscopy. Microridges may be identified on mature cells of buccal mucosa and in cervical smears, either alcohol or spray fixed, and even in smears up to five years old. The features identified with phase contrast may also be observed with scanning electron microscopy. Identification of microridges may be of diagnostic interest in the evaluation of cervical smears.
Optical microscopy, ultracentrifugation, phase analysis and turbidimetric titration methods were used to study phase state and phase equilibria of quaternary water-gelatin-pectin-dextran system in the absence of salts and at pH higher than the isoionic point. It was found that these systems are two-phase ones, contrary to the single-phase behaviour of the ternary water-gelatin-pectin and water-gelatin-dextran systems under the same conditions. The observed phase separation is the result of incompatibility of gelatin with pectin, dextran molecules being distributed practically uniformly between coexisting phases. This phenomenon is rather typical for many water-gelatin-polysaccharide-1-polysaccharide-2 systems under the conditions when all the pairs of biopolymers are compatible. The high compatibility of gelatin with pectin or dextran in the ternary systems under given conditions is due to the formation of weakly bonded interpolymer complexes. The incompatibility of gelatin with pectin in the presence of dextran can be explained by the blockage of the reactive gelatin groups due to their competitive interactions with dextran.