SUMMARY1. The whole-cell patch-electrode technique of Fenwick, Marty & Neher (1982) has been applied to single suspension-cultured mouse fibroblasts. Seals in the range of 10-50 GQ were obtained without special cleaning of the cell membranes.2. Rupture of the membrane patch inside the electrode was accompanied by a shift of measured potential into the range -10 to -25 mV, but in most cases with little change in the recorded resistance. The latter fact implied that (i) the absolute resistance of the cell membrane must be in the same range as the seal resistance and (ii) the recorded potential is a poor measure of actual cell membrane potential.3. Steady-state current-voltage curves (range -160 mV to + 80 mV) were generated before and after rupture ofthe membrane patch, and the difference between these gave (zero-current) membrane potentials of -50 to -75 mV, which represents a leak-corrected estimate of the true cell-membrane potential.4. The associated slope conductivity of the cell membrane was 5-15,US/cm2 (assumed smooth-sphere geometry, cells 13-15 ,sm in diameter) and was K+-dominated.5. With 0-1 mm (or more) free Ca2+ filling the patch electrode, membrane potentials in the range -60 to -85 mV were observed following patch rupture, with associated slope conductivities of 200-400 /ZS/cm2, also K+-dominated.6. Similar voltages and conductivities were observed at the peak of pulse-induced 'hyperpolarizing activation' (Nelson, Peacock, & Minna, 1972), and the two phenomena probably reflect the behaviour of Ca2+-activated K+ channels.7. Both the pulse-induced conductance and the Ca2+-activated conductance spontaneously decayed, the latter over periods of 5-15 min following patch rupture.8. Sr2+, Ba2+, and Co2+ could also activate the putative K+ channels, but only Sr2+ really mimicked Ca2+. Co2+ and Ba2+ activated with a delay of several minutes following patch rupture, and deactivated quickly with a small decrease ofconductance and a large decrease of membrane potential. Evidently, Co2+ and Ba2+ affect channel specificity as well as channel opening and closing kinetics.

A new method of detecting single-molecule fluorescence has been implemented using a conventional optical microscope and a position-sensitive photon-counting apparatus. The samples were solvent-free fluorescent dyes adsorbed on a solid substrate in air at room temperature by spraying microdroplets of a dye solution followed by solvent evaporation. At the concentration used, each droplet contained a very small number of dye molecules. Quantized fluorescence intensity was determined and the number of occurrences of the quantized intensity agreed well with that calculated from Poisson's formula, thus confirming success of the position-sensitive single-molecule fluorescence detection.

We describe a novel method for enumeration of bacteria, based on the principle that small, light emitting particles on a flat surface can be easily and rapidly detected and counted using an ultra-high-sensitivity TV camera. To test this method, we obtained TV images of individual cells of a luminous bacterium on a membrane filter without the use of a microscope. The positions of the luminous points in the TV images were almost the same as the positions of the bacterial colonies after growth. Our results show that the single cells can be efficiently detected and counted by our method if they emit light or can be stimulated to emit light.

No abstract

No abstract