(80:20 [vol/vol]) in a 10-liter fermentor (54). The mineral medium was autoclaved under H2 plus C02 gas at a pressure of 35 kPa for 40 min. After cooling, the medium was sparged with H2 plus CO2. One hour before inoculation, 10 ml of a sterile solution of 20% Na2S 9H20 was added. The inoculum was 2 to 5% of the working volume and was in the exponential growth phase. During growth, the gas pressure inside the vessel was maintained at 140 kPa with an H2 plus CO2 flow rate of 9 to 18 liters h-1. The stirring rate was 240 to 300 rpm. An additional 10 ml of sterile 20% Na2S * 9H20) was added when the A660 of the culture reached 0.5. Cells were harvested in the early stationary phase with a Sharples continuous-flow centrifuge and stored under N2 gas at -20°C. In some experiments, 1-liter portions of the culture were anaerobically harvested throughout growth. A 1-liter stoppered bottle that had been sitting in an anaerobic chamber overnight was connected to the fermentor outlet with tubing and a syringe needle. The culture was forced into the bottle by the pressure in the fermentor. During this procedure, pressure inside the-bottle was vented by using a second syringe needle. Cells were then transferred to 500-ml centrifuge bottles in the anaerobic chamber and centrifuged at 11,000 x g for 20 min at 325

A procedure was developed for the enrichment of auxotrophs in the antibiotic-insensitive archaebacterium Methanococcus. After mutagenesis with ethyl methanesulfonate, growing cells were selectively killed upon exposure to the base analogs 6-azauracil and 8-azahypoxanthine for 48 hr. Using this method, eight independent acetate auxotrophs of Methanococcus maripaludis were isolated. Six of the auxotrophs had an absolute growth requirement for acetate and contained 1-16% of the wild-ype levels of CO dehydrogenase. Three of these six also contained 14-29% of the wild-type levels of pyruvate oxidoreductase and 12-30% of the wild-type levels of pyruvate synthase. Two spontaneous revertants of these latter auxotrophs regained the ability to grow normally in the absence of acetate and wild-type levels of CO dehydrogenase, acetyl-CoA synthase, pyruvate oxidoreductase, and pyruvate synthase. Likewise, a spontaneous revertant of an auxotroph with reduced levels of CO dehydrogenase and wild-type levels of pyruvate oxidoreductase regained the ability to grow normally in the absence of acetate and wild-type levels of CO dehydrogenase and acetyl-CoA synthase. Two additional auxotrophs grew poorly in the absence of acetate but contained wild-type levels of CO dehydrogenase and pyruvate oxidoreductase. These results provide direct genetic evidence for the

Routine glaucoma screening for middle-aged African American individuals is potentially clinically effective but its impact on visual impairment and blindness may be modest. However, we did not assess the impact on visual field loss.

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