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Anaerobic buffer 8 ; , which was modified by the addition of Na2S 9H20 2.1 mM ; and L-cysteine-HCl 1.8 mM ; and adjusted to a pH between 6.8 and 7.0. Preliminary experiments had indicated that N. ovalis could survive for at least several hours in the modified buffer results not shown ; . Six sealed serum bottles 18-ml volume ; , each containing 2 ml of the final hindgut suspension, were incubated at room temperature, after addition to duplicate samples of 1 ml anaerobic buffer containing sodium acetate 50 mM ; or formic acid 217 mM ; as methanogenic substrate. To two bottles, 1 ml of anaerobic buffer without an additional methanogenic substrate was added. The contents of all bottles were adjusted to pH 6.8 prior to incubation. After flushing of the bottles with N2 gas and addition of 0.2 ml of ethane, methane contents in the bottles were determined in the same way as described for the intact cockroaches. To compensate for daily fluctuations, the procedure was repeated four times. All methane values are expressed in volume milliliters ; produced 1 atm [1 atm 101.29 kPa], 25C ; . Metronidazole treatment. In one of the cockroach cultures culture V ; , the antiprotozoal drug metronidazole Flatyl ; was added to the drinking water at a concentration of 0.1 mg ml. Both short-term days 1 to 8 ; and long-term after 3 months ; effects of metronidazole treatment on methane production and on number of N. ovalis cells in hindguts were studied. The short-term effects were studied in cockroaches which were taken from cockroach culture IV and were subsequently treated in a separate container. Determination of ciliate numbers. Ciliate enumeration was done on homogeneous samples obtained by suspending the hindguts of four insects into 1 ml of buffer 8 ; and cutting them into small pieces. After thorough rinsing of the hindgut fragments, ciliates were fixed by addition of formaldehyde to a final concentration of 4%. The number of N. ovalis cells was determined microscopically by using a counting chamber 0.2 mm deep ; . The results obtained by this method were checked several times by counting all N. ovalis cells in a 20-, ul diluted hindgut suspension. Ciliate counting was repeated several times on different days to compensate for possible daily fluctuations. Average hindgut volume for cockroaches from each culture was estimated by determining the total volume of four to six pooled hindguts. Microscopy. The presence of methanogenic endosymbionts was checked by means of Leitz epifluorescence microscopy, as described by Doddema and Vogels 6 ; . The same technique was used to study free-living methanogens in hindgut contents. For transmission electron microscopy, ciliate cells were first isolated from the hindgut content by means of micropipettes. Cells were then fixed at 0C in 3% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 ; and postfixed on ice in 2% OS04 in the same buffer. 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