A new genus of cellulolytic, gram-negative, nonsporeforming, anaerobic bacteria. is described. The colonies produced by these bacteria on cellulose agar were round, clear, translucent, and cream-colored and had an undulate margin. Single cells of the bacterium were straight to slightly curved rods 0.5 to 0.8 pm wide by 4 to 10 pm long and were motile by means of a single flagellum located one-third of the distance from the end of the cell. Among the various substrates tested, only cellulose, cellobiose, and salicin were able to support growth. The major fermentation products of cellobiose or cellulose degradation were acetic acid, hydrogen, and carbon dioxide. The deoxyribonucleic acid base composition of the type species was 38 mol% guanine plus cytosine. The name Acetiuibrio is proposed for this new genus, which is placed in the family Bacteroidaceae. The type species, Acetiuibrio cellulolyticus sp. nov., is named on the basis of its cellulolytic activity. The type strain of A. ceZZulolyticus is CD2 (= NRC 2248).A cellulolytic anaerobe from a methanogenic enrichment culture from municipal sewage sludge (6, 7) is described. This gram-negative, monotrichous rod produces acetic acid, hydrogen, and carbon dioxide from native cellulose, cellobiose, or salicin. No other carbohydrates tested were utilized. The isolate is described here under the name Acetiuibrio cellulolyticus gen. nov., sp. nov. and is assigned to the-family Bacteroidaceae as emended in 1976 (14). MATERIALS AND METHODSMedia. The basal medium has the following composition (mg/liter, wt/vol): NaHCO:i, 2,060; NHIC1, MgSOl. 7Hn0, 120; 61; 21; N(CH,COOH)a, 15; NaC1, 10; MnSOJ -HnO, 5; CoCln. 6H20,1; ZnS04 -7H20,l; CuSo4-5H20,O.l; AlK(SO4)s-12H20, 0.1; H3B03, 0.1; Na2Mo04 -2H20, 0.1; pyridoxine HC1, 0.1; thiamine HC1, 0.05; riboflavin, 0.05; nicotinic acid, 0.05; p-aminobenzoic acid, 0.05; lipoic acid, 0.05; biotin, 0.02; folic acid, 0.02; vitamin B12, 0.005; and resazurin, 1. This medium was found to produce the maximum and most rapid cellulose digestion in the original methanogenic mixed cultures (8). For isolation and culture maintenance, the basal medium was supplemented with 1 g of cellulose per liter prepared from absorbent cotton as described by Hungate (5) and 250 mg of cysteine-hydrochloride and 250 mg of Na2S.9H20 (4) per liter as reducing agents. This medium, referred to as cellulose broth, was prereduced by the Hungate (5) technique, dispensed in 10-ml amounts in 60-ml serum vials (12) under 80% N2-2076 COz and autoclaved at 15 lb/in2 for 15 min. For the preparation of solid media, the basal medium was supplemented with 18 g of agar, 2 g of cellulose, and 680 KnHP04, 296; KHzP04, 180; (NH3)nS04, 150; t Issued as NRCC no. 17999. 0.25 g of cysteine-hydrochloride per liter, and the pH was adjusted to 7.5. The medium was autoclaved aerobically at 15 lb/inL for 15 min and cooled to 50°C, and 0.25 g of cysteine-hydrochloride (20 ml of a 1.25q cysteine-hydrochloride solution adjusted to pH 10 and autoclaved under oxygen free N,) was added aseptically...

Strains of Methanospirillum hungatii, when treated with dithiothreitol at alkaline pH, formed spheroplasts which lysed in the absence of an osmotic stabilizer. Other methanogenic bacteria showed no response to this treatment, including Methanobacterium thermoautotrophicum. Methanobacterium strain M.o.H., Methanobacterium strain G2R, and Methanosarcina barkeri. The reaction with M. hungatii proceeded slowly at pH 8.0, but increased dramatically up to a pH of 10. Spheroplasts did not form in the presence of MgCl2 or if large amounts of FeS precipitate had been deposited on the cells during growth. Ultra-thin sections of M. hungatii GPI confirmed that individual cells, surrounded by a membrane and an inner wall, were contained within an outer wall or sheath. Adhesive material, which stained in the region of the cell spacer, was observed to bind together the inner and outer wall layers. After treatment with dithiothreitol, the spheroplasts, which retained the flexible inner wall and membrane, were released from the rigid outer wall. Examination of the isolated outer cell wall revealed that dithiothreitol (and dodecyl sodium sulfate) dissolved the adhesive material and damaged the cell spacer at the attachment site to the outer wall. It is proposed that dithiothreitol at alkaline pH loosens both the cell spacer and inner wall attachments to the outer wall, thereby allowing the osmotic pressure of the cytoplasm to eject the spheroplast. Resistance of the cells to lysis by Triton X-100 is lost upon spheroplast formation, indicating that protection to the cell is conferred by the outer cell wall.

Bacterial contamination of blood components remains a significant problem in transfusion medicine. The Pall enhanced bacterial detection system (Pall eBDS) detects the presence of bacteria in leucodepleted platelet concentrates by measuring the reduction of oxygen in the sample, due to aerobic bacterial growth. Pooled platelet concentrates were spiked at 10 cfu mL(-1) with 10 organisms (one species per bag). Pall eBDS pouches were inoculated with the spiked platelet concentrates. After 24 and 30 h of incubation, the oxygen level was measured. A further set of pouches were taken from the inoculated platelet concentrates at 24 h. Incubation and reading intervals were as for the initial set of pouches. A sensitivity study was also performed comparing the Pall eBDS with the BacT/ALERT system. Spiking at 10 cfu mL(-1) and immediately sampling into Pall eBDS pouches resulted in 97.6 and 100% detection after an incubation period of 24 and 30 h, respectively. After 24 h of incubation of the spiked platelet concentrates and then sampling into Pall eBDS pouches, 99.1% detection was obtained after incubation for both 24 and 30 h. The sensitivity of the Pall eBDS and BacT/ALERT is similar and in the order of 1 cfu mL(-1). Implementation of either BacT/ALERT or Pall eBDS for routine screening of platelet concentrates has the potential to further increase the safety of the blood supply.

A new mesophilic, cellulolytic species of Bacteroides was isolated from a methanogenic cellulose enrichment culture of municipal sewage sludge and is described. This species ferments only cellulose and cellobiose. The fermentation products are acetic acid, C02, H2, ethanol, and small amounts of lactic acid. The deoxyribonucleic acid base composition is 43 mol% guanine plus cytosine. The name Bacteroides cellulosolvens is proposed. Type strain WM2 is deposited in the National Research Council of Canada culture collection as strain NRCC 2944.A mesophilic coculture consisting of a cellulolytic anaerobe and a saccharolytic anaerobe was isolated from a cellulose enrichment culture started from municipal sewage sludge. These two anaerobes grew on cellobiose agar as a single, slimy colony (6). The saccharolytic member of this coculture, Clostridium saccharolyticum, has been isolated and described previously (13). In this paper, we describe the isolation and characteristics of the cellulolytic microbe and propose for it the name Bacteroides cellulosolvens. MATERIALS AND METHODSMedia. The basal medium used for isolation and maintenance of the strain which we studied had the following composition (in milligrams per liter); NaHC03, 2,000; MgSO, * 7H20, 120; CaC12 -2H20, 61; FeS04 * 7H20, 21; N(CH2COOH)3, 15; NaC1, 10; M n S O 4 * H 2 O , 5 ; cocl2 -6H20, 1; ZnSO4 7H20, 1; c u s o 4 -5H20, 0.1; AIK(S04)2 12H20, 0.1; H3B03, 0.1; Na2Mo04 * 2H20, 0.1; pyridoxine hydrochloride, 0.1; thiamine hydrochloride, 0.05; riboflavin, 0.05; nicotinic acid, 0.05; p-aminobenzoic acid, 0.05; lipoic acid, 0.05; biotin, 0.02; folic acid, 0.02; vitamin BI2, 0.005; and resazurin, l ( 7 ) . This medium was prepared by the procedure of Holdeman et al. (3) and was prereduced by the Hungate technique (3, using 250 mg of cysteine hydrochloride per liter and 250 mg of Na2S -9H20 per liter as the reducing agents. The reduced medium was dispensed under 80% N2-20% C 0 2 in 10-ml amounts into 60-1111 serum vials (11) containing preweighed amounts of insoluble substrates, such as cellulose. The vials of media were then autoclaved at 104 kPa for 15 min. The soluble substrates were filter sterilized and were injected by means of hypodermic syringes into the basal medium after the vials had been autoclaved and cooled. The final pH of all media was 7.0 * 0.2. The isolated strain was maintained at 35°C in basal medium containing approximately 0.5% (wt/vol) cellulose in the form of small squares of 4-ply facial tissue.Isolation. All isolation procedures were conducted anaerobically in serum vials or in an anaerobic chamber.Biochemical tests. The procedures of Holdeman et al. (3) were used for biochemical characterization. Selected substrates were added to the basal medium at concentrations of 1% (wthol). When a test required the addition of a fermentable substrate (e.g., sulfate reduction medium), 0.5% cellobiose was used. The test media were inoculated with 0.05 ml of NH4C1, 680; K2HP04, 296; KH2P04, 180; (NH4)2S04, 150; * Corresponding author. t National Resea...

The molecular weight of lysozyme has been redetermined by the ultracentrifuge as 14,100 ± 500. The molecule appears to be a stubby prolate ellipsoid of rotation, with a maximum length of 90 Å and a minimum equatorial diameter of about 18 Å depending on the degree of hydration assumed. Alternative possibilities of chain orientation within this shape are discussed assuming that the chains are coiled in Pauling–Corey helices.