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We reviewed 100 patients retrospectively following primary ACL reconstruction with quadruple hamstring autografts to evaluate the incidence and factors associated with postoperative stiffness. Stiffness was defined as any loss of motion using the contra-lateral leg as a control. The median delay between injury and operation was 15 months. The incidence of stiffness was 12% at 6 months post-reconstruction. Both incomplete attendance at physiotherapy (p0.005) and previous knee surgery (p0.005) were the strongest predictors of the stiffness. Anterior knee pain was also associated with the stiffness (p0.029). Factors that failed to show a significant association with the stiffness included associated MCL sprain at injury (p=0.32), post-injury stiffness (p=1.00) and concomitant menisectomy at reconstruction (p=0.54). Timing of surgery also did not appear to influence the onset of stiffness (median delays: 29 months for stiff patients; 14 months for non-stiff patients). The rate of stiffness fell to 5% at 12 months postreconstruction, without operative intervention.

Researchers compared different methods of calculating kinetic parameters of friction-braked cycle ergometers, and the subsequent effects on calculating power outputs in the Wingate Anaerobic Test (WAnT). Three methods of determining flywheel moment of inertia and frictional torque were investigated, requiring "run-down" tests and segmental geometry. Parameters were used to calculate corrected power outputs from 10 males in a 30-s WAnT against a load related to body mass (0.075 kg•kg−1). Wingate Indices of maximum (5 s) power, work, and fatigue index were also compared. Significant differences were found between uncorrected and corrected power outputs and between correction methods (p

SummaryThe cellulolytic activity of cell-free extracts of 15 species of entodiniomorphid protozoa grownin vitroand under two conditionsin vitrohas been measured using six different assays with substrates varying in complexity from carboxymethylcellulose to microcrystalline cellulose. Although there were differences between the assays, the highest activities were found inEudiplodinium maggii, Epidinium ecaudatum caudatumandOstracodinium obtusum bilobumwith little or no activity in fiveEntodiniumspp. There was no consistent effect of growth conditions on cellulase content although with protozoa grownin vitrothose grown on grass alone tended to contain more cellulase than those grown on starch and grass.A survey of methods for the release of cellulase from other rumen fractions was made and mild treatment with ultrasound of the fraction suspended in water gave the maximal release of enzyme with the highest specific activity. Using this method 62% of the total rumen cellulase was found in the protozoal fraction and 27% was associated with the plant debris in a normal sheep, fed on hay and oats, with an A-type protozoal population.

SUMMARYThe distribution of carboxymethylcellulase (CMCase) between various rumen fractions has been determined at three times after feeding in sheep containing no ciliate protozoa or five different protozoal populations. The total rumen CMCase was lowest in defaunated animals and tended to be higher in animals containing the amylolyticEntodinium caudatumor a natural mixed protozoal population and highest in sheep containing single cellulolytic species. In animals containing the cellulolytic species,Epidinium ecaudatum caudatumorEudiplodinium maggii, 70% of the CMCase was associated with the protozoal fraction whereas less than 15% was present in a fraction prepared in the same way from sheep containing no protozoa or onlyEntodinium caudatum. The activity associated with the free bacteria was higher in sheep containing onlyEntodinium caudatumthan in defaunated animals or those containing the cellulolytic species. Similar results were obtained 1·5, 6·5 or 24 h after feeding. Almost no activity was present in the cell-free rumen liquor under any condition. A variable amount of CMCase (up to 25% of the total) was associated with the plant debris in the rumen and the specific activity of the enzyme released by sonication was often higherthan that released from the free bacteria under the same conditions.

1 Two isolates of Entodinium longinucleatum have been cultured in vitro for at least six months in the presence of bacteria on a reduced buffered salts medium with the daily addition of starch grains and ground dried grass. 2 Cultures of Entodinium longinucleatum were normally maintained by dilution, twice each week, with an equal volume of fresh medium, but the protozoa would also grow from a 2.5% inoculum with a mean generation time of 1.3 days.

Summary: Washed suspensions of three Epidinium spp., grown in vitro and incubated anaerobically in salts solution, released into the medium protein containing hexokinase and an enzyme which lysed Bacillus megaterium, B. subtilis and Micrococcus lysodeikticus. Material from the lysate, containing bound amino acids, was incorporated by Epidinium spp. at a rate that would allow only very slow growth if it were the sole source of nitrogen. Although there was no massive cell lysis the enzymes were liberated as the result of a release of whole cell contents by an unknown mechanism. In the presence of other rumen protozoa, protein, but no lytic enzyme, appeared in the medium. No good evidence was obtained for the existence in the rumen of a lytic enzyme associated with the presence of Epidinium spp.

The amylolytic activity of the cytoplasmic fraction prepared from 14 single species of rumen ciliate protozoa and two natural mixed populations has been determined using five different assays. All the protozoa contained an a-amylase and the major product of amylose digestion was maltose. The highest activities were found inEremoplastron bovis, Diploplastron affine, Ophryoscolex caudatus and Polyplastron multivesiculatumgrown in vitro on grass and wholemeal flour and the lowest inOstracodinium obtusum bilobumandDiplodinium pentacanthumunder all growth conditions and in culturedEntodinium bursaandE. caudatum. The maximum activities depended on the substrate and withEremoplastronbovis varied from 2·3 (with starch grains) to 48·8 (with amylose) μtmol maltose produced/mg protein per h.The distribution of amylase between various rumen fractions has been determined at three times after feeding in sheep containing no ciliate protozoa or seven different protozoal populations. A method to give maximal release of the enzyme from mixed rumen bacteria was devised and three different assay methods were compared. The total rumen amylase varied by up to 2·6 times and was highest in animals that contained onlyEntodinium caudatumor natural A- or B-type populations. The distribution of amylase activity between rumen fractions varied considerably with the various protozoal populations, that in the protozoal cytoplasm being highest in animals containingEpidinium ecaudatum caudatum(46–53%) and the natural A- and B-type populations (35–57 %) and lowest in the animals containing no protozoa (3–11 %) or onlyOstra-codinium obtusum bilobum(6–21%). The reverse was true for the activity in the bacterial fraction which contained little activity in animals with natural A- and B-type populations and over 58% of the total activity in an animal containing onlyEntodinium caudatum. The lowest activities in all fractions were obtained 1·5 h after feeding. Comparatively little activity was found in the plant debris or clarified rumen fluid fractions from any animal. Using results obtained previously the ratios of carboxymethylcellulase to amylase activities have been calculated and shown to be highest in the protozoal cyto-plasmic fraction ofDiplodinium pentacanthumandOstracodinium obtusum bilobumandlowest in Entodiniumspp.,Diploplastron affineandEremoplastron bovis.

Summary: The depolymerase activity of cell-free extracts of nine species of rumen ciliate protozoa and two mixed protozoal preparations, grown in vivo and in vitro, towards polygalacturonic acid was examined. The highest activity was found with Eremoplastron bovis and Ostracodinium obtusum bilobum while there was none in the spined or spineless forms of Entodinium caudatum and little in Polyplastron multivesiculatum. On the basis of the rapid drop in viscosity, inhibition by EDTA and the production of u.v.-absorbing material, the enzymes from all active species were designated as endopectate lyases (EC 4.2.2.2) although some polygalacturonase may be present. Neither pectin nor polygalacturonic acid supported the survival or growth of any of the protozoal species tested.

SUMMARYFive species of rumen ciliate protozoa, isolated from sheep in which they were the only ciliate protozoon, and mixed rumen ciliates were incubated with labelled bacteria and the rate of uptake and digestion of the bacteria was followed. Evidence is presented that mixed rumen bacteria labelled by incubation with14C-isoleucine,14C-guanine or35S-sulphate were engulfed more rapidly by the protozoa than those labelled with14C-glucose but that, withEntodiniumspp. at least, this apparent lower rate of uptake was associated with an increased rate of digestion. The rate of uptake of mixed rumen bacteria (at 109/ml) varied from 230 to 2670 bacteria/protozoon per hour, depending on the protozoal species, and increased with increasing bacterial population density. Starvation of the protozoa decreased the initial rate of uptake of bacteria by up to 60% and the length of time over which the protozoa would engulf bacteria.Entodiniumspp. andEpidinium ecaudatum caudatumengulfed fewer bacteria when these bacteria were prepared from the same sheep as the protozoa than when they were prepared from sheep with no or other protozoal populations. Of the protozoa tested, onlyEntodiniumspp. and the mixed large rumen protozoa digested mixed rumen bacteria,Eremoplastron bovisandEpidinium ecaudatum caudatumbeing inactive. It is suggested that previous estimates of the rate of uptake and digestion of bacteria based on studiesin vitrowere too high.The non-rumen bacteriaEschericfoia coliandKlebsiella aerogenesand the rumen bacteriumMegasphaera elsdeniiwere not or only poorly taken up by the protozoa.Entodiniumspp. selectively engulfed bacteria of rumen origin and digested two cellulolytic bacteria rapidly. In contrast,Eremplastron bovisengulfed these cellulolytio bacteria more slowly than other bacteria.Entodiniumspp. and the large rumen protozoa digested more bacterial species than the other protozoa butButyrivibrio fibrisolvenswas digested more rapidly than other bacteria by all the protozoa.

SUMMARYThe protozoa in washed suspensions of rumen ciliates were killed by freezing or heating at 49 °C for 5 min before incubation at 39 °C, aeration during incubation or incubation in hypotonic salt solution (30% of ncrmal) and released their intracellular cellulose and amylase during 4–6 h. With the last two treatments it was only necessary to expose the protozoa to the conditions for 2 min to obtain over 50% of maximal enzyme release 4–6 h later. It is suggested that death of the protozoa during exposure to air and/or hypotonic salt solution for short times during drinking and rumination by the host animal could be the explanation for the high postulated protozoal death rate in the rumen. The amylase and cellulase released from the protozoa tested were stable in the presence of mixed rumen bacteria for 1·5 h and could be of importance in the degradation of plant constituents in the rumen.

Washed suspensions of the rumen ciliate protozoon Eudiplodinium maggii grown in vitro and incubated anaerobically engulfed all the bacteria tested except for Bacteroides ruminicola and Klebsiella aerogenes. There was considerable variation (160–9100 bacteria/h/protozoon at an external concentration of 1010 bacteria/ml) in the rate at which the bacteria were engulfed, but Eu. maggii showed some preference for bacteria of rumen origin. Some of the bacteria were digested with the release of soluble materials into the medium. Free amino acids were incorporated from an 0.1 mM solution at rates of 0.13 to 0.84 pmol/h/protozoon. Evidence is presented that Eu. maggii could obtain half the amino acids required for growth by the engulfment and digestion of bacteria and half by the uptake of free amino acids. Eudiplodinium maggii incorporated uridine 5' monophosphate and also hydrolysed this to uridine and then to uracil which was reduced to dihydrouracil. These products all appeared in the medium. Ribose was incorporated by the protozoon and appeared as glucose in protozoal and bacterial polysaccharide; none was incorporated as such into protozoal nucleic acid.

Entodinium longinucleatum grown in vitro in the presence of bacteria engulfed a wide range of bacterial species at rates of 130–3400 bacteria/h/protozoon (from suspensions of 10 bacteria/ml), but showed a preference for Klebsiella aerogenes and Proteus mirabilis which occurred in the growth medium. Some of the bacteria were digested with release of soluble material into the medium. Free amino acids were incorporated by the protozoa in the presence of chloramphenicol at rates of 5·4–15·1 nmol/h/106 protozoa and approximately 40% of the amino acid-carbon was incorporated into protein. There was no appreciable synthesis of protozoal protein from carbohydrate. Evidence was obtained that the protozoa obtained the amino acids required for growth largely from engulfed bacteria.

The rate of uptake of mixed rumen bacteria and free amino acids by washed suspensions of seven species of rumen ciliate protozoa has been followed. By assuming that the behaviour of these protozoa was the same under these conditions as during growth it was shown that Ophryoscolex caudatus could obtain the amino acids for growth by the engulfment of rumen bacteria. However, all the cellulolytic protozoa studied (Diploplastron affine, Diplodinium anacanthum, Diplodinium anisacanthum, Enoploplastron triloricatum, Eremoplastron bovis and Ostracodinium obtusum bilobum) were unable to obtain sufficient amino acids from either source to grow at even 25% of the maximum rate and it is postulated that they might utilize plant protein. O. caudatus grown in vitro did not engulf Klebsiella aerogenes or Escherichia coli but took up other bacteria and a rumen yeast at rates of up to 54000 organisms/protozoon/h from a population density of 109/ml. When grown in vivo it was more selective and engulfed mixed rumen bacteria at only 10% of the rate obtained with protozoa grown in vitro. D. affine grown in vitro did not engulf Bacteroides ruminicola, Esch. coli, Kl. aerogenes or Proteus mirabilis but took up mixed rumen bacteria from a population of 109/ml at a rate of 2200 bacteria/ protozoon/h.

Summary: The rumen ciliate protozoon Entodinium bursa has been grown in vitro in the presence of bacteria and Entodinium caudatum for over a year at population densities of 100 to 200 ml−1. The medium contained potassium phosphate, prepared fresh rumen fluid, cysteine, wholemeal flour (or rice starch), dried grass and a culture of the spineless form of Entodinium caudatum. Entodinium bursa has an obligate requirement for this protozoon and died within 48 h in its absence. During growth from a 2% inoculum, the mean generation time of E. bursa was 6 h. Entodinium bursa engulfed 1.5 to 2.5 E. caudatum organisms h−1, and when E. caudatum was in excess it developed caudal spines for the first time in 17 years; these spined forms were engulfed much less readily than the spineless organisms.

Seven isolates of Ophryoscolex caudatus have been cultured anaerobically in vitro (at a population density of 56/ml) for an average of 18 months each in the presence of bacteria on a reduced buffered salts medium containing prepared fresh rumen fluid with the daily addition of ground wheat and dried grass and with twice weekly dilution of the culture with an equal volume of fresh medium. The ground wheat and dried grass could be replaced by ground wheat coated with β-sitosterol, stigmasterol, ergosterol or α-spinasterol and with β-sitosterol the population density increased to 110/ml. Haemin further increased the population density obtained in the presence of sterol by 9–160%. The population density of cultures of Epidinium ecaudatum caudatum was also increased by sterols and haemin, that of Polyplastron multivesiculatum by sterols only, and some sterols and haemin, under certain conditions, increased that of Entodinium caudatum.

SUMMARY: Conditions are described for the isolation from the rumen and subsequent growth of six species of cellulolytic protozoa: Enoploplastron triloricatum, Eudiplodinium maggii, Diploplastron affine, Epidinium ecaudatum caudatum, Diplodinium monacanthum and Diplodinium pentacanthum. The protozoa were grown in an atmosphere of 95% nitrogen plus 5% carbon dioxide, or pure carbon dioxide, in a potassium phosphate-rich medium containing cysteine, sometimes 10% prepared fresh rumen fluid, and daily additions of powdered dried grass. Population densities of 10 to 6000 protozoa/ml (depending on the species) were obtained in cultures that were diluted with fresh medium twice each week. Extracts of these protozoa digested a [14]cellulose preparation, liberating soluble 14C-labelled compounds.

SUMMARY: Oligotrich protozoa from the sheep rumen, principally Entodinium caudatum, were maintained in culture in vitro and dividing every 2 days at a density of 15,000–30,000 protozoa/ml, for 18 months on a medium of rice starch, dried grass, 10% (v/v) rumen fluid (fresh or autoclaved) and 50 μg. chloramphenicol/ml. The effect on these organisms of varying each of these factors in turn and the use of different growth conditions is described. The protozoa have also been grown from an inoculum of 100–900/ml. to a density of over 50,000/ml. in 10 days.

SUMMARY: 14C-labelled Escherichia coli and other bacteria were used to measure their uptake by washed suspensions of Entodinium caudatum. All the bacteria tested were engulfed by the protozoa with a maximum uptake of 1·1 x 104 E. coli/protozoon at an initial rate of over 200 bacteria/protozoon/min. After 30 min. only 12% of the engulfed bacteria were still viable. 50% of the bacterial carbon was retained by the protozoa and, after breakage of the protozoa and centrifugation of the homogenate, 40% of this carbon was present in the supernatant fluid, principally as protein. Competition experiments where the protozoa were offered two different species of bacteria showed that the protozoa engulfed bacteria in the proportion in which they were present in the medium. The growth of E. caudatum in the presence of rice starch, autoclaved rumen fluid and penicillin was stimulated by several species of bacteria, including E. coli, Clostridium welchii, Lactobacillus casei.

SUMMARY: A spore-forming, mesophilic, sulphate-reducing bacterium has been isolated from the rumen of hay-fed sheep. The organism is an obligately anaerobic Gram-negative rod which, in the presence of sulphate and 0·1% yeast extract, used only L- or D-alanine, DL-lactate, pyruvate or formate, but not 47 other compounds tested, as hydrogen donor for growth. Under these conditions the sulphate could be replaced by sulphite or thiosulphate. The yeast extract was replaceable by ferrous sulphate + p-aminobenzoic acid + biotin. Evidence is presented that alanine and lactate are metabolized via pyruvate which breaks down to form acetate and formate, the latter then being metabolized to form CO2. The growth of the organisms was inhibited when the concentration of sulphide reached 6–7 μmole/ml. and the maximum dry weight was only 50–60 μg./ml. Washed suspensions of the organism in 2·0% yeast extract utilized gaseous hydrogen for sulphate reduction and H2: H2S ratios of 3·2–4·0 were obtained.

SUMMARY: Spined and spineless forms of Entodinium caudatum were obtained by growth in vivo in the presence and absence, respectively, of Entodinium bursa. Washed suspensions of both forms engulfed all the bacteria tested although the spined form took them up 1·3 to 1·9 times more rapidly per unit volume of protozoon than did the spineless form. Butyrivibrio fibrisolvens and Selenomonas ruminantium were rapidly digested by the spined form after engulfment. Free amino acids were taken up on average 3·1 times and glucose approximately 60 times faster per unit volume of protozoon by the spined form. Limited amounts of protein were synthesized by the spined form from glucose and starch but engulfed bacteria and, to a lesser extent, free amino acids were probably the prinicpal sources of protein for growth of both forms.

SUMMARY: [14C]Glucose taken up by Epidinium ecaudatum caudatum was found in the pool, in the protozoal polysaccharide and in the bacteria associated with the protozoa. The amount incorporated into the polysaccharide depended on the square of the glucose concentration. Evidence was obtained that glucose was probably taken up initially into the pool unchanged, and then rapidly converted into glucose 6-phosphate and maltose which were subsequently hydrolysed to glucose. [14C]-Maltose was taken up at 20 to 30% of the rate of [14C]glucose, with 14C appearing initially in maltose and glucose 6-phosphate. 14C from 14C-labelled soluble starch appeared in the pool as maltose, glucose 6-phosphate and glucose in that order, but incorporation into protozoal polysaccharide was poor. Hexokinase, phospho-glucomutase, α-glucan and maltose phosphorylases, glucose 6-phosphatase and maltase activities were found in the protozoa.

Summary: The technique of autoradiography in the electron microscope has been used to determine the site of incorporation of tracer from tritium-labelled glucose, amino acids and bacteria by Entodinium caudatum and the ovine form of Epidinium ecaudatum caudatum. Both protozoa incorporated tracer from glucose principally into protozoal polysaccharide and intracellular bacteria. The results with the bacteria confirmed previous findings that Entodinium caudatum digested Bacillus megaterium rapidly in vesicles in the endoplasm, whereas Epidinium spp. lysed the bacteria in the medium before incorporation. These products were then taken up both by the protozoa and the bacteria attached to their surface. In the presence of Entodinium caudatum label from tritiated Escherichia coli was found principally in the endoplasm, whereas that from glycine was present in the ectoplasm also.

Surmmary Non-multiplying and growing cultures of Entodinium caudatum incubated anaerobically engulfed Escherichia coli bacteria specifically labelled with individual 14C-amino acids and incorporated the amino acids into protozoal protein without conversion to any other amino acid. The protozoal cisternae ‘pool’ and the medium contained the free amino acid and with some amino acids the N-acetyl or N-formyl derivative in addition. The constituents of the pool were probably by-products of the metabolism of the bacteria and not intermediates between bacterial and protozoal proteins. There was no extensive catabolism of the bacterial amino acids by the protozoa, although some of the bacterial leucine, isoleucine and valine was broken down to isovaleric acid, α-methylbutyric acid and isobutyric acid, respectively. The addition to the medium of the 12C-form of the 14C-amino acid present in the E. coli decreased the incorporation of 14C into the protozoa with half of the amino acids tested. The rate of loss of viability of various other bacterial species after engulfment by the protozoa was found to be independent of Gram-reaction, size or natural habitat.

Surmmary Washed suspensions of Entodinium caudatum, grown in vitro, incubated anaerobically in the presence of penicillin and neomycin, incorporated single amino acids into the cell protein without conversion to any other amino acid. The 14C-labelled components of the protozoal ‘pool’ and the medium were also investigated but no extensive catabolism of the amino acids observed. At low external concentrations the amino acids were taken up by an ‘active’ process, but above a critical concentration the amino acids entered the cell by passive diffusion and they have been divided into two groups depending on whether this critical concentration was approximately 0–0001 M or 0–001 M. The rate of amino acid uptake was not altered by the presence of inert particulate matter. Of the 75% of the cell volume occupied by liquid, approximately two-thirds was freely available to amino acids.

Washed suspensions of a strain of Rhodospirillum rubrum S1 grown anaerobically in the light and incubated in the light under argon metabolized many of the 15 amino acids tested and produced ammonia and carbon dioxide as the principal extracellular products. Hydrogen was also produced in the presence of glutamic acid. Of the glutamic acid metabolized 80% of the carbon and 55-70% of the nitrogen were converted into intracellular products. The magnitude of the catabolism of the other amino acids, as measured by ammonia and carbon dioxide production, appears to be an inverse function of the closeness to which the elemental composition of the amino acid approximates to that of cell material. When the organism was grown aerobically in the dark, studies of the metabolism of amino acids by washed suspensions, under aerobic conditions, were complicated by the relatively high rate of endogenous respiration. In the presence of certain amino acids this was decreased by 9% (with alanine) to 45% (with aspartic acid), and glutamic acid, aspartic acid and alanine, at least, were oxidized to completion by the organism.

SUMMARY: Washed suspensions of Entodinium simplex grown in vitro and incubated anaerobically engulfed all the types of bacteria tested. At high bacterial densities Klebsiella aerogenes and Proteus mirabilis, two bacteria normally present in the growth medium, were taken up most rapidly. Entodinium simplex utilized bacterial amino acids for the synthesis of protein and no evidence was obtained for appreciable synthesis of protein from carbohydrate. No intercpnversion of amino acids occurred. Free and bacterial adenine, guanine and uracil were incorporated into protozoal nucleic acid: uracil was partially converted into cytosine. Free amino acids and glucose were taken up by the protozoa by two mechanisms, one predominant at high concentrations and the other at low concentrations. Glucose was incorporated, in intact protozoa, into both polysaccharide and bacteria and glucose had to be taken up by the protozoa before it was incorporated into the bacteria.

1. The N-(2-hydroxyethyl)alanine esterified to phosphatidic acid in anaerobic ciliate rumen protozoa has the l configuration. 2. Labelling experiments with Entodinium caudatum cultures using [(32)P]P(i) [2-(14)C]ethanolamine and (32)P- and (14)C-labelled phosphatidylethanolamine show that phosphatidylethanolamine is the direct lipid precursor of the N-(2-hydroxyethyl)alanine-containing phospholipid. 3. Labelling experiments with [(14)C]starch, [(14)C]lactate and [(14)C]pyruvate with E. caudatum cultures indicate that a three-carbon glycolytic intermediate is probably the precursor of the N-(1-carboxyethyl) grouping which substitutes on the amino group of phosphatidylethanolamine. 4. [(32)P]phosphatidylethanolamine is catabolized by E. caudatum forming initially glycerylphosphorylethanolamine and subsequently glycerophosphate and P(i). A little phosphorylethanolamine formed may possibly arise from bacterial enzymes ingested by the protozoa.

IT has been shown previously1,2 that sheep rumen Entodinia spp., principally Entodinium caudatum, could be grown in vitro, dividing every 48 hr. on a medium of salts, dried grass, rice starch, chloramphenicol and fresh or autoclaved rumen fluid, but that it was necessary to add fresh grass and rice starch to the medium every day. This communication describes the successful maintenance of these Protozoa in the presence of penicillin for periods up to 14 days without daily additions.

SUMMARY: Washed suspensions of Entodinium caudatum grown in vitro and incubated anaerobically incorporated 14C from [14C]glucose into the cell pool as [14C]glucose, maltose and hexose phosphate and into protozoal polysaccharide and intracellular bacteria as a glucose polymer. The uptake of sugars into the pool was by an active process (predominant at low sugar concentrations) probably into the protozoal endoplasm, and by a passive process (predominant at high concentrations) into another part of the cell. Uptake of glucose by the active process was not sufficiently rapid to increase the level of glucose in the pool by more than 17% per hour; this was probably insufficient to increase appreciably the rate of glucose utilization. These protozoa hydrolysed starch to maltose and glucose and the level of these sugars in the pool of protozoa metabolizing starch grains was controlled in part by the inhibitory effect of these sugars on the enzymes that formed them. Studies on the enzymes involved in the metabolism of starch showed that the glucose in starch was hydrolysed to free glucose before phosphorylation. In experiments on the effect of carbohydrates on the survival of protozoa, starch was the most effective in prolonging protozoal life, followed by maltose and glucose in that order. The greater effectiveness of maltose was correlated with the finding that the rate of uptake of maltose carbon was always greater than that for glucose when the two sugars were present in equimolar concentrations. However, the rate for both sugars was markedly decreased in the presence of penicillin and neomycin. Evidence is presented that the protozoal ectoplasm may be freely permeable to sugars in the medium and that there is a barrier between the ectoplasm and endoplasm. 14C from [14C]starch grains was incorporated by the protozoa but there was no synthesis of protozoal protein from carbohydrate.

Washed suspensions of Entodinium caudatum incubated anaerobically in the presence of penicillin and neomycin incorporated 14C-glycine in the cell ‘pool’ as N-acetylglycine and into the cell protein as glycine without conversion to any other amino acid. At low external concentrations the glycine was present in the protozoa themselves and was not free in the gastric sac or present solely in the intracellular or extracellular bacteria. The incorporation reached a constant value after incubation for 24 hr; after this time the uptake of glycine was balanced by an equal loss of N-acetylglycine from the organisms. These suspensions also incorporated 14C from NaH14CO3 into carbon atoms 3 and 4 of protozoal glucose which was probably present as starch in the intact protozoa.

SUMMARY: Populations of over 400 Epidinium ecaudatum caudatum/ml were maintained for over 18 months in the presence of bacteria, in a potassium phosphate-rich medium under 95% N2 + 5% CO2 with the daily addition of wholemeal flour and dried grass. Polyplastron multivesiculatum was also maintained at a population density of 45 to 70/ml under the same conditions, except that it was necessary to add each day a culture of another protozoon such as Epidinium ecaudatum caudatum, E. ecaudatum tricaudatum or Eudiplodinium maggii to give a population density of approximately 100/ml. The epidinia at least were engulfed by the polyplastron up to 10 being taken up each day; the minimum daily requirement for growth was one epidinium/polyplastron. Polyplastron multivesicutum also grew in the absence of other protozoa on a sodium chloride-rich medium, and if these protozoa were inoculated into the potassium phosphate-rich medium in the presence of epidinia, there was a lag of 3 to 5 days before they began to engulf the epidinia. The effect of other variations in the culture conditions is also described.

Cellulolytic activity can be released from rumen Entodiniomorphid protozoa by gentle sonication (Coleman, 1978) and there is evidence that most of this activity is of protozoal rather than bacterial origin (Coleman, 1978). Cellulolytic activity can also be released under the same conditions from a crude mixture of bacteria isolated from the rumen. The purpose of this communication is to compare the cellulolytic activity in fractions (prepared by sonication followed by centrifugation (15,500g, 30 min) to produce a clear, bacteria-free supernatant fluid) from faunated and defaunated sheep. Activities were measured by the release of reducing material from carboxymethylcellulose and the release of dye from cellulose azure (Sigma, type II), but unfortunately with both methods the relationship between product and time or enzyme concentration was a curve with a steadily decreasing slope. To overcome this difficulty a standard curve of product against concentration of an extract of the ciliateEpidinium ecaudatum caudatum was constructed for all experiments. Using this curve, the amount of product formed with other enzyme preparations was converted into concentrations of this extract. The specific activity (activity/mg protein) of this extract was taken to be 100. Fractions were prepared from strained rumen contents by differential centrifugation and after sonication and centrifugation as described above, no activity was found in the 360g min pellet (“protozoal fraction”) from defaunated sheep or in the green or brown layer that was present above the protozoa in the same fraction from refaunated animals. Cellulolytic activity was progressively released from washed rumen bacteria prepared from both types of sheep during sonication for 15 min, at which time the specific activity of the material was 2–3% of that in the protozoon. There was a small but variable amount of activity (specific activity, 0.1–1.0%) in the cell-free rumen liquor. If it is assumed that the activity released from the protozoa and bacteria is actually available to degrade cellulose, an estimate can be made of the comparative importance of protozoa and bacteria in rumen cellulolysis. In a sheep containing 20,000 epidinia/mL as the only ciliate protozoon, the bacteria and cellfree rumen liquor contained only 12 and 2 1/2%, respectively, of the activity in the protozoa. If 65% of the protozoa die and are degraded in the rumen (Leng, 1982), then the protozoal cellulase released could be very important in cellulose degradation.

HUNGATE1,2 successfully cultivated ciliate oligotrich protozoa from the rumen of cattle ; but Sugden3 was unable to maintain protozoa from the sheep's rumen alive for more than fifteen days. This communication describes the successful maintenance of these protozoa from the rumen of a single hay-fed Clun Forest sheep.