The cultivation of the rumen ciliate Entodinium bursa in the presence of Entodinium caudatum.

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.     

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa

C oleman , G.S. & H all , F.J. 1984. The uptake and utilization of Entodinium caudatum , bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa. Journal of Applied Bacteriology 56 , 283–294.
Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum , ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus mega-terium, Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bouis , although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and i>Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro , although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa . Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.     

The effect of sterols and haemin on the growth of the rumen ciliate Ophryoscolex caudatus and some other Entodiniomorphid protozoa

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.     

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa

Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum, ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus megaterium. Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bovis, although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro, although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa. Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.     

Axenic Cultivation of the Cellulolytic Flagellate Trichomitopsis termopsidis (Cleveland) from the Termite Zootermopsis*

SYNOPSIS. Trichomitopsis termopsidis (Cleveland), a cellulolytic hindgut symbiote of the termite Zootermopsis, has been cultivated axenically under anaerobic conditions. The medium consists of cellulose, reduced glutathione, fetal calf serum, yeast extract, and autoclaved rumen fluid or autoclaved rumen bacteria, in a buffered salt solution the composition of which is based on an analysis of Zootermopsis hindgut fluid. The hindgut contents of surface-sterilized termites were inoculated into anaerobic buffer-containing cellulose and serum. Repeated passages yielded mixed cultures of T. termopsidis and termite hindgut bacteria. Flagellates were then inoculated into complete medium containing antibiotics, and after 2 passages, axenic cultures of T. termopsidis were obtained. Various nutritional supplements, including clarified rumen fluid or heat-killed bacteria of several known species failed to support the growth of T. termopsidis when substituted for autoclaved rumen fluid. The flagellates did not grow when any of several carbohydrates were substituted for cellulose. Electron microscopy of flagellates from axenic cultures revealed that cellulose particles and partially digested bacteria were present in food vacuoles. No endosymbiotic bacteria were present in the cytoplasm indicating that T. termopsidis does not depend on living prokaryotes for cellulose digestion. The results suggest that T. termopsidis possesses the enzyme cellulase.     

The metabolism of starch, glucose, amino acids, purines, pyrimidines and bacteria by the rumen ciliate Polyplastron multivesiculatum.

The large rumen ciliate protozoon Polyplastron multivesiculatum grown in vitro engulfed a wide range of bacteria (from a population density of 10(9) bacteria ml(-1)) at a rate of 1500 to 137000 bacteria h(-1) protozoon(-1). No evidence was found for the preferential engulfment of bacteria of rumen origin. Except for Proteus mirabilis none of the bacteria were digested with the liberation of soluble materials into the medium. Glucose and amino acids were taken up rapidly by P. multivesiculatum compared with the rate of uptake by Entodinium caudatam. Glucose was incorporated into protozoal polysaccharide and into bacteria associated with the protozoa and was used for the synthesis of a wide range of amino acids. Evidence showed that bacteria and free amino acids at the concentrations found in the rumen could supply the protein requirements of the protozoa for division at least once each day.     

Axenic Paramecium caudatum. I. Mass culture and structure

To establish and grow Paramecium caudatum in mass axenic culture the culture medium of Soldo, Godoy & van Wagtendonk was modified by substituting phosphatidylethanolamine (PE) for TEM-4T and by a 10-fold increase in folic acid. Population densities of 4000 to 6000 cells/ml and a generation time of 20-26 h are regularly obtained. Optimal growth is obtained with PE-stigmasterol ratios between 40:1 to 400:1. Cells from 1-day-old axenic cultures have many lipid bodies aggregated in clumps (which disappear in 2 to 3 days) as well as foci of rough endoplasmic reticulum bordered by dictyosomes. The latter suggests a very active metabolism. Crystalline sheets found in both food vacuoles and lysosomes presumably play a role in digestion. Axenically grown cells also have abundant Golgi bodies (dictyosomes) and by late log phase become filled with lysosomes.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

The Uptake and Utilization of Bacteria, Amino Acids and Nucleic Acid Components by the Rumen Ciliate Eudiplodinium maggii

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.     

In vitro culture of the seagrass Halophila decipiens

The seagrass Halophila decipiens Ostenfeld was grown axenically in a culture medium consisting of 20% artificial seawater, f/4 nutrients (except that glutamic acid was the nitrogen source), and 1% sucrose (w:v). The culture medium was adjusted to pH 5.0. A root–rhizome layer was created by solidifying a portion of the medium with 0.9% agar (w:v) and 1% activated charcoal (w:v). The rhizome layer also contained the following vitamins: 0.5 mg l⁻¹ nicotinic acid, 0.5 mg l⁻¹ pyridoxine, 0.5 mg l⁻¹ biotin, 0.5 mg l⁻¹ cyanocobalamin and 0.1 mg l⁻¹ of thiamine HCl. The liquid overlay (without vitamins or charcoal) was poured onto the agar-solidified root–rhizome layer. Growth of H. decipiens was not improved by the addition of the auxins indoleacetic acid (IAA), indolebutyric acid (IBA) or naphthaleneacetic acid (NAA) at either of the tested concentrations (10 and 50 μM). At a concentration of 10 μM, the cytokinins 6-(γ,γ-dimethylallylamino) purine (2iP) and benzylaminopurine (BA) stimulated shoot and branch production compared to controls with no cytokinins. Among the tested nitrogen sources, growth was best on 1.7 mM glutamic acid. Cultures grown on 1.7 mM NH₄Cl showed the same growth rates as those grown on glutamic acid, but the leaves were smaller and curled, suggesting incipient ammonium toxicity. Use of nitrate or urea led to mortality of the cultures. Long term axenic culture of H. decipiens appears to require the added vitamins. Hence, H. decipiens is the first seagrass known to need exogenous vitamins. Cultures of H. decipiens died when grown suspended in liquid cultures or in a biphasic medium system without activated charcoal in the root–rhizome layer. The use of more highly charged κ-carrageenan could not replace the use of activated charcoal and agar in the root–rhizome layer.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Culture of the Rumen Holotrich Ciliate Dasytricha ruminantium Schuberg

The successful cultivation of the anaerobic ciliate Dasytricha ruminantium is described. The cultures were established in a salts medium containing 30% clarified rumen fluid. Sucrose and extract of rumen holotrich protozoa were fed once daily for 2 to 4 hr, and Dasytricha was then transferred to medium free from these nutrients. Rumen fluid was essential. Omission of protozoal extract resulted in gradual death of the ciliates. Bovine serum satisfactorily substituted for the protozoal extract, but various rumen bacteria, extract of rumen bacteria, and extracts of plant materials could not. There was a positive correlation between formation of methane in the cultures and growth of the ciliates. It is possible that methane bacteria were ingested, but it is not excluded that survival of both dasytrichs and the methanogenic bacteria depended on a low redox potential of the medium.     

Method for the simultaneous establishment of many axenic cultures of Paramecium

A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmaterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing approximately 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliattes can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Axenic Cultivation of Trichomonas tenax, the Oral Flagellate of Man I. Establishment of Cultures

SYNOPSIS. A technique for the axenic cultivation of Trichomonas tenax , the oral flagellate of man, is presented. The medium employed consists of a nutrient broth supplemented with horse serum and a cell-free extract of chick embryo. Three strains established in this medium have been maintained for 16, 23 and 48 months respectively.
All the cultures were initiated with trichomonads grown in association with Trypanosoma cruzi. Attempts to establish axenic cultures with trichomonads obtained from xenic cultures containing a bacterial flora of unknown composition met with failure. This suggests that the successful cutcome of the process of axenization is to a certain extent dependent upon the type of organism(s) with which T. tenax is associated in culture. Furthermore, these findings may serve to explain earlier failures since not only were all of these attempts made with media lacking tissue extract supplements but all were made using bacteria-trichomonad cultures as a source of trichomonads.     

Effect of pH on viability of Entodinium caudatum, Entodinium exiguum, Epidinium caudatum, and Ophryoscolex purkynjei in vitro

Cultures of Entodinium caudatum, Entodinium exiguum, Epidinium caudatum, and Ophryoscolex purkynjei were grown and transferred in poorly buffered media prepared using different concentrations of sodium bicarbonate and a nitrogen gas phase. By transferring every 12 or 24 h, culture pH was gradually decreased until the protozoa disappeared. The cultures were transferred by placing half of the culture into an equal volume of fresh medium, resulting in pH fluctuations similar to those in the rumen, resulting from fermentation, eating, and saliva production. All four species appeared to maintain their concentrations around pH 5.8, but numbers decreased as pH values fell below 5.6. The four species were similar in that they all survived above pH 5.3. These results differ from previous reports in which Entodinium species appeared to be more tolerant to low pH than all other species of rumen ciliates. No adaptation to low pH was observed in Epidinium caudatum cultures after recovery from pH 5.4 medium containing only one or two viable cells.     

Electron microscopy of the rumen ciliate Entodinium caudatum, with special reference to the engulfment of bacteria and other particulate matter

A study in the electron microscope of thin sections of the rumen ciliate Entodinium caudatum was undertaken in an attempt to elucidate the mode of engulfment of particulate matter. This protozoon engulfed bacteria, polystyrene latex particles and olive oil into membrane-lined vesicles in the protozoal endoplasm. Particles of palladium black were also taken up into the endoplasm, but due to the toxic nature of this material it was not possible to demonstrate vesicle formation with certainty. The initial uptake of bacteria may be into large sacs containing many organisms which were subsequently taken into the endoplasm in vesicles that contained only one bacterium each. The evidence obtained in this investigation has been used to distinguish between two different mechanisms for the digestion of bacteria and utilization of the amino acids from the bacterial protein for the synthesis of protozoal protein.     

The uptake of bacteria and amino acids by Ophryoscolex caudatus, Diploplastron affine and some other rumen Entodiniomorphid protozoa

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.     

Isolation of previously uncultured rumen bacteria by dilution to extinction using a new liquid culture medium

A new anaerobic medium that mimics the salts composition of rumen fluid was used in conjunction with a dilution method of liquid culture to isolate fermentative bacteria from the rumen of a grass-fed sheep. The aim was to inoculate a large number of culture tubes each with a mean of < 1 culturable cell, which should maximize the number of cultures that develop from a single bacterium. This minimizes the effort that has to be put into purifying the resultant cultures. Of 1000 tubes, 139 were growth positive. Of the 93 that were able to be subcultured, 54 (58%) appeared to be pure cultures. The phylogenetic placements of these 54 cultures, together with another 6 cultures obtained from a preliminary study, were determined. Using a criterion of < 93% 16S rRNA gene sequence identity to a previously named bacterium as a proxy for defining a new genus, 27 (45%) of the 60 cultures belonged to 14 potentially novel genera. Many of these had 16S rRNA genes that shared > 97% sequence identity to genes of uncultured bacteria detected in various gastrointestinal environments. This strategy has therefore allowed us to cultivate many novel rumen bacteria, opening the way to overcoming the lack of cultures of many of the groups detected using cultivation-independent methods.     

Development of a membrane dialysis bioreactor and its application to a large-scale culture of a symbiotic bacterium,Symbiobacterium thermophilum

A simple membrane dialysis bioreactor was developed for a large-scale axenic culture of Symbiobacterium thermophilum, a symbiotic thermophile that requires co-cultivation with an associating thermophilic Bacillus strain S for normal growth. The bioreactor consisted of an outer- and an inner-coaxial cylindrical compartment bordered across a dialyzing membrane, which enabled a 1 l-scale dialysis culture with exchange of low molecular metabolites between the two compartments to be performed. Using the bioreactor, growth characteristics of S. thermophilum and Bacillus strain S were assessed under two medium conditions. The growth of S. thermophilum was measured by quantitative PCR because the bacterium formed no visible colonies and gave abnormally low turbidity. In medium containing 2% tryptone peptone, S. thermophilum proliferated up to 4x10(7) cells/ml, and strict dependence on the co-culture with Bacillus strain S was observed. On the other hand, medium containing 0.5% yeast extract not only facilitated the growth of S. thermophilum in the co-culture (6x10(7) cells/ml), but also allowed limited pure growth independent of Bacillus strain S (1x10(7) cells/ml), implying that some component of yeast extract can partially replace the growth requirement of S. thermophilum supplied by Bacillus strain S. Both the oxidative redox potential values and the cell morphology in the independently growing culture suggested the occurrence of marked unbalanced growth possibly caused by significant metabolic changes. The bioreactor is applicable to the analyses of culturing characteristics in symbiotic systems between free-living microorganisms.