Detection and cultivation of intestinal trichomonads of squirrel monkeys (Saimiri sciureus)

Routine examinations of fecal samples from squirrel monkeys suggested that intestinal trichomonads might be common inhabitants of these animals. In pursuit of these observations, microscopic examination of fecal suspensions and cultures have demonstrated a 100% incidence of trichomonads in 30 arbitrarily selected animals from a colony of more than 300 housed in groups of ten. The most prominent species was Pentatrichomonas hominis. A not yet fully characterized tritrichomonad was also found on several occasions. The main obstacle in establishing individual strains in culture was the presence of bacterial and fungal flora in the samples. Nevertheless, abundant cultures were obtained from 28 animals by inoculation of fecal suspensions into tissue cultures with appropriately formulated medium and high concentration of antibiotics. In several unattended cultures maintained at room temperature, the flagellates retained motility for at least 4 months. This long survival may explain the widespread occurrence of the parasites within a confined animal community.     

The potential of biological methane generation from chicken manure

The potential for biological methane generating from the manure of laying hens was investigated in the laboratory. Fresh manure was collected, analyzed, and used to prepare medium for bacterial growth. At 55°C and under anaerobic conditions, methanogenic cultures were initiated by incubating the medium with different inoculations from various natural environments. Since there were no significant differences in gas production among these initiated cultures after 40 days of acclimation, they were mixed to maintain a genetic pool. The mixed culture was then challenged with different retention times (RT) and different volatile solid (VS) concentrations for the selection of optimal conditions and cultures. The conditions were finally selected to be 4-day RT and 6% VS for the maximal rate of gas production. The optimal pH and temperature were determined to be 7.5 and 50°C, respectively. Under such conditions the selected culture produced total gas at a rate 4.5 L/L day and methane (70% of total gas) 3.2 L/L day. The chicken manure therefore was able to support the methane yield at 270 L/kg of VS, a value comparably higher than other kinds of livestock wastes.     

Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation

More than 20% of human granulocyte-macrophage and eosinophil colony-forming cells survived in agar culture for up to 4 days without the addition of exogenous colony stimulating factors (human placental-conditioned medium, HPCM). Survival was reduced slightly but not significantly, by the removal of adherent cell populations. Significant survival occurred even when only 100 cells enriched for colony-forming cells (CFCs) were cultured per dish. When individual colonies, initiated by stimulation with HPCM for 5 days, were transferred to dishes without HPCM, subsequent proliferation was significantly reduced compared with control cultures containing HPCM. Using the fluorescence-activated cell sorter and the fluoresceinated lectin from Lotus tetragonolobus, two populations of marrow cells were obtained, one enriched for day 7 and the other for day 14 colony-forming cells. Two colony-stimulating factors fractionated from HPLCM (CSF^β and CSF^α) have been shown previously to stimulate the day 7 and day 14 colony-forming cell populations, respectively. Developing clones from cultures initiated with CSFβ died between the fifth and tenth day of culture after transfer to dishes with CSFα or CSFβ or to dishes with no stimulus. Cells in clusters initiated with CSFα proliferated significantly between the fifth and tenth day of culture when transfered to CSFα or CSFβ but not when transfered to dishes with not stimulus. These studies provide further evidence for the existence of two subtypes of human granulocyte-macrophage progenitor cells each under the primary control of a specific regulator and indicate that these two regulators can both act on some developing clones of cells.     

Infectivity of Trypanosoma brucei Cultivated at 28 C with Tsetse Fly Salivary Glands*

SYNOPSIS. When transformed procyclic noninfective trypanosomes of several unrelated stocks of Trypanosoma brucei were cultivated in T-30 Falcon flasks at 28 C in a liquid medium containing head-salivary gland explants of Glossina morsitans morsitans some of the organisms developed into forms infective for mice. Infective trypanosomes were detected 7 to 14 days after the cultures were prepared and they persisted for varying periods of up to 88 days when the cultures were terminated. A few of the salivary glands became invaded with parasites about the time infective organisms appeared in the cultures. Using T. brucei TREU 929, it was shown that trypanosomes grown with between 27 and 50 explants were capable of producing infections consistently for prolonged periods. On the other hand, trypanosomes cultivated with 25 or fewer explants rarely infected mice. Infectivity titrations on trypanosome suspensions from cultures of stocks TREU 1275 and TREU 929 revealed that the maximum number of infective organisms was present 26 to 50 days after initiation of the cultures. Control cultures of trypanosomes grown in medium alone were generally not infective but 2 of the 6 stocks gave rise to a few sporadic infections. A few epimastigote-like and metacyclic-like trypanosomes were seen in stained preparations of infective inocula.     

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.     

Endogenous hormone levels in habituated nucellar Citrus callus during the initial stages of regeneration

 Embryogenic nucellar callus cultures of different Citrus species and cultivars growing in hormone-free medium were transferred to medium containing either sucrose or glycerol as the only carbohydrate source. Glycerol has been reported to induce further development of Citrus somatic embryos, while in the presence of sucrose they continue to proliferate in an 'undifferentiated' manner. The endogenous hormone levels of the cultures were evaluated after 2 and 5  days to characterise the initial steps of embryo development. In most cases, differences among treatments were observed only after 5 days of culture. Higher cytokinin levels were found in most of the cultures transferred to the glycerol-containing medium. The effect of ageing sweet orange cultures on their endogenous hormone levels was determined by leaving them in the original culture medium without subculturing for 60 days. While no changes were observed in the free indoleacetic acid and gibberellin contents, lower levels of abscisic acid and cytokinins were found in the aged cultures than in those transferred at the normal interval, every 30 days. The endogenous hormone contents of Citrus callus of different genotypes were compared. Significant differences were observed in the levels of all hormones evaluated, even when the in vitro ontogeny of the different genotypes was very similar. Received: 10 February 2000 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

Postmetacercarial changes in Echinostoma caproni maintained in a defined medium plus calf serum

The present study examined postmetacercarial changes in the excysted metacercariae of Echinostoma caproni maintained in the defined medium Mixture 199 plus 20% calf serum for 7 days at 41 degrees C. The gas phase was atmospheric air. Each culture was inoculated with 25 excysted metacerariae. Cultures were maintained upright in closed 15 ml plastic centrifuge tubes each containing 10 ml of medium plus 200 units of penicillin/ml and 200 micrograms of streptomycin/ml. By 4 days in culture, most metacercariae had voided their excretory concretions. Organisms were clumped or solitary at the bottom of the cultures. Many organisms showed flaring of the oral collar and extension of both the collar and tegumentary spines. By 4 days in culture, posterior protuberances or bumps were noted on many of the organisms and some organisms showed abnormal vesicular growths or blebs at their posterior ends. Some mortality was noted in culture by day 5, but most organisms were still alive when the cultures were terminated on day 7.     

Establishment of embryogenic cell suspension cultures of garlic (Allium sativum L.), plant regeneration and biochemical analyses

Embryogenic cell suspension cultures of garlic (Allium sativum L.) were initiated in liquid medium from friable embryogenic tissue. The optimal parameters for culture maintenance were: (1) an initial cell density of 1–4% (v/v); (2) medium renewal every 14 days and subculturing every 28 days; (3) a low 2,4-dichlorophenoxyacetic acid concentration (0.1–0.3 mg/l). Cultures regenerated during a 14-month period. The cell suspension cultures differentiated embryos following transfer to a semi-solid embryo induction medium, with histological studies confirming and characterising the embryogenic nature of the process. Forty percent of these embryos converted into plantlets, which produced micro bulbs in vitro. The composition of the sulphur compounds of the micro bulbs obtained from cell suspension embryo-derived plantlets differed slightly from those produced by in vitro shoot proliferation-derived plantlets, but after two cycles of multiplication in the field these differences had disappeared.     

Infectivity of Trypanosoma rhodesiense Cultivated at 28°C with Various Tsetse Fly Tissues1

ABSTRACT. Metacyclic trypanosomes developed in populations of procyclic forms of four stocks of Trypanosoma brucei rhodesiense cultivated at 28°C in a liquid medium containing explants of tsetse fly head-salivary glands, alimentary tract, abdominal body wall, or thoracic muscle. The cultures became infective for mice 7–16 days after they were prepared, and infective trypanosomes were present for prolonged periods. In the culture series of stock TRUM 545, infectivity persisted for 138 days when the cultures were terminated. Only one explant of thoracic muscle tissue was required for the production of metacyclic stages in stock TRUM 497 cultures. Infectivity titrations on trypanosome suspensions from cultures of stocks TRUM 497, TRUM 545, and TRUM 567 revealed that only a small proportion of the culture population was infective. Using stock TRUM 530, mice were infected consistently from inoculations of trypanosomes grown in the presence of explants; infectivity of the trypanosomes ceased when the explants were removed from the flasks, but reappeared when they were returned to the cultures. Parasites grown in medium “conditioned” by explants produced sporadic infections in mice. The control cultures of trypanosomes grown in medium alone were generally not infective, but two of the stocks produced occasional parasitemias. Stained samples of infective inocula contained a few epimastigote-like and metacyclic-like trypanosomes.     

In-vitro liquid culture of the entomopathogenic nematode,Steinernema colombiense,in orbitally shaken flasks

Steinernema colombiense, an entomopathogenic nematode species (EPN) was grown in two types of orbitally shaken flasks at 130?rpm and 28°C, containing 10 or 20?mL, respectively of a complex culture medium with an initial EPN-concentration of 1,000 Infective Juveniles (IJ)/mL. At the 10th day, the EPN-concentration was 58,771 individuals/mL with 87% of them in the IJ stage. No significant differences were found between the EPN growth kinetics in both types of flasks. The nematode-population growth was modelled by a re-parameterized Gompertz equation of three-parameters with best-fit values of 3.8 days for the lag time, 33.8 day^-1 for the maximum growth rate, and 57.3 (dimensionless) for the maximum asymptotic growth.     

Effect of Chilling Temperatures upon Cell Cultures of Tomato

The effect of chilling temperatures upon cell cultures of tomato (Lycopersicon esculentum Mill cv `VF36,' and cv `VFNT Cherry,' and L. hirsutum Humb. & Bonpl.) was tested. Doubling times for L. esculentum were 2 to 3 days at 28°C, and 3 to 8 days at 12°C. No growth was observed at 8°C, indicating an abrupt limit to growth between 8 and 12°C. Fluorescein diacetate staining indicated that 80 to 90% of the cells were alive when cells were maintained at 8°C for up to 2 weeks. When cultures kept at 8°C for up to 30 days were transferred to 28°C, growth resumed quickly, and at a rate virtually identical to that for unchilled cells. Similar results were found for cells maintained at 0°C, and for cells of `VFNT Cherry' and of L. hirsutum. Under certain conditions, cultures slowly doubled in fresh weight and cell volume at 8 or 9°C but additional growth at 8°C did not occur, nor could growth be maintained by subculture at 8 or 9°C. The results are contrary to reports that cell cultures of tomato die when exposed to temperatures below 10°C for 1 or 2 weeks. Our observations indicate that chilling temperatures quickly inhibit growth of tomato cells, but do not kill them.     

Effects of initial medium pH on growth and metabolism ofCatharanthus roseus hairy root cultures —A study with31P and13C NMR spectroscopy

Summary Hairy root cultures ofCatharanthus roseus were grown for 26 days in half-strength Gamborg's B5 liquid medium at different initial pH values of 4.2, 5.7, 6.5, and 7.3. Maximum growth was obtained for cultures with an initial medium pH 6.5. The lowest growth rate was found in cultures at initial pH values of 4.2 and 7.3. Roots in cultures at initial pH of 4.2 had a thickened and stunted morphology in contrast to the other cultures. Also, cultures at initial medium pH of 4.2 exhibited an increase in medium pH in the first few days instead of the characteristic acidification. All cultures maintained a cytoplasmic pH of 7.4 throughout the growth cycle. However, vacuolar pH was 5.1–5.2 in cultures of initial pH 4.2, as opposed to 5.4–5.5 for other cultures. Sucrose was hydrolyzed completely to glucose and fructose by day 26 except for cultures at initial pH of 7.3. Glucose was the preferred substrate throughout the growth cycle for cultures with initial pH values of 7.3 and 6.5, after day 20 for an initial pH of 5.7, and after day 26 for pH 4.2.     

Methylcellulose effect on cell proliferation and glucose utilization in chemically defined medium in large stationary cultures

Three different established strains of mammalian cells were grown in chemically defined medium in large cultures. The degree of proliferation of cells of an established strain from human skin in large stationary cultures was significantly greater in the presence of methylcellulose (medium NCTC 135M) than in its absence (medium NCTC 135). The relatively fragile cells of a derivative of monkey kidney LLCMK2 strain were carried in large stationary cultures through 11 transfer generations during 152 days. The presence of methylcellulose was associated with higher cell population levels, proliferation rates, and cell viability. Cells of this strain utilized glucose at an extremely high rate; during two representative periods the rate averaged 1.2 mg/10⁶ cells/day in cultures on medium 135M and 1.9 mg in medium 135. In a 53-day experiment with mouse fibroblast 2071-L cells, the cells in suspension culture during the first 28 days went through the normal lag, logarithmic plateau, and initial decline phases in medium 135M, and then were transferred to large stationary cultures, where they proliferated for 7 days at uniformly high rates in both medium 135 and medium 135M. It appeared that cells of strain 2071-L in such stationary cultures had no need for Methocel as a protective agent. Glucose utilization rates while these cells were carried in large stationary cultures averaged 2–4 times the rates while they were in suspension cultures: about 0.8 and 0.2 mg/10⁶ cells/day, repectively.     

Temperature Effects on Shoot and Root Development From Begonia × cheimantha Petiole Segments Grown in vitro

The effect of different temperatures on the shoot and root formation in isolated petiole segments of Begonia × cheimantha was determined after 10 weeks on a modified White medium containing 0.1 mg/1 NAA and 0.5 mg/1 BA. Temperature proved to be important for the induction of shoot and root formation. At a constant temperature the best plants were obtained at 18 to21°C. If the temperature was higher, fewer cultures survived and the number of roots and shoots were lower. Lower temperatures inhibited the development of plants. A pretreatment at 15 or 18°C for two to four weeks improved the number and size of the shoots developed during a following 24°C treatment. High temperatures throughout the growing period reduced the number of shoots severely. A pretreatment of three days at 24°C or one day at 28°C reduced the shoot number by 50 %. After seven days at 28°C there was not a single shoot in any of the cultures. However, after two weeks at 15 or 18°C it was no longer possible to inhibit the shoot formation by a 24°C treatment. It is concluded that the formation of shoots in petiole segments takes place during the first two weeks after excision, and that high temperature is detrimental to the shoot initiation process.     

Effects of sucrose on photosynthesis and phosphoenolpyruvate carboxylase activity of in vitro cultured strawberry plantlets

Photosynthesis and phosphoenolpyruvate carboxylase activity were investigated in 5, 10 and 28 day-old micropropagated strawberry plantlets (Fragaria x ananassa Duch. cv Kent) rooted in vitro with different levels of sucrose (0, 1, 3 and 5%) on cellulose plugs (Sorbarods). The photosynthetic capability was influenced by the level of sucrose in the culture medium with the largest rates of photosynthesis corresponding to the cultures with 0 and 1% sucrose. The apparent quantum yield and the ratio of variable fluorescence to maximum fluorescence were also reduced in plantlets cultured with 3 or 5% sucrose as compared to those with 0 or 1%. Phosphoenolpyruvate carboxylase activity was largest 5 and 10 days after the onset of culture and decreased in the absence of sucrose in the culture medium. At 28 days after the onset of culture, the activity of this carboxylating enzyme was lower than at the beginning of culture and independent of the concentration of sucrose in the culture medium. Phosphoenolpyruvate carboxylase appears to be an important carboxylating enzyme in micropropagated plantlets.     

Inversely related evolution of growth hormone and prolactin secretions in long-term tissue cultures of human pituitary adenomas from acromegalic patients

Summary Pituitary tumoral tissue from 20 acromegalic patients was cultured for up to 120 d in a medium containing 5 nM cortisol. In all cultures, growth hormone (GH) release decreased. At the beginning of the culture, prolactin (PRL) was detected in 18 adenomas, varying from 0.5 to 1000 ng per flask per day. Thereafter, in 10 cases PRL secretion increased from 3 to 50 times the basal level, most frequently after a lapse of 9 to 30 d. PRL secretion remained low in three cases, undetectable in one case only. When added at 350 nM, cortisol increased GH secretion up to 20-fold and simultaneously decreased PRL secretion by as much as 10% of the basal level. Withdrawing cortisol reversed the situation. Immunocytochemical studies of the tumor at surgery showed, besides GH immunoreactive (IR) cells, PRL-IR cells (from rare cells to 10% of total cells) in 15 adenomas, correlating with the first days of culture PRL levels. In cultured explants, mitoses were never found. In 5 nM cortisol medium, the number of GH-IR cells decreased and PRL-IR cells increased or appeared. With 350 nM cortisol, the number of GH-IR cells increased, and PRL-IR cells were scarce or absent. Immunoreactivities for GH and PRL were found in different cells. Care was taken to exclude cultures containing normal pituitary tissue, and because no mitoses were found, these results suggest that most somatotropic adenomas can reversibly shift their secretion from GH to PRL in culture. This capacity to secrete PRL, hidden or low in vivo, is revealed by the favorable low cortisol conditions present in vitro.     

DEVELOPMENT AND GAMETOPHORE INITIATION IN THE MOSS PYLAISIELLA SELWYNII AS INFLUENCED BY AGROBACTERIUM TUMEFACIENS

Spores of the heterotrichous moss Pylaisiella selwynii Kindb. were sown in a defined inorganic liquid culture medium and incubated at 27 C with a 16-hr photoperiod. They germinated at 7–10 days, and formed a few caulonemal buds at 27–30 days which developed into gametophores by 40 days. Bud formation and gametophore development followed a pattern common to many mosses. Addition of a virulent strain of Agrobacterium tumefaciens (B6) to the moss cultures increased bud formation and hastened the time of their appearance by 5–6 days. With 10⁹ or more bacteria per ml of moss culture medium the percentage of plants with gametophores at day 35 after the spores were sown was 96 % or greater, as opposed to 0–24 % in the controls. The mean number of gametophores per responding plant was also increased from one per plant in controls to 4–6 per plant in inoculated cultures. Addition of the bacterium at day 17–18 of culture was as effective as early additions of the bacterium, suggesting that the moss must become ready to bud before the bacterium can influence its development. The promotion of gametophore formation was directly related to the number of bacteria added and depended upon the presence of viable bacteria. The supernatant from bacterial cultures did not promote gametophore formation. The changes induced by A. tumefaciens were similar to those reported for cytokinins.     

Human T-cell cultures with selective autotumor reactivity

Summary T-cell cultures derived from the blood of 14 patients with solid tumors were propagated with T-cell growth factor (TCGF). The cultures were initiated from lymphocytes exposed to autologous tumor-biopsy cells. TCGF was added either immediately or 3–10 days later. In the former culture type the cell yield on day 7 was considerably higher. The cytotoxic potential of the cultured cells was assayed on two occasions, between days 7 and 10 and between weeks 5 and 8. Cells of all but two cultures had the potential to lyse autologous tumor-biopsy cells.On the population level, cytotoxicity was specific for autologous tumor in those cultures that were driven to growth with TCGF after the 3rd day. These lymphocytes did not lyse allogeneic tumor-biopsy cells. In contrast, all five cultures initiated in the presence of TCGF exhibited a broader cytotoxic potential, i.e., in addition to the stimulator autologous-tumor cells, they also lysed other targets. Another difference between the two culture types was their behavior toward K562. Tested on the 7th day they all lysed K562; however, this function declined in strength or disappeared later in the cultures exposed to TCGF after the 3rd day.Reexposure of the lymphocytes to autologous tumor-biopsy cells after 2 weeks of culture period, but not on the 7th day, induced DNA synthesis. This secondary response was specific inasmuch as allogeneic tumor cells had little or no effect.One of the autotumor restimulated cultures was tested for cytotoxic potential. It increased against the autologous but not against other tumors or K562 cells.     

Sugar-induced adventitious roots in<Emphasis Type="Italic"> Arabidopsis</Emphasis> seedlings

The effects of sugars on root growth and on development of adventitious roots were analyzed in Arabidopsis thaliana. Seeds were sown on agar plates containing 0.0–5.0% sugars and placed vertically in darkness (DD) or under long day (LD, 16 h:8 h) conditions, so that the seedlings were constantly attached to the agar medium. In the sucrose-supplemented medium, seedlings showed sustained growth in both DD and LD. However, only dark-grown seedlings developed adventitious roots from the elongated hypocotyl. The adventitious roots began to develop 5 days after imbibition and increased in number until day 11. They could, however, be initiated at any position along the hypocotyl, near the cotyledon or the primary root. They were initiated in the pericycle in the same manner as ordinary lateral roots. Sucrose, glucose and fructose greatly stimulated the induction of adventitious roots, but mannose or sorbitol did not. Sucrose at concentrations of 0.5–2.0% was most effective in inducing adventitious roots, although 5.0% sucrose suppressed induction. Direct contact of the hypocotyl with the sugar-supplemented agar medium was indispensable for the induction of adventitious roots. Electronic Publication     

Microspore development during <Emphasis Type="Italic">in vitro</Emphasis> androgenesis in triticale

Microspore division was monitored in three triticale (× Triticosecale Wittmack) genotypes over 21 d of in vitro anther culture, on two media differing in their 2,4-dichlorophenoxyacetic acid content. After low temperature (4 °C) pre-treatment for two weeks, all the microspores were still alive, but they began to die from day one of culture. Both genotype and culture medium affected the number of microspores that aborted over time (82 – 97 % by day 21), the number of microspores that underwent the first symmetrical division (> 82 % over all), the number of microspores that attained four or more nuclei, and the number of divisions per 100 alive microspores after 21 d of culture.