A new micro-method for testing plant growth retardants in cell suspension cultures

A microtest sytem for detecting the growth retarding potential of chemosynthetic compounds in cell suspension cultures is presented. The new screening technique involves the cultivation of one millilitre of suspension in small sterile test tubes and indirect monitoring of growth by measuring conductivity changes in the medium. Highly significant correlations were obtained in experiments comparing the effects of a broad range of plant growth retardants on the growth of different suspensions in erlenmeyer flasks measured by cell counting and in test tubes with growth recorded by conductivity changes in the medium. The method described is suitable for all suspension cultures hitherto studied.For Abbreviations, see Materials and Methods     

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.     

Influence of pH on organic acid production by Clostridium sporogenes in test tube and fermentor cultures

The influence of pH on the growth parameters of and the organic acids produced by Clostridium sporogenes 3121 cultured in test tubes and fermentors at 35 degrees C was examined. Specific growth rates in the fermentor maintained at a constant pH ranged from 0.20 h-1 at pH 5.00 to 0.86 h-1 at pH 6.50. Acetic acid was the primary organic acid in supernatants of 24-h cultures; total organic acid levels were 2.0 to 22.0 mumol/ml. Supernatants from pH 5.00 and 5.50 cultures had total organic acid levels less than one-third of those found at pH 6.00 to 7.00. The specific growth rates of the test tube cultures ranged from 0.51 h-1 at pH 5.00 to 0.95 h-1 at pH 6.50. The pH of the medium did not affect the average total organic acid content (51.5 mumol/ml) but did affect the distribution of the organic acids, which included formic, acetic, propionic, butyric, 3-(p-hydroxyphenyl)propionic, and 3-phenylpropionic acids. Butyric acid levels were lower, but formic and propionic acid levels were higher, at pH 5.00 than at other pHs.     

Influence of pH on organic acid production by Clostridium sporogenes in test tube and fermentor cultures.

The influence of pH on the growth parameters of and the organic acids produced by Clostridium sporogenes 3121 cultured in test tubes and fermentors at 35 degrees C was examined. Specific growth rates in the fermentor maintained at a constant pH ranged from 0.20 h-1 at pH 5.00 to 0.86 h-1 at pH 6.50. Acetic acid was the primary organic acid in supernatants of 24-h cultures; total organic acid levels were 2.0 to 22.0 mumol/ml. Supernatants from pH 5.00 and 5.50 cultures had total organic acid levels less than one-third of those found at pH 6.00 to 7.00. The specific growth rates of the test tube cultures ranged from 0.51 h-1 at pH 5.00 to 0.95 h-1 at pH 6.50. The pH of the medium did not affect the average total organic acid content (51.5 mumol/ml) but did affect the distribution of the organic acids, which included formic, acetic, propionic, butyric, 3-(p-hydroxyphenyl)propionic, and 3-phenylpropionic acids. Butyric acid levels were lower, but formic and propionic acid levels were higher, at pH 5.00 than at other pHs.     

Effect of oxygen supply on passaging, stabilising and screening of recombinant Hansenula polymorpha production strains in test tube cultures

Twenty-four Hansenula polymorpha transformants were passaged and stabilised in glucose medium and screened in glycerol medium for recombinant phytase in shaken test tubes. The cultivations were performed under either limited or non-limited oxygen supply. Maximum oxygen transfer capacities of test tubes were assessed by sulfite oxidation. Oxygen-limited glucose cultures resulted in a partially anaerobic metabolism and formation of 4.1 g ethanol l(-1), which was subsequently aerobically metabolised. Non-limited oxygen supply led to overflow metabolism and to accumulation of 2.1 g acetic acid l(-1), reducing the biomass yield. The use of glycerol in the screening main cultures prevented by-product formation irrespective of oxygen supply. Preculturing in glucose medium under non-limited oxygen supply resulted in a 20-h lag phase of the screening main culture. This lag phase was not observed when preculturing was performed under oxygen limitation. Phytase activity was on average 25% higher in cultures passaged, stabilised and screened under limited oxygen supply than in cultures under non-limited oxygen supply.     

A technique for in situ karyotyping of primary amniotic fluid cell cultures.

A time proven technique is described for growing amniotic fluid cell cultures on cover glasses in Leighton tubes and for processing the mitotic cells in situ. Karyotyping the clones in situ eliminates most of the problems caused by somatic chromosome mutations in vitro and by maternal cell growth.     

The growth of Spirillum volutans ehrenberg in mixed and pure cultures

Summary The growth of Spirillum volutans in mixed culture was studied and liquid media were developed in which it grew rapidly and attained populations of over a million cells per ml. By taking advantage of their rapid and unidirectional motility, spirilla were separated from the mixed population by migration in capillary tubes. Pure cultures were achieved by growing the separated spirilla inside of dialysis sacks in contact with mixed cultures on the outside, and also by growth in an asparagine-mineral salts medium supplemented with an extract of Escherichia coli.Dedicated to Professor E. G. Pringsheim on his 80th birthday.This investigation was supported in part by grant G 9882 from the National Science Foundation.     

Ethylene enhances shoot formation in cultures of the peach rootstock GF-677 (Prunus persica x P. amygdalus)

The influence of ethylene on shoot formation from GF-677 (Prunus persica × P. amygdalus) shoot tip explants was studied in vitro. Cultures in test tubes were placed inside 5 1 glass jars and supplemented with various ethylene concentrations (0–10 ppm). Ethylene at 0.1 ppm, applied during the first 2 weeks of culture, increased the number and the length of shoots produced in vitro. Test tubes with cultures sealed with various types of closure accumulated in their atmosphere different levels of ethylene ranging from 0.1 to 1.2 ppm, depending on the type of closure. Test tubes with cotton-wool bungs had the least while those with serum stoppers had the highest amount of ethylene. The maximum number of shoots was recorded in test tubes covered with serum stoppers. The ethylene concentration was related linearly (R=0.974) to the shoot number and exponentially (R=0.975) to the shoot length.Abbreviations BA benzyladenine - IAA indoleacetic acid - HSD honestly significant difference     

Conditions affecting the amphotericin B mediated inhibition of Candida albicans attachment to cell cultures

Sublethal amounts of amphotericin B inhibited the attachment of Candida albicans to cultured mammalian cells. The extent of inhibition was influenced by the concentration of serum and the growth phase of the yeasts used to inoculate the cell cultures. Yeasts which were in their exponential phase of growth or had formed germ tubes were the most sensitive to amphotericin B. Equivalent amounts of amphotericin B inhibited yeast-mammalian cell interactions to different degrees depending upon the culture's tissue origin.     

Maintaining cultures of ectomycorrhizal and plant pathogenic fungi in sterile water cold storage.

Mycelial cultures of 64 isolates of 14 species of ectomycorrhizal fungi and 27 isolates of 15 species of plant pathogenic fungi were grown on agar medium in Petri dishes. Mycelial discs, 8 mm in diameter, were removed from the cultures and stored in sterile distilled water in test tubes at 5 degrees C. Sixty-four, 61, and 41 isolates of the symbiotic fungi were viable after 1, 2, and 3 years storage respectively. Only 19, 10, and 8 isolates of the pathogenic fungi were viable after 1, 2, and 3 years storage, respectively. Time in pure culture before water storage did not affect viability of any fungal species following water storage. After 3 years storage, four fungi (three symbionts and one pathogen) were tested and found to have retained their original growth rates and root-infecting abilities on pine seedlings. The same four isolates, however, maintained on agar slants at 5 degrees C and subcultured every 4 to 6 months, grew slower and did not infect as many feeder roots of pine as the water-stored isolates.     

Myotube-like structures in cultures of rat liver cells

Branching and budding myotube-like structures developed in primary cultures of rat liver cells and in the JH1 cell line derived from them. Elongated uninuclear cells aligned in chains and fused into multinuclear tubes of varying length and thickness. The tubes contained thick and thin filaments running in all directions. The filaments were occasionally linked with M lines and formed incomplete hexagonal patterns resembling those of skeletal muscle myofilaments, but a regular arrangement of the filaments and organelles was lacking. Cross-striation and contractions were never observed. Both uninuclear cells and multinuclear tubes contained numerous lysosomes, myelin figures and lamellated bodies as well as electronlucent or content-filled vacuoles and cisternae of variable size, sometimes reminding the sarcoplasmic reticulum in early stages of its development. Endocytotic caveolae and vesicles were present in all elements. These features together with interdigitated cell processes and specialized cell contacts suggested a possible relationship of the cells to the reticuloendothelial system.     

Use of Shake Cultures in a Semisolid Thioglycolate Medium for Differentiating Staphylococci from Micrococci

The standard diagnostic test for differentiating staphylococci from micrococci is based on the ability of the former to produce acid anaerobically in a glucose-containing growth medium. This test has been modified to provide greater convenience, easier interpretation of results, and better correlation with deoxyribonucleic acid (DNA) base composition. In the modified test, shake cultures in Brewer's fluid thioglycolate medium with 0.3% agar added are observed for growth in the anaerobic zone of the tubes. This test was applied to 125 strains of staphylococci and micrococci, and all except two strains gave results that were consistent with other criteria. Of particular interest were eight strains of Micrococcus saprophyticus and three strains of M. lactis that have a DNA composition of 30 to 37% guanine plus cytosine (GC). All 11 of these cultures produced anaerobic growth and thus would be classified as staphylococci. Strains of M. lactis that have a high GC content in their DNA grew only aerobically. Some cultures of staphylococci produced characteristic band patterns of anaerobic growth and other cultures produced only a few anaerobic colonies from an inoculum of 10(6) to 10(7) cells. These observations suggest some interesting genetic and metabolic capabilities in such cultures.     

How reliable are in vitro clonal cultures? Some comments based on hemopoietic cultures

Clonal cultures in semisolid medium have proved invaluable in analyzing hemopoietic subpopulations and in detecting their specific growth regulators. However, they can be subject to certain deficiencies that an investigator must take care to exclude. These include inabilities of the particular culture system to detect the true stem cells under study or to allow self-generation of clonogenic cells or a full expression of their differentiation potential. Clonal cultures, like conventional cultures, can be subject to significant cell-cell interactions, complicating attempts to characterize the action of a test regulatory molecule. Culture data need to be supplemented by a variety of other data before they can be regarded as valid evidence that a regulatory molecule detected in vitro is likely to function in a similar manner in vivo.     

Secretion of an antibacterial factor during resuscitation of dormant cells inMicrococcus luteus cultures held in an extended stationary phase

A high proportion ofMicrococcus luteus cells in cultures starved for 3–6 months in spent medium following growth to stationary phase in batch culture lost the ability to grow and form colonies on agar plates, but could be resuscitated from dormancy by incubation in liquid medium containing supernatant taken from the late log phase of viable cultures of the same organism (Kaprelyants et al. 1994). In the present work, we found that during the first 50–70 h of such resuscitation the dormant cells actually divide for 10–17 generations in lactate minimal medium containing yeast extract whilst remaining nonculturable on agar plates. Further incubation results in a decrease in the total cell number in liquid medium. The addition of viable (culturable)Micrococcus luteus cells in concentrations of up to 10⁴ ml^–1 to test tubes containing either resuscitating cells or supernatant from these cultures revealed the excretion of a factor or factors which inhibited the proliferation of otherwise viable cells. The maximum production of this factor took place after some 96 h of incubation of starved cells in resuscitation medium. Supernatant from late logarithmic phase batch cultures ofM. luteus abolished the antibacterial effect of starved cultures incubated in resuscitation medium. It is concluded that the stimulating effect of viable cells, and of supernatant taken from batch cultures, on the resuscitation of dormant cells might be connected in part with overcoming the activity of an antibacterial factor causing self-poisoning of dormant cells during their resuscitation.     

Control of pH and inorganic carbon in batch cultures of cyanobacteria

Summary Synechococcus leopoliensis was grown in batch cultures gassed with air or CO₂ in air to test for effects of gassing on pH drift and growth. A method is described whereby pH and inorganic carbon are held constant during rapid growth.     

An improved method of eliminating fungal contamination from monolayer cell cultures

A method is described for eliminating fungal and other forms of contamination from valuable monolayer cell cultures. The method employs the following sequence of operations: several changes of medium, trypsinization and removal of cells to coverglasses in appropriate tubes with medium plus amphotericin B (Fungizone) or other antibiotic, removal of coverglasses to new tubes with medium plus antibiotic, and removal in a few days to a new culture vessel without antibiotic. If microorganisms do not show up in 3–5 days, they have been eliminated. Greater success is achieved by using the same method with coverglass fragments in small culture wells.     

Neural control of early myogenic differentiation in cultures of mouse somites

Neural tubes, with flanking somite streaks, were isolated from mouse embryos ranging in age from 8 to 11 days post coitus (dpc). The somites were further dissected along the neural tube to obtain one somite streak associated with the neural tube and the other free of nerve cells. The two groups of somites (with and without neural tubes) were dissociated to single cell suspension by a brief incubation with EDTA. High-density micro-mass cultures were established from these two groups of cells and the extent of cell differentiation was assayed by staining the cultures with an anti-myosin antibody. The results obtained indicated that during early somitogenesis (8.5 dpc) the presence of cells from neural tube is necessary for the emergence of myosin-positive cells in culture. At later stages (10.5 dpc), however, the total number of myosin-positive cells appearing in culture is largely independent from the presence of nerve cells. At these later stages, the presence of nerve cells inhibited the occurrence of fusion in myogenic cells. Many multinucleated myotubes appeared in cultures of somitic cells in the absence of nerve cells, but very few in their presence. The possible relationship of these data with the appearance of mononucleated differentiated cells in myotomes in vivo and the possible neural control of this stage of myogenesis are discussed.     

Degradation of 2,4-dichlorophenoxyacetic acid by mixed cultures of bacteria

Summary We explored the feasibility of using mixed cultures for herbicide degradation, with the ultimate aim of application for effluent treatment. The present study reports on mixed cultures which were developed to grow aerobically with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon substrate. Degradation of 2,4-D was verified by HPLC and UV-spectroscopic analysis of the residual 2,4-D concentration in the test cultures. Cultures that were initially developed with 2,4-D also grew readily with glucose, but the degradation of 2,4-D was effectively prevented under mixed substrate conditions. Mamor intermediates or metabolites resulting from 2,4-D degradation were not detected with the HPLC methodology except 2,4-dichlorophenol which appeared to accumulate transiently in the growth medium.     

Semielectronic turbidimeter for automated monitoring of bacterial growth in test tubes.

An automated turbidimeter for measuring bacterial growth in ordinary test tubes is described. The device records and prints adsorbance, expressed as Klett units, of 60 cultures every 15 min. Provision is made for either aerobic or anaerobic incubation. The device is adaptable to modification, depending upon local requirements and availability of computation facilities.     

Monitoring of environmental arsenic by cultures of the photosynthetic bacterial sensor illuminated with a near-infrared light emitting diode array

Recombinant Rhodopseudomonas palustris, harboring the carotenoid-metabolizing gene crtI (CrtIBS), and whose color changes from greenish yellow to red in response to inorganic As(III), was cultured in transparent microplate wells illuminated with a light emitting diode (LED) array. The cells were seen to grow better under near-infrared light, when compared with cells illuminated with blue or green LEDs. The absorbance ratio of 525 to 425 nm after cultivation for 24 h, which reflects red carotenoid accumulation, increased with an increase in As(III) concentrations. The detection limit of cultures illuminated with near-infrared LED was 5 microgram/l, which was equivalent to that of cultures in test tubes illuminated with an incandescent lamp. A near-infrared LED array, in combination with a microplate, enabled the simultaneous handling of multiple cultures, including CrtIBS and a control strain, for normalization by the illumination of those with equal photon flux densities. Thus, the introduction of a near-infrared LED array to the assay is advantageous for the monitoring of arsenic in natural water samples that may contain a number of unknown factors and, therefore, need normalization of the reporter event.