A Centrifugation Technique for Preparing Medium-Free Resinous Mounts of Feulgen-Stained Pollen Tubes

Air-dried pollen of Tradescantia paludosa and a colchicine doubled Allium ascalonium-fistulosum interspecific hybrid was sown in culture tubes on an inorganic salt-lactose liquid medium containing 0.02% colchicine. After a 16-20 hr incubation at 22 C, pollen tubes were harvested by centrifugation for 3-5 min at 1100-1400 rev/min and fixed with acetic-alcohol (1:3). Feulgen staining was carried out in the culture tubes with fluid changes made after the centrifugation following each step. Single drops of the final pollen-45% acetic acid suspension were flattened under silicone-treated coverglasses which were removed by the quick freeze technique prior to counterstaining with fast green, dehydration, and mounting in Diaphane or Canada balsam. Medium-free, Feulgen-stained, resin-mounted preparations of well-dispersed pollen tubes with arrested metaphases were obtained.     

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.     

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.     

Multiple loop purification method for selective cultivation of Pentatrichomonas hominis

It is always troublesome having protozoan cultures contaminated with other organisms in the laboratory. The method described here produces high efficiencies of purification for fast moving flagellate protozoa. A human strain Pentatrichomonas hominis was employed in the study to examine the effects of multiple loop tubes on the purification of flagellates. Trichomonads were harvested from a trypticase yeast extract iron-serum-33 (TYI-S-33) medium, adjusted to 2 X 10(5) organisms/ml, and mixed with an equal volume of 2 X 10(6) organisms/ml of bacteria. The isolation was performed at 37 degrees C in TYI-S-33 medium containing a suitable amount of antibiotics (1000 U/ml of penicillin, 1000 micrograms/ml of streptomycin, and 4 micrograms/ml of fungizone). Four days later, 10(6) organisms/ml of protozoa, free of bacteria, were observed at the other end of the single loop and the double loop tubes. About the same amount of flagellates could be found at the other end of the triple loop tube six days after incubation. The traditional U-shaped tubes were used as controls and 10(5) cells/ml of flagellates were recovered in the presence of bacteria two days after incubation. An axenic culture of P. hominis was successfully isolated from the feces of a Formosan rock-monkey, Macaca cyclopsis, by this method. Purified trichomonads were recovered from a double loop purification tube five days after incubation.     

Method for the Simultaneous Establishment of Many Axenic Cultures of Paramecium*†

SYNOPSIS. 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, stigmasterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing ～ 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 ciliates 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 each of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

Perfusion strategy for rosmarinic acid production by Anchusa officinalis

The production of an intracellular secondary metabolite rosmarinic acid (RA) by plant cell suspensions of Anchusa officinalis cultivated with intermittent medium exchange is investigated. Initially, a two-stage perfusion culture method was employed. After being cultured in the batch mode for ca. 6 days in B5 medium plus 3% sucrose, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg/L kinetin (2,4-D B5 medium), Anchusa culture was cultivated to high cell density by perfusion during the growth stage using a hormone-free Gamborg B5 medium supplemented with 6% sucrose. This was followed by a production stage, in which a complete medium exchange into B5 medium plus 3% sucrose and 0.25 mg/L naphthleneacetic acid (NAA) was conducted. The two-stage perfusion culture had a higher maximum culture RA concentration but a lower RA content per cell than the batch stock culture maintained in the 2,4-D B5 medium. Higher culture RA concentration was due primarily to high cell density. The high packed cell volume, however, seemed to reduce the synergistic effect of NAA on RA synthesis. Subsequently, a single-stage perfusion culture method was investigated. The best result was obtained by growing the culture in the batch mode for ca. 10 days using B5 medium supplemented with 3% sucrose and 0.25 mg/L NAA, followed by perfusing the culture with B5 medium plus 6% sucrose and 0.25 mg/L NAA at a constant perfusion rate of 0.1/day. A maximum cell dry weight of 35 g/L and a RA concentration of almost 4 g/L were achieved. This is the highest RA concentration ever reported in the Anchusa culture. (c) 1993 John Wiley & Sons, Inc.     

Clonal Variation in Paramecium. I. Persistent Unstable Clones

Clones of Paramecium of identical serotype when cultured in test tubes may differ in their ability to give rise to subclones of this serotype. Characteristically, stable clones yield progeny indistinguishable from their parents, while from unstable clones diverse subclones with new serotypes can be isolated repeatedly. Stable lines are resistant to changes in culture medium and also are unaffected by most sera. In contrast, the numbers and kinds of serotypes displayed among subclones derived from unstable lines are often affected by these same agents. Stable and unstable clones are interconvertible when the medium from individual cultures is repeatedly and frequently replaced by fresh culture fluid. This effect is very likely a result of the removal of the initial exhausted medium with any cell products rather than the addition of fresh nutrient.     

Analysis of developmentally homogeneous neural crest cell populations in vitro. III. Role of culture environment in cluster formation and differentiation

Optimal conditions to obtain homogeneous neural crest cell populations with different developmental fates in vitro have been developed. If enzymatically cleaned neural tubes are explanted on an agar-coated dish in a medium containing high concentrations of fetal calf serum (FCS; 20%) and chick embryo extract (CEE; 8%), neural crest cell clusters, each containing several thousand cells, form on the explanted neural tubes within 48 h of culture. Under most standard culture conditions, all of these cells differentiate into melanocytes after another 3–5 days in culture. On the other hand, if tubes be placed on a tissue culture dish in a medium containing a ratio of FCS : CEE of 2–3 : 1 (e.g. 7.5% FCS, 3% CEE), two populations of crest cells are obtained: one population is identical to the clusters described above, and homogeneously differentiates into melanocytes; the other is characterized by cells of similar morphology that migrate onto the culture substratum and undergo little overt differentiation. By altering the extent of aggregation of the crest cells in primary culture, their differentiative fate in vitro can be altered. Therefore, the differentiative behaviors of the two subpopulations appear to be due to different early environmental influences in vitro rather than solely to inhomogeneities in the pre-migratory crest cell population.     

A statistical comparison of two culturing methods for enumerating sulfate-reducing bacteria

The addition of alkaline pyrogallol-soaked cotton wool plugs has been used by other workers to remove oxygen from the headspace gas in culture tubes for the growth of sulfate-reducing bacteria (SRB). This study compares the enumeration fo SRB using the most probable number (MPN) method in tubes with and without the pyrogallol plugs. In both cases, the liquid medium contained two iron finishing nails which help reduce the redox potential of the medium. For each of the 25 samples from oil fields, cooling water systems and natural environments, the time-consuming method using pyrogallol plugs in screw cap tubes yielded virtually the same MPN values as the same method without the plugs and using Kaput® closures on the tubes. However, the pyrogallol plug method, which requires approximately 10-fold more technician time, was superior for pure culture enumeration and in cases where SRB greatly outnumber heterotrophs.     

In vitro culture of bovine preantral follicles

Bovine preantral follicles (40-100 microm diameter at collection) were collected from ovaries of slaughtered cows and cultured in vitro with one of the four treatments: follicle stimulating hormone (FSH; 100 ng/ml) alone; FSH plus epidermal growth factor (EGF; 100 ng/ml); FSH plus insulin-transferrin-selenium (ITS; +1%) or FSH plus hypoxanthine (4 mM) in tissue culture medium (TCM 199) supplemented with 10% fetal calf serum (FCS), 0.1 mg/ml sodium pyruvate, 100 IU/ml of penicillin and 100 microg/ml streptomycin. The control culture medium was TCM 199 with supplements without any treatments. Follicles of each size were cultured separately in groups of one to three in 24-well multidishes each containing 500 microl of the appropriate culture medium. Culture commenced at follicle recovery (day 1) and continued for 10 days (harvested on day 11). In each case, half the medium was removed and replaced by fresh medium every third day. Follicle diameters were recorded on days 1, 5 and 11 of the experiment. At the end of the 10-day culture period, half of the follicles were stained with trypan blue to assess their potential viability and half were stained with bisbenzimide plus propidium iodine to estimate various morphological features of the follicles. Follicles of all initial sizes, on all culture treatments, increased in diameter during in vitro cultures with the greatest increases, both in absolute and proportional size, occurring between days 1 and 5 of culture. All of the culture medium supplements caused greater increases in follicle diameters than control medium at both days 5 and 11 of culture for all initial sizes of follicles (p<0.01). The most effective culture supplements for follicles of 40-, 60- and 80-microm initial diameter were FSH alone and FSH+EGF. The size of these follicles at both days 5 and 11 of culture on both the treatments was significantly larger (p<0.01) than follicles cultured in the presence of the other two supplementary treatments. The growth of follicles of 100-microm initial diameter did not differ between culture medium supplements. None of the culture media caused follicle size to increase to the initial diameters of the next larger size category during the 10 days of culture although follicles of 100-microm diameter achieved a diameter of 120 microm, after 4 days of culture.The overall follicular viability and morphology were better with treatments than the controls in all cases; however, there was no significant difference (p>0.05) among them.From this experiment, FSH and FSH plus EGF may be recommended for in vitro culture of smaller (40, 60 and 80 microm) follicles.     

Lily (<Emphasis Type="Italic">Lilium longiflorum</Emphasis> L.) pollen protoplast adhesion is increased in the presence of the peptide SCA

The style of lily produces a specialized extracellular matrix (ECM) in the transmitting tract epidermis that functions to guide pollen tubes to the ovary. This adhesive ECM contains low esterified pectins and a peptide, SCA (stigma/stylar cysteine-rich adhesin). Together they form a matrix to which pollen tubes adhere as they grow through the style. Pollen tubes also adhere to each other but only when grown in vivo, not in vitro. Pollen does not produce detectable SCA, but when SCA is added to an in vitro growth medium, it binds to pollen tubes that have esterified and low-esterified pectins in their walls. Since adhesion of the pollen tube to the stylar matrix requires tip growth, we hypothesized that the pectin wall at the pollen tube tip interacted with the SCA protein to initiate adhesion with the stylar pectin [Lord (2000) Trends Plant Sci 5:368–373]. Here, we use a pollen protoplast system to examine the effect of SCA on protoplast adhesion when it is added to the growth medium in the absence of the stylar pectin. We found that SCA induces a 2-fold increase in protoplast adhesion when it is added at the start of protoplast culture. This effect is less when SCA is added to the medium after the cell wall on the protoplast has begun to regenerate. We show that among the first components deposited in the new wall are arabinogalactan proteins (AGPs) and highly esterified pectins. We see no labeling for low esterified pectins even after 3 days of culture. In the pollen protoplast culture, adhesion occurs in the absence of the low esterified pectin. The newly formed wall on the protoplast mirrors that of the pollen tube tip in lily, which is rich in AGPs and highly esterified pectins. Thus, the protoplast system may be useful for isolating the pollen partner for SCA in this adhesion event.     

Multiplication of human diploid fibroblasts in a synthetic medium supplemented with EGF, insulin, and dexamethasone

Substantial multiplication of human diploid fibroblasts (HDF) has been obtained in medium MCDB 108 supplemented with epidermal growth factor (EGF), insulin, and dexamethasone (DEX). Growth rate is somewhat slower than in serum-supplemented medium. However, large wellformed colonies can be obtained in 14 days, and sequential monolayer subculture is possible up to a total of about ten population doublings. A basal medium that has been optimized specifically for HDF is essential for such multiplication. In addition, polylysine-coated culture surfaces, low temperature trypsinization, and careful removal or neutralization of residual trypsin are also needed. The culture system contains no deliberately-added undefined components, and is chemically defined except for possible roles of contaminants in the materials that are used for its preparation.     

A simple method for cloning leishmanial promastigotes

A method of cloning leishmanial promastigotes is described in which mid-exponential phase cultures are diluted to contain approximately 3 X 10(3) promastigotes per ml. Hanging-drop preparations made from 0.2-0.4 microliter volumes of the diluted culture seen to contain a single promastigote are picked-up in glass capillary tubes. Additional culture medium is taken into the capillaries which are then heat-sealed and incubated at 22 degrees C. Growth of leishmanial promastigotes inside the sealed capillary tubes is followed by direct microscopic observation through the walls of the tubes. When active promastigotes are seen the contents of the tubes are inoculated into small volumes of culture medium. The method is extremely easy to use, requires no specialised apparatus, and has been successfully used with different strains and species of Leishmania, with up to 100 percent of the cloned organisms growing.     

Description of a tissue culture technique using tissue from mussel (Mytilus edulis) for the preparation of chromosomes]

Explants from mantle and foot tissues of adult mussel were grown in culture tubes containing a medium composed of Eagle's Basal Medium supplemented with salts, Hepes buffer, egg yolk and antibiotics. The cultures were maintained at 18 degrees C and pH 7.50, without medium renewal. After 6-7 days, the cultures were stopped and harvested for slide preparation. Numerous metaphase spreads that were good enough for karyotyping were consistently obtained. This method may prove to be a reliable source of actively dividing cells that is a prerequisite for extensive chromosome structure analyses in the bivalves.     

A Simple Method for Fixing, Dehydrating and Embedding Pollen Tubes Cultivated in Vitro for Optical and Transmission Electron Microscopy

A simple method to cultivate pollen tubes in a gelatin medium is presented. After the growth of the pollen tubes in the culture medium, they are fixed, dehydrated, and embedded in resin for ultramicrotomy. The method is easy and does not require the purchase of special materials beyond those needed for the usual techniques for studying biological specimens under transmission electron microscopy.     

Rapid in vitro propagation of cauliflower

A rapid single step method for complete plantlet regeneration in cauliflower is described. Curd explants cultured on Murashige and Skoog (MS) medium supplemented with 1 mg⁻¹ indoleacetic acid (IAA) developed complete plantlets in 25 days. Regeneration potential of curd explants was drastically reduced with increasing storage of cauliflower curds at room temperature beyond 8 days of harvesting. Light intensity of 3000 lux supplied by white fluorescent tubes was optimum to evoke the best response. It was possible to field transfer the hardened plantlets within 35 days of culture initiation.     

Differentiation of erythroleukemic cells in vitro: irreversible induction by dimethyl sulfoxide (DMSO).

The inclusion of DMSO in the media of suspension cultures of Friend erythroleukemia cells results in the erythroid differentiation of these cells. The studies reported here were directed towards answering two questions. (1) How long an exposure to DMSO is necessary to induce the differentiation of these cells; and (2) What is the fate of the differentiating cells when DMSO is removed from the medium. Exposure to DMSO for less than 24 hours failed to produce any detectable evidence of erythroid differentiation. On the other hand, culture in the presence of DMSO for 24 hours followed by culture in DMSO-free medium for four additional days produced a small but detectable increment in the proportion of benzidine positive cells in the culture. Once the differentiation of an individual cell was initiated, the process continued after removal of DMSO from the medium. The cell became progressively more differentiated as evidenced by increases in the intensity of benzidine staining as well as the rate of heme synthesis and heme content. However, when cells which had been induced to differentiate by DMSO were cultured in DMSO-free medium for more than 3--4 days, they became vacuolated and apparently died. This latter phenomenon, as well as the more rapid proliferation of the undifferentiated cells in the culture, accounts for the observation that when new cultures are established from cultures which have been grown in the presence of DMSO for several days, the culture which results ultimately contains only differentiated cells.     

An in vitro assay for assessing the effects of growth factors on Nicotiana alata pollen tubes

 A new method for assessing the effects of test compounds on Nicotiana alata pollen tubes in culture is described. Pollen tubes grow from a cluster of grains placed beneath a thin layer of gelled medium in which test substances are incorporated and from which evaporation is prevented by a covering layer of oil. Pollen tubes can grow to 8 mm in length in 24 h, which corresponds to about 25% of the maximum growth rate in styles. Growth is non-destructively measured. The developmental stages reached by cultured tubes are similar to those of tubes growing in styles; growth changes from being reserve-dependent to reserve-independent, callose plugs form, and the nucleus of the generative cell divides. Because culture volumes are small (10–20 μl per replicate), the effects of known concentrations of microgram quantities of compounds on the growth of pollen tubes can be tested. Received: 25 February 1997 / 21 July 1997     

Effect of fumagillin on in vitro multiplication of Encephalitozoon cuniculi

Encephalitozoon cuniculi (Levaditi, Nicolau & Schoen) is an obligate intracellular pathogenic parasite of rabbits, carnivores, laboratory rodents, and a variety of other mammals. Cell cultures of rabbit and canine cells were infected with rabbit and dog isolates of E. cuniculi. Four days later 5 microgram/ml of fumagillin was introduced into the culture medium. The multiplication of the parasite was inhibited within 48 h and this effect was maintained as long as the antibiotic remained in the medium. There was no effect when spores and proliferative forms of the parasite were incubated with fumagillin before being used for infecting host cells. No infection occurred, however, if the antibiotic was added to the culture medium before introduction of E. cuniculi. On electron-microscopic examination, the treated parasites were found to have severe cytoplasmic swelling, vesicular distortion of the plasma membrane, and marked reduction in cytoplasmic ribosomes. it was concluded that fumagillin blocks multipliation of E. cuniculi in vitro. The drug may be useful for the treatment or prevention of spontaneous encephalitozoonosis.     

Arabinogalactan proteins, pollen tube growth, and the reversible effects of Yariv phenylglycoside

Arabinogalactan proteins (AGPs) are abundant complex macromolecules involved in both reproductive and vegetative plant growth. They are secreted at pollen tube tips in Lilium longiflorum. Here, we report the effect of the (beta-D-glucosyl)3 Yariv phenylglycoside, known to interact with AGPs, on pollen tube extension in several plant species. In Annona cherimola the Yariv reagent clearly inhibited pollen tube extension within 1-2 h of treatment, as demonstrated previously for L. longiflorum, but had no effect on Lycopersicon pimpinellifolium, Aquilegia eximia, and Nicotiana tabacum. With the monoclonal antibody JIM13 we also examined these same species for evidence that they secreted AGPs at their pollen tube tips. Only A. cherimola showed evidence of AGPs at the pollen tube tip as does lily. The Yariv reagent causes arrest of tube growth in both A. cherimola and lily, but its removal from the medium allows regeneration of new tip growth in both species. We show that the site of the new emerging tip in lily can be predicted by localization of AGP secretion. Labeling with JIM13 appeared on the flanks of the arrested tip 1 h after removal of the Yariv reagent from the growth medium. After 4 h, many of the Yariv reagent-treated pollen tubes had regenerated new pollen tubes with the tips brightly labeled by JIM13 and with a collar of AGPs left at the emergence site. During this recovery, esterified pectins colocalized with AGPs. Secretion at the site of the new tip may be important in the initial polarization event that occurs on the flanks of the arrested tube tip and results in a new pollen tube.