Segmented flow generation by chip reactors for highly parallelized cell cultivation

Micro system technology offers convenient tools for the production of handling devices for small liquid volumes which can be used in cell cultivation. Here, a modular system for the rapid generation of cell suspension aliquots is presented. The system is used to produce and analyze high numbers of well-separated culture volumes. Selected clones may be retrieved from the system. Therefore, the principle of segmented flow is applied. Portions of aqueous culture medium containing one cell or very small cell ensembles are separated from each other by a nonmiscible liquid like dodecane, tetradecane or mineral oil. In addition, the alkane separates the culture droplets from the innerside of the walls of chip channels and capillaries. This way, compatibility problems between cell wall surfaces and the chemical character of walls are excluded. The separated culture droplets are guided by micro flow transportation in different channel and chamber topologies. The whole system has the character of a serially operating cell processing system. The aliquot generation can be sped up to frequencies of about 30 Hz in each microchannel. That means, that about 10(5) individual cultural volumes can be produced per hour or about 2 million per day. The survival and the growth of microorganisms has been shown for model organisms as well as for organisms from a natural sample (soil).     

Detecting the Enterotoxigenicity of Staphylococcus aureus Strains

An optimal sensitivity plate method for examining large number of staphylococcal strains for production of the known enterotoxins (A-E) is presented. Small volumes of relatively concentrated enterotoxin are produced by the semi-solid agar, cellophane-over-agar, or sac culture techniques. Detection of the enterotoxin in the supernatant fluid is accomplished with the optimal sensitivity plate method. In this method small plastic petri dishes (50 mm) were used for a modified Ouchterlony of high sensitivity.     

Plate colonization of Methanococcus maripaludis and Methanosarcina thermophila in a modified canning jar

Abstract Conditions have been optimized for colonization of fastidiously anaerobic microorganisms on solidified plating medium incubated in modified canning jars. Two species of methanogenic Archaea, the mesophilic, hydrogen utilizer Methanococcus maripaludis and the moderately thermophilic methylotroph Methanosarcina thermophila , were grown with high efficiency on agar-solidified medium in petri plates. Maximum colony size and plating efficiencies of 50–100% of total cell counts were obtained by optimizing inoculation method, H₂S concentrations, and agar moisture content. The simple modifications required provide a readily available source of inexpensive anaerobic growth vessels for investigations requiring colonization of methanogens on solidified plating medium.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Emulsification and degradation of "Bunker C" fuel oil by microorganisms

An enrichment culture procedure has been used to isolate mixed culture systems which grow upon “Bunker C” fuel oil. When inoculated into a mineral salts aqueous medium containing Bunker C oil, the mixed cultures initiate oil emulsification. Emulsification usually is observed in 24–48 hr. The role of microbes in this emulsification will be discussed. It appears that certain metabolic products produced by the microbe possess properties of surfactants. Bacteria and fungi have been isolated which possess the ability to cause emulsification. Freeze-dried biomass is also capable of emulsifying oil. Chromatographic analyses of biodegraded Bunker C fuel oil show that microorganisms selectively metabolize the n-paraffin fraction.     

Growth and survival of protozoa isolated from a tannery effluent

Industrial effluent from a tannery was used for the growth of algae in a medium containing various inorganic salts. Growth of algal cells became visible after 7 d. Two species of protozoa were observed to proliferate in the algal culture containing no organic supplement in the medium. The culture was kept bacteria-free by the use of antibiotics and was perpetuated for at least 150 d with no decline in the protozoan population. Efficient growth of protozoa in a culture of algae elucidated new modes of nutrition in protozoa. Cr(VI) was added to the medium to check the resistance of algae and protozoa against this heavy metal. Protozoa showed different degrees of resistance. The results indicate the importance of algae and protozoa in the process of bioremediation.     

Colony formation of mammalian cells on agar plates and its application to Lederberg's replica plating

In this paper we describe a new technique of cloning by use of agar plates and its application to replica plating. It was found that most cell lines form colonies on the surface of solid agar, although the plating efficiency and size of colony is dependent on specimens and concentrations of agar and agarose used. When 0.5% Noble-agar was used as substrate, plating efficiencies were obtained comparable to those of conventional cloning techniques in liquid medium and of agar suspension cultures. In some cases, including the primary culture of Yoshida sarcoma, the efficiency of plating was apparently higher than that obtained by the already established procedures. In an experiment with a series of BHK-21 cells, it was found that virally transformed cells could form colonies on agar plate, whereas untransformed and reverted cells could not divide, suggesting that agar plate culture, as well as agar suspension culture, can be used for a selective assay of transformation.Two methods of replica plating were employed. Method I is that devised by Lederberg in which colonies on the master plate are imprinted on pile fabrics and then transferred to the replica plates. With FM3A cells, the fidelity of replica plating was around 95%. Method II is inoculation of clones by applying a glass rod to the replica plates on which positions of inocula were identified by a grid. Fidelity of replica plating of FM3A, L5178Y and YSC cells was 99.7, 100 and 100% respectively.     

Characterization of Esterases Produced by a Ruminal Bacterium Identified as Butyrivibrio fibrisolvens

A method of obtaining clones of Tetrahymena pyriformis on solid medium has been developed. The medium consists of a basal layer of 1.5% agar topped with 2 ml of 0.3% agar in sterile, plastic petri plates (100 by 15 mm). Both agar layers contain either 2% proteose peptone and 0.1% liver extract (complex medium) or defined medium supplemented with proteose peptone. After drying, 0.5 ml of liquid culture is spread evenly over the top agar, and the plates are then sprinkled lightly and evenly with autoclaved dry Sephadex G-25 (fine). Cell colonies can be observed after 5 days of incubation either by viewing with a microscope or without the aid of a microscope after staining. Plating efficiency is high on either complex or defined medium with a number of strains of Tetrahymena, both micronucleate and amicronucleate. Colonies can be picked and transferred to liquid culture for further growth. The existence of clones was demonstrated by plating a mixture of two different drug-resistant mutants. The method should prove useful in selective procedures for the isolation of mutants and for determining survival after treatments such as ultraviolet irradiation.     

An in vitro model for studies of intercellular communication in cultured rat anterior pituitary cells

The formation of intimate associations among different hormone-secreting cells within the rat adenohypophysis may serve as a possible site for physiologic regulation. In this report we describe a high density plating method which enables us to study cell-to-cell interactions within anterior pituitary cell cultures. Trypsin-dispersed pituitary cell suspensions attach rapidly (within 6 hr) and quantitatively (95-97%) to glass or plastic surfaces when plated in medium containing microM calcium concentrations (pH 7.6-7.8). Freshly plated cell suspensions obtained from female pituitary glands contained subpopulations of mammotrophs 49.3%, somatotrophs 30.3%, gonadotrophs 12.6%, corticotrophs 3.4% and thyrotrophs 1.5%. Epithelial cell colonies were formed during a 3-day culture period as the cells flattened and re-established contacts with neighboring cells. Freeze-fracture electron microscopic analysis of these colonies produced morphological evidence for direct intercellular contacts among the hormone-secreting cells. Large areas of tight junctions and small gap junctions were identified on the membranes of the epithelial cells within these colonies. Cells which contained tight junctions usually contained microvilli and morphological signs of active hormone secretion. Small junctional plaques containing tightly packed intramembrane particles were also occasionally found on the membranes of cells which were actively secreting pituitary hormones. The high density plating procedure which is described in this report provides greater opportunity for cell-cell interaction and thus may prove to be a useful model for evaluating the role of intercellular communication within this tissue.     

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca)

Regeneration of white spruce (Picea glauca) somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mgl^–1 2,4-D and 1 mgl^–1 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarose and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5–10% after 35 days culture.     

Lipase production by Trichosporon fermentans WU-C12, a newly isolated yeast

From the soil samples of various locations, 245 strains of microorganisms were isolated by the enrichment culture method using olive oil as a carbon source. Of these microorganisms one deuteromycotinous yeast was the best producer of extracellular lipase, and the strain WU-C12 was identified as Trichosporon fermentans from the morphological and taxonomical properties. When cultivated at 30°C for 4 d in the medium containing 8% (w/v) corn steep and 3% (v/v) olive oil as sources of nitrogen and carbon, T. fermentans WU-C12 produced 126 U/ml of extracellular lipase. When 3% (v/v) tung oil was used instead of 3% (v/v) olive oil, 146 U/ml of the lipase was produced. Although lipase production decreased to 40 U/ml by the addition of 2% (w/v) glucose to the corn steep-olive oil medium, the strain WU-C12 produced 34 U/ml of lipase in the medium containing 2% (w/v) glucose instead of 3% (v/v) olive oil. On the other hand, T. fermentans WU-C12 could grow and produce lipase in the medium containing n-paraffin as a carbon source.     

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.     

Direct detection of the hydantoinase activity on solid agar plates and electrophoretic acrylamide gels

A rapid and simple method to detect hydantoinase-producing microorganisms was developed using a selective agar plate containing 1% hydantoin and 0.005% Phenol Red in the typical Luria-Bertani medium. Conversion of hydan-toin to hydantoic acid decreases the pH and the agar turns bright yellow. The method was used to isolate hydantoinase-producing E. coli transformants in the cloning of hydantoinase gene and screening of the hydantoinase-producing microorganisms from soils. Detection of hydantoinase activity after PAGE also used the same method.     

Formation of organic acids by the fungus Cladosporium resinae in media containing n-alkanes]

The so-called fungus Cladosporium resinae that often occurs in oil fuels ane increases their acidity grows well at the expense of n-alkanes from C11 to C16. On the n-alkane containing media the fungus grows slowly and only under the stationary conditions. During the fungal cultivation on the media containing n-dodecane or kerosene the culture liquid shows acetic acid and other fatty acids, ketoacids (pyruvic and alpha-ketoglutaric) as well as citric and isocitric acids that dominate among nonvolatile acids. Upon nitrogen deficiency in the medium and comparatively good aeration the content of citric acids increases. The culture liquid of the fungus devoid from the mycelium and nonutilized n-alkanes can be used a a nutrient medium for different microorganisms.     

Induction of hypoxia in glass versus Permanox petri dishes

The survival of Chinese hamster ovary cells in culture following graded doses of X rays delivered under aerobic and hypoxic conditions, or treatment with the bioreductive drug SR 4233 under hypoxic conditions, was evaluated as a function of whether cells were plated onto glass or Permanox plastic petri dishes. In the case of treatment with SR 4233, the influence of varying the total volume of medium in the dishes was also studied. While the Permanox petri dishes were sufficient to yield "radiobiological" hypoxia, that is, oxygen enhancement ratios of approximately 3.0 were obtained for X irradiation, they were inferior to glass petri dishes with respect to the hypoxia-selective cytotoxicity of SR 4233. For a 90-min hypoxic exposure to 40 microM SR 4233, the surviving fraction of cells plated on plastic dishes averaged about 50-fold higher than that of cells plated on glass dishes. Although varying the total medium volume did affect the extent of SR 4233-induced cytotoxicity for glass dishes--drug toxicity decreased slightly with increasing medium volume--this was not the case for the plastic dishes, in which the cell survival following a fixed SR 4233 exposure was essentially constant as a function of medium volume. These results suggest, at least for SR 4233, and under these experimental conditions, that Permanox petri dishes are not satisfactory for such studies.     

SIMPLE AND RAPID METHOD FOR SCREENING ANTIMICROBIAL ACTIVITIES OF BIFIDOBACTERIUM SPECIES OF HUMAN ISOLATES

The objective of this research was to develop a rapid procedure for the screening antimicrobial activities of Bifidobacterium species of human isolates. A bifidobacteria selective medium BIM-25 agar was modified by adding of 0.5 g/L cysteine-hydrochloride, 1.5 g/L lithium chloride, 1.0 g/L beef extract, and 5 mL/L Tween 20. This medium was inoculated (45C) with diluted fecal material from 5 subjects and overlaid into 0.1% Tween 20 BHI agar plates. Plates were incubated in anaerobic chamber at 37C for 48 h. Plates were then inverted to allow the two layers of agar to fall into the petri lid. BHI soft agar (0.45%) containing Micrococcus luteus (as indicator) was overlaid onto the other layers in the petri dish. Plates were incubated at 37C overnight and zone of growth inhibition was observed. This method is simple and rapid whereas the original method for screening of antimicrobial activities of bifidobacteria is a more time consuming and cumbersome procedure.     

Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics of intercellular contacts and cell membrane differentiations

Primary monolayer cultures were obtained in 60-mm petri dishes by incubating 3 X 10(6) isolated hepatocytes at 37 degrees C in Dulbecco's medium supplemented with 17% fetal calf serum. The ultrastructure of monolayer cells was examined after various incubation periods. Within 4 h of plating, the isolated spherical cells adhere to the plastic surface, establish their first contacts by numerous intertwined microvilli, and form new hemidesmosomes. After 12 h of culture, wide branched trabeculae of flattened polyhedral cells extend in all directions. Finally, after 24 h of culture, bile canaliculi are reconstituted, and a biliary polarity is recovered: the Golgi elements, which are scattered throughout the cytoplasm in the isolated cells, are reassembled in front of the newly formed bile canalculi, symmetrically in the adjacent cells; lysosomes are concentrated in that region, and microtubules reappear. Concomitantly, plasma membrane differentiations, namely desmosomes and tight junctions, develop. Tight junctions sealing the bile ducts constitute a barrier to the passage of ruthenium red and horseradish peroxidase. De novo formation of these junctions was studied by the freeze-etching technique: 10-nm particles compose a network of anastomosed linear arrays in the vicinity of the bile canaliculi; in the next step of differentiation, the particles fuse, form short ridge segments and finally continuous branched smooth strands, characteristic of the mature tight junction.     

An efficient and rapid procedure for plantlet regeneration from chicory mesophyll protoplasts

An efficient procedure for plantlet regeneration from chicory mesophyll protoplasts has been developed in order to perform protoplast fusion experiments. Protoplasts were isolated from a genotype of Italian red chicory (CH 363) and purified by centrifugation in a solution containing 13% (w/v) sucrose to collect uniform protoplasts in size. After 2 days culture at a density of 2×10⁴ protoplasts ml⁻¹ of liquid medium, protoplasts were cultured following three different procedures: in liquid medium, stratified in semi-solid medium, and embedded in Ca-alginate droplets. Four different media were used and culture procedures were evaluated recording the protoplast viability, protoplast division frequency and plating efficiency for each experiment. The embedding of protoplasts in Ca-alginate droplets enhanced both division frequency and plating efficiency for chicory mesophyll cells. Furthermore, this procedure shortened the cycle of plant regeneration from protoplasts, which could be completed in eight weeks. This revised version was published online in June 2006 with corrections to the Cover Date.     

Canine BFU-e progenitors: adaptation of a reproducible assay and anatomical distribution.

In vitro cloning assays are used increasingly in investigative hematotoxicology and in screening candidate compounds for their hematotoxic potential. To expand these applications, a practical cloning assay for erythroid burst-forming units (BFU-e) that uses a microplasma clot (MPC) system was adapted to the dog, a species used extensively in experimental hematology and drug development. This system offers the advantage over the methylcellulose and soft agar culture systems of allowing specimen fixation and, therefore, morphological and cytochemical evaluation. The distribution of BFU-e among various anatomic sites was assessed using the MPC cloning system, which was modified to optimize the BFU-e growth. BFU-e growth required only erythropoietin (Epo) in the culture medium and there was no need for an exogenous source of burst-promoting activity (BPA). The cloning efficiency was linearly proportional to the plating concentrations of Epo and marrow mononuclear cells (MMC) over a range of 0 to 3 U Epo and 1 x 10(5) to 3 x 10(5) MMC per ml of culture, respectively. Increases in concentrations of Epo and MMC beyond these levels were not associated with linear growth. The addition of transferrin and spleen-conditioned medium containing a mixture of growth factors (including BPA) reduced BFU-e growth. The relative concentration of BFU-e was comparable among samples collected from the iliac crest, femur, and humerus. Serial cultures performed on individual dogs were highly reproducible and there was little variation in BFU-e activity among dogs of comparable age. It was concluded that the MPC system is a practical and reproducible cloning system for early (BFU-e), as well as late erythroid colony-forming units (CFU-e) in the dog. The concentration of BFU-e appears comparable throughout the active marrow; therefore, various anatomic sites can be used interchangeably for serial quantitative analysis of this progenitor.     

近自然纯培养法对细菌培养的初步研究

依据微生物在自然生境中协作生长的基本特性,提出一种对传统纯培养技术的改进思路及方法：设计一种有孔培养皿,皿内覆盖有不允许细菌通过、但营养物质可以自由流动的微孔滤膜。培养时将该培养皿放入被分离微生物所需生境中,可以克服传统纯培养难以提供外源活性物质的缺陷,一定程度上弥补了混合培养法和传统纯培养法的弱点,从而达到增强部分微生物可培养性、甚至培养出未培养微生物的目的。