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1.
An improved organ culture method for adult mammalian lung 总被引:1,自引:0,他引:1
Robert R. Guerrero Donald E. Rounds Jon Booher 《In vitro cellular & developmental biology. Plant》1977,13(8):517-524
Summary An improved method for maintaining adult rat lung in submerged organ culture is described in which the alveoli were inflated
with agar and 200-μm-thick hand-cut sections were mounted in Rose chambers. The conventional single-compartmented Rose culture
chamber was modified by adding a second chamber separated from the first by a gaspermeable membrane. One compartment functioned
as an air reservoir and the other housed the explants submerged in nutrient medium. Visking dialysis membrane used underneath
the explants prevented cell outgrowth and facilitated the exchange of nutrients and waste products at the glass-tissue interface.
Because of the excellent optical properties of the Rose chamber and the thinness of the explants, individual cell types can
be identified in the living tissue. The explants were studied with time-lapse cinematography, light microscopy, histology,
and with erythrosine B for dye exclusion. With this modified system the functional life span of the explants was increased
from 1 week to 1 month.
This study was supported by NHLBI Grant No. HL15098-05. 相似文献
2.
The concept of a self-feeding roller bottle that delivers a continuous supply of fresh media to cells in culture, which is mechanically simplistic and works with existing roller apparatuses, is presented here. A conventional roller bottle is partitioned into two chambers; one chamber contains the fresh culture media reservoir, and the other contains the cell culture chamber. A spiroid of tubing inside the fresh media reservoir acts as a pump when the bottle rotates on its horizontal axis, continuously delivering fresh media through an opening in the partition to the cell culture chamber. The modified bottle proved capable of maintaining steady-state cell densities of a hybridoma cell line over the 10-day period tested, although at lower densities than reached during batch operation due to the continuous volume dilution. Steady-state density proved to be controllable by adjusting the perfusion rate, which changes with the rotation rate of the bottle. Specific antibody production rate is as much as 3.7 times the rate in conventional roller bottles operating with intermittent batch feeding. 相似文献
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The growth of granulopoietic progenitor cells (CFU-C) in diffusion chambers during culture of peripheral blood leukocytes from 10 normal subjects has been studied. At various times after initiation of diffusion chamber culture, cells harvested from the chambers were transferred to agar culture for measurement of CFU-C concentration. Under these conditions colonies could be grown successfully in agar culture provided pronase, necessary for the chamber harvesting procedure, was first removed by careful washing. A marked increase in the number of CFU-C, up to 25-fold the initial value, was observed in 8 out of 10 subjects. Here the growth pattern was similar, independent of the initial CFU-C values, with an immediate rise to a maximum between 6 and 13 days of culture followed by a decrease. In the other two subjects the growth of CFU-C throughout the diffusion chamber culture period was very poor. The growth of CFU-C from a given individual's blood was shown to be reproducible in repeated studies in 2 subjects, one of whom showed a proliferative and the other a non-proliferative pattern. Evidence suggests that the increase in CFU-C in diffusion chambers is the result of both self-renewal of these cells and influx from a more primitive compartment, although the present data do not allow an estimate of the relative magnitude of each. 相似文献
5.
Detection of microbial proteolytic activity by a cultivation plate assay in which different proteins adsorbed to a hydrophobic surface are used as substrates. 总被引:3,自引:0,他引:3
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M B Wikstrm 《Applied microbiology》1983,45(2):393-400
A screening technique for microbial proteases, the thin-layer enzyme assay cultivation technique, was developed. The inner surface of a polystyrene petri dish was coated with protein and then covered with a culture agar medium. The enzymes, produced during growth of the microorganisms, reach the protein-coated surface by diffusion in the agar. Degradation of the protein was visualized by condensation of water vapor on the surface after removal of the agar medium. The wettability of the enzyme-affected protein-coated polystyrene surface was decreased compared with the unaffected protein surface. Enzyme substrates used were fibrinogen, immunoglobulin G, egg albumin, human serum albumin, bovine serum albumin, hemoglobin, mucin, and gelatin. It was possible to use a variety of culture agar media, nonselective as well as selective, in the assay. The technique provides a sensitive, convenient, and inexpensive method for screening various microbial proteases. In addition, the technique can be used for screening proteolytic enzyme activity of specific microbial species in a mixed microbial sample as well as for studies of factors that influence the cultivation conditions for protease production and activity. 相似文献
6.
M B Wikstr?m 《Applied and environmental microbiology》1983,45(2):393-400
A screening technique for microbial proteases, the thin-layer enzyme assay cultivation technique, was developed. The inner surface of a polystyrene petri dish was coated with protein and then covered with a culture agar medium. The enzymes, produced during growth of the microorganisms, reach the protein-coated surface by diffusion in the agar. Degradation of the protein was visualized by condensation of water vapor on the surface after removal of the agar medium. The wettability of the enzyme-affected protein-coated polystyrene surface was decreased compared with the unaffected protein surface. Enzyme substrates used were fibrinogen, immunoglobulin G, egg albumin, human serum albumin, bovine serum albumin, hemoglobin, mucin, and gelatin. It was possible to use a variety of culture agar media, nonselective as well as selective, in the assay. The technique provides a sensitive, convenient, and inexpensive method for screening various microbial proteases. In addition, the technique can be used for screening proteolytic enzyme activity of specific microbial species in a mixed microbial sample as well as for studies of factors that influence the cultivation conditions for protease production and activity. 相似文献
7.
Summary Cecum from chickens, 4 wk old, can best be maintained for 24 h in a serum-free organ culture system using Trowell T8 medium
agar sheet at 25°C. As determined by light microscopy as well as scanning and transmission electron microscopy, mucosal architecture
involving classic ultrastructure of chicken cecal mucosa was preserved. Protein content of cecal explants did not change up
to 48 h of culture. DNA content of cecal explants did not change up to 24 h of culture but decreased significantly to two-thirds
of control in 48 h of culture. Based on the morphologic and physiologic findings, it became evident that this organ culture
system using Trowell T8 medium at 25°C can be successfully used as an in vitro experimental model for as long as 24 h. The
organ culture system could be a useful tool, from the structural integrity of ceca observed in this study, in investigating
mucosal function and mucosal response to drugs, carcinogens, trophic factors, and pathogens. 相似文献
8.
CAS平板覆盖法检测氢氧化细菌铁载体 总被引:3,自引:1,他引:2
【目的】用CAS平板覆盖法检测氢氧化细菌铁载体,解决通用CAS琼脂平板法中十六烷基三甲基溴化铵对真菌和某些细菌的生长抑制问题。【方法】将改良的CAS检测培养基覆盖在长满菌落的无铁培养基上,生长抑制问题因微生物未与十六烷基三甲基溴化铵直接接触而解决。【结果】3株氢氧化细菌SDW-5、SDW-9和AaP-13均能产生单菌落,加入CAS检测培养基1 h后,菌落周围产生明显的铁载体晕圈。【结论】本方法成功解决了生长抑制问题,可以作为检测微生物铁载体的通用方法。 相似文献
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J Yourno J Goldberg D A Nelson W J Williams 《The journal of histochemistry and cytochemistry》1980,28(9):991-996
Soft agar culture CFU-C (colony-forming units in culture) are rapidly classified in situ as eosinophil, macrophage, and neutrophil-monocyte types by whole culture staining with luxol fast blue for eosinophil specific granules and acetoorcein for nuclei. Stained colonies may be picked and examined individually as wet mount cover slip preparations, or the agar culture may be air dried and mounted permanently. The whole culture stain has been variously modified for the enzyme markers alpha-naphthyl acetate esterase and peroxidase and for nuclear staining alone with acetoorcein. 相似文献
12.
Biological control of plant diseases with antagonistic bacteria is a promising alternative to conventional chemical control strategies. In vitro screening for inhibition of mycelial growth of phytopathogenic fungi by bacterial isolates is the first step in selecting putative bacterial biocontrol agents. Dual culture plate assay is the most common method involved in this first-line selection process. However, it needs independent agar plates to test antagonism by a specific bacterial isolate against each of the fungal phytopathogen. Two modified in vitro antagonism tests are proposed here. Antagonistic activity of a putative biocontrol bacterial strain against four different fungal phytopathogens could be assessed in a single agar plate simultaneously. A comparison of the new methods with conventional dual culture plate assay was also done. The proposed methods are easy to perform and results of antagonism are obtained rapidly. Results of fungal inhibition were qualitatively comparable with that generated through dual culture plate assay. Quantity of resources such as agar medium and plates required for the modified antagonistic assays is several folds less than that required for dual culture plate assay. 相似文献
13.
Lima-Nishimura N Quoirin M Naddaf YG Wilhelm HM Ribas LL Sierakowski MR 《Plant cell reports》2003,21(5):402-407
Xyloglucan was extracted from seeds of Hymenaea courbaril and mixed with agar to prepare a solid culture medium used for micropropagation of the Marubakaido apple rootstock (Malus prunifolia Borkh) and cv. Jonagored (Malus domestica). The performance on gels created from a blend of 0.4%agar and 0.2% xyloglucan (w/v) was compared with that on media gelled with a standard concentration 0.6% (w/v) of agar. The growth of shoots and the multiplication rate were higher on the modified culture medium than on the agar-gelled medium. The occurrence of hyperhydric shoots was lower on the modified medium. In the absence of auxin, shoot rooting reached 70% (Marubakaido) and 66% (Jonagored) on the agar-xyloglucan medium and 6.7% and 10.4%, respectively, on the agar medium. When 0.25 microM indole-3-butyric acid (IBA) was added to both media, the modified medium gave better results in terms of rooting percentage and quality of roots than the agar-gelled medium. 相似文献
14.
Regeneration of plants using nutrient mist culture 总被引:2,自引:0,他引:2
Pamela J. Weathers Kenneth L. Giles 《In vitro cellular & developmental biology. Plant》1988,24(7):727-732
Summary A nutrient mist was used forin vitro culture of plant tissue in a novel bioreactor, wherein the tissues were grown on a biologically inert screen within a sterile
chamber which allows excess media to drain away from the tissue. Plants tested includedDaucus, Lycopersicon, Ficus, Cinchona, andBrassica. The latter 4 genera were fully regenerated within the bioreactor. Tissue inocula included callus, anthers, and shoot meristems.
All plants grew at least as well in nutrient mists as in agar and always produced a greater quantity of shoots of a higher
quality and often faster than agar cultures. Cost analysis estimates showed up to a 65% savings in production costs (labor
and materials) could be realized using nutrient mist culture. Nutrient mist culture offers significant improvements in the
micropropagation of plants. 相似文献
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Surface Attachment Induced Production of Antimicrobial Compounds by Marine Epiphytic Bacteria Using Modified Roller Bottle Cultivation 总被引:1,自引:0,他引:1
A modified roller bottle culture method elicited the production of antimicrobial compounds from 2 epibiotic marine bacterial
strains, EI-34-6 and II-111-5, isolated from the surface of the marine alga Palmaria palmata. These isolates, tentatively
identified as Bacillus species, were grown as a biofilm on the surface of nutrient glycerol ferric agar (NGFA) and marine
Columbia glycerol agar (MCGA) on the inside of a rolling bottle. The biofilm was shown to be stable, and the cells were difficult
to remove from the agar surface. The culture supernatant exhibited a different antibiotic spectrum when the strains were grown
using the agar roller bottle method compared with shake flask cultures or nonagar roller bottle cultures. These results suggest
that biofilm formation is an important factor in the production of antimicrobial compounds by these 2 strains, and roller
bottle cultivation also allowed production of these compounds to be increased. The methodology used here has the potential
to allow increased production of useful secondary metabolites such as antibiotics from marine epibiotic bacteria. 相似文献
17.
《Anaerobe》2000,6(5):269-271
Cycloserine cefoxitin fructose agar is a widely used selective isolation medium for Clostridium difficile from stool specimens. Yeasts often colonize in the intestine of C. difficile disease patients and, if colonized heavily, pure culture of C. difficile can be delayed. The aim of this study was to modify cycloserine cefoxitin fructose agar to suppress the growth of yeasts. Antimicrobial activities of three commonly available antifungal agents were tested against recent clinical isolates of Candida species. Amphotericin B was most active in inhibiting all isolates by ≤0.5 mg/L concentration. Cycloserine cefoxitin fructose agar was modified by adding 2 mg/L of amphotericin B. Serial ten-fold dilution of stool specimens from 126 suspected C. difficile -associated diarrhea patients were cultured both on cycloserine cefoxitin fructose agar plates and modified agar plates. Yeasts grew from 60 specimens on cycloserine cefoxitin fructose agar, but none grew on the modified medium. Growth of C. difficile was detected from 37 and 39 of 126 specimens on cycloserine cefoxitin fructose agar and modified medium, respectively. The number of C. difficile colonies was similar on both media. In conclusion, 2 mg/L of amphotericin B supplementation to cycloserine cefoxitin fructose agar can facilitate the isolation of C. difficile from stool specimens which are densely colonized with yeasts. 相似文献
18.
With increased focus on watershed protection under the Surface Water Treatment Rule, indicators that discriminate among sources of microbial inputs (microbial source tracking) are needed to supplement the quantitative information provided by total and fecal coliform measurements for drinking water monitoring. Bifidobacteria are found in the digestive tract and feces of humans and other animals, and also in sewage. Sorbitol is a food additive used exclusively in food intended for human consumption. Therefore, the presence of sorbitol-fermenting Bifidobacteria in environmental waters can be indicative of sources of human fecal contamination. A series of media were evaluated using ATCC cultures of B. breve and B. adolescentis, feces from different animals, and domestic wastewater samples. The media evaluated were Human Bifid Sorbitol agar (HBSA), modified Human Bifid Sorbitol agar, Beerens Medium, modified Beerens Medium, Reinforced Clostridial agar, BIM-25 Medium, and modified BIM-25 Medium. Variables such as sample preservation, incubation time, different pH indicators, plating technique, and discontinuous exposure to sorbitol were also evaluated. A series of biochemical tests were used to confirm positive colonies enumerated on the various media. Membrane filtration and enumeration of sodium sulfite preserved samples on HBSA containing bromocresol purple using loose lidded plates for 48 h provided the best recoveries for presumptive positive colonies. A number of sorbitol-fermenters that were not Bifidobacteria were able to grow on all media tested, resulting in false-positives. Therefore, plating on HBSA should be followed by a confirmation step when monitoring for sorbitol-fermenting Bifidobacteria in environmental waters. A year-long sampling survey of a managed reservoir in Massachusetts provided field validation of the proposed methodology for sorbitol-fermenting Bifidobacteria as a human-related source tracking indicator tool. 相似文献
19.
To arrive at an appropriate bioreactor design and in situ recovery of the products, red beet hairy roots were used as a model system where the levels of betalain pigments (betacyanins and betaxanthins) were followed as secondary metabolite and the peroxidase enzyme as primary metabolite. Medium volume and other kinetic parameters were found to play significant roles by way of directly affecting the biomass yield rather than a specific metabolite. The hydrodynamic stress created on the roots by large culture volume could be minimized by pulse‐feeding of medium in shake‐flasks; and by separating the biomass chamber from the aerated medium reservoir in circulatory fed‐batch bioreactor. Accordingly the bioreactor was modified to provide anchorage and air‐enrichment chamber which resulted in higher formation of both the metabolites than in shake‐flasks. Various down‐stream processing aspects such as in situ release of pigments by non‐destructive methods, followed by adsorption through a column and recovery by desorption were optimized for betalains. A strategy for simultaneous recovery of pigment and peroxidase was worked out using aqueous two phase extraction (ATPE). 相似文献