Effect of room fluorescent light on the deterioration of tissue culture medium

Summary A major cause of tissue culture medium deterioration is exposure to room fluorescent light. Riboflavin and tryptophan present in Dulbecco's modified Eagle's minimum essential medium, when exposed to light, yield toxic photoproducts responsible for loss of the ability of the medium to support clonal growth of human, mouse and Chinese hamster cell lines. Procedures for minimizing medium deterioration are discussed. This work was supported by American Cancer Society Research Grant No. VC-100B and US PHS Research Career Development Award No. 5 K04 GM70537 from the National Institute of General Medical Sciences.     

Note: Influence of hydrocarbons on some Claviceps cultures

Some Claviceps strains producing clavine alkaloids were cultured in shaken flasks in two-phase systems consisting of a water-immiscible organic solvent, n -alkanes and an aqueous culture medium. Culture behaviour was different with light and heavy alkanes but no changes in the proportions of individual alkaloids were found. n -Octane caused a stimulation of sclerotia-like growth and of specific biomass productivity. Increasing medium volumes in flasks brought about deterioration of aeration conditions and reduced production. Optimum fermentation results were restored by adding liquid paraffin to the culture. Heterogeneous systems of the above kinds appear to be suitable for optimization and scale-up of clavine fermentation.     

The isolation of mitochondria from dipteran flight muscle

Procedures for the isolation of mitochondria from dipteran flight muscle have been investigated in an attempt to determine the extent and to identify the causes of deterioration associated with isolation. In the light of the results obtained isolation procedures have been improved by minimising mechanical damage, avoiding the development of anoxic conditions, and by the use of an isolation medium of a more physiological nature, containing the potassium salt of an organic anion as the principal osmoeffector, phosphate as the principal buffer, and low concentrations of free Mg²⁺. The oxidative capacity of mitochondria isolated by the improved method is adequate to support the in vivo requirements of the flight system.     

Senescence of Hydrilla leaves in light and darkness

Senescence of isolated leaves of Hydrilla verticillata (L.f.) Royle was studied in both darkness and light (20 μmol m⁻² s⁻¹). Senescence in the dark followed a general pattern of deterioration, i.e., gradual loss of cellular macromolecules like chlorophyll, protein and RNA with a concomitant rise in α-amino nitrogen, protease activity and tissue permeability. In light, however, an accelerated loss of chlorophyll took place although protein loss and increase in protease activity were retarded. A higher level of α-amino nitrogen in leaves in the light than in darkness could be correlated with lower leaching of free amino acids in light. Light decreased tissue permeability, as evidenced by lower conductivity of the incubation medium. In the light, RNA increased over the initial level. Both soluble and insoluble carbohydrates declined in the dark. The decline of insoluble carbohydrate was retarded by light, whereas soluble carbohydrate showed an initial rise and then declined sharply in the light.     

Effects of ethephon on aging and photosynthetic activity in isolated chloroplasts

Chloroplasts, isolated from the primary leaves of 7-day-old seedlings, were incubated in vitro at 25°C with 2-chloroethylphosphonic acid (ethephon) under light (0.16 milliwatts per square centimeter) and dark conditions. Ethephon at 1 micromolar (0.1445 ppm), 0.1 and 1 millimolar, or 5 microliters ethylene promoted the deterioration of chloroplasts, increased proteolysis, and reduced the chlorophyll content and PSI and PSII during 72 hours under both light and dark conditions. The decline in PSI and PSII occurred prior to a measurable loss of chlorophyll. The loss of photosynthetic activity affected by ethephon was initiated prior to 12 hours of incubation. After 24 hours in light, 0.1 millimolar (1.445 ppm) epthephon significantly reduced PSI and PSII and promoted the total free amino acid liberation in isolated chloroplasts. In darkness the rate of loss of PSI activity was about 50% of that in light. After 24 hours, in light at 1 millimolar epthephon, PSII activity was 55% of the control, yet nearly 90% of the chlorophyll remained, which indicates that the loss of thylakoid integrity was promoted by ethephon. Ethylene injected in the chloroplast medium at 5 microliters (0.22 micromolar per milliliter) reduced PSI by nearly 50% of the initial in 12 hours. In leaf sections floated in 5 microliters per milliliter suspension medium, a 36% loss of chlorophyll of the control in 36 hours was observed. Cycloheximide at 0.5 millimolar masked the effect of 1 millimolar ethephon and maintained the initial chlorophyll content during the 72 hour period.     

Choline, tetraalkylammonium and aminoalcohol as preserves of photosynthetic activities in isolated chloroplasts

Choline chloride, tetraalkylammonium chloride and aminoalcoholshow preservative effects on photosynthetic activities in spinachchloroplasts against deterioration after isolation, when oneof them is present in the medium used for isolating and storingchloroplasts within the concentration range between 0.1 and0.5 M. Any of these chemicals cause uncoupling to some extentif present in the reaction medium. (Received August 23, 1973; )     

Rat hepatocyte primary cultures. IV. Maintenance in defined medium and the role of production of plasminogen activator and other proteases

Primary cultures of rat hepatocytes survived well for up to 4 days in defined medium in the presence of dexamethasone but not in its absence. The loss of viability was accompanied by a loss of ultrastructural features characteristic of hepatocytes. The cultures began producing plasminogen activator and a neutral protease after 24 hr in culture. Dexamethasone inhibited the production of both of these substances. The deterioration of the cultures appeared not to be related to plasminogen activator, but prolongation of survival by a variety of protease inhibitors suggested that the neutral protease might contribute to deterioration.     

亚热带山地草地退化系列上种群更新研究

从种子库,幼苗和种子雨等方面对亚热带山地草地退化系列上5种植物的种群更新进行了研究,结果表明：（1）引入种多年生黑麦草（Lolium perenne)和白三叶（Trifolium repens)种群在轻度和中度退化条件下能够正常更新,但不能忍受草地进一步的退化;（2）本地种对草地退化的胁迫表现出较强的承受能力。簇生卷耳（Cerastium caespitosum)、箭叶蓼（Polygonum sagittatum)和华蒲公英（Taraxacum sinicum)的种群更新分别在中度、重度和极度退化草地上处于最佳状态;（3）若要防治草地退化,就应当地引入种的种群更新受到抑制时采取有效措施。     

IN VITRO CULTURE OF MUSCLE TISSUE FROM NORMAL, HETEROZYGOUS, AND DYSTROPHIC CHICKS

The degree of histological deterioration of the original explant and the extent of cell spreading was evaluated in cultures of pectoralis muscle from 11-day chicks. Although the frequencies of these two parameters varied with the amounts of horse serum and embryo extract added to the medium, cultures from dystrophic chicks, in comparison to those from either normal or heterozygous animals, consistently showed the largest number of explants with the most extreme forms of histological deterioration and cell spreading. At 20 per cent horse serum the cultures from heterozygous chicks showed greater frequencies of the more extensive forms of deterioration and spreading than the normal muscle explants, but at 5 per cent horse serum these two groups appeared similar. Regardless of genetic background, cultures of the pectoralis muscle from 18-day embryos and of the latissimus dorsi muscle from 11-day chicks exhibited comparable high frequencies for the maximal degrees of deterioration and spreading.     

Effect of 2-deoxy-D-glucose on maintenance in culture of neonatal B cell of rat

The effect of 2-deoxy-D-glucose on maintenance in culture of B cells of the neonatal rat was examined by supplementation of Medium 199 containing 5.5 mM glucose with 1 mM 2-deoxy-D-glucose. Islets maintained in medium with 5.5 mM glucose (basal medium) for 7 d underwent remarkable decreases in glucose sensitivity, and the levels of insulin in the medium dropped. By contrast, addition of 2-deoxy-D-glucose promoted a higher insulin content in medium and an increase in the glucose-induced insulin release and biosynthesis. Moreover, the addition of the deoxysugar caused a selective deletion of fibroblasts and prevented the deterioration of islet cells in basal medium, yielding clusters mostly consisting of islet cells at the end of culture.     

Senescent sperm performance in old male birds

Senescence is the deterioration of the phenotype with age caused by negative effects of mutations acting late in life or the physiological deterioration of vital processes. Birds have traditionally been assumed to senescence slowly despite their high metabolic rates, high blood sugar levels and high body temperature. Here we investigate the patterns of age‐related performance of sperm of a long distance migrant, the barn swallow Hirundo rustica, varying in age from 1 to 6 years, analysed by the computer‐assisted sperm analysis equipment. Sperm showed deteriorating performance in terms of linear movement, track velocity, straight line velocity and reduced proportions of rapidly moving, progressive and motile sperm with age. In a second series of experiments, we assessed performance of sperm from the same males in neutral medium and in medium derived from the reproductive tract of females in an attempt to test if sperm of old males performed relatively better in female medium, as expected from extra‐pair paternity being negatively related to male age, but not to female age. Older males showed consistently better performance in female medium than in neutral medium in terms of track velocity, straight line velocity and reduced proportions of rapidly moving, progressive and motile sperm, whereas young males showed better performance in neutral medium. These results provide evidence of declining sperm performance for important reproductive variables not only with age, but also with the sperm of old males performing differentially better in female medium than young males.     

Removal of hydrogen sulfide by Chlorobium thiosulfatophilum in immobilized-cell and sulfur-settling free-cell recycle reactors.

Bioconversion of hydrogen sulfide to elementary sulfur by the photosynthetic bacterium Chlorobium thiosulfatophilum was studied in immobilized-cell and sulfur-settling free-cell recycle reactors. The cells immobilized in strontium alginate beads excreted elementary sulfur and accumulated it as crystal in the bead matrices, which made it possible that the reactor broth remained clear and the light penetrated the reactor deeper than with the free cells. In comparison with the free cells, the immobilized cells required 30% less light energy at a H2S removal rate of 2 mM/(L.h) and showed an activity of 2.4 times that of the free cells. However, in 40 h after the reaction the deterioration of the H2S removal efficiency became significant due to the accumulation of sulfur in the beads. The scanning electron micrograph (SEM) and energy-dispersive X-ray spectrometer (EDS) studies showed that the sulfur in the beads existed within a layer of 0.4 mm from the bead surface. In the sulfur-settling free-cell recycle reactor, about 80% of the sulfur excreted by the free cells could be removed in a settler. The 4-L fed batch reactor with the settler improved the light transmission to result in a H2S removal rate of 3 mumol/(mg of protein.h), 50% higher than that without it. The settling recycle reactor was much better in the removal of H2S than the immobilized-cell reactor because the former was a continuous system with the constant removal of sulfur particles by settling and of spent medium by supplying fresh medium at the same rate as the filtering rate of the reactor broth, while the latter was essentially a batch system where toxic metabolites and produced sulfur could not be removed.     

Gymnoascus arxii's potential in deteriorating woollen textiles dyed with natural and synthetic dyes

So far, very little is known about microbiological deterioration of dyed woollen textiles. In this paper, the influence of the Gymnoascus arxii fungus on woollen textiles dyed with natural and synthetic dyes was studied. What is more, it was analysed whether the enrichment of the culture medium with additional nutrients has any impact on the deterioration of dyed woollen fabrics caused by a strongly keratinolytic strain. The study was carried out by means of a pure culture method over three different time periods, i.e. 1, 2 and 4 weeks. Within a week, the pure Gymnoascus arxii strain led to a severe deterioration in the mechanical strength of the examined woollen textiles, with the raw fabric being the most severely damaged. After the two-week incubation period, only the fabrics coloured in yellow, i.e. the fabric dyed with natural dye weld, and the synthetic yellow textile as well as the textile dyed with natural dye indigo survived, exclusively on the enriched medium. Solely the weld dyed textile withstood the four-week culture on the nutrient-enriched medium. The conducted studies demonstrated a strong influence of Gymnoascus arxii on dyed fabrics leading to their irreversible destruction. It has been also shown that the presence of nutrients in the substrate that are readily available to microorganism may hinder the development of the Gymnoascus arxii strain and thus, prevent textile deterioration.     

Effect of light quality and culture medium on somatic embryogenesis of <Emphasis Type="Italic">Agave tequilana</Emphasis> Weber var. Azul

Somatic embryogenesis in Agave tequilana Weber var. Azul was affected by the interaction between the light regimes applied during the induction phase and the expression phase. When embryogenic calli was exposed to white or red light during the expression phase, an average of two germinated embryos per explant was obtained regardless of the light treatment used for callus induction. Conversely, the highest number of germinated embryos, an average of 18 per explant, was obtained when applying either white or red light during the induction phase and then wide-spectrum light during the expression phase. Culture medium had also a great influence in this process, with embryo germination being reduced by up to 70%, depending on the light treatment, when using Schenk and Hildebrandt (SH) medium instead of Murashige and Skoog (MS) medium.     

The effect of low light flux and nitrogen deficiency on the chemical composition of Spirulina sp. (Arthrospira) grown on digested pig waste

Evaluation of the effect of low light flux and nitrogen deficiency on growth and chemical composition of Spirulina sp. (straight filaments strain, SF) in batch cultures utilizing a complex medium containing sea-water supplemented with anaerobic effluents from digested pig waste, was undertaken. Cultivation was carried out either at a light flux of 66 (lower) or 144 micromol photon m(-2) s(-1) (higher), utilizing bench raceways. Biomass concentration (as dry weight) after 12 days of cultivation in the complex medium was similar (P < 0.05) to the one observed in a chemically defined medium (Zarrouk), regardless of the light intensity. Protein content of the biomass in the complex medium was significantly lower (P < 0.05), compared to the Zarrouk medium, regardless of the light flux. However, biomass from the complex medium was enriched in total lipids (28.6%), when cultures were exposed to the lower light flux. On the other hand, the palmitoleic acid percentage of total fatty acids was significantly higher (P < 0.05) at a higher light intensity and a high level of gamma linolenic acid (GLA) as a percentage of total fatty acids was observed (28.13%) in the biomass harvested from the complex medium at the lower light intensity. Finally, polysaccharide content was significantly higher (P < 0.05) at the high light intensity and a very high content of total polysaccharides (28.41%) was observed in the complex medium.     

Red Light-Dependent CO(2) Uptake and Oxygen Evolution in Guard Cell Protoplasts of Vicia faba L.: Evidence for Photosynthetic CO(2) Fixation

Suspensions of dark-adapted guard cell protoplasts of Vicia faba L. alkalinized their medium in response to irradiation with red light. The alkalinization peaked within about 50 minutes and reached steady state shortly thereafter. Simultaneous measurements of O₂ concentrations and medium pH showed that oxygen evolved in parallel with the red light-induced alkalinization. When the protoplasts were returned to darkness, they acidified their medium and consumed oxygen. Both oxygen evolution and medium alkalinization were inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In photosynthetically competent preparations, light-dependent medium alkalinization is diagnostic for photosynthetic carbon fixation, indicating that guard cell chloroplasts have that capacity. The striking contrast between the responses of guard cell protoplasts to red light, which induces alkalinization, and that to blue light, which activates proton extrusion, suggests that proton pumping and photosynthesis in guard cells are regulated by light quality.     

Preparation of rat synovial membrane for studies of cytokine secretion

The objective of this work was to devise an in vitro system for studies on cytokine secretion by synovial membrane treated as a whole organ with various synoviocyte populations. Synovial membrane from knee joints of WAG rats was dissected and incubated in culture medium without serum for 4 - 48 h. The level of IL-1alpha was determined in synovial lysates and IL-6 in culture medium. The synovial membrane from left and right knee joint of the same rat produced similar amount of cytokines both in lysates and in the medium. Synovial membrane stimulated by LPS for 4 or 24 h gave significantly stronger cytokine response than the membrane from the opposite (control) knee. After 48 h incubation of synovial membrane drastic drop in cytokine level was noted, which indicated on deterioration of the membranes. The test may be useful in studies on factors affecting cytokine secretion by synoviocytes.     

Nutritional control of sexuality in Chlamydomonas reinhardi

1. Cells of Chlamydomonas reinhardi grown in the light or dark on standard medium require an additional exposure to light in the absence of a nitrogen source, in order to become sexually active. As the culture ages, the light requirement decreases. 2. This light requirement is a function of nitrogen depletion, as shown by the observation that cells from cultures grown to maturity on a low nitrogen medium in the light or in the dark, have no additional light requirement for zygote formation. The withholding of no other component of the medium has this effect. 3. In cells requiring light for zygote formation, the light can be supplied before the mating types are mixed, indicating that light is required, not for mating per se, but for the conversion of vegetative cells to gametes. 4. Gametes can be dedifferentiated to the vegetative state by any nitrogen compound which the cells can use for growth; and by further exposure to light in the absence of a nitrogen source, these vegetative cells can again become gametic. 5. Cells grown at different nitrogen levels become gametic at widely different cell concentrations of nitrogen and carbon and C/N ratios. 6. It is postulated that the role of light in gametic differentiation is indirect, providing by photosynthesis, energy for the mating process and carbohydrates to tie up excess nitrogenous reserves; and that the concentration of some particular nitrogen fraction or compound determines whether or not gametic differentiation is initiated.     

培养基与光照对沼兰种子非共生萌发的影响

近年来由于灭绝性采挖和生态环境的恶化,沼兰(Malaxis monophyllos)的自然资源遭到严重破坏,加上沼兰种子具有萌发缓慢和萌发率低的特性,已处于近危乃至易危的状态。为了繁殖和保护沼兰种质资源,利用添加2%蔗糖、3%活性炭和0.8%琼脂的Knudson C(KC)培养基,研究了萘乙酸(NAA)、6-苄基腺嘌呤(6-BA)、椰乳、香蕉泥、土豆泥和光照条件6种因素对沼兰种子非共生萌发的影响;同时通过光学显微镜和扫描电镜观察了种子的外部形态。结果显示,沼兰种皮细胞的平周壁方向结构平坦、没有纹络,但是沿着细胞垂周壁方向细胞壁较厚。通过正交试验设计和冗余分析,建立了沼兰种子非共生萌发的最佳方案,即在含有4.5mg·L–1NAA、10mg·L–16-BA、8%香蕉泥和3%土豆泥的KC培养基上光照条件(光照强度为20μmol·m–2·s–1,光周期为12h/12h)下萌发。该方案的种子萌发率在90%以上。     

Analysis of the cytotoxic effects of light-exposed hepes-containing culture medium

Summary The addition ofN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) to RPMI 1640 medium markedly increases the production of cytotoxic products during exposure of the medium to visible light. The cytotoxicity has been analyzed by measuring uptake of [³H]thymidine by murine thymocytes cultured in preirradiated medium containing 25 mM HEPES. Complete inhibition of thymidine uptake was produced by exposing 50% of the culture medium to light for 3 h before addition of cells. The HEPES-mediated effect requires only that HEPES and riboflavin be exposed to light; other medium constituents are not necessary. Hydrogen peroxide is a principal cytotoxic agent produced in this system. It is demonstrated that most, but not all, of the inhibition of thymidine uptake can be attributed to hydrogen peroxide.