Further studies on autoclaved-induced toxicity in tissue culture media: gauging sugar breakdown by spectrophotometry

Fructose and sucrose were used to investigate cyanide-induced absorption changes after high temperature treatment. By comparing the time-resolved absorbance difference spectra obtained under aerobic conditions with those under aerobic conditions, absorbance changes that are associated with the process of oxygen consumption were identified. The rates of absorbance changes of autoclaved sucrose solution or of autoclaved MS medium (Murashige and Skoog 1962. Physiol. Plant. 15: 473–497) were correlated with those of oxygen consumption measured by polarography and used for determining the toxicity of culture media (Hsiao and Bornman 1991. Physiol. Plant. 81: 55–58). When autoclaved together with sucrose, FeNa-EDTA promotes its degradation. Absorbance change, therefore, is a convenient parameter for measuring not only the extent of carbohydrate breakdown at high temperature but also the relative toxicity of culture media autoclaved under different conditions.     

Determination of oxygen profiles in agar-based gelled in vitro plant tissue culture media

Keeping account of the limited knowledge concerning the relevance of the oxygen status of the medium during in vitro culture, a technique was elaborated to systematically study the oxygen concentration in gelled media. In a first series of experiments, the Oxygen Diffusion Rate (ODR) technique was used to investigate the dissolved oxygen concentration as a function of time at different depths. The results obtained demonstrated that the oxygen concentration in agar media was reduced by 80% during the heating steps included in the preparation procedure. It took about one week to reach an oxygen concentration equal to 90% of the equilibrium value at a depth of 1 cm, irrespective of the brand of agar used. As the recovery of the oxygen concentration at various depths could be nicely modelled by Fick's law, it follows that this process is diffusion limited. In this respect, fluorescence recovery after photobleaching (FRAP) measurements revealed that the diffusion coefficient of oxygen in gelled media was only affected to a very small extent by the presence of up to 2% (w/v) agar. In a final experiment, explants of Ficus benjamina were cultured on a rooting medium. As the oxygen concentration in the gelled medium was not significantly affected by the presence of the biological material, it was concluded that the oxygen uptake by explants from gelled media is negligibly small and hence cannot be considered as being a growth-limiting factor during in vitro micropropagation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Autoclaved and filter sterilized liquid media in maize anther culture: significance of activated charcoal

Summary Medium sterilization techniques (autoclaving, filter sterilization and separate sterilization of medium components), combined with preculture exposure to activated charcoal (AC) were evaluated for effects on maize anther culture response. The addition of AC to filter sterilized medium had no effect on the number of embryo-like-structures (ES) produced. For autoclaved medium, pre-culture AC treatment resulted in a 3-fold increase in ES yield over medium lacking AC. When AC was included, autoclaved medium was more productive than filter sterilized medium. Autoclaved media without AC gave lower response than filter sterilized medium. Separate sterilization of sucrose or FeEDTA was beneficial for media autoclaved in the absence of AC. However, when all components were autoclaved together in the presence of AC, there was no advantage to separate sterilization. The maximum ES frequency (224.6 ES/100 anthers) was obtained with the genotype ETH-M 52 cultured in autoclaved medium which had been exposed to AC (5 g/L) for 96 h prior to culture initiation. It is supposed that the higher ES frequencies observed with AC-treated, autoclaved media were due to the availability of glucose and fructose following heat-induced hydrolysis of sucrose and the AC-mediated adsorption of inhibitory compounds produced during autoclaving.     

Activated charcoal does not catalyze sucrose hydrolysis in tissue culture media during autoclaving

Tissue culture media or aqueous sucrose solutions containing activated charcoal buffered to pH 5.5 and autoclaved did not undergo appreciable sucrose hydrolysis as reported. Rather, the extent of sucrose hydrolysis in media containing activated charcoal was found to be directly proportional to the hydrogen ion concentration (pH). This finding is consistent with the known mechanism of acid-catalyzed hydrolysis of acetals such as sucrose. Several types of charcoal were identified that acidified culture media to the extent that considerable acid-catalyzed sucrose hydrolysis occurred under autoclave conditions, making it appear as though activated charcoal was responsible for catalyzing sucrose hydrolysis. A simple mathematical expression was empirically derived that can be used to predict the extent of sucrose hydrolysis based on the post-autoclave pH of the media. This revised version was published online in June 2006 with corrections to the Cover Date.     

A new method to measure the haemoglobin oxygen saturation by the oxygen electrode

We describe a new polarographic method to measure the haemoglobin oxygen saturation in whole blood, employing up to 10 μl of sample in a standard case. The measurement is done in an anaerobic staineless-steel cuvette (1 ml) recording three oxygen tension values: (1) that of an air-equilibrated buffer before the addition of the sample; (ii) that after the addition of the sample; and (iii) that after the addition of an oxidant. The haemoglobin oxygen saturation is then calculated from the three oxygen tension values, the volume of the reagents, and solubility coefficient of oxygen. This method is simple, inexpensive and accurate, and correlates well with other standard methods.     

高压氧暴露大鼠肺组织内PPAR通路分子变化规律

目的:研究高压氧暴露大鼠肺组织内过氧化物酶体增殖因子活化受体(PPAR)通路分子表达变化规律。方法:27只雄性SD大鼠随机分为高压常氧对照组(0.23 MPa空气)、高压氧时间序列处理组(0.23 MPa纯氧,分别暴露2 h、4 h、6 h和8 h),持续小流量通风,维持舱内O2浓度>99%。应用HE染色观察各组大鼠肺组织形态学变化;应用oligo芯片检测各个时间表达谱;应用RT-PCR验证发生改变的部分PPAR通路基因。结果:与高压常氧对照组相比,高压氧时间序列处理组造成的肺损伤逐渐加重;表达谱芯片的基因本体论(Go)富集分析结果中有一类PPAR通路类,包含有多个PPAR通路分子;RT-PCR结果提示PPARδ-和PPARγ-均在较长时间高压氧暴露的肺组织中上调。结论:高压氧长时间暴露造成的肺型氧中毒可引起PPAR通路的激活。     

Effects of autoclave-induced carbohydrate hydrolysis on the growth ofBeta vulgaris cells in suspension

Media of de Greef & Jacobs (1979) were autoclaved either with all the nutrient components in a single vessel (medium 1) or with the following components in separate vessels: FeNa–EDTA+CaCl₂ (medium 2), FeNa–EDTA+NaH₂PO₄ (medium 3) or sucrose (medium 4). Medium 5 was prepared by autoclaving FeNa–EDTA+NaH₂PO₄ and sucrose in two separate vessels. It was found that the dry mass yield of cell suspensions ofBeta vulgaris was lowest in medium 1, followed by media 2 and 3. There was no significant difference among media 3, 4, and 5.The plot of dry mass yield of the cell suspensions against the rates of cyanide-initiated oxygen consumption which indicate the extent of carbohydrate hydrolysis of the media during autoclaving, indicated the presence of a threshold rate of about 17–20 nmol ml^–1 min^–1. Dry mass yield of the suspensions decreased rapidly when the rate exceeded this value.For media with glucose as the source of carbohydrate, the rate of cyanide-initiated oxygen consumption exceeded the threshold value by a factor of 1.5 to 2, depending on the volume of the media autoclaved.Abbreviations FeNa-EDTA ferric monosodium ethylenediamine-tetraacetic acid     

Influence of cultural and physiochemical factors on ascorbate stability in plant tissue culture media

Ascorbic acid rapidly decays in plant tissue culture media. Within 50 min to 3 h after preparing 100 mM solutions, ascorbic acid was destroyed. Autoclaving, shaking flasks, high light intensity and increasing pH over a range from 4.5–7 accelerated decay. Ascorbic acid was oxidized to dehydroascorbic acid which also underwent decay. Within 11 h and 15 min after adding ascorbic acid both ascorbic acid and its oxidation product, dehydroascorbic acid, disappeared from medium. Since ascorbic acid is rapidly destroyed in plant tissue culture media it may not exert its effect as an intact molecule. Instead its antioxidant/antibrowning role in plant cell, tissue and organ cultures may be mediated by some product of further oxidation.     

Iron toxicity in organotypic cultures of hippocampal slices: role of reactive oxygen species

Free iron has been assumed to potentiate oxygen toxicity by generating reactive oxygen species (ROS) via the iron-catalyzed Haber-Weiss reaction, leading to oxidative stress. ROS-mediated iron cytotoxicity may trigger apoptotic cell death. In the present study, we used iron treatment of organotypic cultures of hippocampal slices to study potential mechanisms involved in iron-induced neuronal damage. Exposure of mature hippocampal slices to ferrous sulfate resulted in concentration- and time-dependent cell death. After iron treatment, markers of ROS formation and lipid peroxidation, i.e. intensity of dichlorofluorescein (DCF) fluorescence and levels of thiobarbiturate reactive substances (TBARS), were significantly increased. Levels of cytochrome c were increased while levels of pro-caspase-9 and pro-caspase-3 were decreased in cytosolic fractions of iron-treated hippocampal slice cultures. Treatment of cultured slices with a synthetic catalytic ROS scavenger, EUK-134, provided between 50 and 70% protection against various parameters of cell damage and markers of oxidative stress. In addition, inhibition of caspase-3 activity by Ac-DEVDcho partially protected cells from iron toxicity. The combination of EUK-134 and Ac-DEVDcho resulted in an almost complete blockade of iron-induced damage. These results indicate that iron elicits cellular damage predominantly by oxidative stress, and that ROS-mediated iron toxicity may involve cytochrome c- and caspase-3-dependent apoptotic pathways.     

Easy method to determine refractive indices of microspheres and in micro-regions of inhomogeneous media

We describe an innovative method which can accurately determine the refractive index (RI) of individual microspheres by immersing the microspheres in a medium and analyzing their phase-contrast microscopic images. Compared with the current techniques for microsphere RI measurement, the method has several advantages: it is simple and easy and it cannot only measure the RI of each individual microsphere but also perform measurement simultaneously on all the microspheres in the same field of view. In measurement, microspheres are not required to be suspended in a specific liquid but in any medium with known RI which is appropriate for the microspheres or even just in atmosphere. By using microspheres with known RI as sensors, the method can also be used for rapid in situ measurement of the local RI of inhomogeneous media. In this paper, we describe the principle of the method and the experiments of using the method to measure the RI of individual microspheres. Its applications for sensing instantaneous RI/concentration/temperature variation in critical situations such as anywhere in mixing flows or living biological specimens are also presented.     

三七总皂甙和银杏叶提取物预防急性氧中毒的实验研究

目的:研究三七总皂甙和银杏叶提取物对急性氧中毒的预防作用及其可能机制.方法:分别给小鼠连续腹腔注射三七总皂甙和银杏叶提取物5 d后,在500kPa高压氧中暴露60 min,观察惊厥潜伏期、惊厥次数、惊厥间隔时间等指标;另外测定高压氧暴露15 min后脑组织中活性氧单位、脂质过氧化物、一氧化氮、谷胱甘肽的含量和过氧化氢酶、谷胱甘肽过氧化物酶、单胺氧化酶的活性.结果:三七总皂甙和银杏叶提取物可以明显延长氧惊厥潜伏期和惊厥间隔时间,减少惊厥次数;降低高压氧暴露后脑组织中脂质过氧化物、一氧化氮的含量,使活性氧单位、谷胱甘肽含量和谷胱甘肽过氧化物酶活性保持在较高的水平;对过氧化氢酶和单胺氧化酶活性的影响则不显著.结论:三七总皂甙和银杏叶提取物可以有效预防急性氧中毒,其机制可能与它们的抗氧化活性有关.     

An electrochemical approach to monitor pH change in agar media during plant tissue culture

In this work, metal oxide microelectrodes were developed to monitor pH change in agar media during plant tissue culture. An antimony wire was produced by a new approach "capillary melt method". The surface of the obtained antimony wire was oxidized in a potassium nitrate melt to fabricate an antimony oxide film for pH sensing. Characterization results show that the oxide layer grown on the wire surface consists of Sb(2)O(3) crystal phase. The sensing response, open-circuit potential, of the electrode has a good linear relationship (R(2)=1.00) with pH value of the test solution. Adding organic compounds into the test media would not affect the linear relationship, although the slope of the lines varied with different ingredients added. The antimony oxide electrodes were employed to continuously monitor pH change of agar culture media during a 2-week plant tissue culture of Dendrobium candidum. The antimony oxide electrode fabricated this way has the advantages of low cost, easy fabrication, fast response, and almost no contamination introduced into the system. It would be suitable for in situ and continuous pH measurement in many bio applications.     

杀虫剂对烟粉虱成虫毒力的微量筛选方法

采用直径为18mm的指形管代替常规浸叶法中的培养皿,研究不同寄主植物叶片和琼脂浓度对烟粉虱Bemisia tabaci(Gennadius)成虫自然死亡的影响,并用吡虫啉和啶虫脒为供试药剂对烟粉虱成虫毒力的筛选方法进行比较。结果表明:选用棉花叶片和14g.L-1的琼脂时,指形管内烟粉虱成虫72h自然死亡率最低,只有(5.4±1.5)%;采用此方法就吡虫啉和啶虫脒对烟粉虱成虫的毒力进行测定,测定结果重复性较好,节省药剂用量,无需对烟粉虱成虫麻醉,操作简便,达到微量活体筛选的目标。     

High tolerance of methanogens in granular sludge to oxygen

This research assessed the effect of oxygen exposure on the methanogenic activity of anaerobic granular sludges. The toxicity of oxygen to acetoclastic methanogens in five different anaerobic granular sludges was determined in serum flasks with effective gas-to-liquid volumes of 4.65 to 1. The amount of oxygen that caused 50% inhibition of the methanogenic activity after 3 days of exposure ranged from 7% to 41% oxygen in the head space. These results indicate that methanogens located in granular sludge have a high tolerance for oxygen. The most important factor contributing to the tolerance was the oxygen consumption by facultative bacteria metabolizing biodegradable substrates. Uptake of oxygen by these bacteria creates anaerobic microenvironments where the methanogenic bacteria are protected. The results also indicate that methanogens in sludge consortia still have some tolerance to oxygen, even in the absence of facultative substrate for oxygen respiration. (c) 1993 John Wiley & Sons, Inc.     

Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: Modulation by culture conditions

Summary Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were studied. Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture. Cells were prelabeled with⁵¹Cr before xanthine plus xanthine oxidase exposure. Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus seemed more sensitive to this oxidant stress. The effect of cell culture age, as indicated by population doubling level (PDL), was examined. Response of low PDL endothelial cells and fibroblasts subjected to oxidant stress was compared with the response of PDL 15 cells. Both low PDL endothelial cells and fibroblasts responded differently to the lytic effect of xanthine oxidase-derived free radicals than did higher PDL cells. Specific activities of the antioxidant enzymes catalase, managanese superoxide dismutase, copper-zinc superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured in both low and high PDL fibroblasts and endothelial cells. Antioxidant enzyme specific activities could only partially explain the differences in response to oxidant stress between fibroblasts and endothelial cells and between low and high PDL cells. Cell culture medium composition modulated the rate of production, and relative proportions of xanthine plus xanthine oxidase-derived partially reduced species of oxygen, i.e. superoxide, hydrogen peroxide, and hydroxyl radical. Serum content of medium was important in modulating free radical generation; superoxide production rates decreased 32%, H₂O₂ became undetectable, and hydroxyl radical generation decreased 54% in the presence of 10% serum. The medium protein and iron content also modulated free radical generation. The data suggest that cell culture media constituents, cell type, and cell culture age greatly affect in vitro response of cells subjected to oxidant stress. Research supported by American Lung Association Fellowship Training Grant and Research Training Grant, the R. J. Reynolds Corporation, and National Institutes of Health Grants HL29784 and 1 HL 23805.     

A cytotoxic substance in insect cell culture spent media

Summary Spent media from five different insect cell lines when inoculated intoTrichoplusia ni (TN-368) cultures produced cytotoxicity resulting in rounding and detachment of cells. The substance in spent medium from the established cell lineCarpocapsa pomonella (CP-169) is believed to be a toxin, based on the failure to serially passage the agent, the early appearance of the cytotoxic effect, and the inability to detect microbes by culturing techniques as well as by electron microscopy. The ability to extract the toxic substance from CP-169 cells indicates that it is cell associated. Biophysical and biochemical properties of the CP-169 cytotoxin are presented.     

A method for estimating oxygen availability of stationary plant cell cultures in liquid media

A method for estimating the oxygen availability in plant cell cultures grown in stationary liquid media (e.g. many protoplast cultures) was developed. The method is based on short-term measurements of respiration rate versus oxygen concentration on a sample of cells, suspended in liquid media. From such data it is possible to estimate the oxygen concentration at the bottom of a stagnant liquid culture, by calculating the amount of oxygen reaching the cells by diffusion. As an example, rape (Brassica napus L. cv. Omega) hypocotyl protoplasts were grown with different oxygen concentrations at the site of the cells, obtained by varying the cell density, the height of the liquid layer and the oxygen content of the gas phase. The number of surviving calli was positively correlated with the estimated oxygen availability in the range between 60 and 350 M O₂, below 60 M all cells died. This indicates that oxygen availability can be a limiting factor in the range usually encountered in protoplast cultures, and that the method can be useful when designing optimal growth conditions for stationary cultures of plant cells.Abbreviations C₁ bulk oxygen concentration in agitated medium - C_o oxygen concentration in medium at the gas-liquid interface, in equilibrium with the gas - C_x oxygen concentration at cell level - D diffusion constant of oxygen in water - K_La oxygen transfer rate - l height of liquid above cells - n number of cells per ml - R_x respiration rate per cell     

Activation of BDNF mRNA and protein after seizures in hyperbaric oxygen: implications for sensitization to seizures in re-exposures

Previous studies have demonstrated that exposure to convulsive doses of hyperbaric oxygen (HBO) increases sensitivity to seizures in re-exposures. Because brain derived neurotrophic factor (BDNF) is induced after a variety of seizures and increases cell excitability, it may contribute to the mechanism of sensitization. In this study, a fast induction in BDNF mRNA 2 hr after seizures and a temporary increase in BDNF protein 1 day after seizures induced by 100% O₂ at 5 atm (gauge pressure) were demonstrated in the rat cortex. To determine whether an elevation in BDNF protein level can modify sensitivity to the toxic effect of HBO, recombinant BDNF (12 g) was injected into cerebral ventricles 30 min prior to exposure. Administration of exogenous BDNF significantly shortened latent time to seizures in HBO exposures. We propose that upregulation of BDNF expression in the brain after seizures may contribute to sensitization to HBO toxicity.     

A continuous-flow culture system for organ culture of large explants of adult tissue: Effect of oxygen tension on mouse molar periodontium

Summary A modified continuous-flow culture system (CFCS) was developed to maintain large explants of periodontium from adult mouse in organ culture. The culture medium was stored in a reservoir outside of the incubator, pumped via polyvinyl tubing into small glass culture chambers that were placed in the oxygenator and then collected in a waste flask. Medium was analyzed for pO₂, pCO₂ and pH during the culture period. Three-molar and singlemolar explants of periodontium were maintained for 48 hr in the CFCS at two different pO₂ ranges: 100 to 120 mm Hg and 400 to 420 mm Hg. [³H]Proline was added 24 hr prior to sacrifice. Light-microscope morphological and radioautographic observations suggested that cell viability and incorporation of [³H]proline, probably into newly synthesized protein, increased with an increase in pO₂ and was related to a pO₂ gradient extending from the periphery to the center of the explants.     

Comparative study with two different enrichments in the culture media used in the disinfectant efficacy assay

Recent changes in Brazilian legislation for commercial disinfectants have been published due to the recent epidemic of nosocomial infections caused by rapidly growing mycobacteria (RGM) in many states of Brazil over the last 8 years. One of these documents requires that all the manufacturers provide evidence of efficacy of sterilizing and disinfectant products, used for semi critical medical devices, against the Mycobacterium bovis BCG Moreau and Mycobacterium abscessus subsp. bolletii INCQS 00594 strains by using the Confirmative in vitro Test for Determining Tuberculocidal Activity of Disinfectants recommended by the Association of Official Analytical Chemists. These changes have caused additional costs and increased problems for importation of enrichment products at national laboratories where disinfectant efficacy assay service is performed. Middlebrook ADC Enrichment (ADC) is provided by a unique manufacturer and used in the official protocol. The aim of the present study was to evaluate an alternative in house low-cost enrichment composed of fetal bovine serum and glucose (FBSG) with ADC for performance of disinfectant efficacy assay against mycobacteria. After obtaining the growth curves for M. abscessus ATCC 19977, M. abscessus subsp. bolletii INCQS 00594, Mycobacterium chelonae ATCC 35752, and Mycobacterium fortuitum ATCC 6841 by using ADC enrichment and FBSG in Kirchners and 7H9 culture media. Through statistical analysis via the Kruskal-Wallis test on the evaluation of microorganism growth rate, it was observed that there was no inhibition of RGM growth by any of the enrichments used. These results suggest that low-cost enrichment FBSG may be used as a potential substitute of ADC for composition of media for mycobacterial growth, including in disinfectant tests.