Increased growth yields of Mycoplasma spp. in the presence of pyruvate

Viable and non-viable African green monkey kidney (Vero) cells after treatment with Clostridium perfringens enterotoxin (CPE) followed by simultaneous double staining with fluorescein diacetate (FDA) and propidium iodide (PI) were counted with a flow cytometer (FCM). Within 1 min the FCM analysed 10 000 Vero cells in a sample for viability. After treatment of Vero cells with CPE for 60 min and staining with FDA-PI for 5 min, a reproducible dose-response curve was obtained between 25 and 400 ng/ml of CPE and percentage viable cell numbers. The FCM analysis proved to be a strong tool for rapid discrimination between viable and non-viable Vero cells treated with CPE in a large number of samples at a time.     

Phytotoxicity of the tetramic acid metabolite trichosetin

Trichosetin, a tetramic acid-containing metabolite produced in the dual culture of Trichoderma harzianum and Catharanthus roseus (L.) G. Don callus, was subjected to phytotoxicity assays. In seedling growth assays, trichosetin inhibited root and shoot growth of all five plant species tested by damaging the cell membrane, as evidenced by the dose-dependent increase in electrolyte leakage and lipid peroxidation. Vital staining of trichosetin-treated Nicotiana tabacum BY-2 cells, with rhodamine 123, showed a weaker green fluorescence compared to controls indicating damaging effects on mitochondria. FDA-PI staining, to determine cell viability, indicated that cells of the trichosetin-treated roots were mostly dead.     

Rapid flow cytometric method for viability determination of solventogenic clostridia

We endeavored to develop a method for viability determination of solventogenic clostridia and to apply it for monitoring acetone–butanol–ethanol (ABE) fermentation. Six fluorescent probes (propidium iodide [PI], ethidium bromide, fluorescein diacetate, carboxyfluorescein diacetate [cFDA], rhodamine 123, bis-(1,3-dibutylbarbituric acid)trimethine oxonol [BOX]) were tested in order to distinguish two subpopulations of live and dead clostridial cells in suspension. Three of them were found to be appropriate (PI, BOX and cFDA) for this purpose. Developed fluorescent staining methods were applied to batch fermentation processes of Clostridium pasteurianum and C. beijerinckii carried out in a laboratory bioreactor under anaerobic conditions. Whereas PI was found to be applicable to both strains, BOX was convenient only for viability determination of C. pasteurianum. Although cFDA can distinguish two cell subpopulations in suspension, it was found to be unsuitable for viability determination under tested conditions, since it reflected more variable esterase activity during sporulation cell cycle than viability. Flow cytometry in combination with convenient fluorescent probe has been proved to be a valuable tool for viability determination. We assume this rapid and simple method can help to obtain more complex and precise information about ABE fermentation.     

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.     

Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures

We have recently described Arabidopsis cell suspension cultures that can be effectively synchronised. Here, we describe procedures that allow clonal-transformed cell suspension lines to be produced using Agrobacterium-mediated transformation, and an optimised and straightforward procedure for the cryopreservation and recovery of both parental and transformed lines. Frozen cultures show 90% viability and rapid re-growth after recovery. We show that the cryopreservation procedure is equally applicable to the frequently used tobacco bright yellow (BY)2 cell suspension culture, and that cell cycle synchronisation capacity of parental lines is maintained after both transformation and recovery from cryopreservation. The techniques require no specialised equipment, and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures and providing security against both contamination and cumulative somaclonal variation. Finally, the ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for high-throughput analysis.     

建立同时分离培养小鼠肝细胞及肝星状细胞的技术

目的:建立一种简便、经济、高产的同步分离培养肝细胞以及肝星状细胞的方法。方法:在参照国内外方法的基础上加以改良,首先采用肝脏原位胶原酶灌注消化的方法,获得总细胞悬液,经多次低速离心分离肝细胞;再用Nycodenz作为分离介质,通过密度梯度离心法从非实质细胞中得到肝星状细胞。通过台盼蓝染色方法鉴定细胞的活力,用倒置相差显微镜、立体显微镜、CK-18、白蛋白免疫荧光细胞化学染色对培养的肝细胞形态以及功能进行检测。使用Desmin、α-SMA免疫荧光细胞化学对肝星状细胞进行鉴定。结果:成功的在体外同步分离、培养肝细胞及肝星状细胞,肝细胞产率为5-6×107/只小鼠,两只小鼠肝星状细胞产率达1×106个。细胞存活率及纯度均可达90%。肝细胞在培养24h后呈不规则铺路石样形态,此为典型的肝细胞形态,其标志分子CK-18以及白蛋白免疫荧光染色阳性。倒置相差显微镜下可见贴壁后的肝星状细胞呈典型的星形细胞形态,且其标志分子Desmin、α-SMA免疫荧光染色阳性。结论:改良的原位灌注以及分离方法可以同时分离并且培养具有高活性和功能的肝细胞和肝星状细胞。     

Culturing embryogenic protoplasts of ‘Hamlin’ sweet orange in calcium alginate beads

Two finely-dispersed, homogeneous and regenerable cell suspension cultures were established from embryogenic callus derived from immature and mature embryos in barley. The quality and viability of suspension cells obtained were determined using differential-interference-contrast microscope and fluorescence microscope. Cell suspension cultures, maintained in modified liquid CC medium, showed a 10-fold increase in dry weight after two weeks with a doubling time of about 3 days. Addition of l-proline and casein hydrolysate in the medium had positive effect on the growth of cell cultures. Subculture interval significantly affected mitotic index. Both cell lines established were able to regenerate plants by somatic embryogenesis, but cell line Z-IM showed much higher regeneration capacity than cell line Z-M. Comparatively high frequencies of variations in chromosome number and structure were found in both lines, and a correlation between karyotype and morphogenic capacity was noticed.Abbreviations KT kinetin - BAP benzylaminopurine - FDA-PI fluorescein diacetate and propidium iodide - 2,4-d 2,4-dichlorophenoxyacetic acid     

Preparation of single cells from aggregated Taxus suspension cultures for population analysis

A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism.     

A new microtitre plate screening method for evaluating the viability of aerobic respiring bacteria in high surface biofilms

Aims: It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms. Methods and Results: It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3′‐[1‐[(phenylamino)carbonyl]‐3,4‐tetrazolium]Bis(4‐methoxy)‐6‐nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. Conclusions: We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. Significance and Impact of the Study: This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.     

Method for selecting cells during various phases of the mitotic cycle

Exponentially growing L5178Y cells in suspension culture were separated according to their position in the cell cycle on the basis of their volume with a velocity sedimentation method in which a linear and continuous ficoll gradient was used. Highly purified populations of G1 and S cells were obtained, containing about 90% G1 phase cells and 80% S phase cells. The method is rapid and a larger number of cells can be easily processed with no loss of viability.     

Differential staining for live and dead sperm of honey bees

ABSTRACT Seven staining techniques were modified and tested for differentially staining the live and the dead sperm cells for the honey bee (Apis mellifera L.). The eosin Y staining method was found to be a simple technique by which the live cells stain bluish purple whereas the dead cells stain bright yellow to greenish yellow. Therefore, it produces a strong contrast between the dead and live sperm cells, and appears to be the most suitable supravital staining method for evaluating the viability of honey bee sperm cells. The significance of supravital staining techniques in assessing the quality of sperm cells during cryopreserving sperm cells is discussed.     

Culture substrate dependence of mouse fibroblasts survival at 4° C

Summary When L-929 mouse fibroblasts grown in Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS) were stored in a monodisperse suspension at 4° C, the viability decreased rapidly from the beginning of storage. The viability in this study was determined by counting electronically the number of cells with the capacity to attach to glass substrate and with the membrane boundary resistant to a proteolytic digestion. When, however, the dissociated cells were preincubated briefly at 37° C, and subsequently stored at 4° C as they were attaching on a glass substrate, the rapid loss of viability could be reduced effectively. A biphasic survival profile consisting of an initial phase of slowly decreasing viability and the subsequent phase of rapidly decreasing viability were then observed. The rapid viability loss occurred not only when the cell suspension was prepared by mechanical dislodging but also after trypsinization or dispase treatment. Such viability loss was also observed when the dissociated cells were not stored at 4° C directly but preincubated in a monodisperse suspension at 37° C in a siliconized plate and then stored at 4° C. The above results show that the rapid loss of viability is associated closely with the fact that the cells were not attached to the substrate but in suspension. This work was supported in part by grants-in-aids from the Institute of Physical and Chemical Research and from the Mitsubishi Foundation.     

Evidence for substantial maintenance of membrane integrity and cell viability in normally developing grape (Vitis vinifera L.) berries throughout development

Fluorescein diacetate (FDA) was used as a vital stain to assaymembrane integrity (cell viability) in mesocarp tissue of thedeveloping grape (Vitis vinifera L.) berry in order to testthe hypothesis that there is a substantial loss of compartmentationin these cells during ripening. This technique was also usedto determine whether loss of viability was associated with symptomsof a ripening disorder known as berry shrivel. FDA fluorescenceof berry cells was rapid, bright, and stable for over 1 h atroom temperature. Confocal microscopy detected FDA stainingthrough two to three intact surface cell layers (300–400µm) of bisected berries, and showed that the fluorescencewas confined to the cytoplasm, indicating the maintenance ofintegrity in both cytoplasmic as well as vacuolar membranes,and the presence of active cytoplasmic esterases. FDA clearlydiscriminated between living cells and freeze-killed cells,and exhibited little, if any, non-specific staining. Propidiumiodide and DAPI, both widely used to assess cell viability,were unable to discriminate between living and freeze-killedcells, and did not specifically stain the nuclei of dead cells.For normally developing berries under field conditions therewas no evidence of viability loss until about 40 d after veraison,and the majority (80%) of mesocarp cells remained viable pastcommercial harvest (26 °Brix). These results are inconsistentwith current models of grape berry development which hypothesizethat veraison is associated with a general loss of compartmentationin mesocarp cells. The observed viability loss was primarilyin the locule area around the seeds, suggesting that a localizedloss of viability and compartmentation may occur as part ofnormal fruit development. The cell viability of berry shrivel-affectedberries was similar to that of normally developing berries untilthe onset of visible symptoms (i.e. shrivelling), at which timeviability declined in visibly shrivelled berries. Berries withextensive shrivelling exhibited very low cell viability (15%). Key words: Apoplast, berry shrivel, compartmentation, DAPI, FDA, fluorescence, fruit ripening, locule, propidium iodide Received 19 September 2007; Revised 16 December 2007 Accepted 26 December 2007     

Cortical cell elution by sedimentation field-flow fractionation.

As a cell sorter, Sedimentation field-flow fractionation (SdFFF) can be defined as an effective tool for cell separation and purification, respecting integrity and viability as well as providing enhanced recovery and purified sterile fraction collection. The complex cell suspension containing both neurons and glial cells of all types, obtained from cerebral cortices of 17-day-old rat fetuses, is routinely used as a model of primary neuronal culture. Using SdFFF, this complex cell mixture was eluted in sterile fractions which were collected and cultured. SdFFF cell elution was conducted under strictly defined conditions: rapid cell elution, high recovery (negligible cell trapping), short- and long-term cell viability, sterile collection. After immunological cellular type characterization (neurons and glial cells) of cultured cells, our results demonstrated the effectiveness of SdFFF to provide, in less than 6 min, viable and enriched neurons which can be cultured for further investigations.     

Fluorescein diacetate used as a vital stain for labeling living pollen tubes

Snapdragon and tobacco pollen treated with fluorescein diacetate (FDA, 10 μg/ml) for 40 min could germinate and grew well in FDA-free medium. The pollen tubes showed bright fluorescence of the protoplasm when they were living. Thus the FDA method can be used, in addition to its previous usage for viability test of cells, also for vital staining of pollen tubes, and may be valuable to the study of other living cells as well.     

Aluminum-induced cell death in root-tip cells of barley

Aluminum-induced cell death was investigated in root-tip cells of barley (Hordeum vulgare). The growth of roots in 0.1-50 mM Al treatments was inhibited after 8 h treatments, and could not be recovered after 24 h recovery culture without Al. Viable detection with fluorescein diacetate-propidium iodide (FDA-PI) staining shows that most of the root-tip cells have lost viability. These results suggest that the irreversible inhibition of root growth after 8 h Al treatments or 24 h recovery culture is mainly caused by cell death. DNA ladders occurred in root tips only after 8 h Al treatments (0.1-1.0 mM), but no apoptotic bodies in root tips were observed. Thus, the cell death caused by Al stress is likely to be Al-induced programmed cell death (PCD). The reactive oxygen species (ROS) in root-tip cells measured by ultraweak luminescence indicated that the oxidation status in root-tip cells basically ceased after exposure to 10-50 mM Al for 24 h, but was very violent in the root-tip cells treated with 0.1-1.0 mM for 24 h. Exposure to 0.1-1.0 mM Al for 3-12 h led to ROS burst. Therefore, our results suggest that 0.1-1.0 mM Al treatments for 8 h induce cell death (Al-induced PCD) possibly via a ROS-activated signal transduction pathway, whereas 10-50 mM Al treatments may cause necrosis in the root-tip cells. These results have an important role for further studies on the mechanism of Al toxicity in plants.     

A Simplified Micro-Method for Cytotoxicity Testing Using a Flat-Type Titration Plate for the Detection of H-2 Antigens

A new micro-method for cytotoxicity testing using a flat-type titration plate has been developed. This micro-method is so simple that it involves no washing and staining procedures. In this method, cell viability is judged on the basis of the refractility change in the lymphocytes seen under the phase-contrast microscope. This micro-method has higher sensitivity and reproducibility than the dye exclusion method and ⁵¹Cr-release assay. Fixation of cells with glutaraldehyde and tight covering of the plate allow us to read the result even after 3 weeks. These advantages of the new method enable us to deal with a large number of samples at one time. We also introduce a rapid technique for the preparation of a cell suspension from the lymph node without killing the animal.     

In situ microscopic cytometry enables noninvasive viability assessment of animal cells by measuring entropy states

Current state of the art to determine the viability of animal cell suspension cultures is based on sampling and subsequent counting using specific staining assays. We demonstrate for the first time a noninvasive in situ imaging cytometry capable of determining the statistics of a morphologic transition during cell death in suspension cultures. To this end, we measure morphometric inhomogeneity—defined as information entropy—in cell in situ micrographs. We found that the cells are partitioned into two discrete entropy states broadened by phenotypical variability. During the normal course of a culture or by inducing cell death, we observe the transition of cells between these states. As shown by comparison with ex situ diagnostics, the entropy transition happens before or while the cytoplasmatic membrane is loosing its ability to exclude charged dyes. Therefore, measurement of morphometric inhomogeneity constitutes a noninvasive assessment of viability in real time. Biotechnol. Bioeng. 2011;108: 2884–2893. © 2011 Wiley Periodicals, Inc.     

A new type of two-color fluorescence staining for cytology specimens.

A new two-color fluorescence staining technique for cervical cytology specimens is described. To permit application of this staining in automated cytology, techniques for specimen collection and cell preparation giving a sufficient number of well-separated cells on slides were used. The staining consists of a combination of a modified Feulgen-acriflavine procedure for DNA and a primulin or stilbene isothiocyanate staining for protein. This results in a bright yellow nuclear fluorescence and a blue cytoplasmic fluorescence. The staining procedure can be completed in about 90 min and is therefore suitable for routine application. Sequential inspection of the yellow nuclear and blue cytoplasmic fluorescence can be done with the two-wavelength excitation method used in fluorescence microscopy. For the application of this method, special vertical illuminators are now available. These illuminators are provided with quickly interchangeable filter sets permitting consecutive visualization of, for example, only the nuclei in the first image and the whole cell in the second image. This procedure opens new possibilities for rapid image-analysis systems.     

Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability.

BACKGROUND: Several staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA-binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual-staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9-PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability. METHODS: Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures. RESULTS: SYTO9-PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP-PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data. CONCLUSIONS: In bacterial viability analyses, the GFP-PI combination provided better distinction between current viability stages of E. coli cells than SYTO9-PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed.