Analysis of plating technique for viability and drug-sensitivity determinations using Rosa cells

Rosa Paul's Scarlet'cell suspension cultures were used as a test system for working out a method of viability and drug-sensitivity determination based on plating efficiency. High plating efficiencies (80–95%) were obtained on a simple synthetic medium when aggregates of a mean size of c . 100 cells/unit from exponential phase cultures were plated at a density of 1500 units/plate in the middle layer (5 ml) of three layers of the agar-solidified medium (total = 30 ml). This 3-layer plating technique produces homogeneous colony growth and simplifies the microscopical evaluation of plating efficiencies. The reduction of plating efficiencies seen when the smaller aggregates of stationary phase cultures were plated was mainly due to low cell density and could be overcome by enriching the medium with various supplements. Reconstitution experiments using mixtures of inactivated and non-inactivated aggregates demonstrated that plating efficiency can be taken as a goodmeasure of viability. The described plating technique was found to be more sensitive and reliable compared to two other methods for determining p -fluorophenylalanine-sensitivity of Rosa cells.     

Cellular stress rather than stage of the cell cycle enhances the replication and plating efficiencies of herpes simplex virus type 1 ICP0- viruses

This lab reported previously that the plating efficiency of a herpes simplex virus type 1 ICP0-null mutant was enhanced upon release from an isoleucine block which synchronizes cells to G1 phase (W. Cai and P. A. Schaffer, J. Virol. 65:4078-4090, 1991). Peak plating efficiency occurred as cells cycled out of G1 and into S phase, suggesting that the enhanced plating efficiency was due to cellular activities present in late G1/early S phase. We have found, however, that the enhanced plating efficiency did not occur when cells were synchronized by alternative methods. We now report that the plating efficiency of ICP0- viruses is not enhanced at a particular stage of the cell cycle but rather is enhanced by specific cellular stresses. Both the plating and replication efficiencies of ICP0- viruses were enhanced as much as 25-fold to levels similar to that of wild-type virus when monolayers were heat shocked prior to infection. In addition to heat shock, UV-C irradiation but not cold shock of monolayers prior to infection resulted in enhanced plating efficiency. We further report that the effect of cellular stress is transient and that cell density rather than age of the monolayers is the primary determinant of ICP0- virus plating efficiency. As both cell stress and ICP0 are required for efficient reactivation from latency, the identification of cellular activities that complement ICP0- viruses may lead to the identification of cellular activities that are important for reactivation from neuronal latency.     

Cryopreservation of adult human hepatocytes obtained from resected liver biopsies

Isolated human hepatocytes have been shown to represent a valuable in vitro model to investigate the metabolism and cytotoxicity of xenobiotics. In addition, human hepatocyte transplantation and artificial liver support systems using isolated human hepatocytes are currently investigated as treatment for acute and chronic hepatic failure. In this regard, human hepatocyte banking by cryopreservation would be of great interest. In the present study, freshly isolated hepatocytes from resected liver biopsies of 28 separate donors (viability: 88 +/- 2%; plating efficiency: 79 +/- 5%) were cryopreserved using two different protocols, stepwise freezing (SF) or progressive freezing (PF), in combination (PF(+), SF(+)) or not (PF(-), SF(-)) with a 30 min preincubation in culture medium at 37 degrees C. Total recovery was higher after PF (38 +/- 3%) than after SF (12 +/- 2%). Preincubation prior to SF had no effect on plating efficiency of thawed hepatocytes (SF(-): 38 +/- 6% versus SF(+): 46 +/- 7%) while preincubation prior to PF increased plating efficiency of thawed hepatocytes (PF(-): 42 +/- 6% versus PF(+): 64 +/- 4%, p < 0.05). In attached cultured human cryopreserved/thawed hepatocytes (CH) from the PF(+) group, albumin production and glutathione content were not significantly different from those of the freshly isolated hepatocyte (FIH) cultures. Cells in CH monolayers appeared smaller than cells in FIH monolayers. In addition, the pattern of cytochrome P450- and UDP-glucuronosyl transferase-dependent isoenzyme activities and GST activity were different, suggesting a variability in the resistance to cryopreservation of the various liver hepatocyte populations. Taken all together, the results of the present study suggest that recovery of human hepatocytes after isolation prior to progressive freezing should allow human hepatocyte banking for use in pharmacotoxicology and cell therapy research purposes.     

Factors affecting the growth of Chinese hamster cells in halt selection media

Factors affecting the efficiency of selection of “reverants” of salvage pathway mutants in media containing amethopterin have been examined. Our V79 Chines hamster cell line was found to require a significantly higher level of thymidine for optimal growth in such media than has been reported for other cell lines. Hypoxanthine (but not glycine) was also required for reversal of amethopterin toxicity, but levels did not differ significantly from those reported elsewhere. Growth in HAT was also dependent on plating density and serum batch. Our modification (VHAT) was compared with published HAT recipies in back selection reconstruction experiments. A sharp fall in EOR (efficiency of recovery) of wild type cells from mixtures with mutants at plating densities greater than 3500 cells/cm² (10⁵ cells/6 cm dish) was observed for VHAT. EOR with other HAT recipes was lower still, and was affected also by the particular mutant used in the mixture.EMS induced “revertants” were isolated from three 8AZ^r mutants by plating in VHAT. All. revertants were however amethopterin resistant, they were also 8AZ resistant and the mobility of residual HGPRT (as measured by polyacrylamide gel electrophoresis) was similar to that of their 8AZ^r parents i.e. dissimilar from that in wild type. The modal chromosome number of V79 wild type cells was 21. No significant deviation from this mode was detected in any of the mutant lines examined. The data indicate that the recovery of colonies in HAT from 8AZ^r mutants does not necessarily indicate that a back mutation in the structural gene for HGPRT has occurred. Thus, the frequency of HAT⁺ colonies cannot be taken as a direct indication of reversion frequencies.     

高含量薯芋皂素植株的细胞克隆

盾叶薯芋(Dioscorea zingibernisis Wright)细胞克隆系的建立采用平板培养法。试验了18种不同的培养外界环境因素对植板率的影响:如培养基pH值,在培养基中添加谷氨酰胺、丝氨酸、柠檬酸、葡萄糖和偶合低聚糖等。其中,以添加偶合低聚糖的植板率最高。获得31个克隆细胞系。经大量培养,测定细胞生长,筛选出18个优良克隆系。以代号为Rc81的克隆系在接种量为0.7g/L(折合干重,下同),培养28d可获得15.1g/L。     

The impact of homologous and heterologous nurse cells on the division capability of sparsely plated cells

The growth-promoting effects of nurse cells of carrot, tomato, patato, maize, bean, carnation and two species of tobacco were studied on carrot, tomato, tobacco and potato cells plated at low densities. In an area immediately below the nurse cells the plating efficiency was very high and found to be independent of cell density. In an area outside the nurse cells, in some cases, the plating efficiency tended to be much higher in combinations with cells from a heterologous source as compared with those from a homologous source. Moreover, in the same area with some combinations the plating efficiency decreased when cell density was lowered, while with other combinations this phenomenon did not occur. This decrease was independent of the absolute value of plating efficiency. In experiments in which the concentration of conditioning factors was presumably changed, no significant difference in the plating efficiency was noticed. We therefore suggest that different plating efficiencies observed with heterologous nurse cells were not due to a higher level of conditioning factors, but rather to the production of different types of conditioning factors that are presumably degraded with different efficiency.     

Plating Efficiency for Primary Hamster Embryo Cells as an Index of Efficacy of Fetal Bovine Serum for Cell Culture

Attachment and growth of mammalian cells plated at low cell density require optimum conditions for the cells to form colonies. Reliability, reproducibility, and validity of the plating efficiency test for evaluating cell culture sera were determined by measuring the plating efficiency of 37 lots of fetal bovine serum obtained from 8 suppliers (5 lots from each of 7, 2 lots from 1 supplier), by using hamster embryo fibroblasts plated at low cell density. The test revealed considerable variation between lots of serum and between suppliers. The five lots from some suppliers had consistently high plating efficiencies, whereas one or more lots from other suppliers had quite low efficiencies. The results were reproducible in repeated tests, and control experiments indicated that the test measured the efficiency of the test serum independently of the efficiency of the serum used for the primary outgrowth of the hamster embryo cells.     

The isolation, culture and division of protoplasts from citrus cotyledons

High yields (2.3 × 10⁵ to 1.3 × 10⁶ protoplasts/g.f.wt.) of isolated protoplasts were obtained from cotyledons of Cirus sinensis (L.) Osb. 'Valencia'. Osmotic potential of the medium and enzyme concentrations were important in obtaining high viability of preparations as indicated by FDA fluorescence. Adding malt extract to a Murashige-Tucker basal medium increased plating efficiencies somewhat, but not the rate or duration of cell division. However, modifying the NAA and kinetin concentration optimized plating efficiencies (up to 20%) of protoplasts and also the rate or duration of cell division. The highest plating efficiency and number of cells per colony were obtained on a defined medium containing NAA (15 μ M ). and kinetin (4.6 μ M ). Coincidence of percentage protoplast viability after 13 days (assessed by FDA fluorescence) with plating efficiency after 21 days indicates that FDA fluorescence is an accurate indicator of citrus protoplast viability.     

A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25 degrees C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22 degrees C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22 degrees C and plating on CIN agar (48 h at 25 degrees C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25°C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22°C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22°C and plating on CIN agar (48 h at 25°C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

Supercritical fluid disruption of Ralstonia eutropha for poly(beta-hydroxybutyrate) recovery

A new method for disruption of Gram-negative bacterium Ralstonia eutropha by supercritical CO(2) for poly(beta-hydroxybutyrate) (PHB) recovery is proposed. The effects of different parameters such as exposure time, pressure, temperature, volume of methanol as a modifier, and culture history on cell disruption efficiency were investigated using Taguchi's statistical approach to determine optimum conditions. The optimum conditions for cell disruption and PHB recovery were as follows: exposure time, 100 min; pressure, 200 atm; temperature, 40 degrees C; volume of methanol, 0.2 mL. The cell culture time was less significant. At optimum conditions, the maximum efficiency of PHB recovery was found to be 89%. The proposed method is comparable with other recovery methods in terms of the percentage of PHB recovery, while it is environmentally more benign.     

Primary cultures of Leydig cells from rat, mouse and pig: advantages of porcine cells for the study of gonadotropin regulation of Leydig cell function

Primary cultures of interstitial cells were prepared from the testis of mice, rats, and pigs. The cells were grown in a defined medium supplemented with low (0.1%) serum and insulin, transferrin and epidermal growth factor. Comparisons of the interstitial cell cultures from the three species were made for plating efficiency, cell survival, maintenance of hCG receptors and maintenance of steroidogenic responsiveness to hCG. The porcine cultures had a higher plating efficiency and higher hCG receptor levels per cell than Leydig cells from either rodent. Additionally, the porcine cells showed an increase in testosterone (T) production with hCG stimulation throughout their lifespan in culture while the rodent cultures showed a decrease in T stimulation with time with no stimulation by day 6 in culture. These data indicate that species differences exist in hCG receptor concentrations per cell, the maintenance of hCG receptors and steroidogenic response in culture. The initial high survival, purity and continued functional response of porcine interstitial cell cultures make them a superior system for the study of gonadotropin regulation of Leydig cell function.     

Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose

A procedure is described for the reproducible establishment of rice cell suspension cultures from callus of embryo origin. Protoplasts were readily isolated from cell suspension of four rice cultivars, including japonica and indica types, when maintained in an amino acid-based culture medium. Sustained protoplast division from two japonica genotypes has been obtained in agarose solidified culture medium. An increase in the agarose concentration from 0.6% to 1.2% (w/v) produced a marked improvement in protoplast survival, division and plating efficiency. Protoplast division and plating efficiency frequencies of up to 26% and 0.5%, respectively, were obtained at the higher agarose level. The protoplast-derived calli were similar in appearance to explant-derived morphogenic callus and produced distinct embryo-like structures.     

Dilution of hypoxanthine-guanine phosphoribosyl transferase and other factors affecting the frequency of 6-thioguanine resistance in Chinese hamster lung cells.

Factors influencing the frequency of thioguanine resistant mutations were examined in Chinese hamster lung cells damaged with a carcinogen, N-acetoxy-2-acetyl aminofluorene. Factors such as inoculum density, expression time, and concentration of selective agent were found to have a profound effect on the mutation frequency.Over a range of doses, a longer expression time is required for mutant cells from a more damaged population to reach their maximum frequency. In order to investigate the elements involved in this phenomenon, the increment in the plating efficiency of treated cells as a function of expression time, spontaneous mutation rate per cell per generation, viability of mutant as well as wild type cells, and half life of HGPRTase were evaluated.There was an observed relationship between induced mutation frequency and plating efficiency of treated cells. When treated cells had recovered from effects of the treatment and arrived at the normal level of plating efficiency, they also yielded the maximum frequency of mutations.The estimated mutation rate was 5.5 × 10^?8 per cell per generation. This number is too small to account for the increment in mutation frequency with the increase in the expression time. The mutation frequency of spontaneous origin was 4 × 10^?6 and that of induction of 10^?5 M NA-AAF was 10^?4. Lower growth rates of mutant cells cannot explain this increase in the number of mutants recovered, either.Continuous diminution in the level of HGPRTase, at 35% daily, interpreted as an important factor responsible for the recovery of mutation frequency during expression time, was observed in non-dividing cells. None of a large number of mutants sampled from those isolated had HGRPT activity. This indicates that they are true mutants and are not a result of phenocopy. Only cells completely deficient in HGPRT activity are recovered in TG selection medium. It is suggested, therefore, that this cell line is suitable for mutagenicity testing in the induction of mutation at the HGPRT locus.     

Analysis of seawaters for the recovery of culturable Vibrio parahaemolyticus and some other vibrios

We investigated the recovery of dormant and injured cells along with the normally culturable cells of Vibrio species with special emphasis on V. parahaemolyticus using both selective and non-selective media at moderate (20 C) and standard (37 C) culture temperatures from a bay water environment. Culture temperatures (20 or 37 C) did not affect the recovery of V. parahaemolyticus but did for other vibrios. We observed similar seasonality of V parahaemolyticus as in most other environmental studies. V. parahaemolyticus and other Vibrio species were recovered in higher numbers by a replica plating method compared to most probable number (MPN) and direct TCBS (thiosulfate citrate bile-salt sucrose) agar counts. Even with the replica plating method, however, vibrios number goes down to a minimum level and V. parahaemolyticus was undetectable during the cool temperature period of the year, although total bacterial cells and CFU on nutrient agar (with 2% NaCl) did not vary so much during the study period.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.     

红花细胞克隆的平板培养

在红花(Cathamus unctorrus)细胞克隆的平板培养中很多因素都能影响其植板率,如培养基的 pH 值,无机盐中的 NH_4NO_3、ZnSO_4、MnSO_4;有机酸中的柠檬酸、琥珀酸、苹果酸及延胡索酸,谷氨酰胺和精氨酸,葡萄糖,椰乳和水解乳蛋白等。向培养基中加入10mg/l 柠檬酸或3mg/l 琥珀酸能显著提高植板率。适量的人参寡糖及黑节草寡糖应用对促进红花细胞克隆生长取得很好效果。常规的高压灭菌最不利于细胞生长,在15磅/cm~2灭菌5分钟效果较好,过滤灭菌效果最好。     

Recovery of yeast cells following the combined action of hyperthermia and ionizing radiation

Consecutive action of elevated temperature (50 degrees C) and gamma-irradiation on yeast cells Saccharomyces cerevisiae was studied. It was shown that yeast cells can recover from lethal thermal and radiation lesions after the combined action of the two factors. The efficiency of recovery does not depend upon the sequence of treatments. Heating (50 degrees C) before or after gamma-irradiation increases the radiation response of yeast when plating the cells on a nutrient agar containing 1.5 M KCl. The synergistic effect decreases with yeast cells kept in water at 28 degrees C before plating. The influence of one factor on the effectiveness of recovery from damages induced by the other was estimated.     

Toxicity of microbial products in cell culture

Cytotoxicity of a mixed pyrogen preparation and its components as well as native and radiodetoxified lipopolysacharides (LPS) was determined with established HEp-2 cell cultures and by measuring plating efficiency. This proved to be more sensitive to the damaging effect of both microbe-containing LPS (Escherichia coli) and purified LPS. Microorganisms without this compound (Lactobacillus casei, Streptococcus faecalis) seem to be more infifferent on cells, evenSaccharomyces cerevisiae has a marked augmenting effect on cell plating. Divergence in nulltoxicity between the established cultures and plating efficiency makes it possible to presume that the primary effect of microorganisms on epithelial cells is medited by LPS, and the subsequent liberation of lysosomal proteinases is responsible for major biological effects.     

Electrostimulated regeneration of plantlets from protoplasts derived from cell suspensions of barley (Hordeum vulgare)

For transformation and somatic hybridisation of barley ( Hordeum vulgare L.), it is necessary to develop an efficient and reliable system for routne plant regeneration from protoplasts. Freshly-isolated cell suspension-derived protoplasts were treated with both rectangular and exponential electric pulses with the aim of increasing plating efficiency as well as to stimulate regenerative capacity. Suspensions were initiated from callus from immature embryos of barley (cv. Dissa). Increasing field strength, capicitance, or number of applied pulses resulted in a decreased protoplast viability and plating efficency. However, the regeneration of albino leaves and albino plantlets from electro-treated protoplasts was stimulated in comparison with controls.