Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log(10) was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.     

Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log₁₀ was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.     

Effects of high cell density on growth-associated monoclonal antibody production by hybridoma T0405 cells immobilized in macroporous cellulose carriers

Relationship between monoclonal antibody (MAb) productivity and growth rate, and effects of high cell density on MAb production of hybridoma T0405 cells immobilized in macroporous cellulose carriers were investigated in continuous and batch cultures. The results showing, that the specific MAb production rate increased with increasing specific growth rate in both suspended and immobilized continuous cultures indicate a positively growth-associated relationship between MAb productivity and growth rate. Moreover, the specific production rate was higher in the immobilized cell culture than that in suspended one at all dilution rates. In order to clarify these phenomena, MAb mRNA expression and cell cycle distribution were investigated in batch cultures with immobilized cells and suspended cells. RT-PCR was used for observation of MAb mRNA expression and a two-color bromode-oxyuridine (BrdU)/propidium iodide (PI) flow cytometry method for determination of cell cycle distribution. The results revealed that MAb mRNA expression reached the peak during the exponential growth phase, suggest a positively growth-associated MAb production. And the immobilized cells continued the MAb mRNA expression until dead phase, which was longer than that in suspended cells. The cell cycle distribution patterns were observed almost the same for both immobilized and suspended cells. Such results may imply that a high cell density state has positive influence on the mRNA expression and on growth-associated MAb productivity of T0405 cells.     

Induction of CD40 expression and enhancement of monoclonal antibody production on murine B cell hybridomas by cross-linking of IgG receptors

The 55-6 murine B cell hybridoma line not constitutively expressing CD40 was treated with increasing amounts of intact anti-mouse surface immunoglobulin G antibody (anti-mIgG) either not preincubated or preincubated for 48 h with lipopolysaccharide (LPS). In vitro, cross-linking of surface immunoglobulin G (sIgG) with the whole molecule of anti-IgG antibodies induced the expression of CD69, CD40, and CD19 surface antigens on 55-6 cells. The effect of sIgG ligation was dose-dependent, and preincubation with LPS enhanced their responsiveness to anti-mIgG stimulation. The expression of these surface molecules reached the maximum value during the first part of the cell cycle, corresponding to the position of the G1 peak of the DNA distribution. Stimulation of cells with anti-mIgG did not induce changes either in the number of viable cells or in the fraction of cells undergoing proliferation (mitosis). However, preincubation of 55-6 cells with LPS for 48 h before stimulation with anti-mIgG increased both the maximum specific growth rate (micromax) and the percentage of cells in the G2/M phase, in comparison with non-preincubated cells. Moreover, on cells preincubated with LPS prior to anti-mIgG treatment, specific IgG2a production rate was enhanced significantly compared to that obtained in control cultures. The correlation between the antibody production rate and the amount of IgG that is detectable on the cell surface was analyzed by flow cytometry. A good correlation between secreted and surface IgG was observed, and the results of cell cycle analyses demonstrated that the 55-6 hybridoma cell line has a substantially higher sIgG content in G1 phase.     

Specific monoclonal antibody productivity and the cell cycle-comparisons of batch,continuous and perfusion cultures

A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G₁ phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of G₁ phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.     

New approach to use ethidium bromide monoazide as an analytical tool

Aims: Ethidium bromide monoazide (EMA) has been determined to cause delay in DNA amplification from dead bacteria at real‐time PCR. However, there is concern that the increasing EMA concentration to suppress amplification from high number of dead bacteria also affects live bacteria. The aim is to disclose a novel application of EMA for food hygienic test. Methods and Results: We performed a low‐dose double EMA treatment. Live or heat‐dead Enterobacter sakazakii (reclassified as Cronobacter spp.) in 10% powdered infant formula (PIF) solution was subjected to a treatment with 20 μg ml^?1 of EMA followed by a treatment with 10 μg ml^?1 of EMA without washing, and direct real‐time PCR. We observed that DNA amplification from 10⁷ cells ml^?1 of dead Ent. sakazakii was completely suppressed within 50 cycles of PCR, whereas 10²–10³ cells ml^?1 of viable cells could be detected. When a 3‐h enrichment step in liquid medium was included after the first EMA treatment, live Ent. sakazakii could be detected at initial levels of 10⁰–10² cells ml^?1. We compared the low‐dose double‐treated EMA‐PCR with the culture method using 80 samples of PIF, and completely correlative results were obtained for both methods. Conclusions: We concluded that the newly developed low‐dose double‐treated EMA‐PCR is a very effective tool for live Ent. sakazakii detection in PIF. Significance and Impact of the Study: We focused on the specific nature of photoreactive compound that residual EMA is cancelled by irradiation. We were successful in treating bacteria with EMA in gradient concentration to increase live and dead distinction ability.     

Rapid detection of viable bacteria by nested polymerase chain reaction via long DNA amplification after ethidium monoazide treatment

In assays to determine whether viable cells of Enterobacteriaceae are present in pasteurized milk, the typical ethidium monoazide (EMA) polymerase chain reaction (PCR) targets a short stretch of DNA. This process often triggers false-positive results owing to the high level of dead cells of Enterobacteriaceae that had initially contaminated the sample. We have developed a novel, direct, real-time PCR that does not require DNA isolation (DQ-PCR) to detect low levels of cells of Enterobacteriaceae regardless of live and dead cells first. We confirmed that the DQ-PCR targeting a long DNA (the 16S ribosomal RNA [rRNA] gene, amplified length of 1514 bp) following EMA treatment is a promising tool to detect live bacteria of all genera owing to the complete suppression of background signal from high levels of dead bacteria in pasteurized milk. However, when identifying viable bacteria in pasteurized milk, commercial PCR primers designed for detecting long stretches of DNA are generally not available. Thus, we treated samples with EMA and then carried out an initial round of PCR of a long stretch of DNA (16S gene, 1514 bp). We then performed another round of PCR, a novel nested PCR to generate short products using commercial primers. This procedure resulted in the rapid detection of low levels of viable cells of Enterobacteriaceae.     

Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability.

BACKGROUND: Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of subpopulations to stimuli in mixed cell preparations; however, in low-viability cell preparations, dead cells interfere with accurate flow cytometric data analysis because of nonspecific binding of antibodies and altered DNA-staining profiles. Light scatter differences between nonviable and viable cells are unreliable, particularly after the cell permeabilization step that is necessary for DNA staining. We developed a method for identification of nonviable cells by fluorescence in cell preparations that are stained simultaneously for cell surface or intracellular immunofluorescence and DNA content. MATERIALS AND METHODS: Nonviable cells that have lost membrane integrity are identified by uptake of 7-amino-actinomycin D (7-AAD). Transfer of 7-AAD from stained nonviable cells to unstained viable cells after permeabilization is prevented by blocking DNA binding with nonfluorescent actinomycin D (AD). Pyronin Y(G) (PY) is used for DNA staining because the orange spectral emission of PY can be separated from the green fluorescein isothiocyanate (FITC) emission and the red emission of 7-AAD, respectively. RESULTS: Application of the method to the analysis of the T-cell leukemia cell line Molt-4f and of cultured human peripheral blood mononuclear cells is presented. In both cell preparations, 7-AAD staining permitted reliable dead cell exclusion. Live, 7-AAD-negative Molt-4f cells showed higher expression levels of cell surface CD4 and of intracellular CD3, showed a higher proportion of cells in the G1 phase of the cell cycle, and showed a lower coefficient of variation of the G1 peak compared with data obtained from all the cells in the preparation. Live, CD8+ lymphocytes from OKT3-stimulated cultures of human peripheral blood mononuclear cells showed a specific proliferative response as measured by DNA content analysis. CONCLUSIONS: The results show that cells stained with FITC-labeled antibodies can be analyzed by single-laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exclusion of dead cells from the analysis of cell preparations with low viability to obtain reliable data on immunofluorescence and cell-cycle distributions.     

Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells

The differentiation between live and dead bacterial cells presents an important challenge in many microbiological applications. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based detection methods cannot differentiate whether positive signals originate from live or dead bacterial targets. We present here a novel chemical, propidium monoazide (PMA), that (like propidium iodide) is highly selective in penetrating only into 'dead' bacterial cells with compromised membrane integrity but not into live cells with intact cell membranes/cell walls. Upon intercalation in the DNA of dead cells, the photo-inducible azide group allows PMA to be covalently cross-linked by exposure to bright light. This process renders the DNA insoluble and results in its loss during subsequent genomic DNA extraction. Subjecting a bacterial population comprised of both live and dead cells to PMA treatment thus results in selective removal of DNA from dead cells. We provide evidence that this chemical can be applied to a wide range of species across the bacterial kingdom presenting a major advantage over ethidium monoazide (EMA). The general application of EMA is hampered by the fact that the chemical can also penetrate live cells of some bacterial species. Transport pumps actively export EMA out of metabolically active cells, but the remaining EMA level can lead to substantial loss of DNA. The higher charge of PMA might be the reason for the higher impermeability through intact cell membranes, thus avoiding DNA loss.     

Discrimination of live, anti-tuberculosis agent-injured, and dead Mycobacterium tuberculosis using flow cytometry

Flow cytometry (FCM) using propidium iodide (PI)/bis-oxonol (BOX) staining can distinguish live, dead, and sublethally injured Escherichia coli by detecting intact vs. nonintact membranes (PI) and membrane potential (BOX). However, live bacteria, especially Mycobacterium tuberculosis , are not likely to be successfully discriminated from injured bacterium by FCM when utilizing the live/dead staining agents currently on the market. As injured cell membranes have integrity like that of live cells and are regarded as such by FCM, the distinction between live and injured cells has depended on the culture method, where injured bacteria cannot grow in general. We have previously shown that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro . In this study, we found that the chromosomal DNA of antibiotic-injured, but not live, M. tuberculosis could be cleaved within 2 h by EMA, and that the resultant decrease in the spaces of DNA base pairs could greatly inhibit the intercalation of SYTO9 in FCM. The percentage value of SYTO9⁺/PI⁻ quadrant from antibiotic-injured M. tuberculosis after EMA treatment decreased by at least 80%, compared with that before EMA, but such a phenomenon did not take place in live cells. FCM (SYTO9/PI) following EMA treatment is a very rapid, simple, and effective method for discriminating live, antibiotic-injured, and dead M. tuberculosis without culture.     

Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of Enterobacteriaceae

The polymerase chain reaction (PCR) can confirm the presence of bacteria, but it is unable to differentiate between live and dead bacteria. Although ethidium monoazide (EMA)- and propidium monoazide (PMA)-based PCR have been evaluated, a quantity of ≥ 10(3)cells/ml dead cells produces a false-positive reading at 40 to 50 cycles (K. Rudi et al., Appl. Environ. Microbiol. 71 (2005) 1018-1024). After confirming the precision of real-time PCR of a long DNA target (16S or 23S ribosomal RNA [rRNA] gene, 1490 or 2840 bp), we evaluated the degree of suppression of an EMA treatment on the 16S/23S PCR using various amplification lengths (110-2840 bp) with heat-killed cells of Enterobacteriaceae (e.g., Salmonella enteritidis). We found that the inhibition rate was proportional to the PCR amplification length; short DNA (110 bp) amplification slightly delayed the threshold cycle (C(T)) of heat-killed cells of Enterobacteriaceae when compared with no EMA treatment. Regardless of the amplification length, the C(T) delay using live cells of Enterobacteriaceae with EMA was negligible. Thus, our real-time PCR of a long DNA (16S or 23S) template following EMA treatment is a rapid viable bacterial assay, which can potentially target all genera, for testing pasteurized milk that may have originally been contaminated with high levels of dead bacteria.     

Differentiation of genes extracted from non-viable versus viable micro-organisms in environmental samples using ethidium monoazide bromide

Differentiation of DNA derived from viable or non-viable microorganisms within mixed microbial communities continues to be one of the greatest challenges in molecular studies of environmental samples. A novel method developed for microbial food pathogens is tested here on environmental samples. This technique involves the use of ethidium monoazide bromide (EMA) for the distinction of live/dead cells. In non-viable cells EMA intercalates into the DNA which prevents amplification by PCR. We adapted and evaluated the EMA technique for soil, elemental sulfur and river biofilm samples. Quantitative PCR determined that EMA suppressed 99.99% of E. coli LKI gfp+ signal in non-viable cultures and 100.00% when the cultures were added to soil samples. The same technique was also successful at suppressing DNA amplification from spiked non-viable cells in elemental sulfur samples by 100.00%, but not in three Saskatchewan River biofilms. In sub Antarctic soil, EMA-Q-PCR was used to detect the prevalence of a functional gene, amoA, and this was closely correlated to nitrification activity measurements. The ability of EMA to differentiate between viable and non-viable populations in soil was confirmed by the similarity of the 16S rRNA denaturing-gradient-gel electrophoresis DNA fingerprint of EMA treated soil and the 16S rRNA cDNA fingerprint of non-EMA treated soil. The EMA technique effectively suppressed amplification of non-viable spiked controls, closely mirrored activity assays and yielded community composition profiles similar to rRNA techniques. The use of EMA in soil effectively suppressed amplification of non-viable organism DNA, however it was not effective in biofilm samples and EMA partially inhibited amplification of viable organism DNA in elemental sulfur samples.     

Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide

The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into "dead" cells with compromised cell membrane integrity. Subsequent photoinduced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.     

Flow cytometry for rapid assessment of viability after exposure to a quaternary ammonium compound

The use of flow cytometry to rapidly assess the viability of Pseudomonas spp. and Staphylococcus spp. after exposure to a quaternary ammonium compound (QAC) was investigated using rhodamine 123 (Rh 123), Stain A (LIVE Stain) accumulating in viable but not in dead cells (Live/Dead Bac light bacterial viability kit, Molecular Probes Inc., Eugene, OR, USA), and Sytox green (Molecular Probes) accumulating in dead but not viable cells. Staining conditions were optimized for each stain. The fraction of viable cells after exposure to benzalkonium chloride was determined by using the three staining techniques and colony counts on agar medium. For all Staphylococcus spp. tested there was a high correlation between the methods based on flow cytometry and colony counts irrespective of which stain was used. Although viable, all Pseudomonas spp. tested accumulated Rh 123 poorly and about 30% failed to accumulate LIVE stain as well. However, the correlation between colony counts and Sytox green labelling of Pseudomonas spp. was high. Our results indicate that flow cytometry together with live or dead cell labelling can be used to study the bactericidal effect of QACs. The methods based on LIVE stain and Sytox green were simpler and less time consuming than Rh 123 labelling. Only Sytox green could be used with all strains of Staphylococcvs and Pseudomonas tested.     

Effect of endogenous proteins on growth and antibody productivity in hybridoma batch cultures

It has been shown that some B-cell hybridomas secrete autocrine factorsin vitro which can influence cell metabolic processes. Rather than screen specifically for suspected cytokines, that may or may not affect our cell line, we have examined the lumped effects of intracellular and secreted factors on cell proliferation and monoclonal productivity in hybridoma batch cultures. Firstly, supplements of total soluble intracellular proteins combined with other intracellular metabolites were found to both decrease the specific growth rate and increase the antibody production rate at higher concentrations in batch culture. This is an important consideration in high cell density cultures, such as perfusion systems, where a reduction of growth by the presence of intracellular factors may be compensated by an increase in MAb production. In addition, flow cytometry data revealed that the average cell cycle G₁ phase fraction was unaffected by the variation in the maximum specific growth rates during the exponential growth phase, caused by the addition of intracellular factors; this suggests that higher MAb productivity at lower growth rates are not a result of cell arrest in the G₁ phase. Secondly, secreted extracellular proteins larger than 10,000 Daltons, which were concentrated from spent culture supernatant, were shown to have no significant effect on growth and specific MAb productivity when supplemented to batch culture at levels twice that encountered late in normal batch culture. This indicates that endogenous secreted cytokines, if at all present, do not play a major autocrine role for this cell line.Abbreviations FBS fetal bovine serum - MAb monoclonal antibody - MWCO molecular weight cut off - SDS-PAGE sodium dodecyl-sulphate-polyacrylamide gel electrophoresis - k _d exponential phase death rate, h^–1 - q _MAb exponential phase specific monoclonal antibody productivity, pg/(cell·h) - t time, h - X _d dead cell density, cells/mL - X _v viable cell density, cells/mL - specific growth rate, h^–1 - _{max app} apparent maximum specific growth rate, h^–1 - _max maximum specific growth rate, h^–1 _max = _{max app + Kd}      

Distinct volume distribution of viable and non-viable hybridoma cells: A flow cytometric study

Light scattering properties of hybridoma cells were examined with flow cytometry. Viable and dead cells form two distinct populations. The distribution of the two populations changes during a batch culture. the concentration of dead cells measured by flow cytometry correlates well to that measured by hemacytometer. The distribution based on small-angle light scattering is similar to the distribution based on volume as measured by Elzone particle counter. It thus appears that viable cells form the population with a larger mean cell volume. The results also indicate that the volume of viable cells decreases during the cultivation while that of dead cells remains relatively constant.     

An advanced PCR method for the specific detection of viable total coliform bacteria in pasteurized milk

Pasteurized milk is a complex food that contains various inhibitors of polymerase chain reaction (PCR) and may contain a large number of dead bacteria, depending on the milking conditions and environment. Ethidium monoazide bromide (EMA)-PCR is occasionally used to distinguish between viable and dead bacteria in foods other than pasteurized milk. EMA is a DNA-intercalating dye that selectively permeates the compromised cell membranes of dead bacteria and cleaves DNA. Usually, EMA-PCR techniques reduce the detection of dead bacteria by up to 3.5 logs compared with techniques that do not use EMA. However, this difference may still be insufficient to suppress the amplification of DNA from dead Gram-negative bacteria (e.g., total coliform bacteria) if they are present in pasteurized milk in large numbers. Thus, false positives may result. We developed a new method that uses real-time PCR targeting of a long DNA template (16S-23S rRNA gene, principally 2,451?bp) following EMA treatment to completely suppress the amplification of DNA of up to 7?logs (10(7)?cells) of dead total coliforms. Furthermore, we found that a low dose of proteinase K (25?U/ml) removed PCR inhibitors and simultaneously increased the signal from viable coliform bacteria. In conclusion, our simple protocol specifically detects viable total coliforms in pasteurized milk at an initial count of ≥1?colony forming unit (CFU)/2.22?ml within 7.5?h of total testing time. This detection limit for viable cells complies with the requirements for the analysis of total coliforms in pasteurized milk set by the Japanese Sanitation Act (which specifies <1?CFU/2.22?ml).     

Relationship between hybridoma growth and monoclonal antibody production.

Factors affecting cell growth and antibody production in a mouse hybridoma were investigated. Antibody was produced during the growth and decline phases of a batch culture with an increase in the specific rate of antibody production during the decline phase. The specific rate of antibody production was also increased in cells arrested by 2 mM thymidine, suggesting that cell proliferation and antibody production can be uncoupled. Reduced serum concentrations resulted in lower cell growth rates but increased antibody production rates. However, this trend was reversed in hybridomas which had been arrested by thymidine, since the highest antibody production rate was associated with high serum concentrations. Likewise, in proliferating cells, the optimum pH for antibody production (pH 6.8) was lower than the optimum pH for cell growth (pH 7.2), whereas in thymidine-blocked cells, the highest antibody production rate was at pH 7.2. High antibody production rates and product yields were also associated with low growth rates in continuous cultures. The possibility that antibody was under cell cycle control was investigated in synchronized hybridoma cultures. Antibody production occurred during G1 and G2 with a decline in the M phase and evidence of a further decline in the S phase. Thus antibody production was not restricted to the G1 and S phase in this hybridoma.     

Control of cellular proliferation in HeLa-S3 suspension cultures. Characterization of cultures utilizing acridine orange staining procedures

Growth control is investigated in detail in fed and unfed HeLa-S3 suspension cultures. Two-step acridine orange staining and flow cytometric analysis indicated declines in cellular red fluorescence (proportional to RNA content) of 40-50% between exponential and plateau phase in both culture types. Cellular green fluorescence (DNA content) assessed simultaneously indicates an increment of cells with Gi-DNA content in plateau phase in the unfed cultures, while fed cultures show a brief increment in G1-phase cells in the transition phase followed by a recovery in plateau phase to a value similar to that of exponential cultures. Temporal declines in the 3H-thymidine pulse-labeling index are observed in both culture systems. These data along with the flow cytometry data indicate a distinct G1-arrest in the unfed plateau cultures and suggest a random arrest of cells about the cell cycle in fed plateau cultures. Acidic acridine orange staining and flow cytometric analysis furthermore indicate the occurrence of a quiescent population comprising approximately 345 of the total cells and consisting of both dead and viable cells in plateau phase unfed cultures. In contrast, fed plateau cultures show approximately 14% quiescent, mostly dead cells. Also, both culture systems show temporal declines in the clonogenic index and a longer cell-cycle transit time in plateau phase relative to exponential phase. These findings confirm earlier work which indicates that the environment has a profound influence on the mode of growth control for mammalian cells in vitro.     

Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR

Ethidium bromide monoazide (EMA) was utilized to selectively allow the real-time PCR (RT-PCR) amplification of a targeted DNA sequence in viable but not dead cells of Vibrio vulnificus. The optimized light exposure time to achieve cross-linking of DNA by the EMA in dead cells and to photolyse the free EMA in solution was at least 15 min. The use of 3.0 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable cells of V. vulnificus. The minimum amount of EMA to completely inhibit the RT-PCR amplification of DNA derived from heat-killed cells was 2.5 microg/ml. Amplification of DNA from dead cells in a mixture with viable cells was successfully inhibited by 2.5 microg/ml of EMA, whereas the DNA from viable cells present was successfully amplified by RT-PCR.