scholarly journals Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death

2008 ◽  
Vol 74 (6) ◽  
pp. 1767-1779 ◽  
Author(s):  
Tania Falcioni ◽  
Stefano Papa ◽  
Josep M. Gasol
Keyword(s):  
Nucleic Acid ◽  
Extreme Environments ◽  
Membrane Integrity ◽  
High Capacity ◽  
Live Cells ◽  
Leucine Incorporation ◽  
Double Staining ◽  
Tetrazolium Chloride ◽  
Physiological Diversity ◽  
Staining Protocol

ABSTRACT Since heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Grégori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662-4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability: active respiration by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20 μg ml−1 of propidium iodide, simultaneous to a 10× concentration of Sybr green I, are best for detecting two separated populations of “live” (green cells) and “dead” (red cells) organisms. During exposure to heat and UVC, we observed that the number of live cells declined concurrently with that of actively respiring cells (CTC positive) and with total leucine incorporation. In seawater mesocosms, the NADS protocol allowed detection of bacterioplankton starvation-related death and flagellate predation. The protocol was also tested in deep profiles in the northwest Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton.

scholarly journals Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining

2001 ◽  
Vol 67 (10) ◽  
pp. 4662-4670 ◽  
Author(s):  
Gérald Grégori ◽  
Sandra Citterio ◽  
Alessandra Ghiani ◽  
Massimo Labra ◽  
Sergio Sgorbati ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Large Fraction ◽  
Membrane Integrity ◽  
Molecular Probes ◽  
Natural Environments ◽  
Double Staining ◽  
Marine Waters ◽  
Intact Membrane ◽  
A Cell

ABSTRACT The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214–218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.

scholarly journals Nucleic‐Acid Structures as Intracellular Probes for Live Cells

2019 ◽  
Vol 32 (13) ◽  
pp. 1901743 ◽  
Author(s):  
Devleena Samanta ◽  
Sasha B. Ebrahimi ◽  
Chad A. Mirkin
Keyword(s):  
Nucleic Acid ◽  
Live Cells ◽  
Nucleic Acid Structures

scholarly journals Evaluation of membrane integrity of frozen/thawed deer spermatozoa: Short communication

2001 ◽  
Vol 49 (2) ◽  
pp. 223-227 ◽  
Author(s):  
Sz. Nagy ◽  
A. Kovács ◽  
T. Zubor ◽  
Z. Zomborszky ◽  
J. Tóth ◽  
...  
Keyword(s):  
Cervus Elaphus ◽  
Red Deer ◽  
Short Communication ◽  
Vas Deferens ◽  
Semen Quality ◽  
Membrane Integrity ◽  
Fallow Deer ◽  
Live Cells ◽  
Dama Dama ◽  
Cauda Epididymidis

A simultaneous live/dead and acrosome staining, originally described for domestic mammals, was successfully applied on red deer (Cervus elaphus) and fallow deer (Dama dama) spermatozoa collected from the cauda epididymidis and vas deferens of shot stags. The staining is simple enough for routine application. Seven classes of spermatozoa were distinguished in the smears of frozen/thawed semen samples. Morphology, including cytoplasmic droplets, was evaluated as well. Percentage of live cells with intact acrosomes and with no other morphological aberrations might be a practical index of semen quality.

scholarly journals Silica@zirconia Core@shell Nanoparticles for Nucleic Acid Building Block Sorption

Nanomaterials ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2166
Author(s):  
Livia Naszályi Nagy ◽  
Evert Dhaene ◽  
Matthias Van Zele ◽  
Judith Mihály ◽  
Szilvia Klébert ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Colloidal Stability ◽  
Sol Gel ◽  
High Capacity ◽  
Building Blocks ◽  
Shell Nanoparticles ◽  
Passive Targeting ◽  
Acid Type ◽  
Ligand Adsorption ◽  
Core Shell Nanoparticles

The development of delivery systems for the immobilization of nucleic acid cargo molecules is of prime importance due to the need for safe administration of DNA or RNA type of antigens and adjuvants in vaccines. Nanoparticles (NP) in the size range of 20–200 nm have attractive properties as vaccine carriers because they achieve passive targeting of immune cells and can enhance the immune response of a weakly immunogenic antigen via their size. We prepared high capacity 50 nm diameter silica@zirconia NPs with monoclinic/cubic zirconia shell by a green, cheap and up-scalable sol–gel method. We studied the behavior of the particles upon water dialysis and found that the ageing of the zirconia shell is a major determinant of the colloidal stability after transfer into the water due to physisorption of the zirconia starting material on the surface. We determined the optimum conditions for adsorption of DNA building blocks, deoxynucleoside monophosphates (dNMP), the colloidal stability of the resulting NPs and its time dependence. The ligand adsorption was favored by acidic pH, while colloidal stability required neutral-alkaline pH; thus, the optimal pH for the preparation of nucleic acid-modified particles is between 7.0–7.5. The developed silica@zirconia NPs bind as high as 207 mg dNMPs on 1 g of nanocarrier at neutral-physiological pH while maintaining good colloidal stability. We studied the influence of biological buffers and found that while phosphate buffers decrease the loading dramatically, other commonly used buffers, such as HEPES, are compatible with the nanoplatform. We propose the prepared silica@zirconia NPs as promising carriers for nucleic acid-type drug cargos.

Development of a Mechanism-Based, DNA Staining Protocol Using SYTOX Orange Nucleic Acid Stain and DNA Fragment Sizing Flow Cytometry

2000 ◽  
Vol 286 (1) ◽  
pp. 138-148 ◽  
Author(s):  
Xiaomei Yan ◽  
Robert C. Habbersett ◽  
Julia M. Cordek ◽  
John P. Nolan ◽  
Thomas M. Yoshida ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Dna Staining ◽  
Staining Protocol ◽  
Dna Fragment ◽  
Nucleic Acid Stain

scholarly journals Activity and Phylogenetic Diversity of Bacterial Cells with High and Low Nucleic Acid Content and Electron Transport System Activity in an Upwelling Ecosystem

2005 ◽  
Vol 71 (12) ◽  
pp. 7737-7749 ◽  
Author(s):  
K. Longnecker ◽  
B. F. Sherr ◽  
E. B. Sherr
Keyword(s):  
Nucleic Acid ◽  
Electron Transport ◽  
Transport System ◽  
Acid Content ◽  
Phylogenetic Diversity ◽  
Electron Transport System ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Nucleic Acid Content ◽  
Leucine Incorporation

ABSTRACT We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.

Plasma membrane changes during the liquid storage of boar spermatozoa: A comparison of methods

2010 ◽  
Vol 58 (1) ◽  
pp. 105-116 ◽  
Author(s):  
Dariusz Gączarzewicz ◽  
Małgorzata Piasecka ◽  
Jan Udała ◽  
Barbara Błaszczyk ◽  
Tomasz Stankiewicz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Live Cells ◽  
Lower Percentage ◽  
Loss Of Function ◽  
Plasma Membrane Integrity ◽  
Sperm Plasma Membrane ◽  
Semen Preservation

Studies were performed on boar semen routinely used at the local artificial insemination (AI) centre. The semen was stored in a Safe Cell Plus commercial extender at 17 °C for nine days. The aim of our research was focused on changes in sperm plasma membrane integrity. The integrity of the sperm plasma membrane and acrosome as well as sperm motility decreased after dilution and during storage of the semen. The highest percentage of live sperm was identified by the eosin-nigrosin method, a lower percentage by the SYBR-14/PI test, and the lowest percentage of live cells was discovered by the hypoosmotic swelling (HOS) test (P < 0.01). There were significant differences between the results of staining methods and sperm motility (P < 0.01). No significant differences were found between the HOS test results and sperm motility. The plasma membrane integrity parameters positively correlated (P < 0.001) with each other and with sperm motility but negatively with aspartate aminotransferase activity. Our findings confirmed that the boar sperm aging changes, which increased during liquid semen preservation, were connected with the loss of function and integrity of the sperm plasma membrane. The employed complementary tests are comprehensive indicators of sperm membrane integrity during long-term semen preservation, and they can help establish the actual number of ‘healthy’ cells. The assays may be used in AI laboratories and should be incorporated into the routine of semen analysis.

scholarly journals Immunocytochemical Phenotyping of Disseminated Tumor Cells in Bone Marrow by uPA Receptor and CK18: Investigation of Sensitivity and Specificity of an Immunogold/Alkaline Phosphatase Double Staining Protocol

1997 ◽  
Vol 45 (2) ◽  
pp. 203-212 ◽  
Author(s):  
Heike Allgayer ◽  
Markus Maria Heiss ◽  
Rainer Riesenberg ◽  
Rudolf Babic ◽  
Karl Walter Jauch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Staining Method ◽  
Bone Marrow Cells ◽  
Double Staining ◽  
Staining Protocol ◽  
Double Staining Method ◽  
Marrow Cells

Phenotyping of cytokeratin (CK) 18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell controls exhibited black uPA-R staining in 15–80% of cases and red CK18 staining in almost 100% of tumor cells. Isotype- and cross-matched controls were completely negative. Bone marrow from healthy donors was always CK18-negative. Reproducibility of CK18-positive cell detection was estimated in a series of specimens from 61 gastric cancer patients comparatively stained with the single alkaline phosphatase-anti-alkaline phosphatase (APAAP) and our double staining method (106 bone marrow cells/patient). In four cases, double staining could not reproduce CK18-positive cells. In 34 cases it revealed fewer or equal numbers, and in 23 cases more CK18-positive cells than the APAAP method. Overall quantitative analysis of detected cell numbers (838 in APAAP, range 1–280 in 106; double staining 808, range 0–253) demonstrated relative reproducibility of APAAP results by double staining of 97%. Correlation of results between both methods was significant ( p<0.001, linear regression). Sensitivity of double staining tested in logarithmic tumor cell dilutions was one CK18-positive cell in 300,000. Specific uPA-R staining was seen on CK18-positive cells in bone marrow from 29 of 61 patients, and also on single surrounding bone marrow cells. To test the specificity of this staining, bone marrow cytospins from 10 patients without tumor disease were stained for uPA-R with the APAAP method. uPA-R expression was confirmed in all 10 cases, with a mean of 6.5% uPA-R-positive cells in 1000 bone marrow cells (SEM 1.2%). These results suggest that our double staining protocol is a sensitive, reproducible, and specific method for routine uPA-R phenotyping of disseminated CK18-positive cells in bone marrow of carcinoma patients.

scholarly journals Survival and Resuscitation of Ten Strains of Campylobacter jejuni and Campylobacter coli under Acid Conditions

2003 ◽  
Vol 69 (1) ◽  
pp. 711-714 ◽  
Author(s):  
P. Chaveerach ◽  
A. A. H. M. ter Huurne ◽  
L. J. A. Lipman ◽  
F. van Knapen
Keyword(s):  
Formic Acid ◽  
Campylobacter Jejuni ◽  
Yolk Sac ◽  
Campylobacter Coli ◽  
Double Staining ◽  
Tetrazolium Chloride ◽  
Viable But Nonculturable ◽  
Viable Cells ◽  
Over Time

ABSTRACT The culturability of 10 strains of Campylobacter jejuni and Campylobacter coli was studied after the bacteria were exposed to acid conditions for various periods of time. Campylobacter cells could not survive 2 h under acid conditions (formic acid at pH 4). The 10 Campylobacter strains could not be recovered, even when enrichment media were used. Viable cells, however, could be detected by a double-staining (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]-4′,6′-diamidino-2-phenylindole [DAPI]) technique, demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; the number of VBNC forms decreased over time. Moreover, some VBNC forms of Campylobacter could be successfully resuscitated in specific-free-pathogen fertilized eggs via two routes, amniotic and yolk sac injecting.

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