Variations of bacterial-specific activity with cell size and nucleic acid content assessed by flow cytometry

ABSTRACT The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

Download Full-text

Using flow cytometry and light-induced fluorescence technique to characterizethe variability and characteristics of bioaerosols in springtime at Metro Atlanta, Georgia

10.5194/acp-2018-1073 ◽

2018 ◽

Author(s):

Arnaldo Negron ◽

Natasha DeLeon-Rodriguez ◽

Samantha M. Waters ◽

Luke D. Ziemba ◽

Bruce Anderson ◽

...

Keyword(s):

Flow Cytometry ◽

Nucleic Acid ◽

Acid Content ◽

Fungal Spores ◽

Epifluorescence Microscopy ◽

Nucleic Acid Content ◽

Rain Event ◽

Bacterial Cells ◽

Sampling System ◽

Spores And Pollen

Abstract. The abundance and speciation of primary biological aerosol particles (PBAP) is important for understanding their impacts on human health, cloud formation and ecosystems. Towards this, we have developed a protocol for quantifying PBAP collected from large volumes of air with a portable wet-walled cyclone bioaerosol sampler. A flow cytometry (FCM) protocol was then developed to quantify and characterize the PBAP populations from the sampler, which were confirmed against epifluorescence microscopy. The sampling system and FCM analysis were used to study PBAP in Atlanta, GA over a two-month period and showed clearly defined populations of DNA-containing particles: Low Nucleic Acid-content particles (bioLNA), High Nucleic Acid-content particles (HNA) being fungal spores and pollen. We find that daily-average springtime PBAP concentration (1 to 5 μm diameter) ranged between 1.4 × 104 and 1.1 × 105 m−3. The BioLNA population dominated PBAP during dry days (72 ± 18 %); HNA dominated the PBAP during humid days and following rain events, where HNA (e.g., wet-ejected fungal spores) comprised up to 92 % of the PBAP number. Concurrent measurements with a Wideband Integrated Bioaerosol Sensor (WIBS-4A) showed that FBAP and total FCM counts are similar; HNA (from FCM) significantly correlated with ABC type FBAP concentrations throughout the sampling period (and for the same particle size range, 1–5 μm diameter). However, the FCM bioLNA population, possibly containing bacterial cells, did not correlate to any FBAP type. The lack of correlation of any WIBS FBAP type with the bioLNA suggest bacterial cells may be more difficult to detect with autofluorescence than previously thought. Ιdentification of bacterial cells even in the FCM (bioLNA population) is challenging, given that the fluorescence level of stained cells at times may be comparable to that seen from abiotic particles. HNA and ABC displayed highest concentration on a humid and warm day after a rain event (4/14), suggesting that both populations correspond to wet-ejected fungal spores. Overall, information from both instruments combined reveals a highly dynamic airborne bioaerosol community over Atlanta, with a considerable presence of fungal spores during humid days, and a bioLNA population dominating bioaerosol community during dry days.

Download Full-text

Using flow cytometry and light-induced fluorescence to characterize the variability and characteristics of bioaerosols in springtime in Metro Atlanta, Georgia

Atmospheric Chemistry and Physics ◽

10.5194/acp-20-1817-2020 ◽

2020 ◽

Vol 20 (3) ◽

pp. 1817-1838 ◽

Cited By ~ 1

Author(s):

Arnaldo Negron ◽

Natasha DeLeon-Rodriguez ◽

Samantha M. Waters ◽

Luke D. Ziemba ◽

Bruce Anderson ◽

...

Keyword(s):

Flow Cytometry ◽

Nucleic Acid ◽

Acid Content ◽

Aerosol Particles ◽

Fungal Spores ◽

Epifluorescence Microscopy ◽

Nucleic Acid Content ◽

Rain Event ◽

Bacterial Cells ◽

Biological Aerosol Particles

Abstract. The abundance and speciation of primary biological aerosol particles (PBAP) is important for understanding their impacts on human health, cloud formation, and ecosystems. Towards this, we have developed a protocol for quantifying PBAP collected from large volumes of air with a portable wet-walled cyclone bioaerosol sampler. A flow cytometry (FCM) protocol was then developed to quantify and characterize the PBAP populations from the sampler, which were confirmed against epifluorescence microscopy. The sampling system and FCM analysis were used to study PBAP in Atlanta, GA, over a 2-month period and showed clearly defined populations of nucleic-acid-containing particles: low nucleic acid-content particles above threshold (LNA-AT) and high nucleic acid-content particles (HNA) likely containing wet-ejected fungal spores and pollen. We find that the daily-average springtime PBAP concentration (1 to 5 µm diameter) ranged between 1.4×104 and 1.1×105 m−3. The LNA-AT population dominated PBAP during dry days (72±18 %); HNA dominated the PBAP during humid days and following rain events, where HNA comprised up to 92 % of the PBAP number. Concurrent measurements with a Wideband Integrated Bioaerosol Sensor (WIBS-4A) showed that fluorescent biological aerosol particles (FBAP) and total FCM counts are similar; HNA (from FCM) moderately correlated with ABC-type FBAP concentrations throughout the sampling period (and for the same particle size range, 1–5 µm diameter). However, the FCM LNA-AT population, possibly containing bacterial cells, did not correlate with any FBAP type. The lack of correlation of any WIBS FBAP type with the LNA-AT suggests that airborne bacterial cells may be more difficult to unambiguously detect with autofluorescence than currently thought. Identification of bacterial cells even in the FCM (LNA-AT population) is challenging, given that the fluorescence level of stained cells at times may be comparable to that seen from abiotic particles. HNA and ABC displayed the highest concentration on a humid and warm day after a rain event (14 April 2015), suggesting that both populations correspond to wet-ejected fungal spores. Overall, information from both instruments combined reveals a highly dynamic airborne bioaerosol community over Atlanta, with a considerable presence of fungal spores during humid days and an LNA-AT population dominating the bioaerosol community during dry days.

Download Full-text

Characterization of the Population of the Sulfur-Oxidizing Symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by Single-Cell Analyses

Applied and Environmental Microbiology ◽

10.1128/aem.01683-06 ◽

2007 ◽

Vol 73 (7) ◽

pp. 2101-2109 ◽

Cited By ~ 35

Author(s):

Audrey Caro ◽

Olivier Gros ◽

Patrice Got ◽

Rutger De Wit ◽

Marc Troussellier

Keyword(s):

Nucleic Acid ◽

Single Cell ◽

Cell Size ◽

Acid Content ◽

Size Analysis ◽

Nucleic Acid Content ◽

Tropical Environments ◽

Multiple Copies ◽

Sulfur Oxidizing ◽

Light Side

ABSTRACT We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 μm in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host.

Download Full-text

THE RIBONUCLEIC ACID AND DEOXYRIBONUCLEIC ACID CONTENT OF MONONUCLEAR LEUKOCYTES OF DIFFERENT SIZES STUDIED BY ULTRAVIOLET CYTOPHOTOMETRY

Journal of Histochemistry & Cytochemistry ◽

10.1177/21.7.628 ◽

1973 ◽

Vol 21 (7) ◽

pp. 628-633 ◽

Cited By ~ 1

Author(s):

ALFREDO MARIANO GARCIA ◽

PATRICIA A. N. SULLIVAN

Keyword(s):

Nucleic Acid ◽

Cell Size ◽

Dna Content ◽

Random Error ◽

Acid Content ◽

Deoxyribonucleic Acid ◽

Nucleic Acid Content ◽

Mononuclear Leukocytes ◽

Total Nucleic Acid ◽

Dna And Rna

Rat mononuclears (lymphocytes and monocytes) were studied for total nucleic acid content by means of ultraviolet cytophotometry. Another set was treated with ribonuclease, and deoxyribonucleic acid (DNA) was measured using the same technique. It was found that total nucleic acid content (DNA and RNA) increases linearly with cell size from about 20 units in lymphocytes having 5 µ in diameter up to around 30 units in cells having 12-14 µ in diameter; this is to say, an almost 50% increase for a 6-7-fold enlargement. After ribonuclease treatment, however, the value of the integrated extinction (DNA) tends to remain constant for different cell sizes. A 650% variation in area is accompanied by a DNA change of less than 6%. The differences between treated and nontreated cells are nonsignificant for populations having up to 7.0-7.5 µ in diameter, which implies that small lymphocytes either have a negligible amount of RNA or that the instrument is not sensitive enough to detect it (less than 7% of the DNA content, this figure being the random error of our technique). These differences become highly significant for mononuclears having 8 µ or more in diameter. Therefore, while DNA tends to be constant and independent from cell size, RNA content tends to be harmoniously inconstant, since it is correlated with cell (and nuclear) size and degree of chromatin diffusion.

Download Full-text

Characteristics and Dynamics of Bacterial Populations during Postantibiotic Effect Determined by Flow Cytometry

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.5.1005 ◽

1998 ◽

Vol 42 (5) ◽

pp. 1005-1011 ◽

Cited By ~ 24

Author(s):

Magnús Gottfredsson ◽

Helga Erlendsdóttir ◽

Ásbjörn Sigfússon ◽

Sigurdur Gudmundsson

Keyword(s):

Flow Cytometry ◽

Nucleic Acid ◽

Fluorescence Microscopy ◽

Acid Content ◽

Quantitative Information ◽

Nucleic Acid Content ◽

Dependent Manner ◽

Postantibiotic Effect ◽

E Coli ◽

Drug Removal

ABSTRACT Changes in bacterial ultrastructure after antibiotic exposure and during the postantibiotic effect (PAE) have been demonstrated by electron microscopy (EM). However, EM is qualitative and subject to individual interpretation. In contrast, flow cytometry gives qualitative and quantitative information. The sizes and nucleic acid contents of Escherichia coli and Pseudomonas aeruginosa were studied during antimicrobial exposure as well as during the PAE period by staining the organisms with propidium iodide and analyzing them with flow cytometry and fluorescence microscopy. The effects of ampicillin, ceftriaxone, ciprofloxacin, gentamicin, and rifampin were studied for E. coli, whereas for P. aeruginosa imipenem and ciprofloxacin were investigated. After exposure of E. coli to ampicillin, ceftriaxone, and ciprofloxacin, filamentous organisms were observed by fluorescence microscopy. These changes in morphology were reflected by increased forward light scatter (FSC) and nucleic acid content as measured by flow cytometry. For the β-lactams the extent of filamentation increased in a dose-dependent manner after drug removal, resulting in formation of distinct subpopulations of bacteria. These changes peaked at 20 to 35 min, and bacteria returned to normal after 90 min after drug removal. In contrast, the subpopulations induced by ciprofloxacin did not return to normal until >180 min after the end of the classically defined PAE. Rifampin resulted in formation of small organisms with low FSC, whereas no distinctive characteristics were noted after gentamicin exposure. For P. aeruginosa an identifiable subpopulation of large globoid cells and increased nucleic acid content was detected after exposure to imipenem. These changes persisted past the PAE, as defined by viability counting. Swollen organisms with increased FSC were detected after ciprofloxacin exposure, even persisting during bacterial growth. In summary, for β-lactam antibiotics and ciprofloxacin, the PAE is characterized by dynamic formation of enlarged cell populations of increased nucleic acid content, whereas rifampin induces a decrease in size and nucleic acid content in the organisms. Flow cytometry is an ideal method for future studies of bacterial phenotypic characteristics during the PAE.

Download Full-text

Nucleic acid content in relation to cell size in the mature larval salivary gland of Drosophila melanogaster

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(52)90381-0 ◽

1952 ◽

Vol 36 (1) ◽

pp. 97-113 ◽

Cited By ~ 40

Author(s):

Elizabeth K. Patterson ◽

Marjorie E. Dackerman

Keyword(s):

Drosophila Melanogaster ◽

Salivary Gland ◽

Nucleic Acid ◽

Cell Size ◽

Acid Content ◽

Nucleic Acid Content ◽

Larval Salivary Gland

Download Full-text

Specific rates of leucine incorporation by marine bacterioplantkon in the open Mediterranean Sea in summer using cell sorting

Biogeosciences Discussions ◽

10.5194/bgd-7-6545-2010 ◽

2010 ◽

Vol 7 (4) ◽

pp. 6545-6588 ◽

Cited By ~ 1

Author(s):

A. Talarmin ◽

F. Van Wambeke ◽

P. Catala ◽

C. Courties ◽

P. Lebaron

Keyword(s):

Nucleic Acid ◽

Mediterranean Sea ◽

Acid Content ◽

Specific Activity ◽

Early Summer ◽

Nucleic Acid Content ◽

Leucine Incorporation ◽

Surface Samples ◽

Wide Range ◽

Specific Activities

Abstract. Cell-specific leucine incorporation rates were determined in early summer across the open stratified Mediterranean Sea along vertical profiles from 0 to 200 m. During the period of our study, the bulk leucine incorporation rate was on average 5.0 ± 4.0 (n=31) pmol leu l−1 h−1. After 3H-radiolabeled leucine incorporation and SyBR Green I staining, populations were sorted using flow cytometry. Heterotrophic prokaryotes (Hprok) were divided in several clusters according to the cytometric properties of side scatter and green fluorescence of the cells: the low nucleic acid content cells (LNA) and the high nucleic acid content cells (HNA), with high size and low size (HNA-hs and HNA-ls, respectively). LNA cells represented 45 to 63% of the Hprok abundance between surface and 200 m, and significantly contributed to the bulk activity, from 17 to 55% all along the transect. The HNA/LNA ratio of cell-specific activities was on average 2.1 ± 0.7 (n=31). Among Hprok populations from surface samples (0 down to the deep chlorophyll depth, DCM), HNA-hs was mostly responsible for the leucine incorporation activity. Its cell-specific activity was up to 13.3 and 6.9-fold higher than that of HNA-ls and LNA, respectively, and it varied within a wide range of values (0.9–54.3×10−21 mol leu cell−1 h−1). At the opposite, ratios between the specific activities of the 3 populations tended to get closer to each other, below the DCM, implying a potentially higher homogeneity in activity of Hprok in the vicinity of nutriclines. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific activities equally high, sometimes higher than the HNA-hs group (2.5–55×10−21 mol leu cell−1 h−1). We then showed that all the sorted populations were key-players in leucine incorporation into proteins. The mixotrophic feature of certain photosynthetic prokaryotes and the non-negligible activity of LNA cells all over Mediterranean were reinforced.

Download Full-text

Flow-microfluorometric analysis of Escherichia coli, Rhizobium meliloti, and Rhizobium japonicum at different stages of the growth cycle

Canadian Journal of Microbiology ◽

10.1139/m77-175 ◽

1977 ◽

Vol 23 (9) ◽

pp. 1165-1169 ◽

Cited By ~ 41

Author(s):

Alan S. Paau ◽

Joe R. Cowles ◽

James Oro

Keyword(s):

Escherichia Coli ◽

Nucleic Acid ◽

Stationary Phase ◽

Cell Size ◽

Acid Content ◽

Rhizobium Meliloti ◽

Growth Cycle ◽

Rhizobium Japonicum ◽

Nucleic Acid Content ◽

E Coli

The applicability of flow-microfluorometry (FMF) to the study of bacterial samples was investigated on cultures of Rhizobium meliloti, Rhizobium japonicum, and Escherichia coli using fluorescent and light-scattering signals. This technique which analyzes individual bacterial cells in a population was used to monitor the relative change in nucleic acid content and cell size during the growth cycle of the three microorganisms which were known to have different growth rates.Early log-phase E. coli cells contained at least eightfold more nucleic acid and were significantly larger than the stationary-phase cells. Cultures of early log-phase R. meliloti cells contained three to four-fold more nucleic acid and were slightly larger than cells in the stationary phase. Rhizobium japonicum had very little change in either parameter. In general, the amount of change in both cell size and nucleic acid content upon initiation of log-phase growth was related to the overall growth rate of the organisms, with E. coli experiencing the greatest change and R. japonicum the least. Results obtained by FMF analyses, therefore, were consistent with observations reported by earlier workers. Cultures of R. meliloti also were used to demonstrate that the intensity of the fluorescent signals was sensitive to digestion by DNase and RNase and to prolonged storage and fixation. The potential use of FMF in the study of microorganisms is discussed.

Download Full-text