Limits...
Rapid, Precise, and Accurate Counts of Symbiodinium Cells Using the Guava Flow Cytometer, and a Comparison to Other Methods.

Krediet CJ, DeNofrio JC, Caruso C, Burriesci MS, Cella K, Pringle JR - PLoS ONE (2015)

Bottom Line: In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer.We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument's automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate.We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
In studies of both the establishment and breakdown of cnidarian-dinoflagellate symbiosis, it is often necessary to determine the number of Symbiodinium cells relative to the quantity of host tissue. Ideally, the methods used should be rapid, precise, and accurate. In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer. We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument's automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate. We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.

No MeSH data available.


Related in: MedlinePlus

Quantification of algal cells using the Guava flow cytometer.The instrument was operated as described in Materials and Methods; all anemones were of strain CC7. (a) Effects of sample type and preparation method on the flow-cytometer plots of the red (chlorophyll) fluorescence vs. the side-scatter of the particles. The square within each plot indicates the region containing Symbiodinium cells with approximately normal chlorophyll concentration and light scatter. The samples of cultured algae (strain SSA02), fixed anemone homogenate, and coral homogenate (panels 1, 5, and 6) were prepared as described in Materials and Methods. The homogenates of aposymbiotic (APO) and symbiotic (SYM) anemones (panels 2 and 3) were prepared using our standard protocol except (i) the anemones had not been frozen and (ii) the symbiotic anemone was initially homogenized by rotor stator in ASW, after which one-fifth volume of 0.1% SDS in ASW was added before needle shearing and further dilution in 0.1% SDS in dH2O. The frozen anemones (panel 4) were frozen in 0.1% SDS in ASW, then thawed, homogenized by rotor stator and needle shearing, and diluted further in the same solution. (b and c) Precision of the method and its ability to detect small differences in algal-cell concentrations. Anemone homogenates were prepared and diluted using our standard protocol. (b) Undiluted and diluted homogenates were analyzed as in Fig 3g except that n = 8 in this case. (c) Serial two-fold dilutions of the homogenate were prepared, and the predicted (solid line) and measured (as mean values ± SEMs; n = 3) counts were compared. Most error bars are hidden by the points themselves with the graphing program used here. (d) Efficiency of automatic mixing by the instrument. Anemone homogenate was prepared by our standard protocol except that 0.1% SDS in ASW was used for homogenization and dilution. Samples were added to all wells of a 96-well plate, which was then analyzed in the usual way, so that individual samples sat for up to 84 min without agitation except for the automatic mixing that preceded the sampling from each well. R2 for the regression shown = 0.04. (e) Quantification of algal cells in two coral nubbins that had been held at 26°C and in two nubbins that had been stressed at 33°C for 3 d (see Materials and Methods). Homogenates were prepared as described in Materials and Methods, and algal cell numbers (see a, panel 6) were normalized to protein concentrations as determined by the BCA assay (see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4546242&req=5

pone.0135725.g004: Quantification of algal cells using the Guava flow cytometer.The instrument was operated as described in Materials and Methods; all anemones were of strain CC7. (a) Effects of sample type and preparation method on the flow-cytometer plots of the red (chlorophyll) fluorescence vs. the side-scatter of the particles. The square within each plot indicates the region containing Symbiodinium cells with approximately normal chlorophyll concentration and light scatter. The samples of cultured algae (strain SSA02), fixed anemone homogenate, and coral homogenate (panels 1, 5, and 6) were prepared as described in Materials and Methods. The homogenates of aposymbiotic (APO) and symbiotic (SYM) anemones (panels 2 and 3) were prepared using our standard protocol except (i) the anemones had not been frozen and (ii) the symbiotic anemone was initially homogenized by rotor stator in ASW, after which one-fifth volume of 0.1% SDS in ASW was added before needle shearing and further dilution in 0.1% SDS in dH2O. The frozen anemones (panel 4) were frozen in 0.1% SDS in ASW, then thawed, homogenized by rotor stator and needle shearing, and diluted further in the same solution. (b and c) Precision of the method and its ability to detect small differences in algal-cell concentrations. Anemone homogenates were prepared and diluted using our standard protocol. (b) Undiluted and diluted homogenates were analyzed as in Fig 3g except that n = 8 in this case. (c) Serial two-fold dilutions of the homogenate were prepared, and the predicted (solid line) and measured (as mean values ± SEMs; n = 3) counts were compared. Most error bars are hidden by the points themselves with the graphing program used here. (d) Efficiency of automatic mixing by the instrument. Anemone homogenate was prepared by our standard protocol except that 0.1% SDS in ASW was used for homogenization and dilution. Samples were added to all wells of a 96-well plate, which was then analyzed in the usual way, so that individual samples sat for up to 84 min without agitation except for the automatic mixing that preceded the sampling from each well. R2 for the regression shown = 0.04. (e) Quantification of algal cells in two coral nubbins that had been held at 26°C and in two nubbins that had been stressed at 33°C for 3 d (see Materials and Methods). Homogenates were prepared as described in Materials and Methods, and algal cell numbers (see a, panel 6) were normalized to protein concentrations as determined by the BCA assay (see Materials and Methods).

Mentions: In seeking a method that offered both high precision and rapid, automated processing of samples, we tested the Guava flow cytometer, which detects and counts particles passing through a microcapillary tube on the basis of their fluorescence and light scattering (www.millipore.com/easycyte); this method seemed likely to work well with Symbiodinium cells given their intrinsic chlorophyll fluorescence. Indeed, analyses of both axenic Symbiodinium cultures and homogenates of symbiotic anemones revealed a cluster of particles with high red fluorescence and a well defined range of light-scattering values (Fig 4a, panels 1 and 3). Such a population was totally absent in a homogenate of aposymbiotic anemones (Fig 4a, panel 2) and can be bracketed for automated counting by the instrument (inset boxes in Fig 4a). Although the precision of such Guava counts was slightly less than that obtained with the Coulter Counter (Fig 2, lanes C and G), it was still far superior to that obtained with the hemocytometer (Fig 2, lanes H and G), and it was sufficient to reliably detect small changes in algal numbers over a wide range of algal concentrations (Fig 4b and 4c). The Guava also requires much smaller sample sizes than does the Coulter Counter (Table 2), and it is much faster to use than either the Coulter Counter or hemocytometer, particularly when large numbers of samples are to be counted (Table 1, line 4). In addition, the automatic mixing that occurs before sampling from each well of the 96-well plate in the Guava means that settlement of the algal cells is not a problem (Fig 4d). This last experiment also shows that any gradual dissolution of the algal cells by the SDS used and/or by autolysis is not a problem on the timescale in which the counts are performed.


Rapid, Precise, and Accurate Counts of Symbiodinium Cells Using the Guava Flow Cytometer, and a Comparison to Other Methods.

Krediet CJ, DeNofrio JC, Caruso C, Burriesci MS, Cella K, Pringle JR - PLoS ONE (2015)

Quantification of algal cells using the Guava flow cytometer.The instrument was operated as described in Materials and Methods; all anemones were of strain CC7. (a) Effects of sample type and preparation method on the flow-cytometer plots of the red (chlorophyll) fluorescence vs. the side-scatter of the particles. The square within each plot indicates the region containing Symbiodinium cells with approximately normal chlorophyll concentration and light scatter. The samples of cultured algae (strain SSA02), fixed anemone homogenate, and coral homogenate (panels 1, 5, and 6) were prepared as described in Materials and Methods. The homogenates of aposymbiotic (APO) and symbiotic (SYM) anemones (panels 2 and 3) were prepared using our standard protocol except (i) the anemones had not been frozen and (ii) the symbiotic anemone was initially homogenized by rotor stator in ASW, after which one-fifth volume of 0.1% SDS in ASW was added before needle shearing and further dilution in 0.1% SDS in dH2O. The frozen anemones (panel 4) were frozen in 0.1% SDS in ASW, then thawed, homogenized by rotor stator and needle shearing, and diluted further in the same solution. (b and c) Precision of the method and its ability to detect small differences in algal-cell concentrations. Anemone homogenates were prepared and diluted using our standard protocol. (b) Undiluted and diluted homogenates were analyzed as in Fig 3g except that n = 8 in this case. (c) Serial two-fold dilutions of the homogenate were prepared, and the predicted (solid line) and measured (as mean values ± SEMs; n = 3) counts were compared. Most error bars are hidden by the points themselves with the graphing program used here. (d) Efficiency of automatic mixing by the instrument. Anemone homogenate was prepared by our standard protocol except that 0.1% SDS in ASW was used for homogenization and dilution. Samples were added to all wells of a 96-well plate, which was then analyzed in the usual way, so that individual samples sat for up to 84 min without agitation except for the automatic mixing that preceded the sampling from each well. R2 for the regression shown = 0.04. (e) Quantification of algal cells in two coral nubbins that had been held at 26°C and in two nubbins that had been stressed at 33°C for 3 d (see Materials and Methods). Homogenates were prepared as described in Materials and Methods, and algal cell numbers (see a, panel 6) were normalized to protein concentrations as determined by the BCA assay (see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4546242&req=5

pone.0135725.g004: Quantification of algal cells using the Guava flow cytometer.The instrument was operated as described in Materials and Methods; all anemones were of strain CC7. (a) Effects of sample type and preparation method on the flow-cytometer plots of the red (chlorophyll) fluorescence vs. the side-scatter of the particles. The square within each plot indicates the region containing Symbiodinium cells with approximately normal chlorophyll concentration and light scatter. The samples of cultured algae (strain SSA02), fixed anemone homogenate, and coral homogenate (panels 1, 5, and 6) were prepared as described in Materials and Methods. The homogenates of aposymbiotic (APO) and symbiotic (SYM) anemones (panels 2 and 3) were prepared using our standard protocol except (i) the anemones had not been frozen and (ii) the symbiotic anemone was initially homogenized by rotor stator in ASW, after which one-fifth volume of 0.1% SDS in ASW was added before needle shearing and further dilution in 0.1% SDS in dH2O. The frozen anemones (panel 4) were frozen in 0.1% SDS in ASW, then thawed, homogenized by rotor stator and needle shearing, and diluted further in the same solution. (b and c) Precision of the method and its ability to detect small differences in algal-cell concentrations. Anemone homogenates were prepared and diluted using our standard protocol. (b) Undiluted and diluted homogenates were analyzed as in Fig 3g except that n = 8 in this case. (c) Serial two-fold dilutions of the homogenate were prepared, and the predicted (solid line) and measured (as mean values ± SEMs; n = 3) counts were compared. Most error bars are hidden by the points themselves with the graphing program used here. (d) Efficiency of automatic mixing by the instrument. Anemone homogenate was prepared by our standard protocol except that 0.1% SDS in ASW was used for homogenization and dilution. Samples were added to all wells of a 96-well plate, which was then analyzed in the usual way, so that individual samples sat for up to 84 min without agitation except for the automatic mixing that preceded the sampling from each well. R2 for the regression shown = 0.04. (e) Quantification of algal cells in two coral nubbins that had been held at 26°C and in two nubbins that had been stressed at 33°C for 3 d (see Materials and Methods). Homogenates were prepared as described in Materials and Methods, and algal cell numbers (see a, panel 6) were normalized to protein concentrations as determined by the BCA assay (see Materials and Methods).
Mentions: In seeking a method that offered both high precision and rapid, automated processing of samples, we tested the Guava flow cytometer, which detects and counts particles passing through a microcapillary tube on the basis of their fluorescence and light scattering (www.millipore.com/easycyte); this method seemed likely to work well with Symbiodinium cells given their intrinsic chlorophyll fluorescence. Indeed, analyses of both axenic Symbiodinium cultures and homogenates of symbiotic anemones revealed a cluster of particles with high red fluorescence and a well defined range of light-scattering values (Fig 4a, panels 1 and 3). Such a population was totally absent in a homogenate of aposymbiotic anemones (Fig 4a, panel 2) and can be bracketed for automated counting by the instrument (inset boxes in Fig 4a). Although the precision of such Guava counts was slightly less than that obtained with the Coulter Counter (Fig 2, lanes C and G), it was still far superior to that obtained with the hemocytometer (Fig 2, lanes H and G), and it was sufficient to reliably detect small changes in algal numbers over a wide range of algal concentrations (Fig 4b and 4c). The Guava also requires much smaller sample sizes than does the Coulter Counter (Table 2), and it is much faster to use than either the Coulter Counter or hemocytometer, particularly when large numbers of samples are to be counted (Table 1, line 4). In addition, the automatic mixing that occurs before sampling from each well of the 96-well plate in the Guava means that settlement of the algal cells is not a problem (Fig 4d). This last experiment also shows that any gradual dissolution of the algal cells by the SDS used and/or by autolysis is not a problem on the timescale in which the counts are performed.

Bottom Line: In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer.We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument's automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate.We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
In studies of both the establishment and breakdown of cnidarian-dinoflagellate symbiosis, it is often necessary to determine the number of Symbiodinium cells relative to the quantity of host tissue. Ideally, the methods used should be rapid, precise, and accurate. In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer. We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument's automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate. We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.

No MeSH data available.


Related in: MedlinePlus