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Differ- ent fluorescent probes are available that have poten- tial applicability in measuring phytoplankton vitality. Often these probes are used in combination with.
J. Phycol. 47, 692–702 (2011)  2011 Phycological Society of America DOI: 10.1111/j.1529-8817.2011.00991.x

FLOW CYTOMETRIC APPLICABILITY OF FLUORESCENT VITALITY PROBES ON PHYTOPLANKTON 1 Louis Peperzak and Corina P. D. Brussaard2 Department of Biological Oceanography, Royal Netherlands Institute for Sea Research ⁄ NIOZ, P.O. Box 59, NL-1790 AB Den Burg, the Netherlands

The applicability of six fluorescent probes (four esterase probes: acetoxymethyl ester of Calcein [Calcein-AM], 5-chloromethylfluorescein diacetate [CMFDA], fluorescein diacetate [FDA], and 2¢,7¢-dichlorofluorescein diacetate [H2DCFDA]; and two membrane probes: bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] and SYTOX-Green) as vitality stains was tested on live and killed cells of 40 phytoplankton strains in exponential and stationary growth phases, belonging to 12 classes and consisting of four cold-water, 26 temperate, and four warm-water species. The combined live ⁄ dead ratios of all six probes indicated significant differences between the 12 plankton classes (P < 0.01) and between individual species (P < 0.05). No specific differences were observed among strains of one species, among species or strains from different origin, nor between cells in exponential and stationary growth phase except for FDA. FDA showed a significant (P < 0.05) drop of 610 nm and was set as the trigger. Median values of green probe fluorescence intensity were calculated from cell clusters selected in FL1*FL4 plots. Living cells and killed controls were measured at least twice from duplicate cultures. Forward light scatter (FLS) was measured as a proxy of cell size (Cunningham and Buonnacorsi 1992). In the case of large cells, a neutral filter was placed in the FLS light path that effectively reduced the FLS signal by a factor 9. Fluorescent calibration beads (Flow Set and Flow Check, Coulter Diagnostics, Brea, CA, USA) were used to standardize the green fluorescence of well-stained FDA-probed strains in exponential growth phase to correlate FDA fluorescence intensity with cell size as FLS. Probe protocols. Vitality probes (all from Invitrogen Inc., Carlsbad, CA, USA) were tested on phytoplankton cells in both exponential and stationary growth phase. Controls consisted of cells from both growth phases, killed by adding 50 lL 18% formaldehyde to 1 mL sample and stored at 4C for at least 1 h. For each probe, a number of initial tests were run on a subset of species to determine probe concentrations and incubation specifics (light or dark, temperature, and incubation time). All tests were performed during fixed intervals, 2–9 h into the light period. The final protocols that stained the test species best were used throughout the study and are summarized in Table 2. Data analysis. The response (live ⁄ dead ratio) of each strain to a specific probe was calculated by dividing the green fluorescence intensity of the living cells by the green fluorescence intensity of killed cells in the case of esterase probes and vice versa for the membrane probes, because the intensity of

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TABLE 1. List of phytoplankton classes (n = 12), species (n = 34), strains (n = 40), and culture temperature (C) used to screen six fluorescent vitality probes. Class

Bacillariophyceae Bacillariophyceae Bacillariophyceae Bacillariophyceae Bacillariophyceae Bacillariophyceae Bacillariophyceae Bacillariophyceae Chlorophyceae Chlorophyceae Cryptophyceae Cryptophyceae Cyanophyceae Cyanophyceae Dinophyceae Dinophyceae Dinophyceae Dinophyceae Dinophyceae Euglenophyceae Euglenophyceae Eustigmatophyceae Pelagophyceae Prasinophyceae Prasinophyceae Prasinophyceae Prasinophyceae Prasinophyceae Prasinophyceae Prymnesiophyceae Prymnesiophyceae Prymnesiophyceae Prymnesiophyceae Prymnesiophyceae Prymnesiophyceae Prymnesiophyceae Prymnesiophyceae Raphidophyceae Rhodophyceae Rhodophyceae

Number

Species

Strain

C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Chaetoceros calcitrans Ditylum brightwellii Phaeodactylum tricornutum Pseudo-nitzschia pungens Pseudo-nitzschia sp. Skeletonema costatum Thalassiosira antarctica Thalassiosira oceanica Dunaliella sp. Nannochloris sp. Rhodomonas baltica Rhodomonas salina Synechococcus sp. 1 Synechococcus sp. 2 Alexandrium minutum Amphidinium carterae Gymnodinium simplex Prorocentrum minimum Scrippsiella sp. Eutreptiella cf. gymnastica Eutreptiella marina Nannochloropsis salina Pelagococcus subviridis Micromonas pusilla M. pusilla M. pusilla M. pusilla Pyramimonas sp. Tetraselmis suecica Chrysochromulina polylepis Emiliania huxleyi Isochrysis galbana Phaeocystis antarctica (nonflags) Phaeocystis globosa (nonflags) P. globosa (flags) P. globosa (flags) P. globosa (nonflags, flags) Heterosigma akashiwo Dixoniella grisea Porphiridium cruentum

CCMP 1315 Bigelow 358 U. Wollenzien, UTCC162 G. Casteleyn, UG Belgium B. Bontes, NIOZ CCMP781 B. Bontes, NIOZ CCMP1006 CCMP1320 CCAP 251 ⁄ 2 Hagmeier, Helgoland CCMP 1319 G. Nieuwland, NIOZ CCMP1334 CCMP 113 CCMP1314 A. Noordeloos, NIOZ Biol. Hel. Damm E66 A. Noordeloos, NIOZ CCMP1594 CCMP390 CCAP 849 ⁄ 4 CCMP 1429 Mp38, Roscoff Mp1545, Roscoff Mp449, Roscoff Mp450, Roscoff A. Buma, NIOZ CCMP883 Oldenburg T. Ietswaart, NIOZ CCMP 1323 B. Bontes, NIOZ CCMP627 RIKZ Ph91mf, NIOZ Pg2 NIOZ Pg5 Pg6-1, L. Peperzak, NIOZ K. de Boer, RUG U. Wollenzien, CEME U. Wollenzien, CEME

15 15 15 15 4 15 4 22 15 15 15 15 15 15 15 22 15 15 15 15 15 15 15 15 15 22 22 4 15 15 15 15 4 22 15 15 15 15 15 15

The 40 strains belong to four cold-water, four warm-water, and 26 temperate species. The strain numbers (1–40) appear in the results of statistical analyses.

TABLE 2. Final probe incubation protocols. Probe

Calcein-AM CMFDA DiBAC4(3) FDA H2DCFDA SYTOX-Green

Working stock solvent

Probe added (lL Æ mL)1)

Final (lM)

Incubation

T

Acetone Acetone EtOH Acetone Acetone Milli-Q

10 10 10 10 10 10

10 10 2 10 10 0.5

L L D L L D

CT CT RT CT CT RT

Incubation time (min)

60 60 10; ++, ratio 5 £ 10; +, ratio 2 £ 5; 0, ratio