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MERC+GSI – DRAFT Test Plan
9
second 250 ml sub-sample will be stained using CMFDA (5-chloromethylfluorescein diacetate)
as a selective live/viable indicator. Samples stained
with CMFDA, are incubated and observed
on a Sedgewick Rafter slide using a Leitz Laborlux S modified for epifluorescence. Cells are
scored as live when showing strong fluorescence signature under excitation (some cells also
showed motility).
However, it is also widely accepted that these direct count and staining
techniques have limitations (Lugol’s does not selectively stain live or dead, various algal species
take up CMFDA differently, and other particles in a sample can fluoresce). Therefore, analyses
of chlorophyll are also conducted as supporting information. (C) A third sub-samples is filtered
(Whatman GF/F 0.7 µm pore, 2.5 cm diameter membrane) and frozen (-80
o
C) until analysis of
total active chlorophyll-a by the CBL/UMCES Nutrient Analytical Services Laboratory using US
EPA Methods 445.0 for extractive/fluorometric techniques (see Appendix D). (D) Finally a
fourth sub-sample is used to determine chlorophyll levels after allowed to regrow under
favorable conditions. Algae specific vitamins, minerals, and nutrients (Guillard 1975, F/2
formulation) are added to a sub-sample from each mesocosm and are placed in a standard algal
culture light-dark regimen for six days, prior to extractive chlorophyll-a analysis. An increase in
chlorophyll, or positive regrowth, indicates that viable phytoplankton were in the samples,
whereas chlorophyll levels at or below detection limits of the laboratory analytical method
suggests that there was no viable phytoplankton. Although precise abundances of cells/ml
cannot be determined for diverse communities of phytoplankton using these types of regrowth
experiments, this is a conservative method used to determine the presence/absence of living
organisms.
GSI - For live analysis of organisms 10 – 50 µm in size at least 1 l of unfiltered water is
taken from each of the triplicate control and treatment sample collection mesocosm/tub (a 3 l
integrated sample, Table 2 page 27). Analysis occurs on-site within 1.5 hours of sample
collection, with samples stored in coolers during the interim. Prior to analysis, samples are
concentrated through a 10 µm plankton net and stored in a 25 ml sample container. Next, a 1.5
ml subsample is transferred to a 2-ml sample container, with 4 µl of FDA stock solution added.
The subsample is then allowed to incubate in the dark for 5 minutes. For analysis, the
concentrated algae sample is mixed and immediately transferred to a Sedgwick-Rafter cell,
covered and placed on the stage of microscope that is set for simultaneous observation using
brightfield and epifluorescence. At least 100 entities are then counted and identified along the
horizontal transects, aiming for at least 100 entities (i.e., unicellular organism, colony or
filament). Single cell entities and cells comprising colonial and filamentous entities are
characterized as follows: alive = cells showing obvious green fluorescence from cell contents;
dead = cells showing no or very little evidence of green fluorescence from cell contents (note: for
entities containing multiple cells, all cells must be confirmed as dead to fulfill this category); and
ambiguous = entities that cannot be clearly identified as alive or dead (should be uncommon).
Entities that are less than 10 µm in all visible dimensions or greater than 50 µm in minimum
dimension are not counted. Records are kept of transect lengths and widths so that the total
counted area may be calculated later. Counting and measurement of entities follows standard
procedures for individuals (length and width), colonies (e.g., number of cells, cell length and
width) and filaments (e.g., number of cells, cell length and width or total filament length if cells
cannot be discerned). The remaining concentrated sample in the 25 ml bottle is archived using a
preservative (formalin or Lugol’s) for long-term storage.