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MERC STDN Test Plan
7
collection. (A) One sub-sample from the initial 2 l will be fixed with standard Lugol’s solution,
and placed in a 250 ml amber Nalgene bottles to determine total cell abundances under an
inverted compound microscope using grid settlement columns and phase contrast lighting. (B) A
second 250 ml sub-sample will be stained using a combination of CMFDA (5-
chloromethylfluorescein diacetate) and FDA (fluorescein diacetate) as a selective live/viable
indicator. Samples stained
with CMFDA+FDA, 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
and FDA 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.
Quantifying Viable Indicator Pathogens:
A 1 l sample of water for each mesocosm/tub (a 5 l integrated sample) is collected to
determine concentrations of total heterotrophic bacteria and three specific indicator pathogens,
E.
coli
, intestinal
Enterococci
, and toxigenic
Vibrio
cholerae
(Table 2 page 19). Total heterotrophic
bacteria are enumerated by spread plate method using NWRI agar according to
Standards
Methods for the Examination of Water and Wastewater
(21
st
edition, 2005). The presence and
abundance of
E. coli
and intestinal
Enterococci
is determined using a commercially available
chromogenic substrate method (IDEXX Laboratories, Inc.; Noble et al. 2003) and 10 ml and 100
ml water sample aliquots. Additionally, concentrations of culturable
E. coli
and intestinal
Enterococci
are determined using a standard USEPA method, namely, membrane filtration on
mTEC agar (
E. coli
) (1 ml, 10 ml and 100 ml) and mEA agar (
Enterococcus
) (10 ml and 100
ml). Abundance of total and toxigenic
V. cholerae
are calculated by filtration and selection on
TCBS agar and enumerated using species-specific RNA colony blot (500
μ
l to 1 ml) and
ctxA
DNA colony blot (1-10 ml). Viable toxigenic
V. cholerae
is assayed with a commercial DFA kit
specific for serogroup O1 (New Horizons Diagnostics) using monoclonal antibodies tagged with
fluorescein isothiocyanate (FITC) (Hasan et al. 1994).
Data Analysis:
Although multiple mesocosms, samples, and measures from each tank will be taken, to
avoid pseudo-replication, the unit of replication for statistical analyses is each trial (n = 5 or 6).