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MERC – DRAFT Test Plan
6
evaluations. Total counts and general taxonomic classification will be conducted under a
dissecting microscope at 25X, except for some taxa, which will be removed and identified using
a compound microscope. Larval forms of invertebrates will be identified to higher taxonomic
levels such as order (e.g., Decapoda) suborder (e.g., Balanomorpha) or class (e.g., Bivalvia).
Adults will be identified to species in most cases.
Quantifying Viable Organism 10 - 50 µm in size:
Two liters of unfiltered water for each mesocosm will be collected immediately after
filling, to determine concentrations of organisms in this size class using four distinct methods (A
– D below). All samples will be held in amber Nalgene bottles and transported on ice to
laboratories where analyses occur within 3 hours of 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 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 a
conservative indicator of viable organisms. (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. (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 48 hours,
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 one-liter sample of water for each mesocosm is collected to determine concentrations
of total heterotrophic bacteria and three specific indicator pathogens,
E. coli
, intestinal
Enterococci
, and toxigenic
Vibrio
cholerae
. 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