Download Bacterial Enumeration in Florida Bay Using Epifluorescent Microscopy

Bacterial Enumeration in Florida Bay Using Epifluorescent Microscopy
Matthew Rogers, Susan Dailey, and Joseph Boyer
Southeast Environmental Research Center, Florida International University, Miami, FL
Throughout the past 70 years, the freshwater that once passed through the Taylor and
Shark sloughs has been diverted influencing not only the chemistry, but also turbidity,
salinity and sediment material deposition. The Florida Everglades Restoration Project
aims to re-establish the freshwater flow to these sloughs, which could impact the Florida
Bay ecosystem. Although all aspects of the Bay may be affected, the microbial
community structure of Florida Bay is of key importance. Long-term monitoring projects
have identified trends pertaining to numerous facets including water quality, seasonal
modeling, and unique theories regarding Florida Bay’s microbial loop dynamics.
Redirecting the flow patterns through the Everglades could change not only the amount,
but the chemical composition of the dissolved organic material entering the Bay as well.
The reaction by the microbial community, especially heterotrophic bacteria, to these
changes must be monitored in order to form hypotheses regarding the impacts to higher
trophic organisms and the health and stability of the Bay.
Seagrass communities and their associated epiphytes are an important source of utilizable
DOC for bacteria in view of the fact that the oligotrophic nature of the Bay holds the
phytoplankton production and biomass relatively low. Although models estimate that 1050% of photosynthetically produced carbon is utilized by bacteria, the allochthonous
dissolved organic material provided by the everglades is of critical importance to the
Bay’s microbial community. The freshwater entering Florida Bay via the Everglades is
generally nutrient poor, but it is high in dissolved organic material (DOM), especially as
dissolved organic carbon (DOC), which potentially fuels the bacterial aspects of Florida
Bay’s microbial loop. Although bacteria, like phytoplankton, are nutrient limited, they
are able to take up nutrients faster, more efficiently, and in greater quantities than the
phytoplankton. The microbial loop may play a significant role in Florida Bay’s nutrient
remineralization when utilizable nutrients are low. Heterotrophic and cyanobacteria
liberate inorganic phosphorus via exudation of an extracellular enzyme measured as
alkaline phosphatase activity (APA). Combining our analysis of bacterial enumeration,
alkaline phosphatase activity, and water quality monitoring, with concurrent laboratory
biodegredation experiments could elucidate the role of heterotrophic bacteria in Florida
Bay’s microbial loop.
There has been ongoing research on the C:N:P ratio of the DOM flowing into Florida
Bay for several years. This ratio can determine whether nutrients are produced,
consumed, or some combination of both by the loop. Our analysis involves the use of
nucleic acid stains and epifluorescent microscopy to estimate bacterial cells l-1 of water
throughout 28 sites in Florida Bay. These counts showed distinct seasonal patterns and
produced an extensive database, which may be compared to future data accumulated as
the restoration project continues.
Since September 2001, we have collected 250ml samples monthly from each of the 28
sites in sterile, lightproof bottles concurrently with those collected for the Water Quality
Monitoring Program. Bottles were immediately stored on ice for transport to the lab
where our laboratory enumeration analysis began within 24 hours of collection. From
each site, a 10 ml aliquot was fixed with a final concentration of 2% formalin by volume.
From this, 0.5ml was stained with the nucleic acid stain DAPI in a filtration tower for 20
minutes. After staining, the sample was vacuum filtered through a 0.2μm polycarbonate
membrane filter, the tower rinsed with sterile water, and the filter affixed between a slide
and cover slip. The slides were then viewed under 365 nm wavelength light and the
fluorescing bacterial cells were counted within a 2000μm square grid. The totals from 10
randomly selected grids per slide were averaged and a cell count l-1 is calculated.
The past 14 months have provided a large data set that complements the 12-year Florida
Bay database. Large spatial and temporal differences were observed among areas of the
Bay. The highest cell counts came from the western Bay, and the lowest were shown to
be in the eastern. Highest counts in each zone were typically associated with the fall
(57% Sept. - Nov.) and to a lesser extent the spring (25% Apr.-May). Monthly low counts
showed less seasonality but the majority of the low counts fell in the winter (36% Dec. –
Feb.) and summer (29% June – Aug.). The range of bacterial cell counts across the
sampling period was highest at site 19 (4.480245 X106 cells/l), and lowest at site 25
(5.89050 X105 cells/l). Site 15 was shown to have the greatest variance, while site 24
had the least. The coefficient of variation ranged from 21.8% to 97.9% but 49.4% was
the average over 28 sites. Further analysis involving the comparison of the monthly
water quality monitoring to bacterial counts will be investigated.
Other methods of enumeration are being compared to the epifluorescent direct count
method presently used. A pilot study using a FACSort flow cytometer was conducted
using the October 2002 Florida sampling. The data produced by the two methods were
highly correlated (r2 = 0.8071). We intend to use this technology to enumerate bacteria
while investigating protist-bacteria relationships in Florida Bay. With further calibration,
the adoption of flow cytometry will greatly reduce sample processing time and expedite
proposed research regarding the grazing pressure on Florida Bay’s bacteria.
Understanding the role of protist grazing may provide a more complete picture of the
factors controlling the Bay’s bacterial dynamics and the transfer of materials and energy
to higher trophic levels.
The primary objective regarding Florida Bay research has been to study the effects of the
modification of overland flow through the everglades for agricultural demand and
seasonal flooding control. The continued research on the microbial aspects of the Bay
could be a powerful tool in assessing the impact of the restoration of the Everglades.
Tying the Water Quality Monitoring Network data with our concurrent bacterial analysis
may elucidate the mechanisms that control bacterial abundance across the Bay.
Rogers, Matthew, Southeast Environmental Research Center, OE-148, Florida
International University, Miami, FL 33199, Phone: 305-348-, Fax: 305-348-4096,
[email protected], Question 2