Techniques for enumerating protozoa in saturated subsurface sediments

Publication Year:
1992
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Repository URL:
https://scholars.unh.edu/dissertation/1701
Author(s):
Bunn, Amoret L
Tags:
Engineering; Civil; Hydrology; Biology; Microbiology
thesis / dissertation description
Three techniques were investigated for the enumeration of small (2-5 $\mu$m in diameter) flagellates and amoebae in sediments collected within and outside of a wastewater contaminated ground water plume at the U.S. Geological Survey Toxic Substances Hydrology Research site, located on Cape Cod, MA. An epifluorescent direct count technique was developed to enumerate DAPI stained protozoa on polycarbonate membrane filters. These estimates were compared to the those from the Darbyshire liquid media MPN and Singh solid media MPN techniques. In Fall 1991, sediment samples were collected to investigated the variability of the hold time of cores, total and encysted protozoan populations (MPN techniques only), and sites.The population estimates changed significantly (with 95% confidence) from 1 to 28 days hold time: 1.22 $\times$ 10$\sp4$ to 7.71 $\times$ 10$\sp3$ protozoa/gdw for the epifluorescent technique; 2.94 $\times$ 10$\sp4$ to 3.82 $\times$ 10$\sp4$ total MPN/gdw for the Darbyshire MPN technique; and 6.85 $\times$ 10$\sp2$ to 1.74 $\times$ 10$\sp5$ total MPN/gdw for the Singh MPN technique. The epifluorescent technique had the lowest variability of all techniques. The encysted population did not exceed 42% of the total population by either MPN technique.Protozoan populations by all enumeration techniques were significantly higher from three cores at a contaminated site compared to those from three cores at an uncontaminated site. The largest source of variation for the protozoan estimates was the cores for the epifluorescent and Darbyshire MPN techniques and the subsamples within the cores for the Singh MPN technique. The maximum probable error calculated for each enumeration technique based on the components from the sampling of the contaminated site were: 6.07 $\times$ 10$\sp3$ protozoa/gdw for the epifluorescent technique; 5.56 $\times$ 10$\sp4$ total MPN/gdw for the Darbyshire MPN technique; and 5.86 $\times$ 10$\sp4$ total MPN/gdw for the Singh MPN technique. The changes over the hold time were within the detectable difference for the epifluorescent and Darbyshire MPN techniques. However, the significant increase in the Singh MPN estimates over time was not explained by the errors in the sampling technique and should be further investigated.