Detection of Escherichia coli and Enterobacter aerogenes in water samples collected from two sites, one site near South Shore Water Treatment Facility, the other farther from the Facility, on Lake Michigan, in Milwaukee, Wisconsin

Stephani Duelge

Melissa Unruh

BI 341, Fall 2002

Research Report

November 2, 2002

Abstract

Water was collected from two sites in Milwaukee County on the shoreline of Lake Michigan to determine if there is a difference in the number fecal coliforms in the water. The locations selected were located 3.24 km and 13.35 km upstream from the South Shore Water Treatment Facility. The water was collected from the two sites on four different days. The water was inoculated into lactose broth and observed for gas production and the amount of gas production was recorded. The lactose broth was used to inoculate plates that are selective for fecal coliforms. Inoculating lactose broth and nutrient agar slant with the plates containing the selective media was the way we performed a confirmatory test. The broth was measured again for gas production and the nutrient agar slant was used to make gram stain slides to confirm the presence of gram-negative rods that also ferment lactose, or coliforms. We found no significant difference between levels of coliforms between the two sites (p = 0.421).

Keywords: fecal coliforms, Lake Michigan, South Shore Water Treatment Facility, Escherichia coli, Enterobacter aerogenes

Introduction

In Milwaukee, Wisconsin and the surrounding areas, much of the fresh water used in homes and industry originates from Lake Michigan. However, Lake Michigan is also the dumping point for much of the wastewater. In order to ensure that water is safe for human consumption, water is treated locally at the Jones Island and South Shore Water Treatment Facilities. We hypothesized that water samples near to, and far from, the South Shore Water Treatment Facility will show different levels of fecal coliforms bacteria.

Ideally, direct detection of pathogens gives the best measure of the prevalence of pathogens in water, however applications of these methods are limited by time, expense, and difficulty adapting microbiological techniques to field applications (Islam, et al, 2000). Instead, a common practice, and the one used by the Milwaukee Metropolitan Sewerage District (MMSD), is to test for the presence of fecal coliform bacteria. Fecal coliform bacteria are defined as facultative anaerobes that ferment lactose to form gas, and are Gram-negative, non-spore forming rods (Brion, Mao, 2000). The two types of fecal coliform bacteria are Escherichia coli and Enterobacter aerogenes. E. coli is considered a good sewage indicator because it is not normally encountered in water or soil, is easily identified, and does not form endospores, so it does not survive for extended periods of time in water (Islam, et al, 2000). When it is found, it is safe to assume that water is unfit for consumption due to fecal contamination. Also, tests for the nonpathogenic E. coli can imply that other pathogenic intestinal bacteria are present, when those pathogens may be difficult to identify from a water sample (Islam, et al, 2000). Use of fecal coliforms as indicators has reduced the incidence of waterborne disease since the onset of water treatment in the early twentieth century (Brion, Mao, 2002).

The purpose for this experiment was to determine if levels of fecal coliform bacteria were higher nearer to the South Shore Water Treatment Facility than levels away from the facility. The Department of Natural Resources has set safe limits of fecal coliform and total coliform bacteria levels of 400 individuals per 100 mL of water (Shafer, 2001).

Methods

For this experiment, it was decided to test the numbers of fecal coliform bacteria in Lake Michigan on two sites. The first site was near the South Shore Water Treatment Facility, in Oak Creek, Wisconsin. The outlet pipe for the facility was 3.218 km from the actual plant. The site chosen for sampling was 0.4023 km from the actual plant, due to restricted access to the water. This made a straight-line distance from the outlet pipe of 3.243 km. This was called the South Shore site in all future references. The second site was 13.355 km from the actual facility, making the straight-line distance from the outlet 13.737 km. This was called the Sheridan Park site in all future references. Water samples were collected on three consecutive days, one from each site (see table 1). A fourth sample was collected non-consecutively, due to sample contamination on Sunday, October 27, 2002.

Table 1: Locations, dates, and times of sample collection.

Site	Date Collected	Time Collected
South Shore	Thursday, October 24, 2002	12:15 p.m.
Sheridan Park	Thursday, October 24, 2002	12:42 p.m.
South Shore	Friday, October 25, 2002	11:44 am
Sheridan Park	Friday, October 25, 2002	12:22 p.m.
South Shore	Saturday, October 26, 2002	11:31 a.m.
Sheridan Park	Saturday, October 26, 2002	12:09 p.m.
South Shore	Monday, October 28, 2002	5:04 p.m.
Sheridan Park	Monday, October 28, 2002	5:42 p.m.

For collection, 200 mL glass bottles with caps were sterilized with caps on by autoclaving at 121˚C for 15 minutes. Sample bottles were fully submerged in the lake water before caps were removed. Water was allowed to fill the bottle, expelling any air left in the bottle before caps were replaced. Only after the cap was tightened was the bottle removed from the water. By collecting samples aseptically, the chance of sample contamination from the non-water environment was decreased greatly. Because the water samples collected from Lake Michigan were non-potable, samples needed to be tested immediately after collection. When this was not possible, samples could be stored for 8 hours at 8˚C. However, in this procedure, all samples were cultured within a 2-hour time period.

In preparation for the cultures the media was prepared as follows:

Single Strength Lactose Broth (SSLB):

17 grams of dehydrated lactose broth media was dissolved in 500 mL of water. From this, 100 culture tubes with inverted Durham tubes were filled with 5 mL of the broth. All tubes were capped and autoclaved at 121˚ C for 15 minutes. Tubes were refrigerated to prevent contamination.

Double Strength Lactose Broth (DSLB):

8.5 grams of dehydrated lactose broth media was dissolved in 125 mL of water. From this, 25 culture tubes with inverted Durham tubes were filled with 5 mL of the broth. All tubes were capped and autoclaved at 121˚ C for 15 minutes. Tubes were refrigerated to prevent contamination.

Endo Agar Plates:

14 g of dehydrated Endo agar media was dissolved in 500 mL of water and brought to a rolling boil. The media was then autoclaved at 121˚C for 15 minutes. 20 mL of the agar was poured into Petri plates that were sterilized under UV light and allowed to cool. The Endo agar plates were capped and stored upside down in the refrigerator for future use.

Levine EMB Agar Plates:

15.5 g of dehydrated Levine EMB agar media was dissolved in 500 mL of water and brought to a rolling boil. The media was then autoclaved at 121˚C for 15 minutes. 20 mL of the agar was poured into Petri plates that were sterilized under UV light and allowed to cool. The Levine EMB agar plates were capped and stored upside down in the refrigerator for future use.

Nutrient Agar Slants:

14.6 g of dehydrated nutrient agar media was dissolved in 400 mL of water and brought to a rapid boil. 8 mL of the agar was poured into culture tubes, and the tubes were capped. The media was then autoclaved at 121˚C for 15 minutes. The tubes were promptly removed from the autoclave and tipped on the side to form a slanted surface for bacterial growth. The slants were refrigerated for future use.

Immediately after the collection of the samples were brought back to Alverno College for culturing. From each sample, three sterile DSLB tubes were inoculated with 10.0 mL of the sample, three sterile SSLB tubes were inoculated with 1.0 mL of the sample, and three sterile SSLB tubes were inoculated with 0.1 mL of the sample. All pipettes used in transfer were sterile, and all work was done in the sterile UV hood.

All tubes were then incubated at 34˚C for 48 hours. After 48 hours, all tubes were examined for gas production, a sign that lactose-fermenting bacteria were present. The inverted tubes were 5 cm long, and to determine the percent gas produced, the gas bubble was measured, and then the measured length was divided by 5 cm and then multiplied by 100. When 10% or more gas was present in two or more tubes, the water was presumed to contain levels of fecal coliforms unfit for human consumption.

From this data, the number of bacteria present in each sample was determined. The most probable number (MPN) of coliform bacteria present in 100 mL of water was decided using Appendix A, Table VI, “MPN Determination from Multiple Tube Test,” (Benson, 2002). This table gives the 95% confidence that the MPN is as shown in table 10 in the results section.

After determination of the MPN, the tube showing the highest percentage of growth was chosen for inoculation of Endo agar plates and Levine EMB agar plates. Endo agar contains a fuschin-sulfite indicator. When fecal coliforms grow on Endo agar plates, colonies appear red, while other bacterial type colonies remain colorless. Levine EMB agar contains methylene blue, which inhibits the growth of gram-positive bacteria. Coliform bacteria form nucleated colonies on the media, and E. coli colonies exhibit a greenish-metallic sheen.

Plates were inoculated by streaking the plate in a quadrant method, to provide good isolation of colonies. All plates were then incubated at 34˚C for 24 hours. After 24 hours, the plates were checked for coliform growth.

After plates were examined for coliform growth, one sterile SSLB tube, and one sterile nutrient agar slant was inoculated from a coliform colony on each plate. The SSLB Durham tubes and nutrient agar slants were incubated at 34˚C for 24 hours.

After 24 hours, the SSLB Durham tubes were examined for gas production, as a double check for the presence of coliform bacteria. However, the absence of gas does not rule out the presence of coliforms since the tubes must incubate for 48 hours before a negative result can be confirmed. Also, after 24 hours, slides were prepared using the growth from the nutrient agar slants. First, a drop of distilled water was placed on a clean slide, and one loopful of bacteria was placed in the water and allowed to air dry. After drying, the smear was heat fixed with a Bunsen burner. Because the confirmation of fecal coliforms requires the identification of gram-negative rods, smears were gram-stained and examined under a microscope

This was the final confirmation stage for this experiment. All tubes and plates were autoclaved to kill any remaining live bacteria and cleaned and disposed of properly.

Results

Tables 2-9 show the amount of gas formed by lactose fermenting bacteria in the Durham tubes. In order for the tube to be positive for lactose fermenting bacteria, 10%, or more gas must have been produced within 48 hours from inoculation. Because we were interested in comparing two sites, even the tubes that displayed less than 10% gas formation were considered in subsequent comparisons.

Day 1				Day 1
South Shore				Sheridan Park
Broth	Tube 1	Tube 2	Tube 3	Broth	Tube 1	Tube 2	Tube 3
DSLB 10mL	52%	56%	58%	DSLB 10mL	100%	80%	62%
SSLB 1.0mL	22%	14%	18%	SSLB 1.0mL	70%	30%	22%
SSLB 0.1mL	0%	0%	22%	SSLB 0.1mL	12%	24%	16%
Table 2				Table 3

Day 2				Day 2
South Shore				Sheridan Park
Broth	Tube 1	Tube 2	Tube 3	Broth	Tube 1	Tube 2	Tube 3
DSLB 10mL	28%	28%	32%	DSLB 10mL	40%	40%	32%
SSLB 1.0mL	8%	10%	36%	SSLB 1.0mL	12%	30%	32%
SSLB 0.1mL	2%	8%	56%	SSLB 0.1mL	38%	8%	6%
Table 4				Table 5

Day 3				Day 3
South Shore				Sheridan Park
Broth	Tube 1	Tube 2	Tube 3	Broth	Tube 1	Tube 2	Tube 3
DSLB 10mL	6%	34%	24%	DSLB 10mL	0%	16%	26%
SSLB 1.0mL	2%	0%	0%	SSLB 1.0mL	0%	0%	0%
SSLB 0.1mL	0%	0%	0%	SSLB 0.1mL	0%	0%	0%
Table 6				Table 7

Day 4				Day 4
South Shore				Sheridan Park
Broth	Tube 1	Tube 2	Tube 3	Broth	Tube 1	Tube 2	Tube 3
DSLB 10mL	52%	22%	28%	DSLB 10mL	20%	78%	50%
SSLB 1.0mL	12%	12%	12%	SSLB 1.0mL	16%	8%	26%
SSLB 0.1mL	100%	0%	2%	SSLB 0.1mL	10%	6%	8%
Table 8				Table 9

Table 10

The above table (10) shows the MPN, determined in the manner presented in the methods section of this report, is listed for each site, by the day the sample was collected. Day 3 for both sites shows a significantly reduced number of coliforms present per 100 mL of water compared to the other three collection dates.

Figure 3: Bar graph showing the MPN at both sites on days 1-4.

This graph (figure 3) shows the difference between the most probable numbers of coliforms present at both sites on all days samples were collected. Note that the greatest difference was seen between days, not between sites. The t-test results calculated to determine the significance of the MPN between both sites is 0.421.

Day 1			Day 1
Growth on Endo and Levine Agar Plates			Growth on Endo and Levine Agar Plates
South Shore			Sheridan Park
	Endo Plate	Levine Plate		Endo Plate	Levine Plate
DSLB 10 mL	positive	positive	DSLB 10 mL	positive	positive
SSLB 1.0 mL	positive	negative	SSLB 1.0 mL	positive	positive
SSLB 0.1 mL	positive	positive	SSLB 0.1 mL	positive	positive
Table 11			Table 12

Day 2			Day 2
Growth on Endo and Levine Agar Plates			Growth on Endo and Levine Agar Plates
South Shore			Sheridan Park
	Endo Plate	Levine Plate		Endo Plate	Levine Plate
DSLB 10 mL	positive	positive	DSLB 10 mL	positive	negative
SSLB 1.0 mL	positive	positive	SSLB 1.0 mL	positive	negative
SSLB 0.1 mL	positive	positive	SSLB 0.1 mL	positive	negative
Table 13			Table 14

Day 3				Day 3
Growth on Endo and Levine Agar Plates