Swim Guide E. Coli Bacteria Analysis

Every Wednesday from Memorial Day to Labor Day, our volunteers and staff collect water samples from our sites across the mountain region. Samples are kept cold and are returned to our regional offices to be processed in our laboratories. Volunteers are essential to the program, helping MountainTrue publish data from 1,167 samples at 90 locations over the course of the 15-week period in 2022. After processing in the lab, the weekly E. coli results are published on, a free smartphone app and website that allows us to inform the public if our rivers and lakes are safe to swim. 

We recruit volunteers seasonally for the Swim Guide program through newsletters, press releases, and outreach events. Volunteers are trained through virtual and onsite training to ensure samples are collected and stored according to our standard operating procedures. 

Fecal coliform bacteria, such as E. coli, are indicator organisms present when other pathogens are in the water. If fecal coliform counts are high in the river, there is a greater chance that pathogenic organisms are also present. E. coli is found in the guts of warm-blooded animals, birds, and humans. The bacteria are shed in the feces of all of those organisms. E. coli does not occur naturally outside of the bodies of warm-blooded animals, so it is an accurate indicator of fecal contamination of our waters. These bacteria can enter rivers through agricultural and stormwater runoff, direct seeps such as leaking septic tanks and other sources described above. In general, E. coli levels are much higher during and after rain events. A person or pet has a much higher chance of getting sick from swallowing disease-causing organisms or from pathogens entering the body through the mouth, nose, ears, or cuts on the skin. Monitoring E. coli levels within our rivers is a valuable tool for equipping the public with the data to make informed decisions before recreating to avoid infections or illness from contaminated waters. 

Samples are collected using a sterilized 100mL IDEXX sample bottle with sodium thiosulfate and stored on ice until processed by MountainTrue staff the same day. Samples are labeled with the date, time collected, and site name. Incorrectly labeled samples are discarded. A colilert reagent is added to the water sample and allows us to detect both total coliforms and Escherichia coli (E. coli) through enzymatic reactions within 18-24 hours. The IDEXX process is U.S. EPA-approved and included in Standard Methods (9223B)  for Examination of Water and Wastewater. After the Colilert reagent is added to the 100mL water sample and agitated, the sample is poured into the Quanti-Tray/2000 and run through the Quanti-Tray Sealer. The sealed Quanti-Tray is labeled with the site name, date & time collected/processed, and placed in the IDEXX incubator for 18-24 hours (depending on the Colilert product) at 35±0.5॰C. 

After the incubation period, samples are read by putting the Quanti-Tray/2000 under a fluorescent light. The wells positive for E. coli will “glow” and appear with a yellowish-blue fluorescence. The glowing wells are marked and tallied, with the number of positive large and small wells referenced in the Most Probable Number (MPN) table. The MPN table calculates the “Most Probable Number of Colony Forming Units” or MPN/100mL. Swim Guide marks the pass/fail of samples based on the EPA’s recreational standard of 235 MPN/100mL. The EPA estimates that at that concentration, 36 per 1,000 people engaging in primary recreation will contract an illness from surface water. Pass/Fail results are updated every Friday on to inform the public about local water quality. We use the data generated from our Swim Guide Program to identify sites for follow-up sampling. Determining the location and source of E. coli pollution in our streams and rivers is one of the many ways we monitor our waterways.

Dilutions are helpful in expanding the reading range of the Quanti-Tray. This technique is primarily used for sites that repeatedly max out the Quanti-Tray/2000’s range of 2,419 MPN/100mL. In watersheds such as the French Broad, samples are regularly diluted due to very high E. coli concentrations. Samples are diluted before adding the Colilert reagent with sterile deionized (DI) water. The standard dilutions are 1:1 (50mL of sample + 50mL of DI water) or 1:10 (10mL of sample + 90mL of DI water). Final E. coli levels in samples are corrected based on the conversion factor of the dilutions. 

Bacteria levels are represented by a red, yellow, and green color coding system, indicating the risk of E. coli exposure from water bodies across the region.

Green: Primary Recreation, an average of less than or equal to 235 MPN/100mL
Yellow: Secondary Recreation, an average of 236-406 MPN/100mL
Red: Increased risk for recreation, an average of 407 MPN/100mL and above

Stream Health Analysis

MountainTrue’s Stream Health analysis is focused on how well a stream or river supports important aquatic life. MountainTrue combined data from multiple sources to formulate our water quality rankings for each stream section and includes the following data:

VWIN: Chemical data — part of the Volunteer Water Information Network (VWIN) — from the Environmental Quality Institute. Data collected includes pH, alkalinity, turbidity, TSS, conductivity, ortho-P, ammonia-N, nitrate-N, sediment score, and nutrient score to establish site rating. 

SMIE: Aquatic insect (a.k.a., benthic macroinvertebrate) data — part of the Stream Monitoring Information Exchange program (SMIE) — from the Environmental Quality Institute. Learn more about SMIE here

303d: The term "303(d) list" or “list” is short for a state's list of impaired and threatened waters (e.g., stream/river segments, lakes). States are required to submit their list for EPA approval every two years. For each water on the list, the state identifies the pollutant causing the impairment when known. In North Carolina, we used the DEQ Department of Water Resources' (DWR) 2022 Final Integrated Report. For sites in Georgia, we used the DNR Environmental Protection Division's (EPD) 2022 305(b)/303(d) List Documents.

AAS: In the Hiwassee River basin, chemical data from the Georgia Adopt-A-Stream (AAS) volunteer water quality monitoring program. Data collected includes water temperature, dissolved oxygen, pH, and conductivity. Turbidity was also collected at some locations.

TVA Ecological Health Ratings: The health ratings are based on dissolved oxygen, chlorophyll, fish, bottom dwellers, and sediment. 

The VWIN and SMIE data compiled from EQI provided site scores consistent with the 10-point grading scale used for the stream health data results. MountainTrue staff used other data sources (i.e., 303d, TVA, AAS) to convert the variables to 0-100 on the 10-point scale. This was accomplished by using “if” and “then” statements to transform information from the data sources according to the scale used in this report. 

The grading scale is as follows: 

A (90-100) - Excellent
Streams with excellent water quality, low pollution levels, and healthy aquatic insect and fish populations.

B (80-89) - Good
Streams with good water quality but some impacts from pollution or development. Aquatic life and fish populations were relatively healthy.

C (70-79) - Good-Fair
Streams with average water quality. There are some concerns about pollution inputs and development impacts. Generally, aquatic life and fish populations were healthy but could become negatively impacted in the future.

D (60-69) - Fair
Streams with below-average water quality. Pollution is a concern, and aquatic life and fish populations were not as healthy as they should be.

F (<60) - Poor
Streams with poor water quality. Pollution levels were often high, and aquatic life and fish populations were impacted.