Every Tuesday morning over the course of the warm weather months, during a time when taking a dip at Puffer’s Pond is a popular pastime, a town conservation staff member arrives to collect water from both the north and south beaches, placing samples into small plastic bottles.

Those 100-milliliter vials are then driven directly to the Wastewater Treatment Plant, a couple miles away at 100 Mullins Way in Hadley, at the western edge of the University of Massachusetts campus, where staff at the plant process the samples and, 24 hours after being placed into an incubator, will identify whether the water has high levels of E. coli.

For much of this summer, including the week of Independence Day, the tests have shown E. coli exceeding state limits, forcing the beaches to be closed and putting Puffer’s Pond off limits for swimming. As of Wednesday morning, 66 beaches across the state were listed as closed, many for similar bacterial contamination, including Lake Wyola in Shutesbury. Musante Beach in Northampton was also listed as closed, but had reopened on Sunday.

As in previous weeks, the latest tests posted to the town of Amherst website show Puffer’s Pond water with unsafe levels of E. coli, a problem that appears to be growing in frequency in recent years. Last summer, the beach remained closed from late July until the last test was completed in early September.

Inside the treatment plant Wednesday, in a lab overlooking the complex where sewage is processed, Andrew Brace, the plant’s chief operator and division director, and Trisha Sullivan, the wastewater treatment operator 2, explain the process that costs, with materials, around $100 per week. They use much of the same equipment that ensures effluent heading to the Connecticut River, and water reused for UMass purposes, meets various standards.

The water samples delivered by Conservation Land Manager Brad Bordewieck the previous day were already in the midst of their 24-hour incubation period.

After using alcohol to ensure all surfaces in the lab are sterilized, Brace and Sullivan begin by placing the water in vials with sodium thiosulfate tablets, which removes any chlorine possibly present in the water. Then, they place so-called “nutrient broths,” designed to help bacteria grow, into the water samples. The water is then deposited into trays that are put into an IDEXX machine, which seals them airtight. Finally, these trays are put into an incubator cabinet that is set at 35 degrees Celsius — 95 degrees F.

At the end of the incubation period, Brace and Sullivan have 49 large wells and 48 small wells to examine on each tray. They can soon begin to tell if the E. coli levels will be high, with wells with contaminated samples turning yellow under natural light and also turning blue under an ultraviolet lamp showing showing total coliform.

Their final examination includes looking at a matrix sheet to get the total coliform readings and determining whether the water samples are in compliance with the Code of Massachusetts Regulations.

“Puffer’s has total limits on E. coli and coliform,” Sullivan said, explaining that the pond water can’t exceed 235 colony-forming units, or CFUs, per 100 milliliters.

In addition, samples can’t exceed a geometric mean of 126 colony-forming units, or CFUs, per 100 milliliters over samples from the five most recent days. There’s also a formula for calculating the rolling geometric mean, Brace said.

When complete, the tests from Tuesday show 214.3 CFUs per 100 milliliters at the south beach and 248.1 CFUs per 100 milliliters at the north beach. But the geometric mean remains high at the south beach, at 213.04 CFUs per 100 milliliters, though lower at the north beach, 124.44 CFUs per 100 milliliters. However, if either the daily or five-day mean standard is exceeded at either beach, no swimming is allowed.

All the information is placed on a chain of custody record, where Brace and Sullivan write down the levels of E. coli, check off if it is an acceptable level or if it has exceeded limits, and then send it off the Department of Public Works and Town Hall so it can be posted on the website as a water analysis report.

“Sending the email is the most time-consuming part,” Sullivan said.

On the website for Puffer’s Pond, the town has frequently asked questions and answers.

“High counts of E. coli indicate possible contamination by feces (poop), increasing the risk of exposure to pathogens that can cause illness. Young children, the elderly, and those with compromised immune systems are most vulnerable. Swallowing contaminated water poses the highest risk of E. coli infection.”

There is also information about what the town is doing to improve the situation.

Assistant Town Manager David Ziomek said this week that tests will be done upstream this month, during both dry conditions and following storm events, understanding that when there is wet weather, more impurities from runoff end up in the pond.

This testing will include a dozen or so locations along Cushman Brook, which feeds Puffer’s Pond out of Shutesbury and Leverett and extends below the Silver Bridge and the swimming hole, as well as some of the tributaries along the brook. Beneath dam, the waterway becomes the Mill River.

Although this will allow town officials to better understand the circumstances, and learn more about the conditions before and after rain, Ziomek said he is skeptical that a single cause of the high E. coli levels will be identified, such as a failed residential septic system.

“There’s probably not going to be a point-source solution,” Ziomek said.

With the concerns about the pond, Ziomek said the town has applied to the state Dam and Seawall Repair or Removal program to begin design work and assessment of the circa 1895 dam that keeps the pond in place, along with the nearby dike.

There is also talk about dredging, which will have to be done at some point. Last done in 1986, 100,000 cubic yards of material were removed at the time.

Officials hope to soon look closely at the dam and remove the increasing amount of sediment likely building up against it and at the bottom of the pond that can compromise the health of the habitat, Ziomek said.