E-coli lab write up

ENGR 113Q1 Fall 2020e-coli lab

 

Introduction:

Escherichia coli (E. coli) bacteria normally live in the intestines of healthy people and animals. Most varieties of E. coli are harmless or cause relatively brief diarrhea. But a few particularly nasty strains, such as E. coli O157:H7, can cause severe abdominal cramps, bloody diarrhea and vomiting.

Early symptoms of E. coli O157:H7 infection

• Stomach cramps (abdominal pain)
• Diarrhea that often is bloody.
• Fever of about 100 F to 101 F (37.7 C to 38.3 C)
• Loss of appetite.
• Mild dehydration.

Some people, especially children under 5 and the elderly, can become very sick fromE. coli O157:H7. The infection damages their red blood cells and their kidneys. This only happens to about 1 out of 50 people, but it is very serious. Without hospital care, they can die.

However, we are not primarily interested in E. coli but are more interested in the other pathogens potential present when we find E. coli. These can be much worse so E. coli functions as an indicator for other pathogens.

An ideal indicator organism should be:

1. The organism should be present whenever enteric pathogens are present
2. The organism should be useful for all types of water
3. The organism should have a longer survival time than the hardiest enteric pathogen
4. The organism should not grow in water
5. The organism should be found in warm-blooded animals’ intestines.
6. The organism should be cheap to identify and identifications should be fast

The presence of any strain of E.coli in waters is a strong indication of recent sewage or animal water contamination. As with coliforms, the presence of any strain of E.coli indicates other harmful strains or other organisms could be present and could pose a threat to human health.

Description of lab procedure:

In this lab we will test several sources of water with which we can come in contact. These can be river water, well water, drinking water and so on.

We will usemicroplate trays to provide a rapid, convenient and accurate test for qualitative and quantitative measurement of the total Coliforms and E.coli bacteria.

 

The method will detect viable Coliforms and E.coli bacteria and contains selective media to stimulate the growth and indicators for chromogenic and fluorogenic enzymes. The substrates react with the enzymes produced by the Coliforms (Beta-D-galactosidase) and the E.coli (Beta-D-glucoronidase) resulting in a formation of color, and a fluorescence emission by the Coliforms and E.coli respectively. Coliform positive test results in a distinctive brightcolor, which enables analysis of brownish, turbid or rust-filled water.

Marked wells have E-coli contamination

 

Above are some of the resutls from previous water testing and we can see that bottled water, water from the bathroom was clean but that water from Crick Creek was contaminated (a lot).

 

We will fill the quanti trays with our collected sample water, seal the tray and put the tray in an incubator for 24 hours. The next day we will read the trays under a fluorecent light and mark cells that are contaminated. Based on the probability table we can now predict the amount of E. coli present in the water. Based on the results we can determine if we need to close a beach or chlorinate a well and so on.

Lab writeup

A brief lab write-up needs to be handed in after reading the cells.

 

 

MATERIALS FOR E. COLI AND ENTEROCOCCI ANALYSIS:

 

• Two Quanti-Trays
• IDEXX Quanti-Tray® sealer
• Incubator
• One snap pack of Colilert
• One snap pack of Enterolert
• 2 100mL water samples
• Light Box (6 watt Fluorescent bulb)
• Incubator Thermometer
• MPN Table

 

 

DEFINITIONS AND IMPORTANT INSTRUCTIONS:

 

IF a water sample has some background color, compare inoculated Colilert sample to a control blank of the same water sample.

 

• Water Sample: For the purpose of this method, a sample taken from one of the following sources: drinking, surface, ground, storm runoff, industrial or domestic wastewater.

 

 

E.COLI SAMPLE ANALYSIS PROCEDURE

 

1. Turn on sealer so it will be ready when you are
2. Shake sample bottle approximately 25 times
3. Pour off access water until it reads 100mLs of water (look at the line on the side of the bottle for reference)
4. Add the contents of one snap pack (Colilert, Colisure, or Colilert-18) to 100mL water sample
5. Shake bottle until completely dissolved
6. Label Quanti-Tray with Date and time that you will be putting into incubator
7. Pour sample/reagent mixture into a Quanti-Tray
8. Seal in an IDEXX Quanti-Tray® sealer
9. Place sealed tray in 35° ± 0.5°C incubator for 24 hours (18 hours if using the Colilert-18)
10. Observe the Quanti-Tray for any color changes and count the wells that have changed color.
11. Observe the Quanti-Tray under a IDEXX viewing box and UV Lamp and count the number of wells that are a fluorescent blue
12. Refer to the MPN table provided as you will obtain the Most Probable Number

 

Result Reading

 

Appearance	Result
Less yellow than the comparator	Negative for total coliforms and E. coli
Yellow equal to or greater than comparator	Position for total coliforms
Yellow and fluorescence equal to or greater than the comparator	Positive for E. coli

 

 

ENTEROCOCCI SAMPLE ANALYSIS PROCEDURE:

 

1. Do steps 1 and 2 the exact same as the coli procedure
2. Add the contents of one snap pack of Enterolert to 100mL water sample
3. Shake bottle until completely dissolved (might take longer than Colilert)
4. Pour Sample/reagent mixture into a Quanti-Tray
5. Label Quanti-Tray with Date and time that you will be putting into incubator
6. Seal in IDEXX Quanti-Tray® sealer
7. Place sealed tray in 41 ± 0.5°C
8. Turn sealer off
9. Observe any Quanti-Tray for color changes and count the small and large wells that have changed color (the color change will be a lot harder to see than the colicoliforms) and refer to the MPN table
10. Observe the Quanti-Tray under an IDEXX viewing box and UV lamp. Count the number of wells (large and small) that are fluorescent blue
11. Refer to the MPN table provided as you will obtain the most probable number

 

 

 

 

 

 

There’s ‘poop in the water’ at America’s dirtiest beaches

Chris Woodyard

USA TODAY

Editor’s note: After this story was published July 23, Environment America, which produced the study, said it found inaccuracies due to calculation and methodology errors. At that time, USA TODAY explained the problem in an editor’s note and deleted the chart that accompanied the story. On Aug. 2, the group republished its study, saying it had corrected the errors. We have updated this story and have revised the chart to conform with changes in the study. Read more aboutwhat mistakes the group made here.

Error due to:

One was not taking into account that some agencies use a different measurement method for water sampling.

The other was a calculation error involvingthe benchmark number for E. coli, one of the bacteria that come from human or animal feces.

The organization said the changes should not diminish the study’s overall importance.

“The report still has great value – and a big regret is the errors distracted from the overall message of the importance of protecting our water,” said John Rumpler, clean water program director for Environment America.

Under the new calculations, 2,580 of 4,523 beach sites tested nationwide, about 57%, were found to have water pollution levels at least one day in 2018 that exceeded the strictest safety guidelines from the Environmental Protection Agency. That’s down from 2,627 beach sites in the earlier, uncorrected report.

Part of the article:

The Environment America Research and Policy Center study looked at the number of days in 2018 that the water had bacteria counts exceeding Environmental Protection Agency standards, which can potentially sicken swimmers.

Most days of the year, beaches are safe, but researchers found bacteria counts can spike on certain days, and fecal matter from people or animals is the chief culprit. It comes when sewers overflow or when fecal waste on streets washes into streams or creeks that flow to lakes or the ocean. A co-author of the study said more work needs to be done to clean up lakes and oceans.

“It’s hard to believe that 47 years after we passed the Clean Water Act that we are still concerned with poop in the water when people want to go swimming,”

South Shore Beach near Milwaukee would be considered potentially unsafe to swimmers on 34 of out the 60 days in which samples were taken.

Los Angeles’ recreation department referred inquiries to the California Coastal Commission, which issued a statement that said curtailing pollution is a high priority: “Our $45 billion a year coastal economy depends on keeping the water and beaches clean.”

Wisconsin

• South Shore Beach, Milwaukee, (34, 60)  (unsafe days, # samples)
• McKinley Beach, Milwaukee (26, 61)
• Eichelman Beach, Kenosha, (21,51)
• Maslowski Beach, Ashland  (14, 32)
• Nicolet Beach, Door (14, 59)

Group discussion: Why was the error such a big deal?