In the confirmatory test, a sample is taken from the most dilute (lowest amount of water) lactose fermentation tube and streak plated on EMB agar. This is important confirmation of the lactose fermentation tube results, since false positives in these tubes are possible. For example, acid from chemicals in the water could turn the broth yellow. Similarly, mishandling of the tubes could cause a false bubble to be trapped in the Durham tube.
Eosin Methylene Blue (EMB) agar plates are both selective and differential. They are selective because they contain the dyes eosin Y and methylene blue, which inhibit the growth of Gram-positive bacteria. So, only Gram-negative bacteria will show good growth on EMB.
They are also differential since they contain lactose and the mentioned dyes, which give a reddish color originally. If the bacteria on the EMB agar plate ferments lactose, it will produce acid waste products that will turn the bacteria purplish-pink or a shiny metallic green. The pink color indicates slow fermentation of lactose, while the shiny metallic green indicates fast fermentation of lactose.
Good growth with pink or green color will confirm that coliforms are present in the water sample.
Slide 12
In this sample, the streak on the far left is clear, showing the red color of the agar behind it. Since this bacteria had good growth, it is indeed Gram-negative. However, the color indicates that it does not ferment lactose. It is not a coliform. Note: any color other than pink or green is also negative for lactose fermentation. Other common colors may include white or tan, depending upon the species of bacteria.
The streak in the middle is purplish-pink. The good growth indicates that the bacteria is Gram-negative while the pink color means it ferments lactose. It is a confirmed coliform.
The streak on the far right is bright, metallic green. The good growth indicates that the bacteria is Gram-negative while the green color means it ferments lactose. It is a confirmed coliform.
If there is no growth on a plate, that means any bacteria present is Gram-positive. That sample would be negative for coliforms.
Slide 13
Confirmation of coliforms on an EMB agar plate tells you that your lactose fermentation tube results are reliable. So, your MPN estimate (coliforms per 100 mL) is likely to be accurate.
However, if there are no coliforms on the EMB culture, this tells you that your lactose fermentation tubes may have been false positives. So, your MPN estimate is likely to be wrong.
Slide 14
If you are interested in reading more, visit https://www.epa.gov/your-drinking-water
Slide 15
Slide 16
Sewage Treatment
In the U.S., sewage is treated to kill or remove contaminants before it is released.
Fortunately, treatment of sewage helps prevent fecal contamination of water.
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Water Treatment•In the U.S. drinking water is monitored for fecal contamination and treated…
Water Treatment
In the U.S. drinking water is monitored for fecal contamination and treated…
Additionally, drinking water can be tested for fecal contamination. In lab, we will conduct a common type of test for fecal contamination of water.
Drinking water is also routinely tested for other contaminates (such as lead). In the U.S. this information is publicly available at: https://www.epa.gov/your-drinking-water (You are not responsible for the information on this site.)
In many cities around the world, drinking water may also be treated. Treatments include filtration and disinfection. Minerals such as fluoride may be added.
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Monitoring Fecal Coliforms•Coliforms are normal bacteria of the intestines, and are not necessarily dangerous.–Why monitor coliforms in water?Because coliforms (normal intestinal bacteria) are an indication that the water has been contaminated with feces, which could spread disease.
Monitoring Fecal Coliforms
Coliforms are normal bacteria of the intestines, and are not necessarily dangerous.
Why monitor coliforms in water?
Because coliforms (normal intestinal bacteria) are an indication that the water has been contaminated with feces, which could spread disease.
In testing for fecal contamination of water, we will try to determine if our water sample contains coliforms – normal microbiota of the intestinal tract. These bacteria themselves are not dangerous. However, the presences of coliforms means that there is feces in the water. As we saw, fecal contamination of water can spread many different types of diseases.
Looking for coliforms, rather than true enteric pathogens (microbes that cause disease in the intestines), is also a more reliable way to detect fecal contamination. Under normal (non-epidemic) conditions, only a small fraction of people will shed pathogenic microbes in their feces. In addition, there are many different types of enteric pathogens. So, it would be very difficult and unreliable to detect the small amount of diverse enteric pathogens that might be present in contaminated water. Coliforms, however, are abundant in all feces.
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•Consists of two parts:–Presumptive test: uses multiple lactose fermentation broth tubes to detect coliforms–Confirmatory test: uses Eosin Methylene Blue (EMB) agar platesto confirm the presence of coliformsMultiple Tube Technique/Most Probably Number Method
Consists of two parts:
Presumptive test: uses multiple lactose fermentation broth tubes to detect coliforms
Confirmatory test: uses Eosin Methylene Blue (EMB) agar plates to confirm the presence of coliforms
Multiple Tube Technique/Most Probably Number Method
The test we will conduct in lab is called the Multiple Tube Technique or Most Probably Number Method. Both of these names refer to the same test. This test has two parts:
Presumptive test – uses multiple lactose fermentation broth tubes to detect coliforms. (This is how the test got the name “Multiple Tube Technique.”)
Confirmatory test – uses Eosin Methylene Blue (EMB) agar plates to confirm the presence of coliforms. (This ensures that presumptive test results were not false positives.)
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Multiple Tube Technique/Most Probably Number Method•Presumptive test -uses multiple lactose fermentation tubes to test for the presence of coliforms.–Why might it be more accurate to test water in many tubes instead of just one or a few?With more tubes, there is more of a chance that you will find the coliforms if they are present.Multiple tubes at different dilutions also allows for an estimate of how many coliforms are present.
Multiple Tube Technique/Most Probably Number Method
Presumptive test - uses multiple lactose fermentation tubes to test for the presence of coliforms.
Why might it be more accurate to test water in many tubes instead of just one or a few?
With more tubes, there is more of a chance that you will find the coliforms if they are present.
Multiple tubes at different dilutions also allows for an estimate of how many coliforms are present.
In our presumptive test, we will use nine lactose fermentation tubes:
3 with 10 mL of water
3 with 1 mL of water
3 with 0.1 mL of water
Repeating the test over multiple tubes makes it less likely that we may miss coliforms in a contaminated sample. In other words, there is a greater chance of reliably detecting coliforms if they are present.
Testing different amounts of water will allow us to get an estimate of the likely number of coliforms per 100 mL of water. This number is called the MPN, which stands for Most Probable Number (of Coliforms). That is why this same method is also referred to as the Most Probably Number Method.
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Lactose Fermentation Tubes•Is a differential media –contains the carbohydrate lactose, the indicator bromocresolpurple, and a small, inverted Durham tube.–If the organism cultured ferments lactose, it will produce acidic waste products. The indicator will turn from purple to yellow at acidic pH.–If the organism produces gas when fermenting lactose, an air bubble will be present in the Durham tube.
Lactose Fermentation Tubes
Is a differential media – contains the carbohydrate lactose, the indicator bromocresol purple, and a small, inverted Durham tube.
If the organism cultured ferments lactose, it will produce acidic waste products. The indicator will turn from purple to yellow at acidic pH.
If the organism produces gas when fermenting lactose, an air bubble will be present in the
Durham tube.
The lactose fermentation broth tubes are very similar to the glucose fermentation broth tubes we used in lab before. Both are differential broths.
Lactose fermentation tubes contain the carbohydrate lactose and the indicator dye bromocresol purple which gives it a purple color originally, but can change to yellow with an acid pH. If lactose is fermented by the bacteria, acidic waste products are made and the broth turns yellow.
This broth also includes a special Durham tube – a tiny glass tube that is upside-down (inverted) in the broth. This Durham tubes starts out filled with broth. However, if carbon dioxide gas is produced from fermentation of lactose, some of this gas will be trapped in the Durham tube, forming a bubble.
If there are coliforms growing in the lactose fermentation tube, they will be positive for both lactose fermentation and gas production.
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Lactose Fermentation Tube ResultsNegative for lactose fermation and gas productionPositive for lactose fermation and gas production
Lactose Fermentation Tube Results
Negative for lactose fermation and gas production
Positive for lactose fermation and gas production
The example on the left shows a purple tube that is negative for lactose fermentation. You will also notice that there is no bubble in the Durham tube, which means there is no gas production. The bacteria in this tube cannot ferment lactose or produce gas from lactose fermentation.
The example on the right shows a yellow tube that is positive for lactose fermentation. Although this example shows a tube that is completely yellow, even a partial change to yellow (any amount of yellow) is a positive result for lactose fermentation. You will also notice that there is a large bubble at the top of the Durham tube, which means it is also positive for gas production. The bacteria in this tube are able to ferment lactose and produce gas from lactose fermentation. This tube likely contains coliforms.
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Multiple Tube Technique/Most Probably Number Method•Confirmatory test -uses Eosin Methylene Blue (EMB) agar platesto confirm the presence of coliforms.–Why might it be necessary to confirm the results of the lactose fermentation tubes?Some tube results may be false positives.
Multiple Tube Technique/Most Probably Number Method
Confirmatory test - uses Eosin Methylene Blue (EMB) agar plates to confirm the presence of coliforms.
Why might it be necessary to confirm the results of the lactose fermentation tubes?
Some tube results may be false positives.
In the confirmatory test, a sample is taken from the most dilute (lowest amount of water) lactose fermentation tube and streak plated on EMB agar. This is important confirmation of the lactose fermentation tube results, since false positives in these tubes are possible. For example, acid from chemicals in the water could turn the broth yellow. Similarly, mishandling of the tubes could cause a false bubble to be trapped in the Durham tube.
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Eosin Methylene Blue (EMB) Agar•Is a selective media –contains the dyes eosin Y and methyleneblue, which inhibit the growth of Gram positive bacteria.•Is a differential media –contains the carbohydrate lactose and eosin Y and methyleneblue indicators.–If the organism cultured ferments lactose, it will produce acidic waste products. The indicators turn bacteria green or purplish pink at acidic pH.(Green = fast fermentation, pink = slow)
Eosin Methylene Blue (EMB) Agar
Is a selective media – contains the dyes eosin Y and methylene blue, which inhibit the growth of Gram positive bacteria.
Is a differential media – contains the carbohydrate lactose and eosin Y and methylene blue indicators.
If the organism cultured ferments lactose, it will produce acidic waste products. The indicators turn bacteria green or purplish pink at acidic pH. (Green = fast fermentation, pink = slow)
Eosin Methylene Blue (EMB) agar plates are both selective and differential. They are selective because they contain the dyes eosin Y and methylene blue, which inhibit the growth of Gram-positive bacteria. So, only Gram-negative bacteria will show good growth on EMB.
They are also differential since they contain lactose and the mentioned dyes, which give a reddish color originally. If the bacteria on the EMB agar plate ferments lactose, it will produce acid waste products that will turn the bacteria purplish-pink or a shiny metallic green. The pink color indicates slow fermentation of lactose, while the shiny metallic green indicates fast fermentation of lactose.
Good growth with pink or green color will confirm that coliforms are present in the water sample.
11
EMB Results
EMB Results
In this sample, the streak on the far left is clear, showing the red color of the agar behind it. Since this bacteria had good growth, it is indeed Gram-negative. However, the color indicates that it does not ferment lactose. It is not a coliform. Note: any color other than pink or green is also negative for lactose fermentation. Other common colors may include white or tan, depending upon the species of bacteria.
The streak in the middle is purplish-pink. The good growth indicates that the bacteria is Gram-negative while the pink color means it ferments lactose. It is a confirmed coliform.
The streak on the far right is bright, metallic green. The good growth indicates that the bacteria is Gram-negative while the green color means it ferments lactose. It is a confirmed coliform.
If there is no growth on a plate, that means any bacteria present is Gram-positive. That sample would be negative for coliforms.
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Multiple Tube Technique/Most Probably Number Method•If the EMB culture does not confirm the presence of coliforms, is the estimate of coliforms per 100mL in the water source reliable?No, because your results were due to something other than coliforms.
Multiple Tube Technique/Most Probably Number Method
If the EMB culture does not confirm the presence of coliforms, is the estimate of coliforms per 100mL in the water source reliable?
No, because your results were due to something other than coliforms.
Confirmation of coliforms on an EMB agar plate tells you that your lactose fermentation tube results are reliable. So, your MPN estimate (coliforms per 100 mL) is likely to be accurate.
However, if there are no coliforms on the EMB culture, this tells you that your lactose fermentation tubes may have been false positives. So, your MPN estimate is likely to be wrong.
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Government Limits for Coliforms•In the U.S. the Environmental Protection Agency (EPA) sets limits for the amount of coliforms in drinking water.–The maximum contaminant health goal (a recommendation) is 0% coliform-positive tubes.–The maximum coliform limit (a legal limit) is less than 5% coliform-positive tubes.
Government Limits for Coliforms
In the U.S. the Environmental Protection Agency (EPA) sets limits for the amount of coliforms in drinking water.
The maximum contaminant health goal (a recommendation) is 0% coliform-positive tubes.
The maximum coliform limit (a legal limit) is less than 5% coliform-positive tubes.
If you are interested in reading more, visit https://www.epa.gov/your-drinking-water
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You Should Be Able To:1.Identify what type of bacteria are detected in the MTT/MPN technique. Explain why it is important to monitor this type of bacteria in drinking water.2.For lactose fermentation tubes and EMB, know whether these are selective or differential. a.Know what type of bacteria can grow in these media.b.Know what characteristic(s) are being differentiated. c.Be able to interpret results from each of these types of media.
You Should Be Able To:
Identify what type of bacteria are detected in the MTT/MPN technique. Explain why it is important to monitor this type of bacteria in drinking water.
For lactose fermentation tubes and EMB, know whether these are selective or differential.
Know what type of bacteria can grow in these media.
Know what characteristic(s) are being differentiated.
Be able to interpret results from each of these types of media.
You Should Be Able To:3.Know how to use lactose fermentation tube results to estimate the most probable number of coliformsper 100ml water. (Chart will be provided on exam.)4.Explain why it is important to use multiple tubes in the MTT/MPN method.5.Explain why it is important to confirm your results on EMB agar. Be able to use EMB results to determine whether your coliformestimate was reliable or unreliable.
You Should Be Able To:
Know how to use lactose fermentation tube results to estimate the most probable number of coliforms per 100ml water. (Chart will be provided on exam.)
Explain why it is important to use multiple tubes in the MTT/MPN method.
Explain why it is important to confirm your results on EMB agar. Be able to use EMB results to determine whether your coliform estimate was reliable or unreliable.
Water Quality
Diseases Spread by Fecal Contamination of Water•Cholera•Giardia•Dysentery•Hepatitis A•And many more…
Diseases Spread by Fecal Contamination of Water
Cholera
Giardia
Dysentery
Hepatitis A
And many more…
Disease can spread when people drink, prepare food, or bathe in water contaminated with pathogenic microbes. The most common source of pathogenic microbes in water is fecal contamination (feces in the water). Many types of microbes can spread through fecal contamination of water including:
Cholera – a diarrheal disease caused by the bacteria Vibrio cholera from human feces
Giardiasis – another diarrheal disease caused by protozoan Giardia species from animal feces (animals such as beavers can be a reservoir for this microbe)
Dysentery – a broad category of diarrheal diseases caused by a wide variety of microbes
Hepatitis A – a disease that can cause digestive distress and liver problems caused by the Hepatitis A virus from human feces
There are also many other examples.
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Sewage Treatment•In the U.S., sewage is treated to kill or remove contaminants before it is released.
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