EX 24 - EXAMINATION OF WATER - MOST PROBABLE NUMBER
EXAMINATION FOR COLIFORMS IN WATER
What are coliforms?
Coliforms are rod-shaped Gram-negative, facultative, non-spore forming, lactose fermenting bacteria that can be either motile or non-motile. Coliforms find their home in the digestive tract of mammals and can be found in plants, water and soil. E. coli is an example of a coliform bacteria. While the majority of E. coli species are beneficial to humans as they remain in the digestive tract. |
E. coli
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How do we test for lactose fermentation?
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Bacteria that have the ability to ferment lactose, will produce 1) acid and 2) gas when incubated at 35–37°C in the presence of lactose. |
Why do we test for coliforms in water?
In the United States, the Centers for Disease Control and Prevention (CDC) mandates that pubic water supplies be continuously monitored for the presence of coliforms. When coliforms are present in the water supply, they indicate that the water has likely been contaminated with feces or soil. If they are present in drinking water, the CDC will shut down the facility until the problem is cleared up.
How is a positive coliform test interpreted?
A positive coliform test indicates that there is a high probability that the water supply may carry pathogenic organisms that can lead to a variety of waterborne illnesses if consumed. A positive test for total coliforms always requires more tests for fecal coliforms or E. coli.
Are coliform bacteria harmful?
The vast majority of coliform bacteria are harmless or even beneficial to humans. Even though they usually do not cause disease, their presence indicates that pathogenic bacteria may be present!
Which coliforms are harmful?
Some sushi contains raw fish.
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There are a small number of coliform bacteria that can cause disease. For example, a strain of E. coli called E. coli O157:H7, can cause serious illness in humans. Illness from E. coli O157:H7 typically result from contaminated foodstuffs that are consumed raw or undercooked. Outbreaks of E. coli O157:H7 have very rarely come from drinking water.
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How do we test for coliforms?
The test for coliforms in water in done in three distinct phases:
1) The Presumptive Test
2) The Confirmation Test
3) The Completed Test
Phase 1) The Presumptive Test
Directions:
Set One:
- Obtain three test tubes containing double-strength lactose broth.
- Add 10 ml of water to each of these tubes.
- Since 1 ml of water = 1 gram, and we have 10 ml in our tubes, we have 10g of our water sample in each tube of our Set One.
Set Two:
- Obtain three centrifuge tubes containing single-strength lactose broth.
- Add 1 ml (1000 ul) of water to each of these tubes.
- Since 1 ml of water = 1 gram, and we have 1 ml in our tubes, we have 1g of our water sample in each tube of our Set Two.
Set Three:
- Obtain three centrifuge tubes containing single-strength lactose broth.
- Add 0.1 ml (100 ul) of water to each of these tubes.
- Since 1 ml of water = 1 gram, and we have 0.1 ml in our tubes, we have 0.1g of our water sample in each tube of our Set Three.
Watch this Video for a Demonstration of the Inoculations. Remember that the "sample" we are using is water.
- Incubate all tubes overnight for ~18 hours at 42 degrees Celsius.
- After the incubation, the tubes may be placed in the refrigerator (4 degrees Celsius) until the lab period.
- At the next lab period, examine each of the tubes for the presence of gas.
- You will use an MPN table to calculate the Most Probable Number (MPN).
- Then we will go on to the confirmation test, because right now, all we know is that we have bacteria that ferment lactose. They may not be Gram negative rods.
What is the most probable number (MPN) method?
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The most probable number method is also known as the method of Poisson zeroes. It is a method for quantifying, to some degree of accuracy, the presence of something that is difficult to directly observe. |
In order to find the MPN, count the number of tubes in each set that were positive for gas (lactose fermentation:
Set One) 10 ml in double-strength lactose broth
Set Two) 1 ml in single-strength lactose broth
Set Three) 0.1 ml in single-strength lactose broth
Example: If you have 2 tubes with gas in the first and last set, 3 tubes with gas in the second set and 2 tubes with gas in the last set, using the table, you should find that your MPN is 43. That means that you have ~43 organisms per 100 ml.
Example: If you have 2 tubes with gas in the first and last set, 3 tubes with gas in the second set and 2 tubes with gas in the last set, using the table, you should find that your MPN is 43. That means that you have ~43 organisms per 100 ml.
Phase 2) The Confirmation Test
We will use two media which are selective and differential. Each group of two people will work with one of each plate. Use the water sample to streak for isolation on the plates.
EMB is selective because it has eosin and methylene blue, which only grow Gram negative organisms. It is differential because it shows lactose fermentation. Colonies with a black center is a positive confirmation test, since only coliforms will form colonies with black nuclei on EMB agar. When they show a green metallic sheen, that is positive for lactose fermentation.
MacConkey’s agar is selective because it has crystal violet and bile salts, which only grow Gram negative organisms. It is differential because it shows lactose fermentation by turning a pink color. Pink is positive for lactose fermentation.
Whether the confirmation tests were positive or not, we go on to the completed test, because right now, all we know is that we have Gram negative bacteria that ferment lactose. The may not be rods.
EMB is selective because it has eosin and methylene blue, which only grow Gram negative organisms. It is differential because it shows lactose fermentation. Colonies with a black center is a positive confirmation test, since only coliforms will form colonies with black nuclei on EMB agar. When they show a green metallic sheen, that is positive for lactose fermentation.
MacConkey’s agar is selective because it has crystal violet and bile salts, which only grow Gram negative organisms. It is differential because it shows lactose fermentation by turning a pink color. Pink is positive for lactose fermentation.
Whether the confirmation tests were positive or not, we go on to the completed test, because right now, all we know is that we have Gram negative bacteria that ferment lactose. The may not be rods.