1. WaterBy: Andrew Paek Nirav Parekh

2. Background  Water is an essential part of our well being  We use it for nourishment, cooking, cleansing, and many other things.  Because of water’s importance, there are methods used to purity water to make safe for human consumption.  Federal regulations are put in place to ensure this safety.

3. Purification  To purify water…  1) Raw water (untreated water) is pumped into a sediment basin where large particles such as sand settle to the bottom.  2) The sediment free water is then pumped into the coagulation basin where the anionic polymers and chlorine added to the sediment basin, which helps form larger insoluble particles out of the suspended tiny particles in the water.  3) The water is filtered to remove remaining solutes, suspended particles and microorganisms

4. Disinfection  Filtered water must be disinfected before it is released as drinking water  Chlorine is the most common disinfectant  Kills microorganisms within 30 minutes  Neutralizes organic compounds  Improves water taste and smell

5. Regulations  There are certain bacteria known as coliforms which are used to indicate if water is contaminated.  Based on the Safe Drinking Water Act, water is only considered safe if the average number of coliform bacteria does not exceed 1 CFU/100 mL  Water utilities report results to the EPA if they don’t meet standards  The public is also informed  Outbreaks are reported to the CDC

6. Hypothesis  We predict that water from various drinking sites on campus will meet federal regulations. As of now, there isn’t any kind of public announcement that the water on campus isn’t safe to drink, so it can be assumed that the water on campus meets federal drinking regulations.  We predict that we will not find any coliforms in the water samples intended for drinking.

7. Methods  Obtained three 100 mL samples of water at five sites on this campus:  Dorm water fountain  Dorm shower  Dorm sink  Outdoor fountain  Diner drinking water  These samples were filtered, incubated, and the number of colonies were counted

8. Membrane Filtration Method  1) Shake each 100ml sample in their containers at least 25 times.  2) Pour 100ml of each sample into the filter apparatus.  3) Allow the water to filter for at least an hour.  4) Pour each of the 2ml ampules of the m- coliblue24 broth into each sample.

9. Rational for MF method  The m-coliblue24 broth is a type of media that selects for coliforms. It’s also a differentiatial media which detects between E.Coli and other coliforms.  The membrane filtration method allows us to count the number of colony forming units which allows us to determine if the water samples meet federal regulations.

10. Plating and Enterotubes  We inoculated the Eosin-methylene blue plates with unique colonies found from the filters.  From the pure colonies we inocoluated enterotubes which tests for various characteristics of the colonies found in the water samples.  These methods are used to identify the coliforms found in the water samples.

11. Expected Results  For the MF technique:  Total coliforms would appear as red colonies.  E.coli would appear as blue colonies.  For the eosin-methylene blue media:  Only coliforms should be able to grow.  Lactose fermenters such as E.coli would appear with a green sheen.

12. Results  No coliforms were detected in these water samples:  Dorm water fountain  Dorm shower  Dorm sink  Diner drinking water  Colonies were found from the outdoor fountain.

13. Filters from the Outdoor fountain Filter 1 5 brown and 3 pink/purple colonies Filter 2 1 brown and 5 pink/purple colonies About 80 tiny purple colonies. Filter 3 No colonies observed.

14. Filter 2

15. EMB Plates Outdoor Filter 1 Control E.Coli

16. Enterotube Results

17. Analysis  Since no coliforms were present in the sites where people drink water, we can conclude that the drinking water here on campus is safe according to federal regulations.  DON’T DRINK WATER FROM THE OUTDOOR FOUNTAINS.  The isolated colonies found on the filter could possibly be from the genus Pseudomonas, which doesn’t produce acids or gas from glucose. These are normally found in soil and water.

18. Implications of coliforms  We’re interested in finding bacteria called coliforms, which are facultatively aerobic, gram- negative, nonspore-forming, rod-shaped bacteria which are able to ferment lactose and produce gas.  Their presence could indicate that the water contains fecal matter.  Coliforms are not necessarily responsible for serious illnesses.  If the water is dirty enough to have fecal matter, chances are that there are pathogens in the water as well.

19. Implications (cont.)  Some of the main microorganisms which cause serious waterborne illnesses:  Typhoid fever – Salmonella typhi  Cholera – Vibrio cholerae  Legionellosis – Legionella pneumophila  Giardiasis – Giardia Intestinalis  Amebiasis – Entamoeba histolytica

20. Frequency of disease in the US  In the year 2001-2002 there were 6 outbreaks caused by the bacteria Legionella which resulted in:  Illnesses in 80 people  41 hospitalizations  Four deaths  This was the most prevalent cause of waterborne illness out of the other causes including chemicals, parasites, viruses, and other bacteria.

21. Legionellosis  An intracellular parasite that grows in alveolar macrophages and monocytes.  Leads to pneumonia or a mild infection known as Pontiac fever  Symptoms:  High fever  Cough  Muscle aches and headaches  Treatment  Antibiotics – rifampin and erythromycin  Prevention  The bacteria can be killed in the water supply through hyperchlorination or heating the water to temperatures greater than 65 degrees Celsius.

22. Protocol Evaluation  Water samples were not dechlorinated which could have inhibited growth.  As seen on the control, some bacteria may not have grown on raised parts of the filter.

23. Comments on the results  In the enterotube that tested the sample from the outdoor fountain, only the last test was positive. This is very unusual since coliforms should be able to ferment some kind of sugar being facultative anerobes. This colony should be tested again for identification.  Only two out of six colonies (including the control) from the filter membranes were able to grow on the EMB agar. The process which the m-coliblue24 broth and the EMB agar select for coliforms may be different between each other. This would cause one media to select against some colonies which may have grown in the other media.

24. Overall significance of the Project  Based on our tests on the water samples across campus, we can conclude that the water is safe to drink.  We can also conclude that water in outdoor fountains contain fecal matter. So it’s advised that nobody drinks or touches it.

25. THE END  Bibliography  Madigan, Michael and Martinko, John. Brock Biology of Microorganisms. Upper Saddle River, NJ: Pearson Prentice Hall. 2006  Centers for Disease Control and Prevention. Surveillance for waterborne-disease outbreaks associated with recreational water — United States, 001–2002 and Surveillance for waterbornedisease outbreaks associated with drinking water — United States, 2001–2002. In: Surveillance Summaries, October 22, 2004. MMWR 2004:53(No. SS-8).