1. What Lives In Your Water? MdBioLab Activity

2. Basic Learning Goals Students will understand the connection between water quality and Human health Waterborne illness Students will learn laboratory techniques identical to those used by EPA and scientists

3. Cholera Severe Bacterial Infection Targets Small Intestine – Profuse watery diarrhea (gallons per day) – Vomiting – Leads to dehydration and electrolyte loss 20% of body weight in 24 hrs http://commons.wikimedia.org/wiki/File:Cholera_hospital_in_Dhaka.jpg

7. Vibrio cholerae Causative agent of cholera Symptoms caused by bacterial toxin (CTX or cholera toxin) http://dhiez.files.wordpress.com/2008/05/cholera.jpg

8. Cholera Kills in Developing Nations Primarily passed in contaminated drinking water and shellfish Harbored in zooplankton – outbreaks often follow zooplankton blooms – Water Temperature Dependent Last outbreak in US in 1911 – Water chlorination, ozone, UV, or cloth filter and boiling June 23, 2010; Kenya http://www.nation.co.ke/News/regional/Crisis%20feared%20as%20cholera%20outbreak%20kills%2060/-/1070/945376/-/t2lpkm/-/

9. Cholera Outbreaks http://gamapserver.who.int/mapLibrary/Files/Maps/Global_CholeraCases0709_20091008.png

10. Oithona spp female with eggs Acartia tonsa Eurytemora affinis, female with eggs Source: T. Rawlings

12. OLD SARI NEW SARI

16. Susceptibility to Cholera People with Type O most susceptible, AB least susceptible Heterozygous carriers of Cystic Fibrosis gene have some protection – Similar to sickle cell trait in malaria Malnourishment or immunocompromized http://en.wikipedia.org/wiki/Cholera 1915

17. Vibrio spp in the Chesapeake Bay Vibrio infect cuts – “hand swollen to the size of a catchers mitt” Infected shellfish cause GI illness Public health websites suggest to protect yourself against infection: – Avoid swimming 48 hours after any heavy rainfall. – Do not swim with an open cut or wound. – If you get cut while in the water, wash it thoroughly and cover with a waterproof bandage. – Try not to swallow water while swimming. Chesapeake Bay Foundation. 2009. Bad Water 2009

18. Fecal Bacteria “Where do the bacteria come from? There are about 180 failing septic tanks in the Severn River’s suburbanized watershed, according to the Maryland Department of the Environment (MDE). But a far more significant source of bacteria in the river is pet waste, which produces an estimated 69 percent of the E. coli bacteria in Voith’s section of the Severn River, with wildlife contributing 24 percent, livestock three percent, and humans three percent, according to an April 2008 MDE analysis of pollution in the Severn River. About 41 percent of dog owners in the area admit they do not pick up after their animals most of the time, the report says. “ Chesapeake Bay Foundation. 2009. Bad Water 2009 Disgusting Picture Warning

19. Our GI Microflora There are 10-100 times more microbes in your gut than cells in your body with your DNA Source: www.nature.com

20. This is a two-way street Benefits the bacteria – We keep them warm – We protect them from the environment – We send food Benefits Us – Bacteria help fight off pathogenic bacteria (the bad guys) – Bacteria help us digest our food – Bacteria produce useful products for us (e.g., Vitamins) – Bacteria also interact with our body systems to help us keep balanced

21. The Operative Word is BALANCE When pathogenic bacteria invade us, we usually get sick When good bacteria end up in the wrong place, we sometimes get sick When bacteria get into improper balance (“dysbiosis”), we can get sick When our immune system gets confused about the good guys, we can get sick http://commons.wikimedia.org/wiki/File:Balanced_scales.svg

22. THE REST OF THESE SLIDES APPLY TO THE ENVIRONMENTAL FOCUS AS WELL

23. One Member of the GI Microflora Enterococcus faecalis – Part of normal flora of all mammals and birds – About 10M Enterococci per gram of human feces. – Gram-positive cocci, facultative anaerobe – Tolerate a wide range of growth conditions including salt and oxygen Enterococcus faecalis infecting lung tissue. Source: Wikipedia

24. Opportunistic pathogen Can cause: – Bladder infections – Endocarditis (infection of heart lining) – Bacteremia (bacteria in blood) – Peritonitis (infection in abdominal cavity) – Meningitis (brain infection) Most cases are hospital-acquired (“nosocomial”) infections Hard to treat – Naturally antibiotic resistant to penicillins – Acquired resistance to many other antibiotics

25. E. Faecalis is a Good Indicator Organism in the Environment Stays alive but doesn’t grow in environment So… numbers stay constant So…counts are representative of volume of pollution sources Scanning Electron Micrograph of Enterococcus faecalis. Sources: CDC Public Health Image Library (PHIL), Photo by Janice Haney Carr http://phil.cdc.gov/Phil/details.asp

26. Culturing Bacteria in the Lab We create the optimal growth conditions – Temperature – Nutrients – pH Selective media – Contains chemicals that only allow one species to grow Example of bacterial growth on selective media. Photo courtesy of Hornor Lab, Anne Arundel Community College, Arnold, MD.

27. Our Activity Step 1- Collect water samples – Field trip or Homework Students should work in pairs Will require a “collection kit” – Clean plastic bottles – Gloves – Ziplocs for ice and containment of sample http://ian.umces.edu/imagelibrary/albums/userpics/10025/normal_iil_ian_bf_395.JPG

28. Our Activity Step 2- Filter water samples and culture overnight – 2 different volumes 10 ml 100 ml Allows for best opportunity to get a countable plate of 20-60 colonies http://www.umesc.usgs.gov/aquatic/drug_research/capabilities.html

29. Our Activity Step 3- (Next Day) Count Colonies Example of bacterial growth on selective media. Photo courtesy of Hornor Lab, Anne Arundel Community College, Arnold, MD.

30. Equipment Setup Completely assembled filtration apparatus Water samples in ice bucket Field data sheet Sterile 10 ml syringe Beaker with ethanol holding forceps Sterile paper filter

31. Sterile Technique Forceps removed from ethanol, flamed THEN handed to students

32. Place Filter 1 Peel cover off filter (best done by instructor or partner) Grab edge with sterilized forceps

33. Place Filter 2 Place paper filter grid side up on top of metal screen Paper must completely cover screen to get proper filtration

34. Reassemble Filtration Apparatus Place filter funnel on top of paper filter Clamp glassware in place

35. 10 ml Sample Wet filter with 10 ml sterile, distilled water – Water removes static from syringe When the water has suctioned through filter, apply 10 ml of water sample to filter

36. Wash Filter Funnel With clean syringe, wash the sides of the funnel to get any splashes

37. Remove Filter Unclamp filter funnel Flame forceps Grab edge of filter and break vacuum seal

38. Place on Plate Hold plate tilted downward and away Place filter at bottom edge of plate Roll onto media to minimize bubbles Cover and incubate 24 hrs

39. Repeat for 100 ml Place new filter on filtration apparatus Wet filter and suction through Pour 100 ml into funnel Wash sides of funnel Place filter on media

40. After Incubation This is what the students will see after a 24 hour incubation at 41˚C (chicken body temperature) Left-hand plates came from Patuxent River Right-hand plates came from Warehouse Creek off South River Top plates are 10 ml, bottom plates are 100ml samples

41. POST-LAB ACTIVITIES

42. Reporting Results

43. Land Use Impervious Surface Farming

44. Civic Engagement Opportunities Information can be reported to local water quality monitoring agencies Community Associations to encourage picking up after pets Service projects to fence streams from livestock

45. Curriculum Materials Provided at MdBioLab Website Instructor’s Manual – Biomedical – Environmental Science Student Handout Field Data Collection Sheet Powerpoint Slides (with speakers notes) – The ones shown today – A set to show to students with a Biomedical focus – A set to show to students with an Environmental Science focus