What Lives In Your Water?MdBioLab Activity

Basic Learning Goals

• Students will understand the connection between water quality and– Economics of Maryland• Fishing• Tourism

• Students will learn laboratory techniques identical to those used by EPA and scientists

Water cycle

Conceptualization of the water cycle. Source: USGCRP website

http://chesapeake.towson.edu/landscape/impervious/all_watersheds.asp

Stream Orders

Stream Length in Mid-Atlantic

http://www.epa.gov/bioiweb1/pdf/EPA-620-R-06-001MAIAStateoftheFlowingWaters.pdf

http://www.chesapeakebay.net/watersheds.aspx?menuitem=14603

Chesapeake Bay Watershed

Chesapeake Bay Sub-watersheds

http://pubs.usgs.gov/circ/circ1316/circular1316.pdf

Why Should I Worry?

• Cholera is a huge killer worldwide– Almost nonexistent in

the US due to our water treatment policies

– Kills millions of people every year in the developing world

http://en.wikipedia.org/wiki/Cholera

1915

Cholera Outbreaks

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

Vibrio spp in the Chesapeake Bay• Cholera caused by bacteria

Vibrio cholerae• 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

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

Sources of Fecal Pollution

http://www.ars.usda.gov/Main/docs.htm?docid=11769

Sources of Fecal Pollution

http://static.gotpetsonline.com/pictures-gallery/dog-pictures-breeders-puppies-rescue/english-shepherd-dog-pictures-breeders-puppies-rescue/pictures/english-shepherd-dog-0003.jpg

http://jakst.files.wordpress.com/2009/07/cat.jpg

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

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

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

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.

Water Does Not Have To Look Dirty To Be Dangerous!!

LAB ACTIVITIES

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 samplehttp://ian.umces.edu/imagelibrary/albums/userpics/10025/normal_iil_ian_bf_395.JPG

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

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.

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

Sterile Technique

• Forceps removed from ethanol, flamed

• THEN handed to students

Place Filter 1

• Peel cover off filter (best done by instructor or partner)

• Grab edge with sterilized forceps

Place Filter 2• Place paper filter grid

side up on top of metal screen

• Paper must completely cover screen to get proper filtration

Reassemble Filtration Apparatus

• Place filter funnel on top of paper filter

• Clamp glassware in place

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

Wash Filter Funnel

• With clean syringe, wash the sides of the funnel to get any splashes

Remove Filter

• Unclamp filter funnel• Flame forceps• Grab edge of filter and

break vacuum seal

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

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

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

POST-LAB ACTIVITIES

Reporting Results

Land Use

• Impervious Surface• Farming

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

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