What Lives In Your Water?MdBioLab Activity

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

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

Vibrio cholerae

• Causative agent of cholera

• Symptoms caused by bacterial toxin (CTX or cholera toxin)

http://dhiez.files.wordpress.com/2008/05/cholera.jpg

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/-/

Cholera Outbreaks

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

Oithona spp female with eggs

Acartia tonsa

Eurytemora affinis, female with eggs

Source: T. Rawlings

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2-year Study

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

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

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

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

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

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

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

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.

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

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