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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Test Group
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