University of Maryland
The MdBio Foundation
Anne Arundel Community College
Howard Hughes Medical Institute
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Understand the importance of scientific research in
improving human health
Students will
• Learn about watershed ecology, sources and health
consequences of bacterial contamination, and factors
that affect levels of bacterial contamination (e.g.,
patterns of land use, rainfall)
• Design simple, hypothesis-driven investigations and
will test their hypotheses with water samples they
collect near their schools
• Compare their results to those obtained by students
state-wide via a project website
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Severe Bacterial
Infection
Targets Small
Intestine
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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
“The world has seen seven global cholera
outbreaks since 1817, and the current one
seems to have come to stay. Rising
temperatures and a stubbornly persistent, toxic
bacteria strain appear to have given the disease
the upper hand.”
By Doug Struck, DailyClimate.org
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Causative agent of
cholera
Symptoms caused by
bacterial toxin (CTX
or cholera toxin)
http://dhiez.files.wordpress.com/2008/05/cholera.jpg
Scanning Electron Micrograph of Vibrio cholerae O1
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Primarily passed in
contaminated drinking
water and shellfish
Harbored in
zooplankton
outbreaks often follow
zooplankton blooms
 Water temperature
Dependent

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June 23, 2010; Kenya
Last outbreak in US was
in 1911

Water chlorination,
ozone, UV, or cloth
filter and boiling
http://www.nation.co.ke/News/regional/Crisis%20feared%20as%20cholera%20outbreak%20kills%2060/-/1070/945376/-/t2lpkm/-/
http://gamapserver.who.int/mapLibrary/Files/Maps/Global_CholeraCases0709_20091008.png
Cholera outbreak in Haiti
December 30, 2010
• First reported case: 2nd week of October, 2010
• As of December 30, 2010: reported hospitalized cases:70,865
• Reported Deaths: 2,761 deaths
Overall fatality rate of 2.1 per cent nationwide.
The fatality rates in South- East reached to 12-13% in early December.
http://ochaonline.un.org/tabid/6412/language/en-US/Default.aspx
Climate Change and Health
Climate can have a profound influence on health, both directly and
indirectly
Direct effects include deaths and illnesses related to excessive heat or
cold exposure
Indirect effects via waterborne vector borne diseases influenced by:
occurrence and geographic distribution of known vectors, e.g.,
mosquitoes, rodents and copepods have been implicated with diseases,
such as
Malaria,
Dengue fever
Cholera
Encephalitis &
Haemorrhagic fever.
Hanta virus
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People with Type O
are most susceptible,
AB least susceptible
Heterozygous carriers
of Cystic Fibrosis gene
have some protection
Malnourishment or
immunocompromized
1915
http://en.wikipedia.org/wiki/Cholera
Sunlight
Precipitation
Temperature
Nutrients
Disease
Outbreak
Phytoplankton: micro &
macro-algae
Zooplankton &
Animal larvae
Increase of
V. cholerae population
Phage – another player?
Infectious dose is now readily
available
Infectious Dose of Bacteria that Can Cause
Cholera
(Cash et al, 1974)
Inoculum
Symptom
10,000 with antacids
Mild diarrhea
1,000,000 with food
Severe diarrhea
>1,000,000 with water
Severe diarrhea
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Vibrio’s infect cuts
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“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
“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).
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 200
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There are 10-100 times
more microbes in
your gut than cells in
your body with your
DNA
Source: www.nature.com
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Benefits the bacteria
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We keep them warm
We protect them from
the environment
We send food
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Benefits Us
Bacteria help fight off
pathogens
 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
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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
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Enterococcus faecalis
Part of normal flora
of all mammals and
birds
 About 10 million
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
Coliform bacteria include genera that originate in feces
(e.g. Escherichia) as well as genera not of fecal origin
(e.g. Enterobacter, Klebsiella, Citrobacter).
Basics of fecal coliforms
In general, increased levels of fecal coliforms provide a
warning of failure in water treatment, a break in the
integrity of the distribution system, or possible
contamination with pathogens. When levels are high
there may be an elevated risk of waterborne
gastroenteritis. Tests for the bacteria are cheap,
reliable and rapid (1-day incubation).
http://en.wikipedia.org/wiki/Fecal_coliform
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Can cause:
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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
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Naturally antibiotic resistant to penicillins
Acquired resistance to many other antibiotics
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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
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Step 1- Collect water
samples
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
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Step 3- (Next Day)
Count Colonies
Example of bacterial growth on
selective media. Photo courtesy of
Hornor Lab, Anne Arundel
Community College, Arnold, MD.
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Completely
assembled filtration
apparatus
Water samples in ice
bucket
Field data sheet
Sterile 10 ml syringe
Beaker with ethanol
holding forceps
Sterile paper filter
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Forceps removed
from ethanol, flamed
THEN handed to
students
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Peel cover off filter
(best done by
instructor or partner)
Grab edge with
sterilized forceps
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Place paper filter grid
side up on top of
metal screen
Paper must
completely cover
screen to get proper
filtration
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Place filter funnel on
top of paper filter
Clamp glassware in
place
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Wet filter with 10 ml
sterile, distilled water
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Water removes static
from syringe
When the water has
suctioned through
filter, apply 10 ml of
water sample to filter
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With clean syringe,
wash the sides of the
funnel to get any
splashes
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Unclamp filter funnel
Flame forceps
Grab edge of filter
and break vacuum
seal
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Hold plate tilted
downward and away
Place filter at bottom
edge of plate
Roll onto media to
minimize bubbles
Cover and incubate 24
hrs
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Place new filter on
filtration apparatus
Wet filter and suction
through
Pour 100 ml into
funnel
Wash sides of funnel
Place filter on media
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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
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http://www.youtube.com/watch?feature=pla
yer_embedded&v=o7kB7-UN7m4