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
June 23, 2010; Kenya
• Last outbreak in US 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/-/
Cholera Outbreaks
http://gamapserver.who.int/mapLibrary/Files/Maps/Global_CholeraCases0709_20091008.png
Oithona spp female with eggs
Eurytemora affinis, female with eggs
Acartia tonsa
Source: T. Rawlings
OLD SARI
NEW SARI
•
Cases of Cholera Per 1000
Population
2-year Study
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
Control
Sari
Test Group
Nylon
Susceptibility to Cholera
• People with Type O
most susceptible, AB
least susceptible
• Heterozygous carriers of
Cystic Fibrosis gene
have some protection
1915
– Similar to sickle cell trait
in malaria
• Malnourishment or
immunocompromized
http://en.wikipedia.org/wiki/Cholera
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. “
Disgusting
Picture
Warning
Chesapeake Bay Foundation. 2009. Bad Water 2009
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