Microbiological Examination of
Water
January 17, 2007
Dr. Paul F. Vendrell
Agenda
• Water-borne diseases
• Pathogen indicators
– Coliform
– Streptococcus
– Enterococcus
• Enumeration Methods
– Membrane filter
– Multiple tube fermentation
• Surface Water Standards
• How much water can wild
birds contaminate?
• Survival and Transport
Water borne pathogens
1991 Cholera Epidemic
• Bacteria 1,000,000 cases/10,000 deaths
• Virus
• Protozoa
• Helmiths
• Spirochete
• Rickettsia
• Algae
Bacteria
• Enteritis, diarrhea, and dysentery
– Campylobacter
– Cholera
– E. coli 0157:H
– Salmonella
– Shigella
• Enteric fever
– Typhoid
– Paratyphoid
• Paralysis
– Botulism
Bacteria…….continued
• Eye, ear, and skin infections
– Miscellaneous bacteria
• Urinary tract infections
– E. coli
– Others
Viruses
• Enteritis, diarrhea, and dysentery
– Rotavirus
– Norwalk
• Flu like (liver damage)
– Hepatitis A
– Hepatitis E
• Paralysis
– Polio
Protozoa
• Giardia
• Cryptosporidia
• Amoeba
Helmith
• Round worm
• Tape worm
• Hook worm
• Whip worm
Others…..
• Algae
– Mycrocystis
– Dinoflaggelates
• Fungi
• Water-related
diseases
– Malaria
– Schistosomiasis
– Yellow fever
– Dengue fever
Problems
• Numerous water borne pathogens
• Individual pathogen numbers may be too
low to detect in a reasonable sized water
sample
• Isolation and detection of some
pathogens can take several days, weeks,
or months
• Absence of one particular pathogen does
not rule out the presence of another
Indicator Organism Concept
• Correlated to the presence of
pathogens
• Population large enough to isolate in
small water samples (100 mL)
• Rapid
• Inexpensive
• Safety, not culturing pathogens
Coliform Group (total coliform)
• Enterobacteriaceae • Coliform genera
– Facultative anaerobe – Enterobacter
– Gram negative – Klebsiella
– Non-spore forming – Citrobacter
– Rod shaped – Escherichia
– Ferment lactose
– Produce gas and acid
within 48 h @ 35 C
Coliform Group
• Total coliform
• Fecal coliform
– All total coliform total coliform
criteria
– Grows at 44.5 C fecal
coliform
• Escherichia coli
– Individual species E. coli
– Enzyme specific
Streptococcus and Enterococcus
• Fecal Strep • Enterococcus
– S. faecalis • Fecal Streps that
– S. faecium survive in 6.5%
– S. avium sodium chloride
– S. bovis – S. faecalis
– S. equinus – S. faecium
– S. gallinarum – S. avium
– S. gallinarum
Membrane Filter Methods
• Filter water through a 0.45 μM
membrane filter
• Place membrane on selective media
• Incubate
– 35 C total coliform
– 44.5 C fecal coliform
• Count colonies
Multiple Tube Fermentation Methods
• Serial dilution to extinction
• Inoculate multiple tubes (5 or 10) of media
with across the increasing series of dilutions
• Incubate
– 35 C or
– 44.5 C
• Count positive growth tubes
• Use Most-Probable-Number (MPN) table to
estimate density
Enzyme Substrate or Chromogenic
Substrate Method
• Used with the Presence-Absence, the Multiple
Tube Methods, or Quanti-TraysTM
• Total coliform have the enzyme
– β-D-galactosidase which hydrolyses
– ortho-nitrophenyl- β-D-galactopyranoside (ONPG)
– Yellow when hydrolyzed
• E. coli has the enzyme
– β-glucuronidase which hydrolyses
– 4-methylumbelliferyl-β-glucuronide (MUG)
– Fluoresces when hydrolyzed
ONPG-Total Coliform
MUG- E. coli
Georgia EPD Fecal Coliform Standard
for Water Contact Activities
• Geometric mean (GM)
– GM= (Y1 * Y2 * Y3 * Y4)1/4
– At least 4 samples
– Over a 30-day period
– At least 24 hours apart
Fishing and Drinking--Fecal Coliform
Standards…cont.
• May thru October
– GM not to exceed 200 MPN/100-ml
– No individual samples exceeding 4,000
MPN/100-ml
• November thru April
– GM not exceeding 1,000 MPN/100-ml
– No individual sample exceeding 4,000
MPN/100-ml
EPAs E. coli Criteria
Illness Geometric Single
Rate/1000 Mean/100mL Sample/100mL
8 126 235
9 206 300
10 206 383
11 263 490
12 336 626
13 429 799
14 548 1021
Look-out for that DIRTY-BIRD!!!
Worse Case Gull and Goose Dropping
Dropping Fecal Fecal
Weight Coliform Coliform
(wet-g) (CFU/wet-g) Load
(CFU)
Ring-Billed o.54 1.52 x 109 8.21 x 108
Gull
Canada 9.98 2.42 x 107 2.41 x 108
Goose
From: Alderisio, K. A. and N. DeLuca. 1999. Seasonal enumeration of fecal coliform bacteria from feces
of ring-billed gulls (Larus delawarensis) and Canada Geese (Branta canadesis). App. Environ.
Microbiology. p. 5628-5630.
How much water could one Gull
dropping increase to 200 MPN/100 ml?
• Gull
– 8.21 x 108 Fecal Coliform/dropping
– 4.105 x 105 liters
– 1.084 x 105 gallons
–3.99 acre inches
–1.45 x 104 feet3
How much water could one Goose
dropping increase to 200 MPN/100 ml?
• Goose
– 2.41 x 108 Fecal Coliform/defecation
– 1.205 x 105 liters
– 3.183 x 104 gallons
– 1.17 acre inches
10 geese defecating during a day of feeding
123 acre feet @ 200/100mL
100 gulls defecating one time
33 acre feet @ 200/100 mL
Factors that determine the fecal coliform
concentration in water
Survival/Die-off
Source Transport
Factors Affecting Bacterial Survival/Die-off
• Drying
• Temperature
• pH
• uV Radiation
• Competition
• Predation
• Toxic substance
Factors Effecting Transport
• Precipitation
• Slope
• Runoff
• Soil type
• Surface features
• Deposition proximity to water
• Relationship with sediment
