Overview of Microbial Indicator Monitoring Lab Methods
Transcript of Overview of Microbial Indicator Monitoring Lab Methods
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Overview of Microbial Indicator
Monitoring Lab Methods
Jim Ferretti, USEPA Region 2
DESA, Laboratory Branch
May 23, 2018
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Water Contamination and Public Health
• 1854- John Snow mapped and correlated incidence of cholera with proximity to public water pumps
• 1880’s- Robert Koch reported microbial disease agents
• 1880’s- Theodore Escherichsuggested “Bacillus coli” as an indicator of fecal contamination
Street map of area surrounding Broad Street pump
London, 1843
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A brief history of bacterial assays
• 1904 – assays for E. coli using
glucose broths (Eijkman)
• 1920’s – multiple tube
fermentation with lactose
broths (Leiter)
• 1950’s- membrane filtration
(Goetz and Tsuneishi)
• 1988 – defined substrates
developed (Edberg et al.)
• 2000’s- Real Time PCR
Phenol red lactose broths
Methods
• Membrane filter using m-endo broth (left)
• Quanti-tray using Colilert Medium (right)
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What is a pathogen?
• Pathogens are organisms capable of causing disease
• The following are some of the “bad guys”:
• Protozoa: Giardia, Cryptosporidium
• Bacteria: Salmonella typhi, Legionella, Shigella, Vibrio cholerae, Vibrio vulnificus
• Virus: Hepatitis, Polio
• How do they make you sick?
• They produce poisons (toxins) that result in fever, headache, vomiting, and diarrhea and destroy body tissue
USDA NIFSI Food Safety in the Classroom©
University of Tennessee, Knoxville 2006
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Are all bacteria pathogens?
• No, most are harmless
• Some are even helpful
• Examples of helpful bacteria:
USDA NIFSI Food Safety in the Classroom©
University of Tennessee, Knoxville 2006
makes cheese, yogurt, & buttermilk and produces vitamins in your intestine.
Lactobacillus:
makes pepperoni, salami, & summer sausage
Pediococcus:
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Example of Common Pathogens
Salmonella E. coli O157:H7
Staphylococcus aureus Campylobacter jejuni
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Examples of waterborne pathogens- Bacteria
Name of micro-organisms Major diseasesMajor reservoirs and primary
sources
Bacteria
Salmonella typhi Typhoid fever Human feces
Salmonella paratyphi Paratyphoid fever Human feces
Other Salmonella Salmonellosis Human and animal feces
Shigella spp. Bacillary dysentery Human feces
Vibrio cholera Cholera Human feces and freshwater
zooplankton
Enteropathogenic E. coli Gastroenteritis Human feces
Yersinia enterocolitica Gastroenteritis Human and animal feces
Campylobacter jejuni Gastroenteritis Human and animal feces
Leptospira spp. Leptospirosis Animal and human urine
Various mycobacteria Pulmonary illness Soil and water
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More Examples of waterborne pathogens - Viruses
Name of micro-organisms Major diseasesMajor reservoirs and primary
sources
Enteric viruses
Polio viruses Poliomyelities Human feces
Coxsackie viruses A Aseptic meningitis Human feces
Coxsackie viruses B Aseptic meningitis Human feces
Echo viruses Aseptic meningitis Human feces
Norovirus Gastroenteritis Human feces to fomites and water
Rotaviruses Gastroenteritis Human feces
Adenoviruses Upper respiratory and
gastrointestinal illness Human feces
Hepatitis A virus Infectious hepatitis Human feces
Hepatitis E virus Infectious hepatitis; miscarriage and
death Human feces
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More Examples of waterborne pathogens - Protozoans
Name of micro-organisms Major diseasesMajor reservoirs and primary
sources
Protozoa
Acanthamocba castellani Amoebic meningoencephalitis Human feces
Balantidium coli Balantidosis (dysentery) Human and animal feces
Cryptosporidium homonis, C.
parvumCryptosporidiosis (gastroenteritis)
Water, human and other mammal
feces
Entamoeba histolytica Amoebic dysentery Human and animal feces
Giardia lamblia Giardiasis (gastroenteritis) Water and animal feces
Naegleria fowleriPrimary amoebic
meningoencephalitis Warm water
Helminths
Ascaris lumbricoides ascariosis Animal and human feces
Adapted from Ashbolt, 2004
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Protozoa Intestinal Parasites &
Water Borne Diseases
Giardia lamblia Cryptosporidium parvum
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Difficulties With Routine Testing
Of Pathogens in Water
• present in low numbers
• limited survival time
• numerous pathogens to analyze
• time and cost prohibitive
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INDICATOR ORGANISM
• Exposure to fecal contaminated water does not
always translate into infection. However, the higher
the fecal bacterial levels in water, the higher the
chances of pathogens to be present in significant
numbers too.
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Common Indicator: Bacteria
• Total Coliforms
• Fecal Coliforms
• E. coli
• Enterococci E. coli
Total
Coliforms
Fecal
Coliforms
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Indicator Organisms
• Present when pathogens are present in water
• Absent in uncontaminated water
• Present in higher numbers than pathogens in
contaminated water
• Better survival in water than pathogens
• Easy to analyze
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Coliform bacteria are defined as rod-shaped Gram-negative non-
spore forming and motile or non-motile bacteria which can ferment
lactose with the production of acid and gas when incubated at 35–37°C
COLIFORMS…………………
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Coliform Genera
►Escherichia- human and animal feces
►Enterobacter- environment, feces
►Klebsiella- environment
►Citrobacter- environment
►Serratia- environment
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Indicator Bacteria Regulated Based on Risk/Human
Epidemiological Data
➢ Drinking Water 0 CFU/100 mLs (E.coli)
➢ Shellfish Beds 14 CFU/100 mLs (Fecal)
➢ Bathing Beaches 126 CFU/100 mLs (E. coli)
➢ Secondary Contact 770-1500 (Fecal)
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COMMON TECHNIQUES TO
MEASURE INDICATOR
BACTERIA
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Approved Drinking Water Methods
[40 CFR 141.21 (f)]
• Membrane Filter Method - total coliforms, fecal
coliforms, E. coli
• Multiple Tube Fermentation Method- total
coliforms, fecal coliforms, E. coli
• Defined Substrate Tests – Colilert, Colilert 18,
Colisure
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Bacterial Measurement
• Membrane Filtration Methods
• Quantify bacteria numbers by filtering water, growing
bacteria on selective or deselective media, and counting
• Multiple Tube Fermentation
• Estimate bacterial numbers based upon a color change or
amount of gas produced through a specific bacterial
metabolic process
• Defined Substrate Methods
• Use a substrate that fluoresces and/or changes color to measure
bacterial density (ex, Colilert)
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Total Coliforms (m-Endo broth)
Total Coliform
• Red colony wiih a
metallic sheen
within 24±2 hours
at 35±0.2oC
Total Coliforms(http://dl.clackamas.cc.or.us/wqt111/unit-8-coliformtest.htm)
Membrane Filtration
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Fecal Coliforms (m-FC broth)
• Blue colonies
for fecal
coliforms.
Gray to cream
colored are
non fecal
coliforms
Fecal Coliform
• Blue colony
within 24±2 hours
at 44.5±0.5oCFecal Coliforms(http://dl.clackamas.cc.or.us/wqt111/unit-8-coliformtest.htm)
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If Coliform bacteria are present in the
sample, they will appear as red colonies and if
E.coli in present in the sample,
they will appear as blue colonies.
mColiBlue24 MF Test for Total Coliforms and E. coli
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Multiple Tube Fermentation Method
• Step 1 presumptive phase
• Use lauryl tryptose broth
• Grow “stressed” organisms
• Confirm positives
• Calculate MPN
Growth
Gas
http://www.bact.wisc.edu/Microtextbook/index.php?module=Book&func=displaychapter&chap_id=55&theme=printer
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Lauryl Tryptose
Broth
Brilliant Green Broth
EC Media
EC w/Mug
Common Media for MTF Tests for Total, Fecal, and E. coli
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Enzymatic Methods
• Coliform specific enzymes
• -Galactosidase (lacZ) –total coliforms
• Ability to breakdown lactose
• -D-Glucuronidase (uidA) –fecal coliforms
• Ability to breakdown complex sugars
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Enzymatic Methods
• Presence / Absence Tests
• like MTF but with enzymes
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Colilert® - Total colifoms & E.coli
•ONPG (sugar analog) metabolized by coliform bacteria
•MUG tests for presence of enzyme found ONLY* in E. coli
•Results are easily read:
•Yellow indicates Coliform bacteria
•Fluorescent Blue/green indicates E. coli
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Molecular Methods
• Nucleic Acid based techniques
• Polymerase Chain Reaction (PCR)
• phylogenetic primers
• enzyme specific primers
• detection limit
• viability
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Thank you!