Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
and E.coli
Presented by:
Kalina Warren
Summary of Presentation
• Background information
– Total coliform group
– Fecal coliform group
– Escherichia coli (E.coli)
– Other pathogenic bacteria
• Test methods
– Membrane filter technique
– Multiple-tube fermentation
technique
– Enzyme substrate coliform test
• Colilert and Colilert - 18
• US Rules
• World-wide perspective
Total Coliform Group
• Consists of several genera of bacteria of fecal and non-fecal origin
• Definition based on the method of detection
– Ferment lactose with gas and acid formation within 48 h at 35oC
– Produce dark red colony with a metallic sheen within 24 h on an Endo-type
medium containing lactose
• Considered an indicator organism
– A criterion of the degree of pollution and thus of sanitary quality
• Belongs to the family of Enterobacteriaceae
• Facultative anaerobic (do not have to have oxygen)
• Gram-negative
• Non-spore forming
• Rod-shaped
• Includes four genera that have at least some species of fecal origin
• Posses the enzyme β-galactosidase (nickname beta-gal) that catalyzes the
hydrolysis of β-galactosides into monosaccharides
Total coliform group, cont.
• Citrobacter
– Use citrate as a carbon source
– Found everywhere including in soil, water, wastewater, human intestine
– Rarely source off illness (except urinary tract & infant meningitis)
• Enterobacter
– Highly motile
– Part of normal flora of human intestinal tract
– Several strains can be pathogenic and cause urinary tract infection
• Escherichia
– Inhabitants of the gastrointestinal tracts of warm-blooded animals
– Provide a portion of the microbial–derived vitamin K
– E.coli – most numerous from this group
• Klebsiella
– Widely distributed in nature
– Occurring in soil, water, grain, vegetation, wood pulp, other…
– Opportunistic pathogens
– Can cause pneumonia, urinary tract infections, other infections
– Associated with coliform re-growth in large water distribution systems
– Klebsiella pneumoniae ( from feces) – 60 – 80 % shows as positive in FC tests
• Others
– Budvicia, Erwinia, Leclercia, Serratia
– Found only in the environment (no fecal origin)
Fecal coliform group
• Subset of total coliform
• Resides in the intestinal tract of warm-blooded
animals (including humans)
• Outside of a host, FC are short-lived
• Contains pathogens (disease causing e.g., E.coli)
and non-pathogenic bacteria
• Indicates fecal contamination and potential
presence of pathogens
• Incubation temperature 44.5 ± 0.2oC
Escherichia coli
• E.coli is a sub-group of fecal coliform group
• It is naturally found in the intestines of warm-blooded
animals, such as cows, chicken, pigs, dogs, cats, birds,
and people; comprise of up to 1% of bacterial biomass
• Enters the environment through feces.
• There are thousands of different strains of E. coli.
Fortunately very few strains are pathogenic.
– More than one strain of E.coli may coexist in the intestinal tract;
they displace one another 3-4 times a year
– Pathogenic strains may cause one of three types of infection:
urinary tract, neonatal meningitis, intestinal diseases
• Possess the enzyme β- glucuronidase and is capable of
cleaving the fluorogenic substrate, MUG, with the
corresponding release of the fluorogen.
Other pathogenic bacteria regulated by
the wastewater program
– Salmonella
• Belongs to family of Enterobacteriaceae
• Usually does not ferment lactose, instead most produce
hydrogen sulfide
• Causes typhoid fever and food-borne diseases
• Lives in the intestinal tract of humans and other animals
including birds
• Present in small numbers compared to coliform
– Fecal Streptococci
• Found in gastrointestinal tract of warm-blooded animals
• Enterococci are a subgroup of FS
– Valuable indicator of fecal contamination of recreational waters
Testing Methods
Sample Collection
• Use appropriate sampling container (sterile) with
Na2S2O3 to remove chlorine
• Type: Grab (not composite)
• Use gloves while sampling
• Sample should be representative of flow
• After collection place it on ice or in a refrigerator
• Deliver to a laboratory ASAP, but no later than
6 hours of collection
• Maintain records
Membrane Filter Technique
• Pour medium onto absorbent pad in petri dish
• Shake sample vigorously
• Measure appropriate volume
• Filter sample or sample aliquot through a membrane filter, diameter
47 mm, mean pore diameter 0.45 um
• Rinse funnel with approx. 30 mL of dilution water
• Place filter on a pad in petri dish with a selected medium
• Incubate inverted plate
• Count colonies
• Record results
• Perform confirmatory tests when needed
• Run appropriate blanks, known positive and known negative
Total coliform, SM 9222B
• Culture medium
– LES Endo agar
– M-Endo agar or broth
• Incubation
– 35 ± 0.5oC
– 22 to 24 hours
• Produce dark, red colony with
metallic sheen
• Many background colonies usually are observed
• At least three sample dilutions should be setup
• Up to 80 TC colonies can be counted if no more than
200 total colonies (including background) are noted
Fecal coliform, SM 9222D
• Most commonly used method
• Culture medium
– M-FC broth (96 h shelve life)
– M-FC agar (2 weeks shelve life)
• Incubation
– 44.5 ± 0.2oC
– 24 ± 2 hours
• Count blue colonies; can be
different size and shade
• Few background colonies usually are observed due to selectivity of
medium, especially if rosalic acid is added
• At least three sample dilutions should be setup
• Up to 60 FC colonies can be counted
Confirmation procedures, SM
9020B. 9
• Verify positives monthly by picking
at least 10 colonies from a plate
• To determine false negatives, pick
atypical colonies
• For FC test use tubes with:
– Lauryl Tryptose Broth
(LTB) at 35 ± 0.5oC
– EC broth at 44.5 ± 0.2oC
• Adjust counts based on percent of
verification
• Tips
– Each colony is confirmed in a separate tube
– LTB and EC tubes can be confirmed simultaneously
• Keep good records
Calculation of Coliform Density - MF
• If one dilution is used for calculation
• Use the second formula only if no plate yield colony count in ideal
range
Multiple Tube Fermentation
Technique
• In general, this method is much more time consumig
• Set up 5 tubes with appropriate presumptive media for each of
three sample dilutions
– 5 x 10 mL
– 5 x 1 mL
– 5 x 0.1 mL
• Add a sterile water control
• Incubate
• Swirl each tube gently and examine growth
• Look for gas and acidic reaction
• Re-incubate for additional 24 h if no gas or acidic reaction is evident
• Using sterile 3-mm loop or wooden applicator transfer growth from
a positive presumptive medium to a tube with confirmation medium
• Incubate
• Examine growth, look for gas
• Incubation in both culture mediums can be done simultaneously
• Calculate results using SM Table 9221.IV.
Multiple-Tube Fermentation Technique,
Total Coliform Procedure, SM 9221B
• Presumptive medium – LTB
• Incubation
– 35 ± 0.5oC
– 24 ± 2 hours, examine and re-incubate negative tubes
• Use brilliant green lactose bile broth
(BGBB) for confirmed phase
• Incubation
– 35 ± 0.5oC
– 24 ± 2 hours
• Use EC (for FC) or EC-MUG for E.coli
• Incubation
– 44.5 ± 0.2oC
– 24 ± 2 hours
• Calculate results using SM Table 9221.IV.
Multiple-Tube Fermentation Technique,
Fecal Coliform Procedure, SM 9221E
• Mostly used for biosolids (EPA 1680 or 1681)
– Special sample preparation required
• Presumptive media – LTB
• Incubation
– 35 ± 0.5oC
– 22 to 24 hours
• Inoculate EC broth tubes using
positive LTB tubes
• Incubation
– 44.5 ± 0.2oC
– 24 ± 2 hours
• Presumptive and confirmatory test
can be run simultaneously
• One step method
– A-1 broth
• Incubation
– 3 hours at 35 ± 0.5oC
– 21 ± 2 hours at 44.5 ± 0.2oC
• Calculate results using SM Table 9221.IV.
Control Cultures for Microbiological
Tests
Group Positive Negative