Presented by:
Kalina Warren

• 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

• 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 35 o C
– 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

• 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)

• 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 ± 0.2
o C

• 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.

– 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

• Use appropriate sampling container (sterile) with
Na
2
S
2
O
3 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

• 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

• Culture medium
– LES Endo agar
– M-Endo agar or broth
• Incubation
– 35 ± 0.5
o C
– 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

• Most commonly used method
• Culture medium
– M-FC broth (96 h shelve life)
– M-FC agar (2 weeks shelve life)
• Incubation
– 44.5 ± 0.2
o C
– 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

• 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 ± 0.5
o C
– EC broth at 44.5 ± 0.2
o C
• 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

• If one dilution is used for calculation
Coliforms/100 mL = coliform colonies x 100 mL sample filtered
Example: 25x100/ 10 = 250 coliforms in 100 mL of sample

• If more than one plate use to calculate density
• Coliforms/100 mL = (sum of colony count from plates x 100 sum of filtered volumes
Example: [( 15+6+0) x 100] / (50+25+10) = 25 coliforms in 100 mL
• Use the second formula only if no plate yield colony count in ideal range

• 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.

• Presumptive medium – LTB
• Incubation
– 35 ± 0.5
o C
– 24 ± 2 hours, examine and re-incubate negative tubes
• Use brilliant green lactose bile broth
(BGBB) for confirmed phase
• Incubation
– 35 ± 0.5
o C
– 24 ± 2 hours
• Use EC (for FC) or EC-MUG for E.coli
• Incubation
– 44.5 ± 0.2
o C
– 24 ± 2 hours
• Calculate results using SM Table 9221.IV.

• Mostly used for biosolids (EPA 1680 or 1681)
– Special sample preparation required
• Presumptive media – LTB
• Incubation
– 35 ± 0.5
o C
– 22 to 24 hours
• Inoculate EC broth tubes using positive LTB tubes
• Incubation
– 44.5 ± 0.2
o C
– 24 ± 2 hours
• Presumptive and confirmatory test can be run simultaneously
• One step method
– A-1 broth
• Incubation
– 3 hours at 35 ± 0.5
o C
– 21 ± 2 hours at 44.5 ± 0.2
o C
• Calculate results using SM Table 9221.IV.

Group Positive Negative
Total Coliform
Fecal Coliform
Escherichia Coli
E.coli
E.aerogenes
E.coli
E.coli
Staphylococcus aureus
Pseudomonas sp.
Enterobacter aerogenes
Enterobacter aerogenes
Fecal Streptococci Enterococcus faecalis Staphylococcus aureus
Enterococci Streptococcus faecalis S.mitis/salivarius

• Federal Register Notice- August 16 th , 2005
– Comments on proposal ended on October 17 th , 2005
– Promulgated on March 26 th ,2007
– Effective date –April 25 th , 2007
– http://www.epa.gov/EPA-WATER/2007/March/Day-26/w1455.htm
• Multiple Tube Fermentation, SM 9221F
– The procedure is used as a confirmatory test after prior enrichment in a presumptive medium for TC
• Enzyme substrate coliform (multiple tube/multiple well)
– SM 9223.B
– Colilert ® , Colilert ® -18 ( IDEXX)
• E.coli, membrane filtration single step
– mColiBlue 24 (HACH)
• Membrane filtration, SM 9222G
– This method is preceded by SM 9222B for TC
• Testing for Enterococci

Colilert & Colilert-18
Information provided by IDEXX

MUG Positive Reaction
Colilert & Colilert-18
Information provided by IDEXX

Add Colilert to sample and shake to dissolve
Pour mixture into a
Quanti-Tray
Information provided by IDEXX

cont.
Seal and then incubate at
35°C for 24 hours
Count positive wells and refer to MPN table
Information provided by IDEXX

Information provided by IDEXX

Information provided by IDEXX

• Indiana
• Oregon
• Regions of California
• Virginia
• Arizona
• Tennessee
• Utah
• Maryland
• Pennsylvania
• Kentucky
Information provided by IDEXX

• Enumerate TC and E.coli on one petri dish
• Read and confirm results in 24 hours
• E.coli colonies are blue
• Other coliforms are red

• Method 1603: Escherichia coli (E.coli) in Water by Membrane
Filtration Using Modified membrane-Thermotolerant Escherichia coli
Agar (Modified mTEC) (September 2002) - Method 1603 is a revised membrane filter (MF) procedure, a single-step method that uses one medium, modified mTEC Agar, and does not require the transfer of the membrane filter to another medium or other substrate
• Method 1604: Total Coliforms and Escherichia coli in Water by
Membrane Filtration Using a Simultaneous Detection Technique (MI
Medium) (September 2002) - This document (EPA 821-R-02-024) is identical to the February 2000 version of the MI Agar Method
(Membrane Filter Method for the Simultaneous Detection of Total
Coliforms and Escherichia coli in Drinking Water, EPA-600-R-00-13), with one exception, the addition of MI Broth. The MI Agar Method was approved for use in compliance monitoring of drinking water and source water in support of the National Primary and Secondary
Drinking Water Regulations (Federal Register, Vol. 64, No. 230, 1
December 1999, p.67450-67467) becoming effective 3 January
2000. On 6 November 2001, MI Broth was approved as a minor modification. This method, now designated as Method 1604, has been approved for use in monitoring ambient water.

• Applicable to the NPDES facilities
– Check with your State environmental department for non-NPDES facilities
• Code of Federal Regulations
– Title 40 – Protection of Environment
– Chapter 1 – Environmental Protection Agency
– Subchapter D – Water Programs
– Part 136 – Guidelines Establishing Test Procedures for the Analysis of Pollutants
– 136.3 Identification of Test Procedures
– Table 1A Approved Biological Methods for Wastewater and Sewage Sludge
• http://ecfr.gpoaccess.gov

• The Council of European Communities and the World Health
Organization have replaced fecal coliforms to E.coli in standards and guidelines.
– Source: Domestic Wastewater Treatment In Developing Countries, Published in
2004, Written by Duncan Mara
• Scientific studies suggest that E.coli is the preferred indicator of fecal contamination. E.coli is approximately equivalent to 90% of the fecal coliforms.
– Source: A compendium of standards for wastewater reuse in the Eastern
Mediterranean Region, World Health Organization

Contact information
Kalina Warren
Florida Department of Environmental Protection
Central District
Wastewater Compliance/Enforcement Section
407-893-7875
Kalina.warren@dep.state.fl.us