Testing for Fecal Coliforms

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 Endotype 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 -galactosidase (nickname beta-gal) that
catalyzes the hydrolysis of -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)

Highly motile
Part of normal flora of human intestinal tract
Several strains can be pathogenic and cause urinary tract infection

Inhabitants of the gastrointestinal tracts of warm-blooded animals

Provide a portion of the microbialderived vitamin K
E.coli most numerous from this group

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

Budvicia, Erwinia, Leclercia, Serratia

Found only in the environment (no fecal origin)

Enterobacter
Escherichia
Klebsiella

Others

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 - 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
Coliforms/100 mL = coliform colonies x 100
mL sample filtered

Example: 25x100/ 10 = 250 coliforms in 100 mL of sample

Calculation of Coliform Density

MF, cont.
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

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

35 0.5oC
22 to 24 hours

Presumptive media LTB

Incubation

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

3 hours at 35 0.5oC
21 2 hours at 44.5 0.2oC

Incubation

Calculate results using SM Table 9221.IV.

Control Cultures for

Microbiological Tests
Group

Positive

Total Coliform

E.coli
E.aerogenes

Fecal Coliform

E.coli

Enterobacter aerogenes

E.coli

Enterobacter aerogenes

Escherichia Coli

Fecal Streptococci
aureus
Enterococci

Negative
Staphylococcus aureus
Pseudomonas sp.

Enterococcus faecalis Staphylococcus

Streptococcus faecalis

S.mitis/salivarius

Recently Approved Methods for

E.coli and Enterococci
Federal Register Notice- August 16th, 2005

Comments on proposal ended on October 17 th, 2005

Promulgated on March 26th,2007
Effective date April 25th, 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

Enzyme Substrate Coliform

by
multiple tube / multiple well

ONPG Positive
Reaction
Colilert & Colilert-18

Information provided by IDEXX

MUG Positive Reaction

Colilert & Colilert-18

Information provided by IDEXX

Quanti-Tray
Demonstration

Add Colilert to sample

and shake to dissolve

Pour mixture into a

Quanti-Tray

Information provided by IDEXX

Quanti-Tray Demonstration
cont.

Seal and then incubate at

35C for 24 hours

Count positive wells and

refer to MPN table
Information provided by IDEXX

E.coli Positive Tray

Information provided by IDEXX

Quanti-Tray/2000 Scientific
Basis

Information provided by IDEXX

States or regions approving E.coli and

Colilert for Waste Water Testing

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

New Member Filtration

Methods for E.coli

M-ColiBlue 24

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

EPA Methods for E.coli

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-02024) 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-600R-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.

Federal Regulations / List of

Approved Biological Methods
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

World-wide Prospective
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