1. Testing for Fecal Coliforms and E.coli
Presented by:
Kalina Warren

2. 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

3. 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

4. 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)

5. 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

6. 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.

8. 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

10. Testing Methods

11. 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

12. 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

14. Total coliform, SM 9222B Culture medium Incubation
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

15. 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

16. 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

17. 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

18. 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: [( ) x 100] / ( ) = 25 coliforms in 100 mL
Use the second formula only if no plate yield colony count in ideal range

19. 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
Examine growth, look for gas
Incubation in both culture mediums can be done simultaneously
Calculate results using SM Table 9221.IV.

20. 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
24 ± 2 hours
Use EC (for FC) or EC-MUG for E.coli
44.5 ± 0.2oC
Calculate results using SM Table 9221.IV.

21. 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
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
Calculate results using SM Table 9221.IV.

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

26. Recently Approved Methods for E.coli and Enterococci
Federal Register Notice- August 16th, 2005
Comments on proposal ended on October 17th, 2005
Promulgated on March 26th,2007
Effective date –April 25th, 2007
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

27. Enzyme Substrate Coliform by multiple tube / multiple well

28. ONPG Positive Reaction Colilert & Colilert-18
Coliforms have an enzyme galactosidase and if present will eat the glactopyranoside (sugar molecule) of the ONPG. o-nitrophenyl is then hydrolzed and yields a yellow color indicating the presence of coliforms.
Information provided by IDEXX

29. MUG Positive Reaction Colilert & Colilert-18
E.coli has a specific enzyme glucuronidase and will start to eat the sugar part (glucuronide) of the MUG. When this happens, the 4-methylumbelliferyl is hydrolized and under a UV light, will fluoresce.
Information provided by IDEXX

30. Quanti-Tray Demonstration
1. Carefully separate one pack from the strip taking care not to accidentally open the adjacent pack.
2. Ensure all the powder is in the bottom of the pack.
3. Open the pack by snapping back the top at the scoreline.
4. Add the powder to the water sample.
5. Place the cap on the vessel to seal securely. Shake to dissolve the reagent
6. Carefully open the top of the quanti-tray, by squeezing the plastic inwards and using the tab to gently pull apart from the foil. (Do not put finger inside the tray). Pour the sample mixture into the tray.
Note. On the vessel, there is a score line indicating 100ml. Fill the vessel with the miniscus of the sample touching the scoreline. Adding more than 102 ml of sample will result in liquid coming out of the tray during sealing. The tray is designed with an overflow well that allows up to 105 ml of volume which includes the Colilert reagent (approx. 3 ml).
Add Colilert to sample and shake to dissolve
Pour mixture into a Quanti-Tray
Information provided by IDEXX

31. Quanti-Tray Demonstration cont.
1.Align the tray with the openings of the rubber insert and gently push it into the sealer until it grabs. Remove the tray from the other side.
2. Place the tray into a 35°C ±0.5 °C incubator for 24 hours.
3. Interpret the results as follows: Note: The overflow well at the top of the tray is counted an one well.
- If no yellow color, the test is negative.
- If any of the wells have a yellow color equal to or greater than the comparator, the presence of total coliforms is confirmed. Count the number of yellow positive wells.
- If yellow is observed, check for fluorescence by placing a 6 watt 365 nm UV light within 5 inches of the tray in a dark environment. If fluorescence is equal to or greater than the comparator, the presence of E.coli is confirmed. Count the number of positive yellow and fluorescing wells. Note: The color and fluorescence of positive wells may vary. Refer to the MPN table provided to determine the Most Probable Number (MPN)/100ml.
Seal and then incubate at 35°C for 24 hours
Count positive wells and refer to MPN table
Information provided by IDEXX

32. E.coli Positive Tray Information provided by IDEXX
Fluorescence under a UV light, 365 nm, 6 watt bulb
Information provided by IDEXX

33. Quanti-Tray/2000 Scientific Basis
Information provided by IDEXX

34. States or regions approving E
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

35. New Member Filtration Methods for E.coli

36. 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

37. 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 ) 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 ) becoming effective 3 January 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.

38. 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

39. 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

41. Florida Department of Environmental Protection Central District
Contact information
Kalina Warren
Florida Department of Environmental Protection
Central District
Wastewater Compliance/Enforcement Section