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 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 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

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 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 24 ± 2 hours Use EC (for FC) or EC-MUG for E.coli 44.5 ± 0.2oC 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 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.

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

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 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 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

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

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

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

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

Quanti-Tray/2000 Scientific Basis Information provided by IDEXX

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

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-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.

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

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