Training-Workshop on Wastewater Analysis ROBINSONS LAND CORPORATION LABORATORY Robinsons Place Manila, Ermita

Testing for Fecal Coliforms and E.coli

Summary of Presentation
Background information
Total coliform group Fecal coliform group Escherichia coli (E.coli) Other pathogenic bacteria

Test methods
Multiple-tube fermentation technique

RLC LABORATORY Training-Workshop in Wastewater Analysis 2012

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
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Coliform Group
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
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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
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Coliform group, cont.

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
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Coliform group, cont.

Escherichia Inhabitants of the gastrointestinal tracts of warm-blooded animals Provide a portion of the microbialderived vitamin K E.coli most numerous from this group Others Budvicia, Erwinia, Leclercia, Serratia Found only in the environment (no fecal origin)
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Fecal coliform group

Subset of total coliform Resides in the intestinal tract of warmblooded 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
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Escherichia coli
E.coli is a sub-group of fecal coliform group It is naturally found in the intestines of warmblooded animals, such as cows, chicken, pigs, dogs, cats, birds, and people; comprise of up to 1% of bacterial biomass Enters the environment through feces.

RLC LABORATORY Training-Workshop in Wastewater Analysis 2012

Escherichia coli (cont.)

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.
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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

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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
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Testing Method
Multiple-tube Fermentation Technique (MTFT) In general, this method is time consuming Applicable to wastewater, drinking water and surface water.

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MEDIA, REAGENTS, MATERIALS

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Media
Lauryl Tryptose Broth (LTB) Brilliant Green Bile Broth (BGB) EC Mug

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MTFT (cont)
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
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MTFT (cont)
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 SMEWW Table 9221.IV.
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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.

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Control Cultures for Microbiological Tests

Group
Total Coliform

Positive
E.coli E.aerogenes
E.coli

Negative
Staphylococcus aureus Pseudomonas sp.
Enterobacter aerogenes

Fecal Coliform

Escherichia Coli
Fecal Streptococci Enterococci

E.coli
Enterococcus faecalis Streptococcus faecalis

Enterobacter aerogenes
Staphylococcus aureus S.mitis/salivarius

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References
SMEWW 21st Edition. 2005. APHA, AWWA, WEF

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