Microorganisms The Coliform Group Bacteria S D Spence

Microorganisms (The Coliform Group Bacteria) S. D. Spence

Microorganisms w single-celled w generally microscopic w include bacteria, protozoa, algae, fungi, viruses w may or may not be disease causing

Implications of Microbial Contamination of Water w potential for transmission of disease causing microorganisms (pathogens) via water contact - oral fecal route w contact includes ingestion, primary contact, and secondary contact

Water Borne Diseases/Pathogens w Bacteria • • Vibrio cholera - cholera Yersinia enterocolitica - gastroenteritis Escherichia coli - gastroenteritis Shigella sp. - gastroenteritis Listeria monocytogenes - flu-like symptoms Salmonella sp. - gastroenteritis, typhoid Campylobacter jejuni - gastroenteritis (animals sources)

Vibrio cholerae

Yersinia enterocolitica

E. coli

Shigella

Clostridium perfringens

Water Borne Diseases/Pathogens w Viruses • • Polio Virus - paralytic poliomyelitis Rotaviruses - gastroenteritis Norwalk Viruses - gastroenteritis Hepatitis Type A and E - liver inflammation

Polio Virus

Rotavirus

Norwalk Agent

Water Borne Diseases/Pathogens w Protozoa • Entamoeba histolytica - amoebic dysentery • Giardia lamblia - intestinal disease • Cryptosporidium - intestinal disease

Giardia lamblia

Difficulties With Routine Testing Of Pathogens in Water w present in low numbers w limited survival time w numerous pathogens to analyze w time and cost prohibitive

Testing of Indicator Organisms w need an indicator of potential pathogen contamination of water which is easy, reliable, inexpensive, quick, etc.

Requirements of Indicator Organisms w w w present when pathogens present in water absent in uncontaminated water present in higher numbers than pathogens in contaminated water better survival in water than pathogens easy to analyze

Coliform Bacteria - Indicators of Fecal Contamination w w w bacteria in the family Enterobacteriaceae generally include the genera Enterobacter, Klebsiella, Citrobacter, and Escherichia present in the intestinal tract of numerous organisms gram negative, non-spore forming rods ferment lactose with acid and gas production in 48 hours at 35 °C

Total Coliform Bacteria inhabit the intestinal tract of animals w sources: fecal material, soil, water, grain w some capable of reproduction in the environment w non-fecal examples include: Klebsiella sp. , Citrobacter sp. , Enterobacter sp. w

Fecal Coliform Bacteria w w w subset of the total coliform group separated from non-fecal coliforms by growth at 44. 5 °C sources: fecal material capable of limited survival and growth in the environment primary example is Escherichia coli (E. coli)

Can Stagnant Water Result in Positive Coliform Results? w presence of coliform bacteria indicates influence from a source - human activity, fecal material, soil, water, grain w stagnation of water - environment for reproduction of bacteria allowing great enough numbers to be detected

Coliform Sample Preservation

Coliform Sample Preservation w w sodium thiosulfate added to chlorinated samples no additives for nonchlorinated samples w recommended-drinking water samples iced w required-source water samples iced

Coliform Sample Holding Times and Analyses Methods

Source Water vs. Drinking Water w Drinking water and source water have different requirements for coliform analysis methods and holding times

Coliform Sample Holding Times w drinking water - 30 hours [40 CFR 141. 21(f)] w surface/source water - 8 hours [40 CFR 141. 74 (a)(1)]

Effects of Holding Time on Samples w coliform numbers may decline w coliform numbers may increase w hetertrophic (non-coliform) bacteria may increase and inhibit detection of coliforms in the sample

Heterotrophic Interference w Non-coliform bacteria which are better than coliforms at surviving in the environment may be present in the sample. If these organisms are given a chance to grow, they may out-compete coliforms during analysis and may limit the analysts ability to detect coliforms. The effect of HI is an underestimation of coliform numbers or a false negative result.

Holding Times Are Critical w greater chance of heterotrophic interference in untreated source water - hence, shorter allowable holding time

Coliform Methods

Choosing a Laboratory for Testing w laboratory must be certified by the State or EPA to conduct analyses of compliance drinking water and surface/source water samples [40 CFR 141. 28(a), 40 CFR 141. 74(a)]

Certified Laboratory w w w granted certification by State or EPA meets QA/QC requirements analyzes blind audit samples audited every 3 years documentation of certification certified for specific methods

Approved Drinking Water Methods

Approved Source Water Methods

Approved Drinking Water Methods [40 CFR 141. 21 (f)] w Membrane Filter Method - total coliforms, fecal coliforms, E. coli w Multiple Tube Fermentation Method- total coliforms, fecal coliforms, E. coli

Approved Drinking Water Methods [40 CFR 141. 21 (f)] w Presence/Absence Method -total coliforms, fecal coliforms, E. coli w w Colilert Method - total coliforms, E. coli Colisure Method - total coliforms, E. coli

Membrane Filter Method 4 to 5 day test w metallic green colonies on endo mediumpresumptive coliform positive w growth/gas in LTB and BGBB - confirmed total coliform positive w growth/gas in LTB and EC medium - confirmed fecal coliform positive w growth and gas in LTB and growth and fluorescence in EC + MUG - confirmed E. coli positive

Membrane Filter Method (Endo agar with coliform growth)

Multiple Tube Method 5 day test w growth/gas in LTB- presumptive coliform positive w growth/gas in BGBB - confirmed total coliform positive w growth/gas in EC medium - confirmed fecal coliform positive w growth and fluorescence in EC + MUG confirmed E. coli positive

Multiple Tube Fermentation Method (Total Coliforms)

Fecal Coliform/E. coli Multiple Tube Method EC medium + MUG

Presence Absence Method 5 day test w growth/acid in PA broth - presumptive coliform positive w growth/gas in BGBB - confirmed total coliform positive w growth/gas in EC medium - confirmed fecal coliform positive w growth and fluorescence in EC + MUG confirmed E. coli positive

Colilert and Colisure Methods 1 day test w color change in culture media - confirmed total coliform positive w production of fluorescence under 366 nm UV light - confirmed E. coli positive

Colilert Method

Useful References w Standard Methods for the Examination of Water and Wastewater, 18 th Edition, 1992, American Public Health Association, 1015 Fifteenth Street NW, Washington, D. C. , 20005 w Total Coliform Rule (40 CFR 141. 21) w Surface Water Treatment Rule (40 CFR 141. 74)

Photo References w Microorganism photos scanned from Microbiology Concepts and Applications, Pelczar et al. , Mc. Graw-Hill, Inc. , 1993 w Media vials and plate photos scanned from Products and Analysis 1999, Hach Company, P. O. Box 608, Loveland Colorado, 80539 -0608