This document discusses microorganisms and coliform bacteria as indicators of water quality. It describes various pathogens that can be transmitted through contaminated water and issues with directly testing for all pathogens. Coliform bacteria are discussed as reliable indicators of fecal contamination in water that are easier to detect than pathogens. The document outlines approved methods for testing drinking water and surface water for total coliforms and E. coli, including membrane filtration, multiple tube fermentation, and presence-absence tests. Holding times and certification of laboratories to conduct these analyses are also summarized.

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

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

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

4. Water Borne Diseases/Pathogens
 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)

5. Vibrio cholerae

6. Yersinia enterocolitica

7. E. coli

8. Shigella

9. Clostridium perfringens

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

11. Polio Virus

12. Rotavirus

13. Norwalk Agent

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

15. Giardia lamblia

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

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

18. Requirements of Indicator
Organisms
 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

19. Coliform Bacteria - Indicators of
Fecal Contamination
 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

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

21. Fecal Coliform Bacteria
 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)

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

23. Coliform Sample Preservation

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

25. Coliform Sample Holding Times
and Analyses Methods

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

27. Coliform Sample Holding Times
 drinking water - 30 hours
[40 CFR 141.21(f)]
 surface/source water - 8 hours
[40 CFR 141.74 (a)(1)]

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

29. Heterotrophic Interference
 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.

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

31. Coliform Methods

32. Choosing a Laboratory for
Testing
 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)]

33. Certified Laboratory
 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

34. Approved Drinking Water Methods
Analyte Method Reference
Total Coliform Membrane Filter SM 9222 C
Total Coliform Multiple Tube SM 9221 B
Total Coliform Colilert SM 9223
Total Coliform Colisure Millipore
Total Coliform Presence/Absence SM 9221 D
Fecal Coliform Fec Col Multiple Tube SM 9221 E
E. coli EC + MUG 40 CFR 141.21 (f)(5)(i)
E. coli Nutrient Agar + MUG 40 CFR 141.21 (f)(5)(ii)
E. coli Colilert, Colisure SM 9223, Millipore

35. Approved Source Water Methods
Analyte Method Ref
Total Coliform Membrane Filter SM 9222 C
Total Coliform Multiple Tube SM 9221 B
Total Coliform Colilert SM 9223
Fecal Coliform Multiple Tube SM 9221 E
Fecal Coliform Membrane Filter SM 9221 E

36. Approved Drinking Water
Methods [40 CFR 141.21 (f)]
 Membrane Filter Method - total coliforms, fecal
coliforms, E. coli
 Multiple Tube Fermentation Method- total
coliforms, fecal coliforms, E. coli

37. Approved Drinking Water
Methods [40 CFR 141.21 (f)]
 Presence/Absence Method -total coliforms,
fecal coliforms, E. coli
 Colilert Method - total coliforms, E. coli
 Colisure Method - total coliforms, E. coli

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

39. Membrane Filter Method (Endo
agar with coliform growth)

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

41. Multiple Tube Fermentation
Method (Total Coliforms)

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

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

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

45. Colilert Method

46. Useful References
 Standard Methods for the Examination of
Water and Wastewater, 18th Edition, 1992,
American Public Health Association, 1015
Fifteenth Street NW, Washington, D.C., 20005
 Total Coliform Rule (40 CFR 141.21)
 Surface Water Treatment Rule (40 CFR
141.74)

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