# Presentation: 094214

## Microorganisms (The Coliform Group Bacteria)

- S. D. Spence

## Microorganisms

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

## 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

## 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)

## Vibrio cholerae

## Yersinia enterocolitica

## E. coli

## Shigella

## Clostridium perfringens

## Water Borne Diseases/Pathogens

- 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

- Protozoa
*Entamoeba histolytica* - amoebic dysentery
*Giardia lamblia* - intestinal disease
*Cryptosporidium* - intestinal disease

## Giardia lamblia

## Difficulties With Routine Testing Of Pathogens in Water

- present in low numbers
- limited survival time
- numerous pathogens to analyze
- time and cost prohibitive

## Testing of Indicator Organisms 

- need an indicator of potential pathogen contamination of water which is easy, reliable, inexpensive, quick, etc.

## 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

## 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

## 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. *

## 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*)

## 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

## Coliform Sample Preservation 

## Coliform Sample Preservation 

- sodium thiosulfate added to chlorinated samples
-  no additives for nonchlorinated samples
**recommended**-drinking water samples iced
**required**-source water samples iced

## Coliform Sample Holding Times and Analyses Methods 

## Source Water vs. Drinking Water

- Drinking water and source water have different requirements for coliform analysis methods and holding times

## Coliform Sample Holding Times

- drinking water - 30 hours                          [40 CFR 141.21(f)]
- surface/source water - 8 hours                                [40 CFR 141.74 (a)(1)]

## 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

## 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.

## Holding Times Are Critical

- greater chance of heterotrophic interference in untreated source water - hence, shorter allowable holding time

## Coliform Methods 

## 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)]

## 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

## Approved Drinking Water Methods

## Approved Source Water Methods

## 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*

## 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*

## 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

## Membrane Filter Method (Endo agar with coliform growth)

## 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

## Multiple Tube Fermentation Method (Total Coliforms)

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

## 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

## 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

## Colilert Method

## 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)

## 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