Coliform Water

Testing Investigation

Issabella Santini

Arizona State University

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http://www.cleanwaterstore.com/blog/sources-of-coliform-bacteria-contamination-in-home-well-water/http://www.cleanwaterstore.com/blog/sources-of-coliform-bacteria-contamination-in-home-well-water/

Figure 1. Clean Water Store (2014)

Background on Coliform Bacteria

• Known characteristics
• Gram Negative
• Rod Shaped
• Facultatively Anaerobic
• Ferment Lactose

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(New Hampshire Department of Environmental Services, 2003)

Background

• Total coliform bacteria can be pathogenic and cause disease if they contaminate sources such as water supply.
• E.g. fecal coliforms
• Escherichia coli
• Contamination is often caused by
• Sewage
• Animal contamination

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(New Hampshire Department of Environmental Services, 2003)

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Testing

• In Lab
• Membrane filter technique
• Isolation via M-Endo Plate
• Differential Testing
• At home testing kits
• Usually specifically test for Escherichia coli
• As well as nitrates, nitrites, etc.
• Testing for total coliform
• Utilized to detect total environmental coliform
• If there is low environmental contamination there is a lower chance of fecal coliform to be present.

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Bacteria Test Kit. Home Depot. http://www.homedepot.com/p/PurTest-Bacteria-Test-Kit-700/202708273?cm_mmc=Shopping%7CTHD%7CG%7C0%7CG-BASE-PLA-D26P-Plumbing%7C&gclid=CjwKEAjwrMzHBRDW3saA88aT80MSJACbvo1TVO56bsaFdXp4yitG3h2NwyK34EeqvKr1gDNz1k62rBoCTHjw_wcB&gclsrc=aw.ds

Membrane Filter Technique

Water Microbiology, 2013.

https://www.slideshare.net/HamaNabaz/water-microbiology-21923074 slide 64

• Water sample is filtered, ensuring that there is little to no debris.
• In this experiment, 100 mL of the sample poured through the filter
• Water went through the filter with the use of the vacuum pump.
• Once drained, filter is carefully lifted on transferred to a M-Endo agar plate for isolation of the bacteria.

M-Endo Plate Isolation

Used as a medium to isolate coliforms after membrane filtration technique.

Hardy Diagnostics Product Catalog.

https://catalog.hardydiagnostics.com/cp_prod/product/1640002enk-m-endo-media-determination-of-ie-colii-and-coliforms-individually-sterile-packaged-25ml-ampoules-50-pack-by-sartorius-stedim-biotech-membrane-filtration-microbiology

Local Water Quality - Oak Creek, Arizona

• Study showed an annual increase in fecal coliform concentration in water.
• Usually occurred during the summer seasons
• Contamination concentration was greater than 800 CFU per 100 mL
• Water quality affected by
• Sediment agitation
• Recreational activity
• Monsoon storms
• Fecal contamination
• Indigenous wildlife feces
• Failed sewage systems

In This Investigation

• Do coliforms exist in the pond water in Riverview Park?
• If there are, what species of coliform are in the pond water?
• What is the concentration of coliforms per 100 mL of water?
• Is the water potable based on these results?

Hypothesis

The water sample taken from Riverview Park contains total coliforms.

• The contamination concentration is higher than 1 CFU per 100 mL making the water non-potable.

Prediction - The species of coliform is likely Escherichia coli due to frequent animal exposure. → fecal coliforms

Testing the Hypothesis

• Membrane Filter Technique
• M Endo plate for isolation of bacteria
• Capsule Stain
• Gram Stain

Differential Testing

• Indole
• Motility
• Methyl Red
• Voges Proskauer
• Citrate

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• Glucose
• Sucrose
• Lactose
• Mannitol
• Xylose

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Resulting Data

M Endo Plate for Isolation

CFU - Number too high to count → Water is non potable due to CFU being greater than 1 CFU per 100 mL

Red color indicates that the bacteria ferments lactose.

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Morphology - Gram Stain

• Resulting stain shows pink stain indicating the bacteria is gram Negative.
• Morphology - Rods

Capsule Stain

Differential Tests - Methyl Red and VP

• Methyl Red Test
• Negative
• Indicates that the bacteria does not perform mixed acid fermentation.

• VP Test
• Positive
• Indicates the bacteria does produce 2,3 butanediol in the presence of glucose

Indole and Motility Test on SIM media

• Test results showed:
• Motility - Positive
• Indole production - Negative
• Sulfide production - Positive

Citrate Test

Xylose - Positive Sucrose - Positive Mannitol - Negative Lactose - Positive Glucose - Positive

Results

Table 1. Differential Tests comparing different bacteria to experimental results. Information obtained from Bergey’s Manual.

Discussion

Using bioinfo.bisr.res.in

Percentage values on similarity based on test results could be found.

Enterobacter Aerogenes was the organism to have the highest percentage similarity.

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Discussion

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• Test Results indicate the most probable bacteria in unknown sample is Enterobacter Aerogenes.
• Bergey’s Manual Group 5
• Facultatively Anaerobic Gram negative Rods
• The only results that did not match with the Enterobacter Aerogenes expected results were the hydrogen sulfide production and Mannitol fermentation test.
• Could be due to contamination of other species of bacteria.
• Public pond water

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Conclusion - Were the hypotheses supported?

The sample taken from Riverview Park contained total coliforms.

The CFU of the sample was too high to count, therefore fails the “1 CFU per 100mL of water” parameters. The water in non potable.

The water is indeed non potable and contains a high concentration of total coliforms, predominantly Enterobacter Aerogenes. But no clear indication e Coli.

References

N. (2003). Environmental Fact Sheet. Retrieved April 17, 2017, from https://www.des.nh.gov/organization/commissioner/pip/factsheets/wwt/documents/web-18.pdf

C. C., & R. D. (1999, June). The impact of sediment fecal coliform reservoirs on seasonal water quality in Oak Creek, ARIZONA. Retrieved April 17, 2017, from http://www.sciencedirect.com/science/article/pii/S0043135498004370

D. (2016, April). Coliform Bacteria and Drinking Water. Retrieved April 15, 2017, from http://www.doh.wa.gov/portals/1/Documents/Pubs/331-181.pdf

Bergey, D. H., & Holt, J. G. (2000). Bergey's manual of determinative bacteriology. Philadelphia: Lippincott Williams & Wilkins. Print.

B. (n.d.). D-Bite: Database of Biochemical Tests of Pathogenic Enterobacteriaceae Family. Retrieved April 18, 2017, from Database of Biochemical Tests of Pathogenic Enterobacteriaceae Family

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