Water Quality Analysis via Indicator Organisms
Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba -The University of Arizona
Demonstrating Author: Luisa Ikner
Water quality analysis monit…
1. Colilert Presence – Absence (P – A) Test
- Open the 100 mL plastic Colilert bottle. The bottle includes a small amount of powdered reagent that is necessary for the proper reactions, do not discard this powder.
- Add 100 mL water sample into Colilert bottle.
- Open the pillow tube containing the nutrient substrate and pour the contents into the water sample inside the Colilert bottle.
- Cap and seal the Colilert bottle. Shake the bottle vigorously, repeatedly inverting the bottle until the substrate is completely dissolved.
- Incubate the reagent/water sample mixture inside the bottle at 35 ± 0.5 °C for 24 h.
- Observe the yellow color change in the reagent/water sample mixture. The yellow color indicates coliform is present. Clear water or no change in color indicates that coliforms are absent.
- Expose the reagent/water sample to ultraviolet light and observe the blue fluorescence. Blue fluorescence indicates that E. coli is present. No fluorescence indicates that E. coli is absent (Figure 3).
Figure 3. P-A test negative (left), coliform positive (middle), and E. coli positive (right).
2. Colilert MPN: Quanti-tray 2000
- Open the Colilert bottle, and 100 mL water sample into Colilert bottle.
- Open the pillow tube containing nutrient substrate and pour contents into the water sample inside the Colilert bottle.
- Cap and seal the Colilert bottle. Shake the bottle vigorously, repeatedly inverting the bottle until the substrate is completely dissolved.
- Carefully open Quanti-tray 2000 by squeezing the edges at the top of the tray and pulling back the paper tab. Keep squeezing so that the tray is open.
- Pour the reagent/water sample mixture into the tray, and then incubate the sample inside the tray at 35 ± 0.5 °C for 24 h.
- Observe the yellow color change in the reagent/water sample mixture. Count the number of large and small wells that signal positive presence for coliforms. The yellow color indicates coliform is present. Clear water or no change in color indicates that coliforms are absent.
- Expose reagent/water sample to ultraviolet light and observe blue fluorescence. Count the number of large and small wells that signal positive presence for E. coli. The blue fluorescence indicates that E. coli is present. No fluorescence indicates that E. coli is absent.
- Use the Quanti-tray 2000 MPN sheet (Figure 4) to quantify the concentration for each indicator organism present in 100 mL of water. Use the spreadsheet to compare large: small positive well ratio to enumerate presence for both indicator organisms.
Figure 4. Quanti-tray negative (left), coliform positive (middle), and E. coli positive (right).
Water quality analysis is vital to safeguard the integrity of water resources. The presence of indicator microorganisms is correlated with the presence of fecal matter, which may contain disease-causing pathogens. Indicator organisms can therefore be used to evaluate the safety of water supplies.
Fecal contamination in water poses a significant risk to the health of plants, animals, and humans, as gastrointestinal pathogens are shed in very high numbers in the feces. However, monitoring water samples for each type of unique pathogen associated with fecal pollution is not feasible. Surveying for Indicator organisms provides a simple, rapid, and cost effective way to detect fecal contamination in water resources.
This video will illustrate the principles behind using indicator organisms to evaluate water quality, how to test collected water samples, and the interpretation and quantification of resulting data.
To be used as a water quality indicator, organisms must meet five specific criteria. First, it should be detectable in water where the pathogen is present, and absent when the pathogen is absent. Second, the number of indicator organisms must correspond with pathogen levels. It should also be tougher and persist longer in the environment than the pathogen. Finally, detection should be easy, safe, and inexpensive, and effective across all water types.
Two of the most common bacterial indicator groups are total coliforms and fecal coliforms, typically E. coli. Total coliforms can be found in the mammalian gut, but may also occur naturally in soil and surface water. Fecal coliforms are a subset that reside entirely within the gastrointestinal tracts of mammals and birds and are continuously shed in feces. Coliforms are vulnerable to the same stresses as many common gut pathogens, such as water treatment or low nutrient levels, their presence in a water sample is a useful indicator of the potential presence of pathogens. Both total coliforms and E. coli are readily detected in the laboratory setting.
For detection, chemical substrates are added to the sample that the coliforms metabolize, resulting in a color change. For total coliforms, added ONPG is converted to nitrophenol, turning the water yellow. For fecal coliforms, E. coli converts MUG to a methyl-umbelliferone product that fluoresces blue-green under ultraviolet light. In its simplest application, the substrate test can confirm the presence or absence of coliforms existing in the water at the time of sampling.
In contrast to this qualitative method, the number of total coliforms per sample can be estimated using a specialized partitioned tray. After the reactive substrate is dissolved, the water sample is added to a tray containing large and small wells, and then incubated. Wells exhibiting the color change are counted, and the ratio of small to large wells demonstrating positive colorimetric signals is aligned to a chart that indicates a quantity. US drinking water supplies must contain zero total coliforms per 100 mL.
Now that we are familiar with the principles of using indicator organisms to identify and quantify water contamination, let's take a look at how this is carried out in the laboratory.
Once samples have been collected, bring them into the laboratory for testing. To begin, open a 100-mL plastic bottle. Bottles may contain a small amount of powdered sodium thiosulfate reagent that is used to ensure the neutralization of any chlorine that might be present. Add 100 mL of water sample into the bottle. Open a pillow tube containing nutrient substrate and pour the contents into the water sample inside the bottle. Cap and seal the bottle, then shake vigorously, repeatedly inverting the bottle until the substrate is completely dissolved. Next, incubate the sample-reagent bottle at 35 ?C for 24 h.
Observe the yellow color change in the sample-reagent mixture. Yellow color indicates that coliforms are present. No change in color indicates that coliforms are absent. Finally, expose the sample-reagent mixture to ultraviolet light and observe. Blue fluorescence, in combination with?a yellow color change, indicates that E. coli is present. No fluorescence indicates absence.
Most Probable Number, or MPN, can also be determined for samples. Open a bottle, and add 100 mL?of water sample. Open the pillow tube of nutrient substrate and pour the contents into the water sample in the bottle. Cap and seal the bottle. Shake vigorously, inverting repeatedly until the substrate is completely dissolved. Carefully open the tray by squeezing the edges at the top and pull back the paper tab. Apply constant pressure to keep the tray open. Pour the sample-reagent mixture into the tray and seal. Incubate the tray at 35 ?C for 24 h.
Observe the color change in the sample-reagent mix tray. Count the number of large wells and small wells that have turned yellow to indicate the presence of coliforms. Next, expose the sample-reagent tray to ultraviolet light and observe blue fluorescence. Count the number of large and small wells that signal positive presence of E. coli.
Using the provided MPN sheet, quantify the concentration for each indicator organism present in 100 mL?of water. Find the number of small positive wells along the top of the table, and the number of large positive wells on the left side axis. The intersection of the two will give a figure representing the Most Probable Number, which is the estimated number of organisms per 100 mL.
Total coliform and E. coli detection tests are used to check for contamination in a variety of water samples.
Water that is meant for human consumption, or potable, is routinely tested for contamination. In order for water to be deemed safe, it should contain fewer than 1 coliform per 100 mL. Here, water from a tap was collected, and tested for total coliform or E. coli contamination, as previously demonstrated. The results determined if a water source was safe for consumption.
Another sample commonly tested is treated wastewater. The water must be tested to ensure it is safe for release into the environment or repurposing for human use. As high levels of contamination were expected prior to treatment, the raw sewage sample was diluted to 1:100,000. These samples were then subjected to total coliform and E. coli detection tests, and MPN values calculated. The safe value after processing should be zero detectable indicator bacteria.
You've just watched JoVE's introduction to testing water quality using indicator organisms. You should now understand how to test water samples for E. coli and other coliforms, and how to quantify the degree of contamination present. Thanks for watching!
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Q1: Why are indicator organisms used to test water quality instead of testing for specific pathogens?
Monitoring water samples for each unique pathogen associated with fecal pollution is not feasible due to extensive labor, time, and costs. Indicator organisms provide a simple, rapid, and cost-effective way to detect fecal contamination. Their presence correlates with pathogen presence, allowing water safety assessment without testing for every individual disease-causing microorganism.
Q2: What are the five criteria that make an organism suitable as a water quality indicator?
Indicator organisms must be detectable when pathogens are present and absent when pathogens are absent. Their numbers must correspond with pathogen levels. They should persist longer in the environment than pathogens and be tougher. Finally, detection must be easy, safe, inexpensive, and effective across all water types.
Q3: How do total coliforms and fecal coliforms differ in their origin and distribution?
Total coliforms are found in mammalian guts but also occur naturally in soil and surface water. Fecal coliforms, typically E. coli, reside entirely within gastrointestinal tracts of mammals and birds and are continuously shed in feces. Because fecal coliforms are exclusively from fecal sources, they serve as more specific indicators of fecal contamination than total coliforms.
Q4: What chemical reactions occur when substrate tests detect coliforms in water samples?
For total coliforms, added ONPG is converted to nitrophenol, turning the water yellow. For fecal coliforms, E. coli converts MUG to methyl-umbelliferone product that fluoresces blue-green under ultraviolet light. These color changes and fluorescence signals confirm the presence of indicator organisms in the water sample.
Q5: How is the Most Probable Number calculated to quantify coliform contamination?
After incubating a sample-reagent mixture in a partitioned tray, positive wells are counted and categorized by size. The number of small positive wells is located on the chart's top axis, and large positive wells on the left axis. The intersection provides the Most Probable Number, representing the estimated organisms per 100 milliliters of water sample.
Q6: What safety standards apply to drinking water and treated wastewater regarding coliform levels?
US drinking water supplies must contain zero total coliforms per 100 milliliters to be deemed safe for human consumption. Treated wastewater must also achieve zero detectable indicator bacteria before release into the environment or repurposing for human use. These strict standards protect public health by ensuring pathogenic contamination is minimized.
Q7: What is the role of sodium thiosulfate in water quality testing procedures?
Sodium thiosulfate is a powdered reagent included in sample bottles to neutralize chlorine that might be present in water samples. This neutralization is essential because residual chlorine can interfere with indicator organism detection and compromise test accuracy, ensuring reliable results for water quality analysis.