Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Luisa Ikner
Water quality analysis monitors anthropogenic influences such as pollutants, nutrients, pathogens, and any other constituent that can impact the water’s integrity as a resource. Fecal contamination contributes microbial pathogens that threaten plant, animal, and human health with disease or illness. Increasing water demands and strict quality standards require that water being supplied for human or environmental resources be monitored for low pathogen levels. However, monitoring each pathogen associated with fecal pollution is not feasible, as laboratory techniques involve extensive labor, time, and costs. Therefore, detection for indicator organisms provides a simple, rapid, and cost effective technique to monitor pathogens associated with unsanitary conditions.
Indicators are easily detectable organisms whose presence correlates directly to one or more pathogens contaminating an environment. In order to be considered an appropriate indicator, an organism must meet the five following criterion:
- The indicator organism must be present when the pathogen is present, and the indicator organism must be absent when the pathogen is absent.
- The indicator organism’s concentration must correlate with the pathogen’s concentration. However, the indicator organism should always be found at higher numbers.
- The indicator organism should be able to survive easier and longer in the environment than the pathogen.
- Detection for the indicator organism should be easy, safe, and inexpensive.
- The indicator organism should be effective for all water types.
Most indicators are enteric organisms or viruses, which are commonly found in warm blooded mammalian and avian gastrointestinal systems, giving a direct connection to fecal contamination. However, many indicators can lack effectiveness due to a poor correlation with certain pathogens. Two of the most widely accepted bacterial indicator organisms are Escherichia coli and coliforms due to their fecal linkages, and ease in laboratory analysis.
Colilert is a defined substrate technology (DST) approach for simultaneous detection, specific identification, and confirmation for E. coli and total coliforms in water samples. This laboratory technique utilizes substrate nutrients specific to each indicator organisms’ metabolic pathway, enumerating only desired microorganisms, which release a signal when the bacteria alter the compound. In the presence of a coliform, the ortho-nitrophenyl-β-D-galactopyranoside (ONPG) nutrient is hydrolyzed by the coliform’s β-galactosidase enzyme. The product compound, ortho-nitrophenyl, is a chromogen that releases a color signal, turning the water yellow (Figure 1).
Figure 1. Schematic showing ortho-nitrophenyl releasing a color signal, turning the water yellow.
In the presence of E. coli, the methylumbelliferyl-β-D-glucuronide (MUG) nutrient is cleaved by the bacteria’s glucuronidase enzyme, producing a methylumbelliferone product that fluoresces blue-green under ultraviolet light (Figure 2).
Figure 2. Schematic showing the methylumbelliferyl-β-D-glucuronide (MUG) nutrient cleaved by the bacteria’s glucuronidase enzyme, producing a methylumberlliferone product that fluoresces blue-green under ultraviolet light.
Colilert can be performed as a presence-absence (P-A) test to indicate whether or not the organisms exist in the sample. This test is completed by dissolving the substrate into 100 mL water samples, incubating at 35 ± 0.5 °C for 24 h, and observing the color signals. The indicators’ presence can also be quantified by utilizing a system which determines the most probable number (MPN) for each organism. This procedure involves dissolving the substrate into 100 mL water samples that are sealed into a tray containing 49 large wells and 48 small wells. The tray is incubated at 35 ± 0.5 °C for 24 h, and then the wells containing positive color changes are counted. The ratio of large to small wells containing positive signals is aligned to the MPN chart that provides the quantification for the presence of each indicator organism present. Regulations for drinking water in the United States require that zero coliforms be present in 100 mL of drinking water.
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!
Applications and Summary
Indicator organisms are employed to rapidly and inexpensively determine environmental contamination. Colilert assays are utilized to analyze water quality for drinking, recreational, and wastewater sources. Water quality must meet legal standards set by the Environmental Protection Agency (EPA) and state regulatory departments in order to be accepted as a resource for human and/or environmental consumption.
Colilert assays are also strategically used as mass balance markers within environmental research, and this data can be analyzed along with other environmental assays to measure the correlation between results. Performing a simple P-A Colilert test gives indication whether a sample is contaminated, which can be analyzed alongside research results. If the P-A sample shows that there is contamination in the water, then the water samples being utilized in research may also have contamination that leads to misinterpreted results, while the MPN Quanti-tray provides a baseline quantification for contamination present. For example, the indicator organisms can be used to correlate indicator quantifications with the number of pathogens found in a water sample. If the quanti-tray enumerates low indicator numbers, this suggests that the water sample should also experience similar trends with low pathogen levels.