The Mysterious “Bac-T” Test

Understanding biological testing.

By Eric Duderstadt

As the revised total coliform rule (RTCR) became mandatory in April, all public water systems are required to accept new criteria and treatment techniques for addressing possible sanitary defects.

The group of bacteria for which the rule is named after was previously adopted for such a role, with the implementation of the original total coliform rule (TCR) in 1989.

Since that time the analytical methods used for monitoring coliforms, commonly known as “Bac-T” tests, have jumped to the forefront of bacterial testing in the water industry. The notoriety these methods have gained as a result of these regulatory actions has not been unexpected.

However, the general understanding of biological testing and its ability to provide additional insight into the fouling potential and operational aspects of a well has, in large part, taken a back seat to coliform testing.

Even worse, misconceptions regarding the interpretations of Bac-T testing results have risen, leading to a state of confusion with suppliers, regulators, and consumers alike.

The presence of specific bacteria within our water supplies can undoubtedly pose risks to human health and have been known to do so for hundreds of years. However, bacteria can also be responsible for a number of other undesirable impacts—including foul odors, corrosion, production losses, and tarnished
aesthetic properties.

The ability to account for other species of bacteria responsible for biofouling and assess the overall biological load can be a valuable tool in extending the life of a well and preventing costly maintenance procedures. Increasing demands on produced water quality and a greater emphasis on asset management are resulting in a growing need within the industry for more accurate monitoring of water wells and the fouling mechanisms they are susceptible to.

Beyond the Coliform

“Bac-T” testing, coined from the methods available for coliform detection, has become the industry standard for determining drinking water’s sanitary quality due to regulatory actions put in place by the U.S. Environmental Protection Agency. A group of closely related bacteria, coliforms have earned the role of “indicator” organisms due to their similarities to a variety of bacteria, parasites, and viruses all known to be harmful if consumed.

Simply put, the presence of coliforms in water suggests the probable presence of other known pathogens—diseasecausing organisms.

The water industry has come to rely on this renowned organism as an answer to the impractical alternative of testing directly for the numerous pathogens potentially inhabiting a water supply. Yet, the practicality of this method does come with a few qualifiers. First, “coliform” does not refer to a single species of bacterium. Rather, coliforms are a large group of bacteria, most of which occur naturally in the environment and pose little risk if consumed.

In reality, only a small percentage of coliforms are pathogenic. Thus, the designation between total coliform and fecal coliforms is important to consider. Fecal coliforms, which include Escherichia coli, are the organisms of most sanitary significance as they are commonly found in the feces of warmblooded
animals and accordingly indicate a true contamination event.

Additionally, the presence of coliforms only suggests the potential presence of more problematic organisms; they don’t confirm it. And more important, the absence of coliforms doesn’t confirm a water sample is free of harmful microorganisms.

To illustrate this point, in the most recent Centers for Disease Control and Prevention’s Surveillance for Waterborne Disease Outbreaks Associated with Drinking Water report, Legionella was found to be responsible for 66% of all the drinking water–associated disease outbreaks from 2011-2012. All other non-Legionella bacteria collectively accounted for just 16% of the confirmed outbreaks.

Legionella is a pathogenic group of gram-negative bacteria including the species L. pneumophila,  responsible for Legionnaires’ disease. Yet they are not detected by coliform testing.

Coliform evaluation using the Simplate method. The test method confirms the presence of coliforms and allows for enumeration of the occurrence.

The Biological Fouling Mechanism

As the groundwater industry continues to adapt to the ongoing challenges of production and water quality, developing a better understanding of the fouling mechanisms impacting our wells is essential.

While Bac-T testing gives some indication of the sanitary conditions of water, it does not consider the role bacteria play in fouling outside of the context of potential health risks. In reality, bacteria have the means to influence production, materials, and water quality through a number of tactics.

Perhaps the most influential bacterial fouling mechanism is the formation of biofilm.

Biofilm is a naturally occurring expression of bacteria resulting from the extrusion of a slimy polysaccharide exopolymer. Bacteria exude this slime as a means of attaching themselves to a smooth surface for propagation, nutrient capture, and growth. Biofilms act as suburban communities within a well system, developing in numerous locations and supporting a combination of different types of bacteria, including aerobic (oxygen present) and anaerobic (oxygen absent) organisms.

Unfortunately, the aspects of biofilm that make it advantageous for bacteria are extremely detrimental to well systems. As the biofilm matrix grows, it can physically restrict flow paths and decreases production. Its excellent surface adhesion capabilities make biofilm an excellent catalyst for the accumulation
of mineral scale, further complicating well operation.

Similarly, fine-grained sediments and debris, mobilized towards the well during operation, often become entrapped in biofilm, increasing the fouling potential.

A phenomenon commonly observed in biofilm is stratification within the biofilm matrix—which not only increases the relative density and fouling potential of the biofilm, but also reduces oxygen levels in the deeper layers where anaerobic bacteria can thrive. This layering effect can harbor more harmful organisms, such as coliforms, and severely impede disinfection efforts. The increased stratification can also result in a more stubborn accumulation that challenges removal strategies.

Several types of bacteria are also known to influence the basic principles which cause corrosion. Any mode of corrosion which incorporates microbes that react and cause corrosion or influence other corrosion processes is called “microbiologically influenced corrosion” (MIC). In order for MIC to occur,
the proper environment and necessary nutrients must be available. MIC often occurs in the form of pitting but can also be seen in any number of other forms of corrosion.

By growth and metabolism, microbes can introduce corrosion-assisting constituents such as acids and sulfides into a given system. The most recognized type of MIC has traditionally been associated with anaerobic organisms, which can create acidic conditions through release of compounds created during
metabolic processes. A prominent example of this is the reduction of sulfate (SO2−4) to hydrogen sulfide (H2S) by sulfate-reducing bacteria (SRBs).

Uneven colonization of microorganisms over a surface can also contribute to corrosion by influencing oxygen concentration gradients. Aerobic bacteria located in biofilms near the water interface can create an oxygen gradient during oxygen consumption—leaving oxygen levels depleted in lower portions of the
biofilm matrix near the surface. This results in the surface area under the biofilm becoming anodic to the area exposed to the bulk aqueous phase.

Direct degradation of materials due to metabolic processes carried out by specific bacteria, such as iron-oxidizing bacteria, can also influence corrosion. One of the more recognized species of iron-oxidizing bacteria is Gallionella, which is a naturally occurring bacteria found in a variety of aquatic environments,
including aquifers. Gallionella are a stalked bacterium making use of iron as an energy source and secreting an iron-oxy-hydroxide byproduct. In its attachment to iron-bearing surfaces, Gallionella pits the metal in an effort to secure the iron necessary for energy. All iron-bearing structures, including
stainless steel, are susceptible to this form of pitting.

Degraded water quality is likewise a common result of bacterial fouling. Foul tastes and odors, resembling rotten eggs, can be created by the previously mentioned SRBs and other microorganisms.
Increased turbidity can result from both living and dead bacterial cells in solution, as well as from the detachment or sluffing off of biofilm from surfaces.

Discoloration, most commonly in the form of red water resulting from elevated iron levels, can also be a direct result of bacterial fouling. These unpleasant conditions don’t typically present a health threat, but they can become very costly to remedy during the treatment process.

The heterotrophic plate count testing method provides an assessment of viable biological growth in response to a specific agar.

Outfitting Your Toolbox

Because of the large number and diversity of microorganisms which can be found in water supplies, their ability to impact well fouling is increased and any attempt to monitor their activities must be weighed against the effort required to do so.

Albert Einstein once said, “Everything should be as simple as possible and no simpler.” In that light, identifying the best approach to monitoring bacterial fouling is perhaps easier if considered as “the minimum effort required to obtain the highest return, and no less.”

A point of diminishing returns must be identified, and often that point is when the data, and insight derived from it, does not increase relative to the amount of time and expense employed to conduct the testing.

If we use a basic understanding of bacteria and consider the types that have the greatest impact on our wells, combined with the knowledge of the advantages and limitations of the Bac-T (coliform) test, the selection of testing methods most suitable for monitoring bacteria then becomes more feasible.

The implementation of coliform testing has addressed the need for timely and affordable testing related to the presence of potentially harmful bacteria. But the knowledge of other biological fouling mechanisms implores us to expand our horizons to other types of bacteria which can influence our wells.

Quantifying the total bacterial population offers a good starting point in monitoring biologically based fouling by essentially illustrating the overall biological load within a well. A variety of methods, including heterotrophic plate counts (HPC) and adenosine triphosphate (ATP), are available and offer quick, relatively inexpensive approaches to determining the level of bacteria present. An additional benefit of ATP testing is it is non-reliant on culturing the available bacteria, which often proves to be unconducive to a large percentage of the available bacteria in a laboratory setting.

Once the population size is determined, it can be further dissected into subgroups much in the way a census is conducted. In the world of biological testing this can be done in a number of ways with varying degrees of detail. But in sticking with the approach of gaining the most bang for our buck, a simple designation to begin with is the differentiation between potentially harmful organisms and those that do not pose health risks but can cause other fouling issues.

The latter of these we could term “nuisance” bacteria. Examples of nuisance bacteria include the previously discussed slime-forming bacteria, iron-oxidizing bacteria, and sulfatereducing bacteria. These groups of bacteria are important to account for—not because they can make water supplies unsafe,
but because they can be damaging to the infrastructure of a well, drastically change the quality of water being produced, and cause increased maintenance and treatment costs.

Detection of many of these bacteria can be accomplished through relatively simple scientific testing. Microscopy can be used to not only observe active bacteria, but also detect masses of biofilm accumulations and in some cases actually identify bacteria themselves. One such example of this is ironoxidizing bacteria, many of which are easily identifiable by the structure of the stalks they produce. Simple nutrient characterization assays are also available to assess anaerobic bacteria, including SRBs.

Other tests used to identify specific bacteria include polymerase chain reaction, DNA sequencing, and phenotype microarrays. These methods vary greatly in time and cost which should be considered in determining if they are suitable for your needs.

Conclusion

In order to decipher which biological tests are most appropriate for a well system, it is necessary to first obtain a basic understanding about the microorganisms themselves which inhabit groundwater.
Bacteria are an extremely diverse domain of single-celled organisms that can be found in nearly every environment on Earth.

Testing for bacteria within water supplies has increased in notoriety, accuracy, and availability in a relatively short time span. Yet understanding which methods offer the best approach for evaluating the biological activity within a well is often overlooked due to the requirement of coliform testing
prominence of the Bac-T (coliform) test within the industry. However, the need to monitor other groups of bacteria beyond coliforms is valid due to the great influence they can have within the well environment.

The task of selecting the proper tests for bacterial monitoring can be daunting given the sheer volume of tests available, the levels of information provided, and the costs associated with them. However, the best approach is one that can account for the greatest number of influential organisms within a reasonable
time frame at an acceptable cost.

References

Centers for Disease Control and Prevention. 2015. Surveillance for Waterborne Disease Outbreaks Associated with Drinking Water—United States, 2011–2012. Atlanta, Georgia: CDC.

Duderstadt, Eric. 2013. “Customize Biological Testing for Best Results.” Opflow 39, no. 2: 20-22.

Schnieders, Michael. 2015. “Well Testing: Regulatory vs. Maintenance.” Water Well Journal 69, no. 5: 23-25.


Eric Duderstadt is an environmental biologist with Water Systems Engineering Inc. of Ottawa, Kansas, where he works as a consultant. He earned his bachelor’s degree in biology at Ottawa University in 2007 and has since become certified as a corrosion technician within the National Association of Corrosion Engineers. He also works within the firm’s research department and investigative laboratory centering on microbiology and chemistry. Duderstadt can be reached at eduderstadt@h2osystems.com.

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