Environmental Microbiology: An Interview with an Industry Expert

Microbiologics recently had the opportunity to interview Dr. Edward Askew, the founder of Askew Scientific Consulting, LLC. Edward has been involved in the environmental industry for over 25 years. He became a laboratory supervisor for a wastewater plant in 1995 for the city of Muscatine, Iowa. Edward then became involved with the professional organizations such as the Water Environment Federation (WEF) and the American Water Works Association (AWWA). As a committee member at the state and national level, Edward has been involved with regulatory compliance with federal regulation involving the Clean Water Act (CWA) and Safe Drinking Water Act (SDWA). He is currently the Part 4000 Coordinator for Standard Methods for the Examination of Water and Wastewater and the chair of the Water and Wastewater Community for AOAC International.

Why do we need regulatory oversight?
If we are to have clean water for drinking and a clean environment, then there must be regulatory oversight. Past history, such as the Cuyahoga River fires, caused the public outrage that led to the formation of the Environmental Protection Agency (EPA) and the CWA. But, creation of federal regulations and agencies does not assure protection for the public. Failure to have meaningful regulatory oversight leads to complacency and tragedies such as the recent case of E. coli contamination of drinking water in Walkerton, Ontario.

Case History: The Walkerton, Ontario O157:H7 strain of E. coli bacteria contamination
In 2010, the public consumers of the drinking water were exposed to E. coli O157:H7 and Campylobacter jejuni in their drinking water for weeks. This tragedy was caused by both the drinking water utility management's culture of deceit and sloth and the regulatory agency's failure to have meaningful oversight. Seven people died, and more than 2,300 became ill. Some people, particularly children, may be effected over their lifetime.

What controls needed to be in place?
First of all, the regulators should have performed a more in-depth audit of the sample collection practices at the utility. Utility staff regularly falsified the site the sample was collected due to the culture promoted by the management. A review of utility records and unannounced on-site observation with split samples would have emphasized to the management that their practices were under review.
Secondly, if the inspectors had looked into the chlorine disinfection practices at the utility with emphasis on the chlorine purchases, that would have led to the discovery that the utility was not disinfecting the drinking water as required. This discovery would have lead regulators to discover that operators were falsifying the chlorination/disinfection records.

What were the final outcomes of the tragedy? The deaths and illnesses caused by the utility management's actions have led to greater emphasis on training of drinking water plant operators. The regulators have reviewed their existing agencies and have recommended increasing oversight with inspectors that are technically proficient in the areas they regulate.

What are the major federal and state regulatory rules that control microbiological testing for water?
Current rules for Safe Drinking Water under either Ground Water or Surface Water refer to 40 CFR Part 141 for the statutory analytical methods for chemical and biological analytical methods. The Clean Water rules refer to 40 CFR Part 136 for the statutory methods for chemical and biological analytical methods.

How have they changed over the last few years?
For microbiological methods, improvements such as the sealable bacteria most probable number (MPN) quantification trays used in enzyme substrate tests for E. coli have allowed more laboratories to analyze their water for this indicatory organism. Also, new EPA methods for coliphages and rapid analysis utilizing polymerase chain reaction (PCR) have expanded the regulatory oversight of compliance with the SDWA and CWA.

What do you see as the major analytical concerns for a water laboratory performing bacteria analysis?
Sample collection and preservation of microbiological samples are key to producing accurate and defensible results. The laboratory must have in place procedures that meet the minimum requirements of the regulatory method and they must actively audit these practices, especially in the field.

Holding times must meet the requirements of the regulations. The holding time begins when the sample is collected and ends when the sample is set-up for testing. Exceeding these holding times will produce a biased result which most probably is an under reported value. This result is termed to be a "false negative" and places the laboratory in violation of their regulatory requirements and can put the public at risk.

What minimum quality controls checks should a water laboratory have in place?

Dilution water:
The dilution water should be checked regularly for background bacteria that can produce false positive results and chemical carryover from the laboratory handling procedures that can produce false negative results.

Sterilization:
The autoclave efficiency and the sterilization procedures should be checked regularly. The use of culture strips/ampules along with an autoclave thermometer can determine the efficiency of the autoclave. Also, samples of the equipment and media sterilized should be checked for sterilization efficiency.
Cleaning agents: Cross contamination by cleaning agents used in the laboratory for glassware or countertops must be monitored and eliminated. Chemical test such as the Bromothymol Blue test will indicate cleaning agent carry-over from the glassware washing procedures.

Media and Buffers:
All media and buffer preparation, storage and shelf-life must be monitored and checked on a regular basis. The use of positive bacteria controls for media and buffer viability is key in preventing false negative results.

Temperatures:
All temperatures should be monitored regularly and their values recorded. Procedures need to be in place to determine the accuracy of all temperature recording/indicating devices.

Positive Controls and Negative Controls:
All microbiological tests should have challenge organisms to test whether the analysis will produce a positive or negative result. These controls along with a control blank should be run at a minimum with each sample batch.

Initial and Ongoing Demonstrations of Capability:
The analytical capabilities the analyst should be determined on a continuous basis. The analyst's initial demonstration of capability should be determined prior to any results being reported and the analyst's ongoing demonstration of capability will provide oversight of analyst performance.

Duplicates:
Duplicate reproducibility measured by the Relative Percent Difference will determine the procedure and analyst precision.

Control Blanks:
The dilution water or buffer solution used by the analyst in an analysis batch is analyzed as a unique sample. A positive result would indicate a cross contamination.

Control Standards:
Both Single Blind Performance Tests and Laboratory Internal Controls are needed to establish the analyst's capabilities and determine if the analysis procedure is performing correctly. Organisms that will produce positive or negative results are used.

Root Cause Analysis:
All quality control checks must have limits that must be met if the analysis is considered to be "In Control". If the limits are exceeded, then the analysis is "Out of Control" and a root cause analysis must be performed. The laboratory must have a procedure in place outlining the steps that must be taken to perform the analysis and correct the problem.

What will the future hold for microbiological analysis in a water laboratory?
In the near future, the laboratory will have to address the quality control requirements for PCR analysis. Different routes for cross contamination of water samples need to be identified and prevented. Also, the challenge of equating PCR results to the current membrane filtration/most probable number tests will require the laboratory to educate their clients. On the horizon are the emerging pollutants such as coliphage enumeration for the confirmation of four-log removal under the EPA Ground Water Rule and the move from indicator tests, such as the current E. coli analysis, to pathogen-specific tests. All in all, the future will be both interesting and demanding for the lab.

Thank you Mr. Askew for sharing your expertise with the Microbiologics® Magnified readers.

Microbiologics offers a wide variety of qualitative and quantitative QC microorganism products that are designed for water testing. For example, E3 Epower™ provides a concentration of 10³ CFU per pellet and can be used to perform water tests using the Membrane Filtration Method. EZ-Accu Shot™,  EZ-CFU™ and EZ-CFU™ One Step are ideal because they provide a final concentration of less than 100 CFU per 0.1 mL. KWIK-STIK™ and LYFO DISK® microorganisms are perfect for presence/absence testing.

Helpful Resources:

• Click here to view a comprehensive table of recommended QC strains and regulatory methods for the CWA and SDWA.

• Click here to view illustrated instructions for performing water testing using E3 Epower™ and the Membrane Filtration Method.