New test method for detecting microbes in MWFs

Dr. Neil Canter, Contributing Editor | TLT Tech Beat August 2009

Results are available in 5-10 minutes rather than hours.

KEY CONCEPTS
• The current methodology to determine the level of microbes in a metalworking fluid system is time consuming.
• A new method for detecting ATP quickly and accurately determines the concentration of microbes and can be run in a matter of minutes.
• Preparation of a subsample by filtration through a 10-micron filter can be used to differentiate between bacteria and fungi.

Microbial contamination continues to be an ongoing problem in managing metalworking fluid systems. If left untreated, bacteria and fungi can degrade the components in a metalworking fluid and present health risks to the workers at an end-user facility.

Degradation of metalworking fluids leads to a reduction in fluid performance. Such detrimental issues as corrosion, increased tool wear and replacement and increased parts rejection rates also can occur in systems dealing with microbial contamination. One other factor is the generation of biofilms that can lead to filter plugging.

Determination of the level of microbes is mainly conducted by using dipslides. STLE-Fellow Dr. Fred Passman of BCA, Inc., in Princeton, N.J., says, “The purpose of the test is to detect microbes by placing a sample of the metalworking fluid in a growth medium for 36 to 48 hours. On one side of the paddle, red spots characteristic of bacteria can grow while on the other side fuzzy white spots characteristic of fungi are seen.”

But if you are facing a problem at an end-user facility and need an immediate answer, this test is time consuming and only detects 0.01% to 1% of the microbes in a metalworking fluid system. Passman adds, “Only this small percentage of microbes is likely to elaborate into colonies and, therefore, be seen by this method.”

This problem can be compounded because in many cases a sample from the metalworking fluid system is collected and then mailed to a facility that is managing the system. Even if the sample is not received within 24 hours, this represents an additional loss of time in dealing with the problem.

For a metalworking fluids system that is prone to a quick loss of control due to microbial contamination, the dipslide procedure is not adequate. Passman explains, “In a system that can be subjected to extensive microbial contamination in six to eight hours, the dipslide procedure cannot generate data fast enough to prevent the system from going out of control.”

There is need for a procedure to give the individual maintaining a metalworking fluid system a fast and accurate answer regarding microbial contamination that can also be conducted onsite. Such a procedure has not been available until now.

LUCIFERIN-LUCIFERASE
A test method has been developed to determine the concentration of microbes in a metalworking fluids system with the results generated in only a matter of five to 10 minutes. It was developed by LuminUltra™ Technologies Ltd. And is commercially available in a test kit known as the Quench-Gone Organic Modified (QGO-M) ATP test kit.

Pat Whalen, president of LuminUltra, says, “We have developed a test procedure to measure the primary energy carrier present in all living things, Adenosine Triphosphate (ATP). This technique extracts ATP from the microbes present in the metalworking fluid and detects its concentration as a function of the concentration of light released when the ATP is reacted with the enzyme substrate complex Luciferin-Luciferase.”

Reaction of ATP with Luciferin-Luciferase produces Adenosine Monophosphate (AMP), pyrophosphate and a photon of light. Passman adds, “The ATP test was originally developed to quantify biomass in aqueous systems (1). A variation was used to determine the concentration of microbes in metalworking fluids but was plagued by interference from presence of organic chemicals dispersed or dissolved in the sample.” 

The current test method overcomes these problems by filtering the sample prior to extraction of the ATP. This procedure has been approved by ASTM and is designated as E 2694 (2).

The procedure for using the test kit is shown in Figure 1. The MWF fluid sample is concentrated by filtration through a 0.7-micron, in-line, glass fiber filter. An extraction reagent is used to isolate the ATP, which is then mixed with the Luciferin-Luciferase reagent in a cuvette. The sample is placed in a luminometer that detects light at 420 nanometers. A value in Relative Light Units (RLU) is obtained that can be converted to the actual ATP concentration in Log₁₀ pg (picograms) ATP/milliliter of sample. A picogram is 10^-12 gram.


Figure 1. The procedure for determining the concentration of ATP in a sample involves concentrating the sample, extracting and diluting the ATP and obtaining a measurement in Relative Light Units. (Courtesy of LuminUltra)

The ATP concentration data readily provides a good assessment of how well a metalworking fluid system is being maintained to minimize microbial growth. Passman explains, “A value less than log₁₀ 2 is an indication that a metalworking fluid system is well maintained. Values between log₁₀ 2 and log₁₀ 3.5 mean that the system has moderate microbial contamination and needs to be closely monitored. Systems with values above log₁₀ 3.5 have heavy microbial contamination which needs to be dealt with immediately.” 

This test procedure is a modification of a technique used to determine the concentration of microbes in wastewater and drinking water. Whalen says, “We conducted research in developing this test method for the past 12 years, particularly for use in wastewater treatment which is a most unforgiving application. The procedure has worked well with no incompatibilities.” 

Passman indicates that no incompatibilities have been seen with the three main types of water dilutable metalworking fluids (emulsified oils, semisynthetics and synthetics). The method was vetted in a 12-week case study evaluating 12 metalworking fluid systems that were conducting a variety of large and small chip metal-removal operations. Two technicians ran each test in triplicate.

Passman says, “The coefficient of variation for the ATP test is less than 10%.” 

While the ATP test can quickly provide information on the concentration of microbes, there is a question about how to differentiate between bacteria and fungi. Both microbe types can be present in a system, but a maintenance engineer needs to deal with each of them in a different manner.

Passman answers, “The best approach is to prepare a subsample by filtering the sample through a 10-micron filter. All bacteria are less than 5 microns in size and should pass through this filter. Fungi are larger in size and should be isolated by the 10-micron filter.”

Future work will involve the participation of at least six labs in a full interlaboratory study with the objective of developing an ASTM research report, which will define the precision and bias of the method over the method’s recommended ATP measurement range.

Additional information can be found in a recent paper presented at STLE’s 2009 Annual Meeting & Exhibition (3) and by contacting Passman at bcainc@comcast.net. 

REFERENCES
1. Holm-Hansen, O. and Booth, C. (1966), “The Measurement of Adenosine Tripohosphate in the Ocean and its Ecological Significance,” Limnol. Oceanog., 11 (4) pp. 510–519.
2. ASTM E 2694 (2009), Standard Test Method for Measurement of Adenosine Triphosphate in Water-Miscible Metalworking Fluids.
3. Passman, F., Egger, G., Hallahan, S., Skinner, B. and DeSchepper, M. (2009), “Real-Time Testing of Bioburdens in Metalworking Fluids Using Adenosine Triphosphate as a Biomass Indicator,” STLE Annual Meeting & Exhibition, Lake Buena Vista, Fla., (Manuscript has been accepted for publication in Tribology Transactions).


Neil Canter heads his own consulting company, Chemical Solutions, in Willow Grove, Pa. Ideas for Tech Beat items can be sent to him at neilcanter@comcast.net.