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Adenosine triphosphate (ATP) assays have been used to quantify bioburdens (biomass) in low-organic-compoundcontent fluids (freshwater, seawater, cooling tower water, and similar fluids) since the early 1950s. The original methodology was labor intensive and required considerable laboratory skill. Over the past half-century, the protocol has been simplified substantially, but until recently, chemical interferences made it impractical to use the ATP test in metalworking fluids (MWF). This article presents precision and bias statistics for a new test protocol for ATP in emulsifiable oil, semisynthetic and synthetic MWF at end-use dilutions. Additionally it presents the results of field tests in which ATP data are compared with other MWF condition monitoring data. The field evaluation demonstrates the applicability of the new protocol to MWF bioburden condition monitoring.


Recirculating water-miscible metalworking fluids (MWF) provide an excellent environment for the growth and proliferation of microbes (Passman1). These organisms, primarily bacteria and fungi, can cause two types of problems in the metalworking environment. Uncontrolled microbial activity can degrade fluid performance, by changing the fluid properties (Passman2), selectively attacking MWF constituents, or a combination of both. Moreover, this growth can cause system fouling as evidenced by biomass accumulation on sluice and machine surfaces, filter plugging, or fluid-flow restriction (Passman2). Even in so-called bioresistant MWF, microbe proliferation can pose a health risk (Passman and Rossmoore3).

The key to successful microbial contamination control is effective condition monitoring. Historically, culture methods have been the most commonly used tool for quantifying microbial loads in MWF (Passman1). Much of the time, culture methods are adequate. However, the delay between culturing and data availability is typically 36 h to 48 h and may be as long as 30 d for some microbes (Todar4). Passman5 has previously reported the use of catalase activity as a real-time parameter for measuring microbial loads in MWF. The principal limitation of catalase activity testing is that anaerobic bacteria (those that grow only in the absence of oxygen) are catalase-negative (they lack the catalase enzyme). Consequently, the catalase test does not detect sulfate-reducing bacteria and other anaerobic bacteria that can act as biodeteriogens in MWF.

The ATP test was initially developed for quantifying biomass in aqueous systems (Strehler and McElroy6; Holm-Hansen and Booth7). In the original protocol, ATP was extracted from cells by boiling the sample in a buffer. The extracted ATP was reacted with the enzyme-substrate complex: luciferin- luciferase. In this reaction, the luciferase enzyme catalyzes the ATP-mediated oxidation of luciferin. In the process, ATP is cleaved to yield adenosine monophoshpate (AMP) and pyrophosphate, concurrently releasing a photon of light. The amount of light generated is proportional to the ATP concentration, which is proportional to the biomass.

Unfortunately, organic chemicals dispersed or dissolved in the aqueous phase of complex solutions and emulsions such as MWF interfere with the detection of the light-emitting reaction on which the ATP test depends. Over the past 50 years, numerous improvements have been made to the ATP test protocol. However, chemical interference had remained a problem. Passman et al. have previously reported the use of a new protocol that effectively eliminates the interferences that affect ATP measurement in fuels and fuel associated waters (Passman and Eachus8; Passman, et al.9). This article describes the validation of a new method for determining ATP in MWF.

Materials and Methods

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Dr. Frederick Passman, PhD is a Certified Metalworking Fluids Specialist with over 35 years experience in Environmental & Industrial Microbiology. His company, Biodeterioration Control Associates, Inc. (BCA) provides clients with unparalleled expertise in Microbial Contamination Control.

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