Food for Microbes

According to Valeria Tassone of Star Ecotronics in Milan, Italy, All metalworking fluids contain substances that provide the essential nutrients required for microbial growth, including amines, mineral oil, and fatty oils as well as cations and anions like calcium, sodium and magnesium sulfate.

As she explained to the gathering in Ostfildern, Germany, two types of microorganisms contaminate metalworking fluids: bacteria and fungi. Microorganism growth can create a number of problems that can degrade coolant properties and adversely affect cutting or forming performance, said Tassone.

In addition, stressed Fred Passman of Biodeterioration Control Associates, contaminated fluids increase health risks to exposed workers. Operators have been diagnosed with such diseases as hypersensitivity pneumonitis, asthma, bronchitis and Legionella.

The most common on-site method for detecting microbes in MWFs is the dip slide, which involves immersing a slide into the fluid for few seconds, placing it in a test tube, and incubating for 24 to 48 hours. After incubation, the slide is compared to a standard to determine the count of culturable bacteria and fungi.

Unfortunately, said Passman, who is based in Princeton, N.J., the generation time of common microbial contaminants in metalworking fluids ranges from less than an hour to more than 12 hours. This means that, on the one hand, population densities of faster-growing microbes can increase by more than a thousand-fold during the time normally required for culture methods such as dip slides to generate results.

On the other hand, slower-growing microbes can take a week or longer to form colonies. Invariably, dip slide tests miss these slow growers.

Tassone concurred, noting that the long time between sampling and results can lead to a long delay before corrective action is taken. Also, test evaluation is highly subjective, and the test slides must be disposed as hazardous waste.

Patrick Whalen from LuminUltra in Fredericton, New Brunswick, Canada, added another caution: False negative test results are likely when inoculated media are observed for only 24 to 48 hours. Moreover, it has been well documented that any given culture protocol is likely to fail to detect significant portions of the total population.

Lighting Up Fluids

To overcome the drawbacks of using dip slides, ASTM Subcommittee E 34.50 developed a test based on the bioluminescence phenomenon that can detect the presence of microbial contamination in real time. As the word suggests, said Tassone, bioluminescence is a type of microbiological analysis based on reading the amount of light emitted by the sample.

This procedure does not require special sample preparation, can be carried out at manufacturing sites, and provides immediate information on fluid contamination, she said. Because the technique is able to detect the presence of microbes in just a few seconds, corrective action can be taken quickly before problems arise.

Having reliable results in a short time allows a quicker and more effective response, avoiding potential damage due to bacteria and fungi growth, said Lilliana Russo of Petronas Lubricants, Villastellone, Italy. Fast reaction saves money and time.

Bioluminescence relies on measuring a chemical called ATP (adenosine-5-triphosphate), which transports chemical energy within cells for metabolism. When ATP reacts with the enzyme luciferin-luciferase, the sample emits light in the same way that fireflies emit light when the enzyme reacts with the insects own ATP. There is a direct correlation between the light emitted and the amount of ATP and, therefore, the number of microorganisms in the sample, said Tassone.

One issue that must be resolved when measuring bioluminescence is that ATP is widely present in nature in different forms. Since this natural ATP is chemically identical to that emitted by microorganisms in the sample, detecting only the ATP caused by the microorganisms is a challenge.

Three types of ATP are found in nature: microbial, somatic and free. Microbial ATP is contained in viable microorganisms and is the chemical of interest. Somatic ATP is contained in plant and animal cells. Free ATP is released from damaged animal and plant cells, including those of microorganisms.

However, said Tassone, testing shows that the amount of free ATP in metalworking fluids is negligible; therefore, use of additional chemicals to destroy nonmicrobial ATP is not required.

From Meals to MWFs

Bioluminescence initially was used as a quick way to verify the hygiene of surfaces in the food industry. The method detects total ATP arising from both microbial and nonmicrobial sources. Today, with the development of instruments with improved sensitivity and selectivity, bioluminescence is widely used to certify the sterility of finished products in many industries, including cosmetics and toiletries, and foods and beverages like dairy, wine, fruit juice, infant food, soup and sauces, low-alcohol beverages, etc.

The instruments can detect the presence of a single bacterium in the sample, after an incubation period or a filtration process. According to Russo, Longer incubation provides better assessment of low contamination levels, far below the limit of dip slides. This reduces the number of false positives or undetected contamination. She suggested that this approach may be more appropriate in laboratory and R&D work, allowing the effectiveness of a specific additive or a new formulation to be tested.

Proper sample preparation provides the best conditions for microbes to grow to a detectable level, which for dip slides is about 100 colony forming units per milliliter (CFU/ml). The level of microbes typically exceeds this value in MWFs, so incubation typically is not required and the sample can be analyzed immediately, said Tassone. In fact, she noted, according to the Canadian research institute IRSST, the population density of microorganisms in metalworking fluids – bacteria, fungi, molds and yeasts – can easily reach 1 billion CFU/ml.

In bioluminescence tests, a chemical is added to the sample to release microbial ATP and starts the reaction. Then luciferin-luciferase is mixed with the sample, and a light signal is emitted that is measured by a luminometer and recorded as relative light units (RLU). The light intensity is directly proportional to the concentration of ATP in the sample and, in turn, to the number of microorganisms.

The only consensus standard method for quantifying ATP in MWF is ASTM E 2694, Standard Test Method for Measurement of Adenosine Triphosphate in Water-Miscible Metalworking Fluids. The Colloquium heard Passman and Whalen present the results of a study on the effectiveness of this method. The study showed that ASTM E 2694 provides a simple, rapid, reliable means of measuring biomass in MWF, stated Passman.

ASTM E 2694 can be performed either at manufacturing plants or in laboratory facilities, he said, and the method can be mastered with minimal training and no previous technical expertise.

Benefits, Challenges

Reduced analysis time is the major advantage of bioluminescent analysis of MWFs. The method can provide precise measurements of fungi and bacteria in less than 10 seconds, compared to the 24 to 48 hours needed by the dip slide method.

Petronas Russo summarized the other benefits of bioluminescence:

Can be performed even by personnel with limited chemistry and lab experience.

Easy to instruct operators.

Minimum equipment required.

High sensitivity.

Portable instrumentation.

Corrective action can be taken within minutes after testing.

Easily disposable, unlabeled chemicals are left, and no biological materials. Dip slides, on the other hand, have to be treated and sterilized before disposal, Russo pointed out.

The shorter analysis time allows quick and accurate addition of biocides to maintain the optimum performance of MWFs.

In addition, as Russo showed, the method reduces testing costs compared to using dip slides. According to Petronas estimates, the cost of bioluminescence testing is under 3 per sample – quite a bit less than the cost of a typical dip-slide analysis for bacteria, fungi and yeast.

After two years of testing, Russo stated, our experience shows excellent response, and not a single case of contamination went undetected. In addition, in several cases, the bioluminometer detected microbes when dip slides where negative. This increased our confidence and that of our customers about the reliability of the method.

The current ATP test method overcomes handling and interference issues, Passman said, adding a note of caution. However, it still shares two limitations with historical methods. First, dormant microbes have undetectable concentrations of ATP and will not be detected by the ATP test. Second, the method does not differentiate between bacterial and fungal ATP.

Additional testing or examination might be needed to differentiate between bacteria and fungi, and to determine whether antibacterial, anti-fungal or both types of corrective action are needed, said Whalen.

Russo believes it would be of great interest to find a protocol that detects only fungi. Very high RLU levels are often related to fungal contamination. Unfortunately, fungi detection is somewhat tricky, and sometimes misleading. She noted that even if lab results are negative, a manufacturing plant can still experience huge problems with fungi. Testing is underway with a focus on detecting fungi, she said.

Russo also added that a method is under investigation to allow tank-side testing, at least for large centralized systems or particularly critical plants and production areas. This will better quantify the level of contamination so that proper action can be taken.