Method for determining microbial contamination in cooling lubricants in real time

Abstract

Method for solubilizing and clarifying water-based cooling lubricants present as an emulsion in order to quantitatively determine in real time the content of microorganisms and their metabolic activity in the area of ​​interest, characterized in that a clarifying reagent consisting of organic and / or inorganic salts together with non-ionic surfactants in the described concentration and composition is added as a formulation to a cooling lubricant, thereby bringing about immediate clarification at room temperature without heating the emulsion, and measurement in clear, transparent liquid can be carried out in real time using optical or physical methods, such as flow cytometry.

Description

[0001] The invention relates to a method for the chemical and physical pretreatment of the cooling lubricant by converting the existing emulsion into a clear solution. This method is used for certain optical and spectrophotometric measurement techniques such as counting chambers / epifluorescence optical counting, flow cytometry, or simply particle counting using the flow impedance method or electrical flow sensing, and other measurement techniques. This enables the quantitative detection of microorganisms in real time within a range of interest for the cooling lubricant, approximately 10^3 to 10^6 germs per ml or g, whereby the metabolic activity can also be determined.

[0002] Cooling lubricants are oil-in-water emulsions in which the oil is dispersed in the water as droplets. With a particle size of 0.1 µm or smaller, they are generally classified as microemulsions, which are thermodynamically stable. Water-in-oil emulsions are practically obsolete in cooling lubricants. Macroemulsions are kinetically stable. Another classification principle for microemulsions is based on the type of surfactant used (anionic, cationic, or non-ionic). Typically, the multi-component system consists of water, oil, a surfactant as an emulsifier, and a co-surfactant. Examples of co-surfactants include butyldiglycol, n-pentanol, or other alcohols or glycols. Oil-in-water emulsions are generally white or opaque in appearance, with the oil droplets forming the inner phase. Water-soluble emulsifiers are used in these emulsions.

[0003] Cooling lubricant (CL) is an emulsion used in the mechanical engineering industry for drilling, milling, and grinding. Cooling lubricants are extremely complex in their composition. They often vary considerably from manufacturer to manufacturer and frequently contain up to 15 different chemical compounds. Depending on the application, they differ in pH value, conductivity, and other parameters.

[0004] Water-miscible cooling lubricants contain emulsifiers, corrosion inhibitors, polar agents, and extreme pressure (EP) additives, microbiocides, solubilizers, and defoamers. The purpose of cooling lubricants is to ensure high feed rates (number of workpieces per unit of time) while achieving a high surface finish for exceptionally long tool life. Therefore, there is a need for both high cooling and good lubrication. The oil to be emulsified is often mineral oil or based on plant esters as a basic microemulsion.

[0005] Cooling lubricants (soluble oil) are typically used at concentrations 5% to 10% higher than usual – most commonly 5%. Because they consist mainly of water and contain organic components (oil), cooling lubricants are subject to biodegradation (microbial deterioration) as microorganisms metabolize the ingredients. If microbial contamination persists at a certain level for a specific period, for example, 10⁶ germs / ml, the cooling lubricant deteriorates; it becomes unusable and loses its properties. In this case, a complete replacement is necessary, which involves significant costs and labor for cleaning. To prevent this, biocides such as thiodiazoles, phenols, or aldehydes are added at a concentration of 0.3–0.4%. Various biocides are possible, including fungicides and bactericides.Several indirect parameters are monitored to assess the performance of cooling lubricants, such as nitrite content, pH value (as buffering capacity / alkali reserve), viscosity, coolant concentration, water hardness, and microbial count. Most cooling lubricant formulations have a pH value between 8 and 9. This pH range already excludes many microorganisms from growth. Biocides stress bacteria, which can be understood as the first step in the development of resistance.

[0006] In large central warehouses, it is therefore crucial not only to record indirect measurements, such as pH changes (since microbiological growth causes changes), but also to directly measure the metabolic activity of microorganisms that affect the metalworking fluid (MWF). If microbiological growth exceeds a level of often around 10⁶ germs per ml within a certain timeframe, the MWF "turns over." This causes the MWF to lose its properties, become unusable, and require disposal. After extensive cleaning, new MWF must be supplied to the central system, a process that incurs enormous costs. Therefore, a biocide or biocide mixture is continuously added, as the biocide is consumed. However, biocides have a strong influence on the physiological status of microorganisms and thus also affect the overall flora, including growth behavior on the agar plate as a phase interface reaction.In our experiments, we observed that no growth was present on the agar plate, but detection occurred in the flow cytometer. Therefore, if no growth is observed with dip slides or on the agar plate, leading to false results, it is crucial to employ a method that characterizes the actual physiological status as a true snapshot of the metabolic activity of the microbes in the cooling lubricant.

[0007] Since cooling lubricant (CL) is a heterogeneous emulsion that is often heavily contaminated with dirt, slime, and metal particles in practice, optical or spectroscopic measurement methods that allow for the real-time determination of microorganisms are not feasible. These levels of contamination are very high during extended periods of CL service life.

[0008] The area of ​​interest where bacteria are present becomes covered or obscured – typical for an emulsion. Staining with fluorescent dyes is only possible after significant prior dilution. This means that real-time determination of bacterial contamination is no longer possible, as pre-enrichment is necessary to obtain a quantitative result in the measuring range of 10⁻³ to 10⁻⁶ bacteria per ml.

[0009] However, pre-enrichment should be rejected, as it reflects a completely false picture of the metabolic activity of the germs.

[0010] While an indirect measurement of microorganisms via ATP content is possible, it is meaningless because the measurement is given in RLU (Refrigerant Units) and cannot be directly converted into germs per ml or g. Furthermore, numerous effects such as quenching, pH influences, and interactions with biocides, detergents, etc., occur due to the chemical composition of the cooling lubricant, significantly distorting the measurement result in RLU. Additionally, cooling lubricants vary considerably from manufacturer to manufacturer.

[0011] Even today, classic methods like agar plates or a modified version, dip slides, are still used. However, these methods don't provide an immediate, real-time assessment of the current microbial count. Dip slides, in particular, require at least 48 hours for a definitive result. Another disadvantage is that a count or a comparison with surface growth density is necessary to obtain a quantitative assessment. Furthermore, the presence of biocides induces physiological states that prevent microbial growth on the agar plate or dip slide, leading to inaccurate results. Therefore, classic methods are not practical and deliver inconclusive results far too late for effective monitoring. An online method providing real-time results would be highly desirable. For this reason, a new method was developed, taking into account previous experience with emulsions.

[0012] Surfactants are amphiphilic, surface-active substances whose molecular structure contains regions with different polarities. These different polarity zones result in hydrophobic / lipophilic and hydrophilic / lipophobic regions.

[0013] A distinction must be made between anionic, cationic, non-ionic, and amphoteric surfactants. Only non-ionic surfactants are used here to avoid premature lysis.

[0014] Surfactants are typically water-soluble, but not uniformly distributed in the solution. At low concentrations, the surfactant concentrates near the interface, while molecular clusters form as the concentration increases.

[0015] The type of micelles that form usually depends on the structure of the surfactant, with a distinction made between spherical micelles, cylindrical micelles and rod micelles.

[0016] The concentration at which micelles form is known as the critical micelle concentration (CMC). Depending on the type of surfactant, the CMC can vary and is temperature-dependent. Micelle formation significantly influences the physical properties of the solution, particularly particle adsorption, conductivity, osmotic pressure, and surface tension.

[0017] In an aqueous medium, the water concentration decreases from the hydrophilic surface of the micelle towards its core, which is almost completely anhydrous and therefore lipophilic. Micelles typically range in size from 5 to 100 nm, thus filling the size gap between macromolecules and nanoparticles on the one hand and, for example, liposomes on the other.

[0018] Detergents at low concentrations form a single layer at the air-liquid interface or phase. At higher concentrations, which is the case here, the monomeric single layers form other structures called micelles. A micelle is a thermodynamically stable, colloidal aggregate of monomeric detergents, where the non-polar, hydrophobic part faces inwards in a circular arrangement, and the polar, hydrophilic part faces outwards in contact with the water.

[0019] In the thermodynamics of multiphase systems, a partial derivative of the Gibbs free energy G can be calculated in liquid-liquid phase equilibrium, where the distribution coefficient is a dimensionless quantity, a measure of the tendency of a substance to be distributed between two different phases in the equilibrium state.

[0020] Both the number of detergent monomers per micelle (aggregation number corresponds to the number of molecules per micelle) and the range of detergent concentration above which micelles are formed, which is referred to as the critical micelle concentration (CMC value), are specific properties of a single surfactant. The cloud point changes noticeably with the addition of electrolytes and foreign surfactants. The HLB value is also significantly influenced by the addition of electrolytes in the form of a salting-out effect of a highly soluble salt such as NH4Cl and NH4CO2H, which also directly affects the stability of an emulsion. This is of interest because a high salt concentration is used to prevent lysis effects by microorganisms.

[0021] However, sodium or potassium salts must not be used here, as these can interact with the microorganisms and promote osmotic exchange through existing ion channels. A high salt ion concentration thus prevents lysis effects in bacterial cells. The critical micelle temperature (CMT) is the lowest temperature at which micelles can form. The CMT corresponds to the cloud point at temperatures below the critical value, but micelles clear again at temperatures above the CMT.

[0022] This was confirmed in our tests, which were conducted with cooling lubricant, milk, and sunscreen. This phenomenon occurs with the selected (non-ionic) detergent at temperatures around 55°C. Generally, a significant increase in the number of aggregates is observed with increasing ion concentration (electrolyte concentration).

[0023] In general, even mild, non-ionic detergents such as Triton-X-100, NP-40, Brij-35, or Tween 80 tend to damage bacterial cell membranes, which can lead to cell lysis and the extraction of soluble protein, often in its native form. With increasing detergent concentration, the membranes undergo several stages of solubilization. The first stage can result in lysis or destruction of the cell membrane. Under certain conditions (pH, concentration, and temperature), even "mixed micelles" containing phospholipids and membrane proteins are formed. Impurities in the ppm range (10-20 ppm) of ethylene oxide and 1,4-dioxane, which were present in the product isodecyl alcohol-11-polyglycol ether, do not significantly affect lysis. However, the exact percentage cannot be determined precisely, as it is not measurable.The methods of microbiology are not comparable to classical analytical methods such as gravimetry, titrometry or photometric methods in terms of quantitative results, because living organisms are subject to different conditions.

[0024] Non-denaturing detergents (non-ionic) such as Triton-X-100 have pronounced non-polar "heads" and do not penetrate water-miscible or water-soluble proteins. However, the lysis effect is undesirable in our application and should be suppressed, which is successfully achieved.

[0025] It is difficult to pinpoint the exact process involved in clarifying an emulsion, as several reactions occur simultaneously. A key factor is the binding of fat molecules, which is sometimes very successful with the addition of a co-surfactant, such as butyltriglycol. Under normal conditions, the stepwise addition of a co-surfactant to a cooling lubricant almost completely clarifies the emulsion; however, this process is not stable, as oil separates out as a phase after prolonged standing at room temperature.

[0026] Laboratory tests revealed the following: For non-ionic surfactants, the HLB value, the cloud point, the critical micelle concentration, and the critical micelle temperature are crucial factors.

[0027] A 20-25% concentration of isodecyl alcohol-11-polyglycol ether was used for milk. The commercial product has a cloud point of 62-66°C at 1% in 10% NaCl solution and an HLB value of 15.1.

[0028] 0.5 ml of pasteurized milk (1.5 to 3.7% fat content) is mixed with 2.5 ml of reagent and heated for 4 minutes at 55°C, as no clarification occurs at room temperature.

[0029] Very good clarification was achieved at a sample-to-surfactant solution concentration ratio of 1:5. With isodecyl alcohol-11-polyglycol ether, the heating during the emulsion clarification step resulted in a loss of approximately 30% of E. coli and Pseudomonas spp. bacteria through lysis.

[0030] In PCR analysis, a non-ionic detergent is used to disrupt cells when the sample is heated to approximately 70°C to isolate the DNA. Heating the sample with a surfactant therefore leads to lysis. Under these conditions, cooling lubricant and sunscreen oil are also clarified.

[0031] However, since any heating above room temperature definitely leads to the lysis of bacteria, the method is unusable and must be discarded.

[0032] However, a method must be used that operates at room temperature or up to a maximum of 30°C – the enrichment temperature for many microorganisms. The lower the temperature, the better – with room temperature (18° to 25°C) being the preferred method.

[0033] Fat-dissolving power corresponds directly to the HLB value. An HLB value of 15 or higher is suitable for milk and products with a higher fat content. However, a high HLB value also always implies a longer aliphatic chain and a higher degree of ethoxylation. This is diametrically opposed to the cloud point, which increases with longer chain length. Therefore, a mixture of several surfactants must be used to solve this problem. Non-ionic surfactants with a high HLB value of 15 or higher and a cloud point lower than 60 (e.g., 45-55) are preferred. This surfactant must be mixed with another surfactant to achieve solubilization at room temperature, which can also be achieved by adjusting the electrolyte concentration.

[0034] Therefore, for further tests, a mixture of Triton-X-114 with a cloud point of 23°C and Walloxen ID 110 / 80 with a cloud point of 63°C was used. However, the HLB value of Triton-X-114 was too low at 12.4 (10.3), which became noticeable in the milk applications. A mixture of Triton-X-114 (10%) with Walloxen (3%) – a total of 10 to 13% – yielded very good results when using NH4Cl. This led to the following procedure: 0.5 ml of the KSS sample was mixed with 0.5 ml of surfactant reagent. Instead of Walloxen, an alcohol ethoxylate such as alkylphenol ethoxylate, alcohol propoxylate, or laury alcohol ethoxylate can also be used. Trade names include, for example, Tergitol from DOW Chemicals 15-S-9 with a cloud point of 60 and an HLB value of 13.3, and LANSPEC EMP906. The fat-dissolving properties generally depend on the degree of ethoxylation, e.g., 10 EO / mol.

[0035] The emulsion cleared abruptly after the reagent was added. Since this 1:1 concentration of the sample, detectable in the measuring instrument, led to aggregation, exhibiting a high background of approximately 10⁷ particles over a measuring range of about 0.8 to 4 µm (the profile of which varies in height and shape depending on the surfactant), the sample was diluted with 1 ml of deionized distilled water. A 0.9% saline solution can also be used, which buffers changes in conductivity. This prevented aggregation. However, this dilution reduced the sample by a ratio of 1:3, which has a positive effect in cases of high contamination / background, as it reduces the background signal. This behavior can also be demonstrated with a photometer in the UV range at approximately 240 nm. The higher the surfactant concentration in the sample, the greater the degree of aggregation observed when the undiluted sample is in a 1:1 ratio.

[0036] However, when clarifying emulsions, it is essential that the cloud point (CMT) is as low as possible, as with Triton-X-114 at 23°C. This means that, as a mixture, the clarifying reagent will achieve clarification even at low temperatures, and this clarification will remain stable over a long period.

[0037] It was found that at elevated temperatures, starting around 40°C, a loss of bacteria through lysis occurs, which did not happen at room temperature. Generally, however, clarification of cooling lubricant samples at room temperature is possible even with a surfactant concentration of 10%.

[0038] Coolant samples from grinding machines often contain very fine particles in very high numbers. This partially binds the solubilizing capacity of the reagent, necessitating the addition of a higher proportion.

[0039] The lytic effect of non-ionic molecules such as butanol and further to non-ionic detergents shows large differences between prokaryotic cells and eukaryotes.

[0040] In bacteria, a significant difference is visible between gram-negative and gram-positive bacteria. The difference lies in the cell wall. a.) its structure itself and b.) because Gram-negative bacteria have a high proportion of lipids (phospholipids) and Gram-positive bacteria a low proportion of lipids. Gram-positive bacteria have teichoic acids in their cell walls, while Gram-negative bacteria have lipopolysaccharides. This leads to different results when the cells are stained and also has different consequences when exposed to detergents. Euckryon bacteria (e.g., yeast) showed no change in bacterial concentration at the same surfactant concentration, ionic strength, pH, and temperature.

[0041] The type of emulsifier or surfactant and its existing concentration are crucial for solubilizing emulsions in order to create a stable microemulsion. Cooling lubricants are sometimes miscible with water in any ratio.

[0042] Using the Gibbs equation Γi=−1 / RT⋅dy / d ln ci The amount of emulsifier dissolved can be calculated.

[0043] The emulsion type, O / W (oil in water) or W / O (water in oil), is not determined by the water content of the emulsion, but by the emulsifier used. The HLB value (according to Griffin) – hydrophilic-lipophilic balance – indicates the degree of equilibrium between hydrophobic and hydrophilic functional groups. This allows for a numerical approximation of a surfactant's fundamental properties. There is a direct relationship between the HLB value and the CMC value. Fat-dissolving ability is often structurally dependent on the molecule used and its functional group – however, non-ionic surfactants often exhibit good fat-dissolving properties – naturally, this depends on the HLB value.

[0044] In our application, an HLB value of 15 shows very good properties for obtaining a clear solution from the emulsion in small excess quantities after heating.

[0045] The amounts of solubilizing reagent used with the same sample quantity (milk, cooling lubricant) are significantly smaller for cooling lubricant than for milk.

[0046] A further advantage is that a dilution of 1:3 (0.5 ml KSS in 1.5 ml total volume of reagent) does not result in a significantly detrimental dilution of the microorganisms to be determined. After addition, the clear solution remains stable almost indefinitely at room temperature and is photometrically stable.

[0047] Since cooling lubricants vary from manufacturer to manufacturer, it is recommended to perform a pre-test to check the clarity and stability of the solution obtained. State of the art

[0048] The current state of the art does not know of any method for sample pretreatment for cooling lubricants where chemical-physical solubilization is carried out to clarify the emulsion in order to then be able to take a measurement directly after sample preparation that allows an accurate result e.g. 5.4 × 10e4 germs / ml.

[0049] There are, however, publications and registered patents dealing with milk, which is also an emulsion. These are not directly applicable to cooling lubricants, though, as the constituents of milk and cooling lubricants differ significantly. Milk, as a natural product, contains, for example, proteins that interact strongly with the emulsion, further solidifying and stabilizing it. Somatic cells, high levels of sodium, potassium, and calcium with correspondingly high conductivity and electrolyte concentration, vitamins, fat, proteins, carbohydrates, and trace elements promote bacterial growth at a pH of 6-7.

[0050] DE 42 18 555 A1 relates to a process for solubilizing and clarifying milk using salts and surfactants in preparation for optical examination. DD 1 19 612 A5 relates to a process for clarifying turbidity in aqueous liquids. AT 59 903 E describes a process for clarifying biological liquids. The processes described in DE 42 18 55 A1, DD 1 19 612 A5, and AT 59 903 E do not explicitly mention cooling lubricants as the subject of investigation. EP 0 584 711 B1 presents a use of alkenyl succinic acid derivatives as cooling lubricants, in which these alkenyl succinic acid derivatives are identified as a relevant component of this cooling lubricant. WO 2005 / 046 635 A2 relates to a process for producing storage-stable multiple emulsions.

[0051]

[28] US patent 3,679,365 A, dated 1972, uses a saponifying reagent and complexing agents such as EDTA and mercaptoacetic acid or ascorbic acid with a dispersing agent consisting of Triton-X-100 in a 50% methanolic solution with glutaraldehyde for fixation. The aim is always to achieve clarification of the milk, thus converting an emulsion into a clear solution. Patent EP 0 246 978 B1 uses a mixture of 0.1 N sodium hydroxide solution with butanone-2, Triton-X-100, and 1% SDS solution (sodium lauryl sulfate). Anionic or cationic surfactants generally lead to the rapid lysis of bacteria. The proposed reagents are toxic and environmentally harmful.In OS DE 17 089 A1 and in the patent application DE 4017398 A1 as well as EP 0 573 054 B1, enzymatic clarification methods or new solubilization reagents are used, whereby complexing agents such as morpholinium salts of citric acid and co-surfactants differ, with the use of non-ionic surfactants emerging as a central measure.

[0052] Unfortunately, it turns out that other effects, particularly in patent EP 0573054, were not taken into account, leading to erroneous results. The proposed non-ionic surfactant n-decyloxy-polyethylene glycol, from the polyoxyethylene alkyl ether group, has a cloud point (CMT) that is too high. This means that the clarification temperature of 55 to 60°C, where the sample is clarified for approximately 3 to a maximum of 10 minutes depending on the type and concentration of the salt, is too high. Bacteria are strongly lysed, and the loss before measurement is too high, leading to inaccurate results.

[0053] However, since micelle formation only occurs within this temperature range, which is necessary for sample clarification, it is accepted that a large proportion of bacteria will be lysed, ultimately leading to low bacterial counts. Investigations in this patent (EP 0 573 054 B1) with this in mind were not conducted. Therefore, the proposed method with the corresponding reagents and temperatures is scientifically untenable, as it leads to inaccurate results. The cited patent, however, only refers to milk as a matrix. Verification with walloxene, an isomeric, similar compound, confirmed this with E. coli and Pseudomenaden.

[0054] Particularly with gram-negative bacteria, such as E. coli and Pseudomonas aeruginosa, a large proportion of the bacteria are lysed by the reagent, leading to a decrease in bacterial count. The specified ratio of sample to reagent solution is also sometimes too high, resulting in an excess of surfactant that reacts with microorganisms and lyses them. Filtration to remove particles through a 0.2 micron nylon filter – as described – is not feasible with a 20-25% surfactant solution and is almost ineffective. Filtration of the same surfactant solution through a 0.1 micron inert nylon membrane resulted in a reduction of particles. Only surfactant solutions with a concentration of approximately 10-15% are more amenable to particle removal.

[0055] In general, non-ionic or zwitterionic surfactants are milder than anionic or cationic surfactants, which strongly denature proteins and lead to cell lysis. However, it was recognized and described early on that Trition-X-100, as a non-ionic surfactant, causes lysis under normal conditions across a wide concentration range, even at high bacterial counts (Journal of Bacteriology, July 1978, p. 153-160, James B. Cornett).

[0056] In general, it should be noted that lysis of microorganisms can also occur with non-ionic surfactants, depending on: 1 of the ion or electrolyte concentration 2 the pH value 3 of the types of electrolytes 4 of the temperature 5 of the surfactant concentration 6 and the contact time itself 7 of the matrix effects - e.g. fat and protein content 8 Membrane potential of bacteria 9 species / genus of microorganism (gram - / +)

[0057] Functionally, the cell wall in bacteria protects the cell from osmotic fluctuations; the protoplasmic membrane acts as an osmotic barrier, and approximately 70% of the intracellular water is osmotically available. However, a significant factor is the high salt concentration in the proposed formulation, which contains anions that are not utilized in ion channels. The high electrolyte concentration prevents an ion gradient from forming, thus preventing osmosis and altering the cell. Peroxide impurities in the surfactant can lead to unwanted lysis and consequently a reduction of the microorganisms being analyzed within the matrix. Therefore, non-ionic surfactants of food grade or approved for use are preferable.

[0058] Grundlegende Publikationen die zu diesem Thema genutzt wurden sind: Cellular Lysis of Streptococcus faecalis induced with Trition-X-100 (James B. Cornett - Journal of Bacteriology, July 1978 p. 153-160). The salting-out of polyethylene glycol emulsifiers - G.E. Mapstone, Dermacult S.A. (Pty.) Ltd., Johannesburg, South Africa. Disaggregation of bacterial cell wall by anionic detergents (F. Shafa , M.R.J. Salton); J. gen. Microbiol. (1960) 22, 187-141. Bacterial Lysis, - Phetica, B.A. (1958) J. gen. Microbiol. 18, 473-480 Piercenet.com - detergents for cell lysis (Themo Scientific) EP 0573054 - method for solubilising milk for purposes of analysis -1999.

[0059] The n-decyclopolyethylene glycol used in patent EP 0573054 was replaced in experiments by a different surfactant, polyoxyethylene isodecyl ether. However, the high cloud point is a disadvantage, as it requires heating to 55 °C for approximately 3-4 minutes to achieve clarification. It should also be noted that the CMC value of some specific non-ionic detergents decreases with increasing temperature.

[0060] 0.5 ml of KSS is mixed with 2.5 ml of solubilizing reagent – ​​in this case, Walloxen ID 110 / 80 – and heated for 3 to 4 minutes at 55°C. The solution becomes clear and remains stable at room temperature for more than 60 minutes without clouding. The loss of lysed bacteria is too high. The approach / method is therefore discarded. Therefore, further tests were conducted with Triton-X-114, cloud point 23°C, HLB 12.4, to avoid the lysis effect at room temperature and also to generally prevent bacterial loss that occurs at higher temperatures. This low HLB value is not suitable for solubilizing milk. However, Triton-X-114 exhibits abnormal behavior, separating into two phases at temperatures above 24°C. This is overcome by adding Walloxen ID 110 / 80. This effect is also observed during aggregation, as after adding 0.5 ml of surfactant reagent to 0.5 ml of KSS, sometimes no aggregation takes place depending on the temperature, and in the ratio 1:1 it could be measured directly without background in the particle counter.

[0061] The invention is based on the objective of using a solubilizing reagent for cooling lubricant that is present as an emulsion. Detection is achieved in small dilutions in real time using flow cytometry or related or similar rapid methods, which enable a detection limit of 5-10 yeasts per ml or g and a few thousand bacteria per ml or g in real time.

[0062] This provides the cooling lubricant industry with access to a method for the first time to monitor relevant microbial loads of approximately 10⁻³ to 10⁻⁶ germs / ml in real time. For the first time, it becomes possible to quantitatively detect metabolically active microorganisms directly under the influence of biocides, which are responsible for and relevant to changes in the cooling lubricant. Online monitoring is also possible. The new method overcomes the previous disadvantages of reagents that, due to heating steps, resulted in a significant loss of bacteria and were therefore unusable. The method is therefore carried out at room temperature, around 20°C. The reagent is stable and can therefore be used in the autosampler of flow cytometers overnight or in continuous operation.

[0063] To solve this problem, the new reagent uses non-ionic surfactants with a low cloud point, such as Triton-X-114 (23°C), or others like Lutensol Fa. 25 (36.5°C cloud point), Brij 30 (4°C in a 10% aqueous solution), C8E3 (35-40°C cloud point), and Genapol X-80 (42°C cloud point). The HLB value should also be above 12 in the mixture of non-ionic surfactants for cooling lubricants and would be optimal for raw milk with an HLB value of 15 or higher. A mixture of three, four, or more surfactants can also be used, but these must preferably form a clear solution at room temperature or a maximum of 30°C. Proceeding at room temperature is advantageous because heating is not necessary when using an autosampler / workstation.This means that the added salt mixture influences the CMC and CMT values ​​in such a way that the cloud point of the mixture should be as low as possible, between room temperature and below 30°C. The lysis effect at higher temperatures should be avoided. A dilution ratio of 1 part cooling lubricant to 1 to 3 parts reagent with water should preferably be used to prevent aggregation, which would falsely indicate an excessively high background level during measurement.

[0064] The salt used can be organic or an inorganic salt with the highest possible solubility, such as table salt. However, the anion must be neither potassium nor sodium to prevent interaction with a biological cell and thus avoid lysis effects. The high salt gradient stabilizes the system and prevents osmotic effects. Preferably, ammonium chloride, ammonium formate, or equally soluble salts are used. They have approximately the same solubility in water as table salt. A mixture of both salts can also be used.

[0065] The following formulation serves as an example. Weigh 18.8 g of NH4Cl into 90 g of warm distilled / deionized water in a beaker and stir with gentle heating on a heated magnetic stirrer to neutralize the negative heat of solution. At approximately 30–40°C, the salt or salt mixture dissolves relatively quickly. After cooling to approximately 20–25°C, 4 g of 80% isodecyl alcohol-11-polyglycol ether are added with vigorous stirring, resulting in a clear solution. Then, 10 g of Triton-X-114 are slowly added with vigorous stirring at room temperature, yielding approximately 100 ml of ready-to-use reagent. If the temperature is too high, a cloudy, milky liquid will form, which will only become clear and transparent upon further cooling.

[0066] As another example, consider 63 g of 95% ammonium formate (containing 5% water) dissolved in 90 g of warm distilled or deionized water. With vigorous stirring, 4 g of isodecyl alcohol-11-polyglycol ether are dissolved, followed by the slow addition of 10 g of Triton-X-114. Mixtures of ammonium formate and ammonium chloride in a 1:1 ratio, in the same molar proportions, can also be used. In our experiments, no differences in solubilization were observed, although these were not further investigated using other methods.

[0067] As another example, ammonium formate and ammonium chloride can be used as a salt mixture – 9 g NH₄Cl plus 30 g HCOONH₄. The salt concentration can be increased to a point where, upon addition of detergents, no dissolution occurs, but rather the salt precipitates again, or two liquid phases are formed. A high salt concentration is advantageous for measurements by impedance / flow impedance, as the current or impedance is not changed as drastically and remains outside the normal measurement range. No improvement in emulsion solubilization was observed.

[0068] As another example, a different netting agent can be used in the same ratio. Dissolve 9.8 g of ammonium chloride in 45 ml of warm distilled water at 40°C and dissolve 2.2 g of alcohol ethoxylate (Lanspec EMP906) in the solution, then add 5 g of Triton-X-114 and dissolve completely at room temperature. The total volume obtained was 60 ml. Upon addition of Netzer LANSPEC, the liquid became cloudy at 40°C and only cleared up to slightly opaque after cooling to 20-24°C. With the addition of Triton-X-114 at approximately 20°C, it became a clear solution with small air bubbles that showed only a slight opaque tint. The mixtures with cooling lubricant from old, ready-to-use solutions from 3 manufacturers: Fuchs, Rhenus, Blaser SwissLube as a 5% cooling lubricant showed the expected picture or result when reagent was added. Fuchs and Rhenus were clear with a yellow tint immediately after the addition of KSS in a 1:1 ratio. Only the Blaser SwissLube product, although solubilized, still showed a high particle content, which gave the impression of slight turbidity. All solutions were clear and stable after the addition of deionized water in a ratio of 1:3 to a maximum of 1:5. The solutions could be measured directly in the particle counter.

[0069] As a further example, a mixture with twice the concentration achieved using the previous methods was prepared. 2.5 g of ammonium formate plus 2.5 g of ammonium chloride were dissolved in warm, distilled water with a total volume of 25 ml. 2.5 g of isodecyl alcohol-11-polyglycol ether (80%) was added while stirring, followed by 5 g of Triton-X-114. The warm solution was milky and cloudy and only became clear after cooling to room temperature. This double-concentrated solution can be used for cooling lubricant formulations containing 10% oil concentrate. This solution has also been used for other applications, such as hazelnut-nougat cream (Nutella), chocolate-covered ice cream, and fruit cream preparations with a higher fat content. Often, adding just one drop to approximately 0.5 g to 1 g of sample is sufficient for the sample to become clear after shaking. This method greatly simplifies sample preparation and keeps the fat bound in solution, thus allowing for direct and rapid measurement.

[0070] Another example is the use of a flow cytometer with an autosampler. The following quantities were tested: Sample volume: 200 µl for injection - for measurement in the device Fluorescence reagent DiSC 3(5) : 30 µl (standard concentration) For clearing, 333 ml of reagent (e.g., from example 39) are added to 666 ml of filtered (0.2 micron filter) deionized water - also known as autosampler cleaning fluid - which rinses the entire system. Clearing reagent volume: 100 µl Template Eppendorf vessels (2.8 ml volume) as template in the autosampler rack with cooling lubricant.

[0071] The clearing reagent can also be used without mixing (dilution), e.g., 100 µl of sample with 100 µl of clearing reagent. However, care must be taken to prevent aggregation, which could interfere with the measurement. Pre-dilution in the system avoids this.

[0072] If the correct order is not followed when preparing the reagent, and Triton-X-114 is first weighed as a 10% solution in water, and then a salt, e.g., ammonium chloride, is added to the slightly warm solution, the liquid will become cloudy. If stirring is stopped, a clear solution with an upper boundary layer is formed – thus, two clear solutions are layered on top of each other, which is due to the anomaly of Triton-X-114. Triton-X-114 behaves almost amphoteric.

[0073] Only by adding isodecyl alcohol-11-polyglycol ether (Walloxen ID 110 / 80) while stirring does a clear solution with approximately 10-14% wetting agent content result. For cooling lubricants, surfactant concentrations of 8-14% are sufficient, with an HLB value of approximately 12 to 14 being adequate. 0.5 ml of used, contaminated cooling lubricant is now being treated at room temperature with 0.5 ml of solubilizing reagent is added and briefly shaken by hand, resulting in immediate clarification. 1.0 ml of water or 0.9% NaCl solution is then added, mixed, and the mixture is placed in an analyzer to measure the microbial content.

[0074] Since cooling lubricants can contain varying proportions of oil and therefore often differ in their particle load, it is advisable to add one or even three parts of the solubilizing reagent to the cooling lubricant and then dilute it with one to three parts water or dilute saline solution, preferably for flow impedance measurement. This reduces the particle background. For example, take 0.5 ml of cooling lubricant, add 1 ml of reagent, and then add 1 ml of 0.9% particle-free saline solution. After shaking, the solution is clear, shows no aggregation, and changes the impedance only slightly. Thus, even with a 1:3 dilution, good sensitivity sufficient for detecting microbial concentrations can be achieved. Using very high excesses of detergent is disadvantageous, as these often form aggregates that must be diluted again with saline solution or water to dissolve the aggregates.

[0075] The invention proposes a technical solution by way of methodology, namely the use of a solubilizing reagent which has the following features and fulfills the following conditions: 1 The reagent is inexpensive and easy to produce without requiring extensive equipment. 2. It is non-toxic and biodegradable. 3 It allows clearing of the emulsion at room temperature without heating and avoids premature cell lysis effects. 4 It minimizes background and aggregation through the dilution step, especially in heavily contaminated samples with a long service life of the cooling lubricant. 5 Despite dilution of sample 1 in 3, it allows a relevant determination of the germ concentration of 10e3 to 10e6 germs per ml in the cooling lubricant of metabolically active microorganisms in order to define the physiological status in real time and thus prevents premature replacement of the cooling lubricant, as timely re-dosing of biocide can take place. 6. Dilution with the clearing reagent shifts any existing basic pH value towards neutral, which must be taken into account when reacting with fluorescent dyes. 7. By using a mixture of non-ionic wetting agents with an appropriate salt concentration, an HLB value and CMC / CMT value are obtained according to the fat content. This allows for the analysis of various emulsions, such as those found in milk, food, cosmetics, pharmaceuticals, and cooling lubricants, enabling real-time microbial count determination. It is important to note that the proportion of non-ionic wetting agents, such as Triton-X-114 with a very low cloud point of approximately 23°C, constitutes the main component and should not fall significantly below 10% of the total formulation. Salts and other wetting agents will alter these values. 8. Interfering particles are removed by simple filtration of the reagent through a 0.2 micron membrane. 9 The solubilizing reagent is stable for a long time under normal conditions when exposed to light.

Claims

[1] Methods for solubilizing and clarifying water-based cooling lubricants that are present as an emulsion in order to quantitatively determine in real time the content of microorganisms and their metabolic activity in the area of ​​interest characterized by , that a clarifying reagent consisting of organic and / or inorganic salts together with non-ionic surfactants in the described concentration and composition is added as a formulation to a cooling lubricant, thereby bringing about immediate clarification at room temperature without heating the emulsion and enabling measurement in clear, transparent liquid using optical or physical methods, such as flow cytometers, in real time. [2] Method according to claim 1, characterized by, that non-ionic surfactants with the lowest possible cloud point, such as Triton-X-114 of 23°C, or other non-ionic surfactants with a low cloud point and the highest possible HLB value above 12, are mixed with isodecyl alcohol-11-polyglycol ethers or chemically similar compounds such as alcohol ethoxylates in specific ratios to obtain a high grease-dissolving power, that emulsions are clarified to obtain a clear, transparent solution, wherein the mixture increases the HLB value to preferably 12-15 or higher. [3] Method according to claims 1 and 2, characterized by, that salts without complexing properties of metal ions, with approximately the same solubility in water as table salt (5.421 mol / l) under normal conditions are selected, which do not contain sodium or potassium as cations but preferably ammonium and preferably chloride, fluoride, nitrate, formate, or acetate as anions, and are present in concentrations of one (1) to ten (10) mol / liter, since lysis effects due to high salt concentrations are avoided and no osmotic gradients to or from cells are permitted. [4] Method according to claim 1, 2, 3, characterized by , that the clarification of the emulsion preferably takes place at room temperature, but temperatures up to a maximum of 37°C are permitted, whereby the pH range should be in the range of the neutral point, i.e., 7 to pH 8.

5. [5] Method according to claim 1, 2, 3, 4, characterized by, that a favorable change in the cloud point is achieved by mixing electrolytes and surfactants - preferably at room temperature and noticeably by using a surfactant mixture with the same cloud point, critical micelle concentration and critical micelle temperature, further emulsions of any kind such as cosmetic products, food, pharmaceutical products and milk can be clarified and thus made accessible to a real-time microbial count determination method. [6] Method according to any one of claims 1 to 5, characterized by , that by increasing the non-ionic surfactant mixture with a turbidity point below 37°C to an HLB value of 15 and above, all types of milk can be clarified well. [7] Method according to claim 1, 2, 3, 4, 5, 6 characterized by, that polyoxyethylene alkyl ethers or n-decyloxypolyethylene glycol or alcohol ethoxylates with a high HLB value are mixed with non-ionic surfactants such as Triton-X-114 with a low cloud point to achieve clarification of the emulsion at room temperature or below 37°C. [8] Method according to any one of claims 1 to 7, characterized by , that after clarification of the sample, it is treated with fluorescent dyes in a known, published form and concentration, whereby the number of cells and their metabolic activity are determined. [9] Method according to any one of claims 1 to 8, characterized by , that an essential component which is added as a surfactant is a polyethylene glycol ether - such as (1,1,3,3-tetramethylbutyl)phenyl polyethylene glycol. [10] Method according to any one of claims 1 to 9, characterized by, that by simply mixing reagent with cooling lubricant in a ratio of 1 to 1 to 1 to 5 and adding water or dilute salt solution, preferably without sodium and potassium ions, aggregation is avoided and thus a measurement can be carried out stably and evaluably with relevant methods.