KR860000012B1 - Method for maintaining the effectiveness of metal-working oils - Google Patents

Abstract

This process comprises collecting a sample of aerobic microorganisms and H2O2 soln. in an injection syringe, measuring the catalase activity by the scale of syringe, and calculating the viable count in the sample from the measured value. The method is applicable for determination of microorganisms contained in water soluble metal processing oils (e.q. cutting oil, rolling oil, heat treating oil) or liquid foods (e.q. flavorings, drinks, wines, dairy products). By knowing the viable count of organisms, it is possible to prevent putrefaction of the oils or foods at an early stage and preserve them in a pref. state.

Description

Preservation method of metal processing

1 to 5 are explanatory diagrams showing specific examples of reaction vessels that can be used in the present invention.

6 to 8 are calibration curves for aerobic microbial cell count.

9 is a graph showing the relationship between temperature and temperature coefficient.

10 and 11 are calibration curves for aerobic bacterial bioburden.

12 is a calibration curve of viable sulfate reducing bacteria.

13 is a cutting device (universal price plate).

14 is a graph showing the relationship between days elapsed and aerobic bacteria.

* Description of parts of the main parts of the drawings

1: Reactor 2: Inlet

3: cylinder 5: rod

6,7: Spirometer

The present invention relates to a preservation management method of metal processing. In other words, the present invention relates to an anticorrosion management method of metal covalent plants, which measures the number of microbial microorganisms in the metal covalent plants to identify the decay and progress and to perform appropriate antiseptic treatment.

In general, water-soluble metal-covalent materials such as cutting oil, rolled oil, and heat-treated oil contain water or air and satisfy the environmental conditions in which microorganisms propagate. Therefore, if the preservative management of this metal processing oil is not properly carried out, a lot of microorganisms may grow and rot.

The structure of rot is not clear, but it can be estimated from various corruption samples and the tendency of microbial diet to rot. First, aerobic bacteria, mold, and yeast decompose and grow components of metal covalent or other oils to be mixed to synthesize suspended matter, pigments, sugars, proteins and the like. On the other hand, anaerobic bacteria, such as sulfuric acid reduction bacteria, can not directly use the components of the metal covalent or mixed oil, but can use the above suspended matter, sugar, protein and the like.

That is, these substances are decomposed into organic acids such as ammonia, amines, volatile fatty acids, and lactic acid by anaerobic bacteria. Organic acids such as lactic acid are good sources of sulfate reducing bacteria, and around them, sulfate ions and iron necessary for the growth of the bacteria are present. Because of this, sulfuric acid reduction bacteria multiply at this stage and generate hydrogen sulfide. Suspensions, pigments, amines, ammonia, volatile fatty acids, hydrogen sulfide and iron sulfide are typical decay substances.

In order to prevent the decay of the water-soluble metal processing oil in the early stage and to keep it in a suitable state, it is necessary to accurately grasp the number of microorganisms of bimetallic covalent.

Conventionally, as a method for measuring the number of viable microorganisms of aerobic microorganisms, such as yeast and bacteria in various samples, a method of obtaining the number of clones generated by culturing a predetermined amount of samples on an agar culture and calculating the number of viable cells from the number of clones Etc. are taken. However, this method has disadvantages such as complicated operation and a long time until a measurement result is obtained.

On the other hand, as a method of measuring the number of viable microorganisms of aerobic microorganisms, a method of rapidly calculating the number of viable microorganisms by measuring catarase activity is also known. (Public notice 55-15999). However, this method requires a special apparatus because the methods such as the Wolbulg detection method and the Ein Polon tube method are applied, and the handling requires skilled techniques.

As described above, the metal covalent metal is easy to rot, and odor occurs when the rot proceeds. The cause of this odor is sulfuric acid reduction bacteria. Therefore, in order to grasp the degree of decay of metal covalently, it is necessary to know not only aerobic microorganisms but also the reproductive status of sulfate-reducing bacteria in this oil.

Conventionally, as a method of measuring the number of sulfuric acid reduction bacteria, a method of coating and incubating metal covalently on an agar culture and measuring the generated colonies has been performed. However, this agar culture method has a drawback such as complicated operation such as the need for culturing under an anaerobic atmosphere and a long time of 2 to 5 days before the measurement result is obtained.

The object of the present invention is to eliminate such drawbacks and to provide a viable microbial count of aerobic microorganisms and sulfate reducing bacteria in metal processing by simple operation that does not require any special equipment or advanced technology in the field of factories and warehouses. It is to provide a metal processing preservation management method to measure the progress of corruption of metalworking share and to measure the progress of corruption.

The measurement of aerobic microbial bioburden in metal covalent reaction is carried out by putting metal covalent and hydrogen peroxide water into a container having a function of visually identifying a change in volume, and measuring the amount of gas generated by filling in the calibration curve. Is done.

The reaction vessel used in the present invention has a function of visually identifying a change in volume, and specifically, a graduated scale is used as a transparent glass or plastic container.

Metal containers can also be used. It is also preferable that the container is capable of collecting the metal-covalent and hydrogen peroxide water in the container by changing its volume. Specific examples of the container include a syringe, and specific examples of the other container will be described in the drawings. 1 to 3 are cylinders (3) having a reaction vessel (1) in which the reaction vessel has an inlet (2), and a graduation (4) formed at the top or side of the vessel, the bottom of which being in communication with the top or side of the vessel. It consists of the cylindrical cylinder 3 and the rod 5 which slidably mounted in this cylinder. 4 shows a reaction vessel composed of a Benedict-Roth type spirometer (so-called spirometer) 6 and a reaction tank 1 connected to the spirometer, and FIG. 5 shows a membrane-type grass intangible. The reaction container which consists of a spirometer 7 and the reaction tank 1 connected with this spirometer is shown.

The injection port 2 of the reaction tank 1 should just be able to seal a stopper with a simple opening, and may be provided with the tube 8 with a stopper as shown. In addition, the number of injection holes 2 and the mounting position may be arbitrarily selected. In the case of one injection hole 2, the sample and hydrogen peroxide water are injected into the same injection hole and reacted. Put them in a separate inlet to mix both at the start of the reaction. As shown in FIG. 5, the pressure vessel 9 is installed in the reaction vessel so that the amount of generated gas can be measured by reading the scale after making the pressure in the container equal to the external pressure at the time of sampling or measuring the generated gas. Can be.

The above method is characterized by determining the viable cell number of the microorganism by using the catarase activity of the aerobic microorganism, and measuring the activity by a very simple operation. That is, the sample containing aerobic microorganisms is diluted as it is or appropriately, the appropriate amount is put into a reaction tank, and the hydrogen peroxide water is added and reacted similarly, and oxygen gas is generated.

As the hydrogen peroxide solution used here, a concentration of about 0.1 to 30% is suitable. The reaction between the metal-covalent and hydrogen peroxide solution is carried out for 5 to 90 minutes at a temperature of 5 to 40 ° C, and preferably 5 to 30 minutes at 20 to 30 ° C. The measurement of the amount of generated oxygen gas is read in the scale 4 of the cylinder 3 in communication with the reaction tank 1, or in the scale 4 indicated in the case of a spirometer such as FIG. 4 or FIG.

In addition, when a syringe is used as the reaction container, since the metal can be directly shared with the metal or the hydrogen peroxide, it is not necessary to use expensive equipment as in the prior art, and also a pipette for injecting it into a measuring container such as a sample. none. In addition, even samples of high concentration can be measured without the troubles experienced with the anipolon tube method. In addition, when measuring the catarase activity, the reaction vessel and the measurement vessel are the same, and since the operation is very simple, no need for a large place or special technique can be easily measured by anyone in the field.

From the amount of oxygen produced in this way, a calibration curve is used to calculate the number of viable microorganisms in aerobic microorganisms.

In the present invention, the reaction vessel serves as a function of a sampling vessel such as a sample, a reaction tank, and an amount of generated gas, and after the reaction of the sample collected in the vessel, the small amount of the generated hand is read visually, so the operation is simple. No skill is required. In addition, there is no restriction | limiting in the sample concentration calculated | required in the case of the Einpollon tube method in this invention, and even a high concentration sample can be measured without a trouble. It is also one of the characteristics of the present invention that a viable microbial count of aerobic microorganisms can be measured in a short time using a reaction vessel that can be transported anywhere without requiring a special apparatus.

As described above, since the number of aerobic microorganisms in the cotton sample according to the present invention can be measured simply and quickly, the quality control of the sample can be completed more and the extension of the pot life can be avoided. For example, in the case of metal-based covalent oil, a type of lubricating oil, in order to extend the pot life as cutting oil, rolling oil, heat treatment oil, etc., a sterilizer is usually added, but it is not economical to use a large amount of sterilizer from the beginning. When the finished metal processing oil flows out into the wastewater, the disinfectant contained in this oil adversely affects the human body or adversely affects the activated sludge in the wastewater treatment system.

In consideration of such a state, it is preferable to add the fungicide when the number of living bacteria in the metal-covalently increased, and to suppress the addition amount as much as possible.

Such a situation is not limited to metal processing, and the same is true for samples such as liquid foods, and according to the practice of the present invention, the quality control of the samples is appropriate and extremely useful for preventing secondary pollution. In addition, the calibration curve was determined by the following method.

Creation of calibration curve

The number of viable bacteria of aerobic bacteria in the metal-covalent sample as a sample was determined by agar plate method, and diluted to various concentrations with respect to the same sample to determine the amount of produced oxygen by the method of the present invention. From both results, the analytical curves of the number of viable cells and the amount of oxygen were prepared. Since the calibration curve differs between emulsion-type metal covalent and soluble metal covalent, it is necessary to prepare it according to each. In addition, the calibration curve of the chemical sorbate may be the same as the sorbable type.

1) In case of emulsion type

Diluted in 5 ml of reaction vessel (see FIG. 1) and varying the viable cell concentration, 1 ml of various metalworking shares and 1 ml of 6% hydrogen peroxide aqueous solution were collected, reacted, and the amount of generated oxygen gas was measured. On the other hand, the viable bacteria count of aerobic bacteria in the solution of the same sample was calculated by the agar plate method. In other words, a predetermined amount of the sample solution was grafted onto a medium (pH 7.0) containing 0.5% meat juice, peptone 1.0%, sodium chloride 0.5%, and agar 1.5%, and cultured at 30 ° C for 24 hours. The number of aerobic bacteria was measured in the water.

Calibration curves are shown in FIGS. 6 and 7. 6 shows the result of reacting 1 ml of 30% aqueous hydrogen peroxide solution and 1 ml of emulsion type sample (metal covalent A) at 21.8 ° C. for a predetermined time, and FIG. 7 shows 1 ml of 6% aqueous hydrogen peroxide solution and emulsion type sample (metal covalent A). It is the result when 1 ml is made to react at 21.8 degreeC for a predetermined time.

2) In case of soluble type

The calibration curve obtained by performing the calibration curve in the same manner as in the emulsion type using a sorbable sample (metallic covalent B) as a sample is shown in FIG.

Next, the measuring method of the viable cell count of the sulfuric acid reduction bacteria which exist in a common state is demonstrated. First, an appropriate amount of metal covalent metal, which is a sample, is diluted as it is or appropriately and put into a container such as a test tube. In this case, the container does not need to be transparent or have a scale. Next, mineral acid, such as hydrochloric acid, sulfuric acid, and nitric acid, is added to bring the pH of the strongly acidic acid. In this case, hydrochloric acid and sulfuric acid are particularly preferable as the acid, and the pH is set to 2 or less and preferably 1 or less to suit the addition of the acid. If sulfides such as iron sulfide are present during metal covalent sharing by the growth of sulfuric acid reduction bacteria, sulfuric acid hydrogen is generated by addition of acid. Then, a test paper coated with a chemical agent sensitive to hydrogen sulfide is provided in a gaseous phase above the container, and the degree of discoloration of the test paper is observed to determine the sulfate reducing bacteria.

As a test paper used in this case, it is suitable to apply metal salt of acetic acid or formic acid. Examples of metal salts of acetic acid include iron acetate, copper acetate, nickel acetate and tin acetate, and metal salts of formic acid include formate, iron formate, formic acid copper, formic nickel, and formic acid tin. Among these, acetate is good, determination by discoloration is easy, and it can carry out correctly.

In order to determine the number of sulfuric acid reduction bacteria, the number of sulfuric acid reduction bacteria in the metal-covalent sharing of the sample is measured in advance by agar culture method, and the relationship between the degree of discoloration of the test paper for the same sample is summarized in a table and used as a conversion table. For example, when the acetate test paper is used under the same conditions as those shown in Examples described later, the number of bacteria can be determined as follows.

[Table 1]

As experience accumulates, the color of the test paper in the conversion table can be further subdivided to make more accurate judgment.

In explaining the simple method of the present invention, a small amount of covalent metal is injected into a test tube, and then an appropriate amount of acid is added to make the oil strongly acidic, and then the test paper is exposed to the gas phase in the test tube and fixed with a rubber stopper.

In this state, it is left to stand for 5-15 minutes and 10 minutes, and the color change of the test paper is visually judged. Then, the sulfuric acid reduction bacterial count is obtained using the mixed acid table as described above.

According to the present invention, it is possible to quickly measure the number of sulfuric acid reduction bacteria in the metal-covalent sample as a simple operation. In addition, the apparatus to be used is also hydrogen and can be measured in a narrow place without mastery of the technology, and the management of metalworking shares can be appropriately performed.

In addition, the method of the present invention can be applied to wastewater treatment, water quality inspection in methane fermentation, and the like.

Taking the example of the case where the practical procedure of the measuring method of the microbial viable count in the above-mentioned metal covalent sharing is suitable as an example, it is as follows.

(a) Determination of aerobic microorganisms.

1. Put the needle on the pad. Measure the liquid temperature of metal covalent.

2. Take 1 ml of hydrogen peroxide.

3. Take 1 ml of metal covalently from the sample.

4. Seal the needle with rubber.

5. Leave the syringe on its side and let stand for 10 minutes.

6. Read the gas generation amount (×) after 10 minutes on the scale.

7. Find the temperature correction coefficient (n) in Fig. 9 and calculate the amount of correction gas (z) from the following equation. z =

8. Obtain the aerobic microbial cell count from the calibration curves of FIGS. 10 and 11.

(b) Measurement of Sulfate Reducing Bacteria

1. Take 3 ml of metal covalent sample from the test tube.

2. Collect 0.5 ml of 5% hydrochloric acid solution with pipette and add it to the test tube.

3. Insert the acetate coating test paper into the rubber stopper and seal the test tube with this stopper.

4. After 10 minutes, observe the color of test paper.

5. Obtain the viable count of sulfate-reducing bacteria in Fig. 12.

After measuring the viability of microorganisms in metal-covalent organisms, the degree of decay is determined, and appropriate preservative treatment is performed on the metal-covalent organisms. Preservatives include addition of a disinfectant, heat sterilization, aeration, and the like. Among these means, an appropriate means is selected for preservative treatment. As a fungicide, an appropriate one may be selected and used from known materials, and as a specific fungicide, hexahydro-1, 3, 5-toris (2-hydroxyethyl) -s-triazine, 5-chloro-2-methyl-4 Isothiazolin-3-one (methylchlorine triazolone), 2-methyl-4-isothiazolin-3-one, 2-mercaptpyriding-n-oxide sodium, α-bromosinnamaldehyde, 1, 2-benzoisothiazolone-3one, hexahydro-1, 3, 5-triethylene-s-triadine, etc. are mentioned. The antiseptic effect is achieved by selecting an appropriate fungicide and adding the appropriate amount to the metalworking share.

In addition, since microorganisms are generally weak to heat, the number of viable bacteria can be greatly reduced by heating at 50 to 60 ° C. for 5 to 30 minutes in consideration of the type of metal covalently shared and the kind of microorganisms.

Aeration is also an effective means for antiseptic measures. In particular, it has the effect of suppressing the growth of sulfuric acid reduction bacteria. As is well known, microorganisms grow at a tremendous rate once they begin to grow. Therefore, once the degree of decay has been identified by measuring the number of microorganisms living in the metal processing plant, appropriate precautions should be taken at precise timing.

According to the present invention, the aerobic microorganisms causing corruption among the various microorganisms propagated in water-soluble metal covalently, in particular, bacteria and sulfate-reducing bacteria occurring in the final stage can be measured simply and quickly, and appropriate preservative treatment can be performed at an appropriate time. have. This makes it possible to properly maintain and share the metal work share.

Next, the present invention will be described in detail by way of examples.

Example 1

In a 5 ml syringe, 1 ml of 6% aqueous hydrogen peroxide solution and 1 ml of metal covalent C (emulsion type) were collected, the injection port was sealed with a rubber stopper, and the syringe was rotated 2-3 times to react at 21.8 ° C for 10 minutes. The amount of generated oxygen gas was 1.0 ml, and the number of aerobic microorganisms obtained from the calibration curve was 5.0 × 10 ⁷ / ml. For comparison, the number of bacteria determined by agar culture was 4.6 × 10 ⁷ cells / ml.

EXAMPLES 2-5

The aerobic microbial viable count was obtained like Example 1 except having changed the kind of metal covalent sharing. The results are shown in the second table. The results from the agar culture method are also shown for comparison.

[Table 2]

D: Soluble type

E, F, G: emulsion type

Example 6

Using the reaction vessel shown in FIG. 1 (10 ml of the reaction vessel, the volume of the cylinder 3 is 5 ml), the tip of the tubing with the cap of the reaction vessel is inserted into a beaker containing the sample (emulsion type metal covalent A). Then, the cap was opened, the rod 5 was then pulled up, and 1 ml of the sample was collected in the reactor 1.

In the same manner as above, 1 ml of 6% hydrogen peroxide was collected from the reactor. Thereafter, the rod was pushed into the cylinder and the stopper was closed. The reactor was slowly shaken and then left to stand. On the other hand, the rod was sometimes rotated so that the pressure in the reaction vessel was equal to atmospheric pressure.

After reacting at 22 DEG C for 10 minutes in this manner, the generated amount of oxygen gas was 1.0 ml. The aerobic microbial cell count obtained from the calibration curve was 5.5 × 10 ⁷ cells / ml. For comparison, the number of viable cells of the same sample determined by agar culture was 5.1 × 10 ⁷ cells / ml.

Example 7

Using one of the reaction vessels shown in FIG. 2 (reactor A30ml, reactor B50ml, cylinder 3 having a volume of 10ml), one end cap of the reaction vessel was opened, and a sample (emulsion type metal covalent B was added to the A portion of the reactor). 5 ml was collected and this stopper was closed. Next, the other end was opened and 6% of the reaction vessel B and 5 ml of hydrogen oxide water were collected. Then, the rod was pushed in, the gas in the cylinder was discharged, the scale was zeroed, and the stopper was closed.

The reaction vessel was tilted and the sample contained in the A portion was transferred to the B portion and mixed with hydrogen peroxide to react. In this case, the cylinder was allowed to stand horizontally, and the rod was sometimes rotated so that the pressure in the reaction vessel became equal to atmospheric pressure.

After reacting at 22 DEG C for 10 minutes in this way, the amount of generated oxygen gas was found to be 6.5 ml. The aerobic microbial cell count obtained from the calibration curve was 7 × 10 ⁷ cells / ml. For comparison, the number of viable cells of the same sample determined by agar culture was 6.5 × 10 ⁷ cells / ml.

Example 8

3 ml of various metal covalently or diluting samples were collected in a small test tube (length 100 mm, internal diameter 12 mm), and a small amount of 5% aqueous hydrochloric acid solution was added thereto to lower the pH to 1.0 or less. Immediately, the test strip was exposed to the in vitro gas phase, and a rubber stopper was allowed to stand for 10 minutes.

Then, the test paper was taken out, the discoloration state was visually observed, and the number of sulfuric acid reduction bacteria was calculated from the conversion table prepared separately. The results are shown in Table 3.

For comparison, the same sample was added with agar 1.5% and the pH was adjusted to 7.4, and the Sturkey medium was injected into a test tube, inoculated with the sample and incubated for 30 hours at 30 ° C. under anaerobic conditions. The results of calculating the number of sulfate-reducing bacteria in water are shown in Table 3. The composition of the sturky medium is the same as described in the literature (A.T.C.C. 12th edition, 331 pages, 1976) except that 1.5% of agar was added.

[Table 3]

Samples A ~ I: Emulsion Coolant

"J ~ L: Soluble coolant

Samples M and N: chemical solution coolant

"O: Rolled oil

"P and Q: Heat-treating oils

Example 9

Emulsion-type cutting oil (extreme pressure; trade name; Demitsu Raphniy Sparkle E, dilution) by metal cutting by the cutting device shown in FIG. 13 (all-purpose price plate; storage tank capacity 400 l, open type; distance between tank and cutting machine 2 m) Cutting process was performed for 100 days using 200 liter of magnification 20 times.

Once a day, the aerobic microbial viable count was measured by the method of Example 1. Since the number of aerobic microbial viable cells was 5 × 10 ⁷ cells / ml on day 14 from the start of operation, 400 ml of a 10% solution of 2-melcap filidin-N oxide sodium was added as a bactericide. It was measured by the same method the next day, the aerobic microbial viable count was 2 × 10 ⁶ / ml.

The process was then repeated to continue cutting while maintaining aerobic microbial viable counts of less than 1 × 10 ⁷ cells / ml. The relationship between the elapsed days and the number of aerobic bacteria living cells is shown in FIG.

Example 10

The cutting process was carried out in the same manner as in Example 9, except that the storage tank (capacity 1500 liter) was used to make the metal share 1000 liter. The viable cell count of sulfate reducing bacteria was measured by the method of Example 8 once daily. On the 20th day from the start of operation, the number of viable sulfate reducing bacteria was 10 ⁶ / ml, so the air in the storage tank was shared with the air under the condition of flow rate 0.5 2.0 VVM (air volume / lopuid volume / minute) Infused in As measured by the same method the next day, the number of viable sulfate reducing bacteria was 10 ⁴ / ml. Subsequently, by continuing this method, cutting was continued while maintaining the number of viable sulfate reducing bacteria at 10 ⁴ / ml or less.

Claims (22)

Anticorrosion management method of metalworking share which measures the progress of decay by preserving the number of microorganisms in metalworking share and preservative treatment. The method according to claim 1, wherein the metal covalently is selected from cutting oil, rolling oil or heat treatment oil. (Correction) The method according to the preceding item 1, wherein the preservative management is carried out by heat sterilization at a temperature of 50 ° to 60 ° C. for 5 to 30 minutes in order to reduce the number of viable cells in metal processing. (Correction) The method according to claim 1, wherein the preservative management is carried out by performing aeration at a temperature of 10 ° to 60 ° C. and a flow rate of 0.5 to 2.0 V.M. (Correction) The method according to the preceding item 1, wherein the preservative management is carried out by adding a 10% solution of the disinfectant 2-mercapto piridine-N-oxysodium to the metalworking share. The number of aerobic microorganisms and the number of sulfuric acid reduction bacteria in the metal-covalent metal is measured, and the measurement of the number of microbial microorganisms is made by reacting the metal-covalent and hydrogen peroxide in a container having the function of visually identifying the change in volume. Calculate the aerobic microbial bioburden in metal covalently from the amount of gas obtained by measurement, and add acid to metal covalently-coated biocides containing sulfuric acid-reducing microorganisms to make it highly acidic, and then apply test paper coated with chemicals sensitive to hydrogen sulfide. The method according to the preceding claim 1, wherein the opening of the container is sealed while being exposed to and the discoloration of the test paper is observed to determine the number of sulfuric acid reduction bacteria in the metal-covalently shared area. The measurement of the number of microorganisms in the microorganism is carried out by putting metal covalent and hydrogen peroxide into a container having a device for determining a change in volume, reacting the metal covalent and hydrogen peroxide, and measuring the amount of gas generated by the reaction. The method according to claim 1, which is carried out by calculating the number of viable microorganisms of aerobic microorganisms from sheep. The method according to claim 7, wherein the container is a syringe. The method according to claim 7, wherein the vessel comprises a reaction vessel having an inlet, a graduated cylinder installed in communication with the reactor at the top or side of the reactor, and a rod mounted slidably within the cylinder. A method according to claim 3, wherein the vessel consists of a Bendict-Roth type spirometer and a reactor connected to the spirometer. The method according to claim 7, wherein the vessel consists of a membrane-type spirometer and a reactor connected to the spirometer. (Correction) The method according to any one of items 7, 8, 9, 10 or 11, wherein the container is made of transparent glass or plastic. (Correction) The method according to item 7, wherein the concentration of the hydrogen peroxide solution is 5 to 30%. (Correction) According to any one of claims 7, 8, 9, 10, or 13, wherein the reaction between the metal covalent and hydrogen peroxide is carried out for 5 to 90 minutes at a temperature of 5 to 40 ° C. Way. (Correction) The method according to any one of the preceding paragraphs (7), (8), (9), (10), (11), or (13), which converts the measured gas amount into an aerobic viable cell number by writing it on a calibration curve. The microbial bioburden was measured by adding acid to the metal-coated metal in the container to make it strongly acidic, and then the opening of the container was sealed while exposing a test paper coated with a hydrogen sulphide-sensitive agent to the atmosphere. The method according to claim 1, which is performed by observing the degree to determine the number of sulfuric acid reduction bacteria in the metal-covalent. The process according to claim 16, wherein the acid is either hydrochloric acid, sulfuric acid or nitric acid. (Correction) The method according to any one of items 16 to 17, wherein the agent sensitive to hydrogen sulfide is a metal salt of acetic acid or formic acid. (Correction) The method according to item 18, wherein the metal salt of acetic acid or formic acid is one of lead acetate, iron acetate, copper acetate, nickel acetate, tin acetate, lead formate, iron formate, formic acid copper, formic acid nickel or tin formate. The method according to claim 16, which is left for 5 to 15 minutes after sealing the opening of the container. The method according to claim 16, wherein the number of sulfuric acid reduction bacteria is determined by comparing the color of the test paper with the calibration curve. Insert the metal covalent oil, which is a water-soluble cutting oil, into the syringe, and then add hydrogen peroxide solution to the syringe for 5-30 minutes at 20 ° to 30 ° C to measure the amount of oxygen gas produced on the scale of the syringe. To obtain aerobic microbial viable count, grasp the progress of decay of the cutting oil, and perform the appropriate preservative treatment.

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