JPH0160240B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for rapidly measuring the amount of produced gas. BACKGROUND OF THE INVENTION Measuring the amount of gas produced by a reaction is used as an analytical means in industry.
For example, a method has been proposed in which the number of viable aerobic microorganisms in water-soluble metal processing oils and foods containing high salt is calculated by measuring catalase activity (Japanese Patent Publication No. 15999/1983). Usually, the amount of produced gas is calculated by measuring changes in volume using an Einhorn tube, syringe, etc., or measuring changes in pressure using a Warburg manometer or other pressure gauge. However, these measurement methods require expensive special equipment, and their handling requires complicated operations and requires skill. The purpose of the present invention is to eliminate these drawbacks and to quickly increase the amount of generated gas in factories, warehouses, and other workplaces using simple operations that do not require any expensive special equipment or advanced technology. The purpose is to provide a method that allows measurement. That is, the present invention is a method for measuring the amount of gas generated by a reaction of a sample, in which a sample is collected in a graduated test tube, a bubble stabilizer is added, and then reacted, the generated gas is made into bubbles, and the bubbles are removed. This is a method for rapidly measuring the amount of produced gas, which is characterized by measuring the volume containing the gas and calculating the amount of produced gas based on the measured value. The samples to which the present invention can be applied are not particularly limited, and include, for example, water-soluble metal processing oils, high salt-containing foods, etc. Specifically, cutting oils, rolling oils, metal processing oils such as heat treatment oils, and miso , high salt-containing foods such as soy sauce, etc. The method of the present invention is a method for measuring the amount of gas generated by a reaction of a sample. First, the sample is taken as it is or diluted and an appropriate amount is taken into a graduated test tube and allowed to react. The reaction is not particularly limited as long as it generates gas, and may be a biochemical reaction. After the reaction, the amount of generated gas can be calculated by visually reading the total volume containing bubbles from the scale of the test tube and subtracting the sum of the amount of the sample and the added bubble stabilizer etc. from this total volume. As described above, the method of the present invention is characterized in that the amount of produced gas can be rapidly measured with a very simple operation. Therefore, the method of the present invention can be used to measure the number of viable aerobic microorganisms in metal processing oil or high salt-containing foods. To calculate the viable aerobic microorganism count here, create a calibration curve in advance for the relationship between the amount of produced gas and the number of viable microorganisms, and calculate from the calibration curve based on the obtained amount of produced gas. . That is, the viable cell count of aerobic microorganisms is determined by utilizing the catalase activity possessed by the aerobic microorganisms. Specifically, a sample containing aerobic microorganisms and hydrogen peroxide solution are collected in a graduated test tube, a bubble stabilizer is added to form gas bubbles, and the volume containing the bubbles is measured. The amount of gas produced is calculated based on the value, and the number of viable aerobic microorganisms is determined from the calibration curve based on the catalase activity thus obtained. A suitable hydrogen peroxide solution for use in measuring catalase activity is one with a concentration of 0.1 to 30%. Although it is necessary to add a bubble stabilizer in the method of the present invention, the timing of addition and the substance to be added are not particularly limited as long as it is before the start of the reaction. However, in order to generate more stable fine bubbles, it is preferable to add it to the hydrogen peroxide solution. The bubble stabilizer is one or more selected from cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants, and has a concentration of 0.01 to 1.0. % is appropriate. The reaction is 5~
The process is carried out at a temperature of 40° C. for 5 to 90 minutes, preferably 10 minutes, while rotating slowly several times to avoid incorporating air. Next, read the total volume containing the generated bubbles with the naked eye from the scale of the test tube, and subtract the sum of the sample, the added hydrogen peroxide solution, and the bubble stabilizer from this total volume to determine the amount of oxygen gas generated.
That is, catalase activity is measured. The amount of produced gas obtained in this manner is converted into the number of viable aerobic microorganisms using a calibration curve. The amount of gas generated is generally measured using the Einhorn tube method or the Warburg pressure method. However, with the Einhorn tube method, when a large sample is taken, the generated oxygen bubbles are dispersed in the liquid. Because it does not collect at the top, measurements must be made at a low sample concentration. Therefore, measurement requires a long time. In addition, in the case of the Warburg pressure method, the presence of air bubbles does not affect the measured value, but
The drawback is that the entire device is large and expensive. Furthermore, a problem common to both methods is that they require skilled techniques in order to obtain accurate measurement results, and are not suitable for on-site measurements such as factories and warehouses. Using the method of the present invention not only eliminates the need for expensive equipment as described above, but also eliminates the need for pipettes and the like for injecting samples and the like into measurement containers. Moreover, even highly concentrated samples can be measured without the troubles experienced with the Einhorn tube method. In addition, since a graduated test tube is used to measure catalase activity, etc., the reaction container and measurement container are the same, making the operation extremely simple.
Anyone can easily perform measurements on-site without requiring a large space or special skills. As described above, according to the present invention, the amount of generated gas can be measured quickly and accurately, and as a result, the number of viable aerobic microorganisms in a sample can be measured easily and quickly, so that the quality control of the sample can be carried out appropriately. can be done. For example, in the case of metal working oil, which is a type of lubricating oil, a sterilizer is usually added to extend the usable life as cutting oil, rolling oil, heat treatment oil, etc., but a large amount of sterilizer is added from the beginning. It is uneconomical to use disinfectants, and furthermore, if used metal processing oil spills into wastewater, the disinfectants contained in the oil may have an adverse effect on the human body or may cause activated sludge in the wastewater treatment system. This will have a negative impact. Considering this situation, it is desirable to add a bactericide when the number of viable bacteria in the metalworking oil increases, and to suppress the amount added as much as possible. Such cases are not limited to metal processing oils, but also apply to high-salt foods such as miso and soy sauce.By implementing the present invention, it is important to properly control the quality of samples and to prevent secondary It is also extremely useful for preventing pollution caused by. Next, the present invention will be explained in detail with reference to examples.
In addition, the calibration curve was created by the following method. Creating a calibration curve When measuring the number of viable aerobic microorganisms in metalworking oil, first determine the number of viable aerobic bacteria in the metalworking oil sample using the agar plate method, and then calculate the number of viable aerobic bacteria in the metalworking oil as a sample using the agar plate method. After dilution, the amount of oxygen produced (catalase activity) was determined by the method of the present invention. A calibration curve for the number of viable bacteria and the amount of oxygen was created from both results. Since there are differences between emulsion type metal working oil and soluble type metal working oil, it is necessary to create a calibration curve for each. Note that the chemical solution type can use the same calibration curve as the solution type. (1) For emulsion type: In a 10 ml graduated test tube, collect 1 ml of various metal working oils diluted with different concentrations of viable bacteria, and add 6% peroxide containing 0.2% synthetic kitchen detergent. 1 ml of an aqueous hydrogen solution was added while passing through the glass wall, the test tube was slowly rotated several times and then left to stand, and the amount of oxygen gas generated was measured by reading the scale. On the other hand, the number of viable aerobic bacteria in the sample solution of the same sample was calculated by the agar plate method. That is, a certain amount of the sample solution was inoculated into a medium (PH7.0) containing 0.5% meat extract, 1.0% peptone, 0.5% sodium chloride, and 1.5% agar, cultured at 30°C for 24 hours, and the number of colonies produced was calculated. The number of aerobic bacteria was determined. Figures 1 and 2
The calibration curve is shown in the figure. In addition, Figure 1 shows the results when 1 ml of 30% hydrogen peroxide aqueous solution and 1 ml of emulsion type sample (metal working oil A) were reacted at 25.0°C for a specified time, and Figure 2 shows the results when 1 ml of 6% hydrogen peroxide aqueous solution was reacted. and emulsion type sample (metal working oil A)
These are the results when 1 ml was reacted at 25.0°C for a predetermined time. (2) In the case of soluble type A calibration curve was created in the same manner as in the case of emulsion type using metal working oil B (solubil type) as a sample. The obtained calibration curve is shown in FIG. Example 1 1 ml of metal working oil C (emulsion type) was collected in a 10 ml graduated test tube, and a commercially available kitchen detergent [trade name: Mama Lemon, manufactured by Lion Co., Ltd., alpha olefin type (anion) + Higher alcohol type (anion) + straight chain alkylbenzene type = 27%]
1 ml of a 6% aqueous hydrogen peroxide solution containing 0.2% was added while running along the wall of the test tube, the test tube was slowly rotated several times, and the reaction was allowed to proceed at 25.0° C. for 10 minutes. The amount of oxygen gas generated was 1.5 ml, and the number of viable aerobic microorganisms determined from the calibration curve (the line for 10 minutes of reaction time in Figure 2) was 3.0 x 10 ⁷ cells/ml. For comparison, the number of viable bacteria determined by the agar culture method is
It was 2.7×10 ⁷ cells/ml. Examples 2 to 5 Example 1 except that the type of metal working oil was changed
The amount of oxygen gas generated was measured in the same manner as above, and the number of viable aerobic microorganisms was determined using a calibration curve. The results are shown in Table 1. For comparison, results obtained using the agar culture method are also shown. 【table】

[Brief explanation of drawings]

Figures 1 to 3 are calibration curves of the amount of oxygen produced and the number of viable bacteria in metalworking oil measured by the method of the present invention.

Claims (1)

[Claims] 1. A method for measuring the amount of gas produced by a reaction of a sample, in which a sample is collected in a graduated test tube, a bubble stabilizer is added thereto, the reaction is made, and the gas produced is made into bubbles, Measure the volume containing air bubbles,
A method for rapidly measuring the amount of produced gas, characterized in that the amount of produced gas is calculated based on the measured value. 2. The method according to claim 1, wherein the reaction is based on catalase activity.