CN111139177A - Convenient and universal microorganism growth curve detection method - Google Patents

Convenient and universal microorganism growth curve detection method Download PDF

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CN111139177A
CN111139177A CN201811329861.9A CN201811329861A CN111139177A CN 111139177 A CN111139177 A CN 111139177A CN 201811329861 A CN201811329861 A CN 201811329861A CN 111139177 A CN111139177 A CN 111139177A
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microorganism
growth curve
culture bottle
pressure
pressure sensor
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黄欣
田贤立
龚佳仪
张杭键
丁奕妍
徐颖
邵杰
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Zhejiang Normal University CJNU
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/08—Flask, bottle or test tube
    • C—CHEMISTRY; METALLURGY
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract

The invention discloses a convenient and fast universal microorganism growth curve detection method. Four cycles of microbial growth are directly available. The problems that a large amount of labor is needed, the operation is complex and the like in the traditional method are solved. And the SAW pressure sensor is adopted to measure the change of the pressure in the culture bottle in real time. Four periods of microbial growth are indirectly obtained. The method does not need to add other substances into the culture bottle, reduces error sources, simplifies detection steps and reduces detection cost. The method has the advantages of simple operation, real-time and quick speed, high result accuracy, and capability of accurately mapping the complete growth curves of different microorganisms, and has important significance for the research of the microorganisms.

Description

Convenient and universal microorganism growth curve detection method
Technical Field
The method is applied to detecting the growth curve of the microorganism, and can be used for conveniently, quickly and real-timely describing the growth cycle of the microorganism.
Background
The distribution of microorganisms in nature is very wide, and a plurality of microorganisms with different quantities and varieties exist in the air, soil, rivers, lakes, oceans and the like. There are also many kinds of microorganisms existing on the body surfaces of human beings, animals and plants and in the cavities and ducts communicating with the outside.
At present, the world knows about more than 8 million kinds of microorganisms, and most of the microorganisms are necessary for the survival of human beings, animals and plants. In nature, the circulation of various elements such as nitrogen, carbon, sulfur, etc. is performed by the metabolic activity of microorganisms. For example, a large amount of nitrogen in the air can be absorbed by plants only by the action of microorganisms, and the microorganisms in the soil can convert animal and plant proteins into inorganic nitrogen-containing compounds for the growth needs of the plants, which are utilized by human beings and animals. Thus, without microorganisms, plants cannot be metabolized, and humans and animals will not survive.
Microorganisms play a vital role in agriculture (e.g., bacterial fertilization, bacterial disease prevention, bacterial growth), industry (e.g., leather production, wastewater treatment, organic phosphorus and toxic substance degradation), food production and safety (food brewing, food storage), health care (pathogenic microorganisms, fungal antibiotics, blood culture), and the like. Therefore, a convenient method for detecting the growth of microorganisms is important.
Currently, the measurement of microbial growth is mainly divided into four categories: 1. dry weight measurement, 2 volume measurement, 3 turbidimetry and 4 physiological index method. Conventional techniques for the detection of microbial growth include the first three techniques that are widely used in experiments due to their versatility and ease of operation. Dry weight measurement: can be measured by centrifugation or filtration. Typically the dry weight is 10-20% of the wet weight. In the centrifugation method, a certain volume of culture solution to be measured is poured into a centrifuge tube, a certain centrifugation time and a certain rotation speed are set, centrifugation is carried out, and the culture solution is centrifugally washed for 1 to 5 times by clear water and dried. This method has no way of distinguishing the dead or alive of the microorganism. Volume measurement: also known as hypha concentration measurement. The growth of the microorganisms is reflected by measuring the amount of hyphae contained in a volume of culture broth. This method is relatively extensive, simple and rapid, but requires setting of consistent treatment conditions, otherwise the deviation is large, and the result has a certain deviation due to inclusion of some solid nutrients in the centrifugal precipitate. Turbidimetry: the growth of the microorganisms causes an increase in the turbidity of the culture. And measuring the light absorption value under a certain wavelength by an ultraviolet spectrophotometer to judge the growth condition of the microorganisms. This method has no way of distinguishing the dead or alive of the microorganism. The above three methods are also called conventional methods, which require a long operation time and involve a large amount of manual operations. These disadvantages make currently available techniques impractical for automatic measurement. Furthermore, invasive procedures can lead to microbial interference and loss of content media, resulting in low precision and low efficiency. Therefore, the conventional methods cannot achieve rapid and accurate analysis of the growth of microorganisms. Physiological index methods; during the growth process of the microorganism, a series of growth-related physiological indexes change along with the growth of the microorganism, and the growth state of the microorganism can be determined by measuring the substances. The technical requirements of the biological indexes are high, and the operation is complex. The method for detecting the growth of microorganisms quickly, accurately, conveniently and universally is found to be the mainstream of the current microorganism detection.
Disclosure of Invention
In order to overcome the defects of the traditional method, the invention designs a method for quickly, conveniently and quickly detecting the growth curve of the microorganism in real time.
The invention provides a microbial growth curve detection method. The culture bottle is filled with microorganisms and culture media, and the culture bottle cap can generate micro deformation to a certain degree due to the change of the air pressure in the culture bottle caused by the metabolism activity of the microorganisms. The SAW sensor above the culture bottle cap detects pressure changes caused by micro deformation and converts the pressure changes into electrical signals. And acquiring and analyzing the electric signals by an acquisition card and a computer to obtain a microorganism growth curve.
Further, the method for detecting the microbial growth curve comprises the following steps:
putting 25ml of sample into a culture bottle containing 225ml of culture medium to prepare a sample homogenizing solution in a ratio of 1: 10;
and step two, establishing an automatic measuring device for the microbial growth curve, which comprises a detection system (1) and a pattern acquisition system, wherein the detection system comprises a sealed culture bottle, a constant temperature module and an SAW pressure sensor, the culture bottle is arranged in the constant temperature module, and the SAW pressure sensor is arranged above a culture bottle cap. The SAW pressure sensor is connected to a pattern acquisition system, the pattern acquisition system is a data acquisition card (2) and a computer (3), and the acquisition card is connected with the computer to realize the description of the microorganism growth curve.
Step three, adding 9ml of culture medium and 1ml of the sample homogenizing solution obtained in the step one into a culture bottle of an automatic microorganism growth curve measuring device, and screwing a sealing cover;
inserting the culture bottle into a constant temperature device, and adjusting the temperature of a constant temperature module to be (37 +/-0.2) DEG C;
fifthly, starting microorganism growth curve drawing software;
specifically, the constant temperature module adjusts the ambient temperature to a state most suitable for the growth of microorganisms.
Specifically, when the SAW pressure sensor is subjected to pressure, the length of a SAW propagation path and the wave speed are changed, and the change of the SAW propagation path and the wave speed causes oscillation frequency shift, so that the pressure can be measured, which is equivalent to the growth condition of microorganisms.
Specifically, the pattern acquisition system analyzes data rapidly in real time to obtain a microorganism growth curve, and the microorganism growth curve is displayed through a display screen of the pattern acquisition system.
In particular, the flasks are tightly sealed to prevent erroneous curves due to air leaks.
The general measurement method for the growth curve of the microorganisms is simple to operate, and the growth curves of different microorganisms can be quickly and automatically obtained in real time.
The invention is technically characterized in that a double-oscillation SAW pressure sensor is adopted to measure the pressure change of a culture bottle cap caused by microorganism metabolism;
by adopting the double-oscillation SAW pressure sensor, because the structures and materials of the two oscillators are the same, the frequency offsets of the two oscillators caused by temperature changes are the same, and can be mutually offset to compensate the temperature changes;
the double-oscillation SAW pressure sensor is small in size and space-saving, common in material and low in price;
the double-oscillation SAW pressure sensor has high sensitivity because the scanning substrate is very thin and the two surfaces are polished, thereby improving the compression strength.
Drawings
FIG. 1 is a schematic view of the structure of the present invention
FIG. 2 is a schematic diagram showing the relationship between the cultivation time of Escherichia coli and the cap pressure of a culture flask
Detailed Description
The invention utilizes the SAW pressure sensor to measure the pressure change on the culture bottle cap caused by the metabolism of microorganisms, thereby obtaining the microorganism growth curve.
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but those skilled in the art will understand that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
As shown in fig. 1, fig. 1 is a schematic structural diagram of the present invention, which is a method for automatically measuring a microorganism growth curve, after a microorganism in a culture bottle performs a metabolic activity for a period of time, atmospheric pressure in the bottle changes, so that a bottle cap of the culture bottle deforms slightly, so that a SAW pressure sensor receives pressure, under the action of the pressure, the length of a SAW propagation path and the wave speed change, and the changes of the SAW propagation path and the wave speed cause oscillation frequency deviation, so that the pressure can be measured, and the microorganism growth curve can be obtained according to the microorganism growth condition.
According to the method for automatically measuring the microbial growth curve, the microbial growth curve is automatically drawn by directly analyzing the obtained data through measuring the pressure change on the culture bottle cap, long operation time is not needed, a large amount of manual operation is not needed for automatic drawing, meanwhile, the SAW sensor is arranged above the culture bottle cap, the non-invasive operation cannot cause mixed bacteria interference and loss of content media, error sources are reduced, and the measuring efficiency and the accuracy of a measuring result can be obviously improved.
The invention has the advantages of various types of materials, small occupied space, low price and convenient operation.
In the present invention, the culture flask is a closed culture flask.
In the structure of the invention, after the sample is injected into the culture bottle containing the culture medium, the initial pressure value F0 applied to the culture bottle cover is immediately measured; when the microorganisms start to metabolize, the gas in the culture bottle is increased, the gas pressure is increased, and therefore the ratio of the real-time pressure value Ft of the culture bottle cover to the initial pressure F0 is larger than 1, the fact that the microorganisms start to grow in the sample can be judged until the monitoring period is finished, the real-time pressure Ft is not increased any more, namely the real-time pressure is kept unchanged, and the fact that the growth period of the microorganisms in the sample is finished is judged.
In the embodiment of the invention, the initial pressure value F0 is a known value, and the measurement principle of Ft has been described above and will not be described herein again.
In the invention, the pressure generated by the deformation of the bottle cap on the SAW pressure sensor is measured, the advantages of high sensitivity and high pressure resistance of the SAW pressure sensor are fully utilized, and the accuracy of the detection result is improved.
In the invention, the thermostatic module keeps the sample in the flask at its optimum growth temperature (typically 35-40℃., preferably 37℃.).
Temperature is one of the important factors for the growth of microorganisms, and the change in temperature has a great influence on the metabolism of microorganisms. When the microorganism is at the optimal growth temperature, the growth and metabolism speed is the highest, the metabolism duration is the shortest, and when the culture temperature of the microorganism is lower than or higher than the critical growth temperature, the microorganism may not grow and even die.
Multiple experiments prove that when the temperature of the constant temperature module is 35-40 ℃, the growth and metabolism of microorganisms are vigorous, the metabolism time is short, and when the temperature of the constant temperature module is 37 ℃, the growth and metabolism of the microorganisms are most vigorous, and the metabolism time is shortest.
The application of the microorganism growth curve detection method provided by the invention can be equivalent to four microorganism growth periods by measuring the change of the pressure of the culture bottle cap, so that other substances are not required to be added into the culture bottle, the error sources are reduced, the detection steps are simplified, the detection cost is reduced, the specificity of the detection result is ensured, and the detection efficiency and the accuracy of the detection result can be obviously improved.
The present invention will be further described with reference to the following specific examples.
Example 1
The embodiment provides a method for detecting a microorganism growth curve, which comprises the following steps:
putting 25ml of sample into a culture bottle containing 225ml of culture medium to prepare a sample homogenizing solution in a ratio of 1: 10;
and step two, establishing an automatic microorganism growth curve measuring device which comprises a detection system and a pattern acquisition system, wherein the detection system comprises a sealed culture bottle, a constant temperature module and an SAW pressure sensor, the culture bottle is arranged in the constant temperature module, and the SAW pressure sensor is arranged above a culture bottle cap. The SAW pressure sensor is connected to an acquisition system, the pattern acquisition system is a data acquisition card and a display, and the acquisition card is connected with the display to realize the description of the microorganism growth curve.
Step three, adding 9ml of culture medium and 1ml of the sample homogenizing solution obtained in the step one into a culture bottle of an automatic microorganism growth curve measuring device, and screwing a sealing cover;
inserting the culture bottle into a constant temperature module, and adjusting the temperature of a constant temperature device to be (37 +/-0.2) DEG C;
and step five, starting the microorganism growth curve drawing software, immediately measuring the initial pressure value F0 of the culture bottle cap, judging the growth condition of the microorganisms in the sample according to the value of Ft, and judging that the growth of the microorganisms stops if the Ft does not change any more within a period of time.
According to the method for detecting the microbial growth curve, the closed culture bottle is adopted for microbial culture, other substances do not need to be added into the culture bottle in the detection process, the detection steps are simplified, error sources are reduced, and the detection cost is reduced; in addition, the method for detecting the microbial growth curve provided by the embodiment is equivalent to four microbial growth periods by measuring the pressure change of the culture bottle cap, and is simple and convenient to operate.
Verification example 1
In the verification example, escherichia coli is inoculated in a culture medium as an example, the accuracy of the microbial growth curve detection method provided by the invention is verified, and the test methods of the initial pressure F0 and the real-time pressure Ft adopted in the verification example are the same as those in example 1, and are not repeated herein.
The method for detecting the microbial growth curve provided by the verification example comprises the following steps:
(a) inoculating Escherichia coli into a 50mL culture bottle, wherein 1/3 gas space is reserved above the culture bottle, and the concentration of the Escherichia coli in the culture bottle is 3 × 104 cfu/mL;
(b) the blood culture bottle is placed into a constant-temperature incubator at 37 ℃ for culture, the pressure change of a culture bottle cap is monitored in real time, the pressures of 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h and 30h are recorded according to the culture time of microorganisms, and the measured pressures in different periods are shown in table 1:
TABLE 1 pressure data sheet for different incubation times
Figure BSA0000173731490000051
According to the relation between the microorganism culture time and the culture bottle cap pressure in the table 1, a rectangular coordinate system drawing is established by taking the culture time as a horizontal coordinate and taking the culture bottle cap pressure as a vertical coordinate, and a graph 2 is obtained, and as can be seen by combining the table 1 and the graph 2, the pressure applied to the culture bottle cap is increased along with the increase of the microorganism culture time, and a growth curve of the microorganism can be further obtained according to the change of the pressure. The method for detecting the microbial growth curve provided by the verification example of the invention can be used for rapidly detecting the change condition of the pressure of the culture bottle cap, so that the determination of the microbial growth cycle is carried out, other substances are not required to be added into the culture bottle, the error sources are reduced, the detection steps are simplified, and the detection cost is reduced.
The medium and flask used in this verification example were the same as those used in example 1, and are not described herein again.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (4)

1. The method for measuring the microbial growth curve is characterized by comprising a detection system and a pattern acquisition system, wherein the detection system comprises a sealed culture bottle, a constant temperature module and an SAW pressure sensor, the culture bottle is arranged in the constant temperature module, and the SAW pressure sensor is arranged above a culture bottle cap. The SAW pressure sensor is connected to an acquisition system, the pattern acquisition system is a data acquisition card and a computer, and the acquisition card is connected with the computer to realize the description of the microorganism growth curve.
Adding 9ml of culture medium and 1ml of sample homogenizing solution into a culture bottle of an automatic microorganism growth curve measuring device, and screwing a sealing cover;
inserting the culture bottle into a constant temperature module, and adjusting the temperature of the constant temperature module to be (37 +/-0.2) DEG C;
opening the data acquisition card and acquiring data;
and starting the microorganism growth curve measuring device.
2. A method for mapping a microorganism growth curve according to claim 1, wherein if the microorganism exists in the sample, the metabolic activity of the microorganism causes an increase in the gas pressure in the flask, so that the pressure applied to the flask cover is changed, and the pressure Ft/F0 monitored in real time is greater than 1, it is determined that the microorganism starts to grow in the sample. And if the real-time pressure Ft does not increase any more until the monitoring period is finished, namely the real-time pressure Ft keeps unchanged, judging that the growth of the microorganisms in the sample is finished. At this time, a curve of the growth of the microorganism is obtained.
3. The SAW pressure sensor for a method of measuring a microbial growth curve of claim 1, wherein said SAW pressure sensor is mounted directly above a cap of a flask, and said microbial growth curve is obtained without adding other substances to the interior of said flask. The method can not cause the interference of mixed bacteria and the loss of content media, reduces error sources, and can obviously improve the measurement efficiency and the accuracy of measurement results. And the operation is simple and convenient, and the result is obtained in real time.
4. The SAW pressure sensor for the microorganism growth curve measuring method according to claim 1, wherein the two oscillators have the same structure and material, and the frequency deviation of the two oscillators caused by the temperature change is the same, so as to cancel each other and compensate the temperature change; the volume is small, the space is saved, the materials are common, and the price is low; high sensitivity and high pressure resistance.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61234324A (en) * 1985-04-11 1986-10-18 Anritsu Corp Stress sensor
CN104673664A (en) * 2015-02-15 2015-06-03 武汉迪艾斯科技有限公司 Blood sample culture device
CN105754852A (en) * 2016-04-21 2016-07-13 东北石油大学 Equipment and measuring method for simulating growth curves of microorganisms in high-temperature and high-pressure oil reservoirs
CN106124616A (en) * 2016-06-12 2016-11-16 肖小玉 A kind of novel microbial fertilizer detection device

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61234324A (en) * 1985-04-11 1986-10-18 Anritsu Corp Stress sensor
CN104673664A (en) * 2015-02-15 2015-06-03 武汉迪艾斯科技有限公司 Blood sample culture device
CN105754852A (en) * 2016-04-21 2016-07-13 东北石油大学 Equipment and measuring method for simulating growth curves of microorganisms in high-temperature and high-pressure oil reservoirs
CN106124616A (en) * 2016-06-12 2016-11-16 肖小玉 A kind of novel microbial fertilizer detection device

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