CN110408675A - An Experimental Method for Measuring Chemotaxis of Bacteria - Google Patents

Abstract

The invention discloses a kind of experimental methods for measuring Chemotaxis of Bacteria, the agar containing attractant is drawn using the pipette tips of liquid-transfering gun, attractant makes the bacterium for experiencing attractant enter pipette tips after spreading, it reuses liquid-transfering gun to get the agar in pipette tips, passes through ATP evaluation experimental result in measurement agar；It is simple to operate, security risk is not present, and experimental material is easy to get, while evaluating into the cell relative number in pipette tips by the concentration of measurement ATP, compared with the mode for directly counting number under the microscope, operation is more simple and convenient, and experimental result is more acurrate.

Description

An Experimental Method for Measuring Chemotaxis of Bacteria

technical field

The invention relates to the technical field of bacterial tropism experiments, in particular to an experimental method for measuring bacterial chemotaxis.

Background technique

Chemotaxis (also known as chemotaxis) is a type of tropism, which refers to the movement of body cells, bacteria and other unicellular and multicellular organisms according to certain chemical substances in the environment. Tendency is very important for bacteria to find energy source substances (such as glucose) and pathogenicity, so that bacteria tend to places with higher concentration of energy source substance molecules, or stay away from toxic places (such as phenol).

At present, when measuring the chemotaxis ability of bacteria to different attractants in the laboratory, capillary glass tubes are often used to absorb agar containing attractants, then put the capillary glass tubes into the bacterial culture solution, and take them out after a certain period of time. Count the cells entering the capillary under a microscope to determine the chemotaxis ability of the bacteria; this method has the following problems: the first is to use the capillary glass tube to absorb the attractant, which is difficult to take out in the capillary glass tube, and when observing The capillary glass tube can only be broken to release the bacteria entering the capillary glass tube completely, which takes time and effort, wastes equipment and has certain safety risks; the second is to directly count the bacteria entering the attractant under a microscope The number is time-consuming and labor-intensive, and prone to errors.

Therefore, how to make the measurement experiment of bacterial chemotaxis easier and more accurate is a technical problem that those skilled in the art need to solve.

Contents of the invention

The object of the present invention is to provide a kind of experimental method of measuring bacterial chemotaxis, it utilizes the tip of pipetting gun to suck and beat out the agar that contains attractant, and evaluates the relative number of the cell that enters in the tip by measuring the concentration of ATP , the operation is simpler and more convenient, and the experimental results are more accurate.

In order to solve the problems of the technologies described above, the invention provides a kind of experimental method of measuring bacterial chemotaxis, comprising:

S1: Rinse and resuspend the bacteria with motility in the first centrifuge tube;

S2: Use a pipette gun to draw agar containing a preset concentration of attractant into its tip, insert the tip into the first centrifuge tube, and immerse the tip of the tip in the bacterial suspension;

S3: After standing still for a preset time, take out the pipette tip from the first centrifuge tube;

S4: using the pipette gun to release the agar in the pipette tip into the second centrifuge tube;

S5: Measure the relative number of cells entering the agar, and then determine the chemotaxis ability of the bacteria to the corresponding chemoattractant.

Preferably, measuring the relative number of cells entering the agar specifically includes: measuring the concentration of ATP in the agar by using a kit to measure the relative number of cells entering the pipette tip.

Preferably, resuspending the bacteria with motility in the first centrifuge tube after rinsing specifically includes:

Centrifuge and rinse 1mL of bacteria that have been cultured to the logarithmic growth phase twice with PBS buffer;

Resuspend the bacteria in the first 1.5 mL centrifuge tube using 100 μL of PSF buffer containing 0.3% methylcellulose.

Preferably, before using a pipette gun to draw the agar containing a preset concentration of attractant into its tip, it also includes:

Cut off the tip of the pipette tip to a preset length.

Preferably, after inserting the gun head into the first centrifuge tube, it also includes:

Put a cap on the nozzle of the first centrifuge tube.

Preferably, between steps S3 and S4, the method further includes: wiping off bacteria on the surface of the pipette tip with an alcohol cotton ball.

Preferably, the attractant includes Glc, Met, Ser, Pro and PC.

Preferably, the range of the preset time is 25-35 minutes.

Preferably, all experimental operations are performed in a biological safety cabinet, and the experimental temperature range is 36-38°C.

In the experimental method for measuring bacterial chemotaxis provided by the present invention, the tip of the pipette is used to absorb the agar containing the attractant. The agar in the tip is shot out, the operation is simple and convenient, there is no safety risk, and the experimental materials are easy to obtain; at the same time, the relative number of cells entering the tip is evaluated by measuring the concentration of ATP. Compared with the direct counting method under the microscope, The operation is simpler and more convenient, and the experimental results are more accurate.

Description of drawings

Fig. 1 is the flow chart of a kind of embodiment of measuring bacterial chemotaxis experimental method provided by the present invention;

Fig. 2 is the structure schematic diagram that gun tip is inserted in the first centrifuge tube during the operation process of measuring bacterial chemotaxis experiment;

Fig. 3 is a schematic diagram of the concentration of ATP in agar under different attractants measured by the experimental method provided by the present invention.

The markings in the attached drawings are as follows:

The first centrifuge tube 1, the gun head 2, and the tube cover 3.

Detailed ways

The core of the present invention is to provide an experimental method for measuring bacterial chemotaxis, which utilizes the tip of the pipette to draw and discharge the agar containing the attractant, and evaluates the relative number of cells entering the tip by measuring the concentration of ATP , the operation is simpler and more convenient, and the experimental results are more accurate.

In order to enable those skilled in the art to better understand the solution of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Please refer to Fig. 1 to Fig. 3, Fig. 1 is the flow chart of a kind of embodiment of the method for measuring bacterial chemotaxis provided by the present invention; A schematic diagram of the structure in a centrifuge tube; Figure 3 is a schematic diagram of the concentration of ATP in the agar under different attractants measured by the experimental method provided by the present invention.

The experimental method for measuring bacterial chemotaxis provided by the specific embodiment of the present invention mainly includes the following steps:

S1: resuspend the bacteria with motility ability in the first centrifuge tube 1 after rinsing;

During the specific operation, a certain volume of bacterial culture solution can be taken from the bacterial culture dish, and washed twice by centrifugal washing with PBS buffer solution (ie, phosphate buffered saline solution), and then use PSF buffer solution containing 0.3% methylcellulose Resuspend the bacteria in the first centrifuge tube 1.

In the experimental method provided by the present invention, bacteria are preferably cultured to the logarithmic growth phase of the bacteria, the population cells in the logarithmic phase have the advantages of relatively consistent physiological characteristics, balanced growth of cell components and constant growth rate, and are the best choice for metabolism and physiological research. It is a good material, and it is also the best material as a strain.

In addition, the present application does not specifically limit the type of bacteria, and any type of bacteria such as Spiroplasma can be selected.

S2: Use a pipette gun to draw agar containing a preset concentration of attractant into its tip 2, insert the tip 2 into the first centrifuge tube 1, and immerse the tip of the tip in the bacterial suspension.

It should be noted that when the pipette tip 2 is inserted into the first centrifuge tube 1, the tip of the pipette head 2 is higher than the liquid level of the bacterial suspension, and only the tip of the pipette tip 2 is immersed in the bacterial suspension.

In addition, there are many types of attractants, and the present application does not specifically limit the types of attractants. The attractants used by different bacteria are not completely the same, and the attractant can be selected according to the type of bacteria; the concentration of the attractant in the agar can also be selected according to The experiment needs to be set by itself, and this application does not make specific restrictions on it.

S3: After standing still for a preset time, take out the tip 2 from the first centrifuge tube 1;

S4: using a pipette gun to release the agar in the pipette tip 2 into the second centrifuge tube.

During the specific operation, after utilizing the pipette gun to suck agar into its pipette head 2, the pipette pipette 2 can be directly squeezed into the first centrifuge tube 1, so that the tip of the pipette head 2 filled with the attractant is immersed in the In the bacterial suspension in the first centrifuge tube 1; after standing for a preset time and taking out the tip 2 from the first centrifuge tube 1, first install the tip 2 on the pipette gun, wipe off the tip with alcohol cotton ball After the bacteria on the surface of 2 are removed, the agar in tip 2 is punched out with a pipette gun.

During the standing process, the chemical substance in the tip of the gun tip 2 diffuses to the surroundings, and the bacteria that feel the chemical substance will move towards the tip of the gun tip 2 and enter the agar inside the gun tip 2 .

Wherein, in order to avoid the pollution of the agar by the external environment when standing still, the operation process is carried out in a biological safety cabinet. After the tip of the gun head 2 is inserted into the first centrifuge tube 1, the mouth of the first centrifuge tube 1 can be covered. Tube cover 3, after reaching the standing time, open the tube cover 3 and take out the gun tip 2.

In addition, in order to ensure that the tropism of the bacteria can be accurately determined, the resting time should not be too short or too long. Preferably, the resting time can be set at about 30 minutes.

S5: Measure the relative number of cells entering the agar, and then determine the ability of the bacteria to chemotaxis to the corresponding chemoattractant.

It should be noted that bacteria are single-cell microorganisms, so the relative number of cells entering the tip 2 is the relative number of bacteria.

Preferably, the concentration of ATP in the agar can be measured by a kit to measure the relative number of cells entering the tip 2, and then determine the chemotaxis ability of the bacteria to the corresponding chemoattractant.

Among them, ATP refers to adenosine triphosphate (referred to as adenosine triphosphate), which is the most direct energy source in organisms. Since the ATP content in each bacterial cell is roughly the same and relatively stable, the number of bacteria can be calculated based on the ATP content. . At present, the ATP concentration is generally determined by the luciferin-luciferase bioluminescent method.

In the experimental method for measuring bacterial chemotaxis provided by the present invention, the tip 2 of the pipette is used to absorb the agar containing the attractant. The operation is simple and convenient, there is no safety risk, and the pipette gun can be reused; and the relative number of cells entering the tip 2 is evaluated by measuring the concentration of ATP, compared with the method of directly counting under a microscope , the operation is simpler and more convenient, and the experimental results are more accurate.

It should be noted that, in order to ensure the accuracy of the experimental results, the entire experimental process needs to be carried out in a biological safety cabinet, and the experimental temperature is set at about 37°C to ensure the activity of bacteria.

On the basis of the above-mentioned specific embodiments, in the experimental method provided by the present invention, before utilizing the pipette gun to suck the agar, the tip of the pipette head 2 can be cut off to a certain length to increase the length of the pipette head 2. The opening is sized for agar suction and agar dispensing.

In the experimental method provided by the present invention, it should be noted that the present application does not specifically limit the specifications of the bacterial culture solution used in the experiment, the volume of agar, the pipette gun, the first centrifuge tube 1, etc., and can be determined according to actual conditions. Adjust the selection. In a specific embodiment, 1 mL of bacterial culture solution can be taken from the bacterial culture bottle, and after rinsing, the buffer solution is used to resuspend the bacteria in the first centrifuge tube 1 of 1.5 mL, and the volume of the bacterial suspension after the resuspension is about At the same time, use a 200mL pipette gun, cut off the tip of the used pipette tip 2 by about 13mm, use the pipette gun to draw about 10mL of agar containing the attractant into the pipette tip 2, and then put the agar-filled pipette The tip of tip 2 was inserted into the spiroplasma suspension in the first centrifuge tube 1, and after incubation at 37° C. for 30 minutes, the agar at the tip of tip 2 was recovered and the ATP concentration in the agar was measured.

When adopting the experimental method provided by the invention to measure the chemotaxis ability of a certain bacterium to different attractants, it is only necessary to select successively the agar containing different attractants to carry out the experiment, and other experimental conditions are the same; , and sequentially select Glc (glucose), Met (methionine), Ser (serine), Pro (proline) and PC (lecithin) as attractant, PSF buffer solution (the formula of 100mL PSF buffer solution is 100mL PBS 0.5% fructose and 5% sorbitol) were added to the buffer solution as the reference material, the measured ATP concentration in the agar was shown in Figure 3.

In the description of the application documents of the present invention, in this specification, relational terms such as first and second are only used to distinguish one entity from several other entities, and do not necessarily require or imply that there is a relationship between these entities There is no such actual relationship or sequence.

In addition, each embodiment in the description is described in a progressive manner, each embodiment focuses on the differences from other embodiments, and the similar parts of the various embodiments can be referred to each other. As for the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and for relevant details, please refer to the description of the method part.

The experimental method for measuring bacterial chemotaxis provided by the present invention has been described in detail above. In this paper, specific examples are used to illustrate the principle and implementation of the present invention, and the descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (9)

1. An experimental method for measuring bacterial chemotaxis, characterized in that, comprising: S1: Rinse and resuspend the bacteria with motility in the first centrifuge tube; S2: Use a pipette gun to draw agar containing a preset concentration of attractant into its tip, insert the tip into the first centrifuge tube, and immerse the tip of the tip in the bacterial suspension; S3: After standing still for a preset time, take out the pipette tip from the first centrifuge tube; S4: using the pipette gun to release the agar in the pipette tip into the second centrifuge tube; S5: Measure the relative number of cells entering the agar, and then determine the chemotaxis ability of the bacteria to the corresponding chemoattractant. 2. the experimental method of measuring bacterial chemotaxis according to claim 1, is characterized in that, measuring and calculating the relative number of cells entering in the agar specifically comprises: measure the concentration of ATP in the agar by the kit, measure and calculate entering the described pipette tip The relative number of cells within. 3. the experimental method of measuring bacterial chemotaxis according to claim 2, is characterized in that, the bacterium with motility is resuspended in the first centrifuge tube after rinsing and specifically comprises: Centrifuge and rinse 1mL of bacteria that have been cultured to the logarithmic growth phase twice with PBS buffer; Resuspend the bacteria in the first 1.5 mL centrifuge tube using 100 μL of PSF buffer containing 0.3% methylcellulose. 4. the experimental method of measuring bacterial chemotaxis according to claim 2, is characterized in that, before utilizing pipette gun to draw the agar that contains preset concentration attractant in its gun tip, also includes: Cut off the tip of the pipette tip to a preset length. 5. the experimental method of measuring bacterial chemotaxis according to claim 4, is characterized in that, also comprises after described gun head is inserted in described first centrifuge tube: Put a cap on the nozzle of the first centrifuge tube. 6. The experimental method for measuring bacterial chemotaxis according to claim 5, characterized in that, between steps S3 and S4, further comprising: wiping off the bacteria on the surface of the pipette tip with an alcohol cotton ball. 7. The experimental method for measuring bacterial chemotaxis according to claim 1, wherein the attractant comprises Glc, Met, Ser, Pro and PC. 8. The experimental method for measuring bacterial chemotaxis according to claim 1, characterized in that, the preset time range is 25-35min. 9. The experimental method for measuring bacterial chemotaxis according to claim 8, characterized in that, all experimental operations are carried out in a biological safety cabinet, and the range of experimental temperature is 36-38°C.