Gradient-based microbial streak planting method and planting system

Abstract

The present application relates to a gradient-based microbial streaking planting method and planting system, the scheme includes reading a streaking pattern, the streaking pattern includes lines of planting tracks on the surface of a culture medium, the lines also have a computer-readable variation feature in the direction of extension; during the automatic streaking process, the streaking pen is controlled to streak and plant on the surface of the culture medium according to the line plane position, and the planting intensity is adjusted at the corresponding position of the line according to the variation feature. The lines of the streaking pattern designed in advance reflect the streaking track, and the variation feature of the lines, which can be recognized by the computer, reflects the planting intensity; when the planting pen is controlled to move along the recognized lines on the surface of the culture medium to streak and plant, the planting intensity is also controlled according to the variation degree of the recognized variation feature, so that the inoculation intensity is controllable. By controlling the intensity of the streaking, the intensity of the streaking at the end of the streaking is lighter, the single colony is ensured to be generated more, and the quality of the streaking planting is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a gradient-based microbial streaking planting method and planting system, belonging to the technical field of automatic microbial planting, used for inoculation before microbial culture; in particular, it relates to a method and system for automatically streaking bacteria on culture medium. BACKGROUND

[0002] Plate streaking separation method is a commonly used method for single colony separation of microbial samples, but due to its complicated steps, manual operation is still the main method in the clinic, which is labor-intensive, and the number of microbial detection personnel in the laboratory is relatively small, making it difficult to complete the processing of a large number of samples.

[0003] Automatic processing can greatly reduce the workload of medical personnel and reduce labor costs. The WASP system of the Italian COPAN company and the InoqulA system of BD are the main automatic streaking products abroad, among which the WASP system is a microbial automatic streaking instrument based on an inoculation ring, and InoqulA uses magnetic beads to roll and inoculate. At present, domestic companies such as Wuhan Di'ais, Jinan Baibo Biology, and Shanghai Bexi Biology have automatic plate streaking equipment. The sample processing system of Di'ais is based on magnetic bead technology, which can take samples and streak without opening the cover; ET-2000 of Baibo Biology can be set for different samples, and different streaking methods can be selected for different samples; Hawk-2200 is a full-automatic microbial streaking instrument developed by Yunluhe Biology Technology, which mainly uses damping force and sensor technology to control the inoculation force of solid culture medium to prevent the culture medium from being broken; Bexi Biology uses a mechanical arm to replace traditional manual microbial sample processing and streaking inoculation, and uses an intelligent vision system to monitor the streaking situation.

[0004] Magnetic bead rolling inoculation can achieve closed cover streaking and customize plate streaking patterns, but its cost is high and its versatility is low. Therefore, there is an urgent need for an inoculation ring streaking scheme that can control the inoculation force, produce a large number of single colonies, and customize the streaking pattern. SUMMARY

[0005] The purpose of the present application is to provide a gradient-based microbial streaking planting method and planting system to solve the problem that the existing automatic plate streaking inoculation scheme is difficult to control the streaking force, thereby resulting in a small number of single colonies.

[0006] To achieve the above-mentioned purpose, the scheme of the present application includes:

[0007] The gradient-based microbial streaking and planting method technical scheme of the present application reads a streaking pattern, the streaking pattern includes lines reflecting the moving track of a streaking and planting pen on the surface of a culture medium, and the lines also have a change degree that is computer-readable in the extension direction of the lines; in the automatic streaking process, the streaking and planting pen is controlled to move and streak and plant on the surface of the culture medium according to the line plane position, and the planting intensity is adjusted on the position of the corresponding planting track of the lines according to the change degree.

[0008] On the basis of automatic streaking, the lines of the pre-designed streaking pattern reflect the streaking and planting track, and the change degree of the lines such as color, thickness, etc. can reflect the planting intensity that is computer-recognizable and readable; when the planting pen is controlled to move along the recognized lines on the surface of the culture medium to perform streaking and planting, the planting intensity is also controlled according to the change degree of the recognized change feature, so that the inoculation intensity is controllable. By controlling the intensity of the streaking, i.e. the thickness of the streaking, the streaking intensity at the end of the streaking is lighter, and the thickness degree is thinner, so that the single colony production is maximized, the streaking and planting quality is improved, and the success rate of the streaking and planting is improved. At the same time, the streaking pattern and the streaking intensity can be customized according to actual needs.

[0009] Further, the planting intensity is embodied by the displacement of the streaking and planting pen in the longitudinal direction, and the planting intensity decreases when moving away from the surface of the culture medium, and the planting intensity increases when moving close to the surface of the culture medium.

[0010] The intensity of the streaking is reflected by the depth of the streaking, the scheme is simple and reliable, and easy to control.

[0011] Further, the streaking pattern also includes a process of initializing the streaking intensity before being generated, and the streaking intensity initialization includes that the change degree and the longitudinal displacement in the longitudinal displacement range of the streaking and planting pen have a set corresponding relationship; the longitudinal displacement range is that the longitudinal maximum displacement of the streaking pen is in the process from contacting the surface of the culture medium to be planted to the surface of the culture medium to be planted being punctured.

[0012] Different culture medium surfaces have different maximum streaking intensities, i.e. the culture medium surface can tolerate different streaking depths before being broken, so in order to adapt to different culture media, the corresponding relationship between the streaking intensity and the change degree of the change feature applied to the current streaking pattern is initialized according to the maximum streaking intensity that the culture medium surface can withstand before the streaking pattern is generated, which can increase the application range of the method of the present application, improve the consistency and success rate of the streaking and planting, and avoid that the streaking is too shallow or breaks the surface of the culture medium under different culture media.

[0013] Further, the maximum longitudinal displacement is determined by the following way: the line planting pen is lowered from a position away from the surface of the culture medium to be planted towards the surface of the culture medium, the resistance during the lowering of the line planting pen is detected, when the line planting pen contacts the surface of the culture medium, the displacement of the line pen at this time is recorded as the contact time displacement according to the change of the value of the force gauge; the line planting pen continues to lower until the surface of the culture medium is ruptured and the value of the force gauge changes suddenly, the displacement of the line pen at this time is recorded as the piercing time displacement, the piercing time displacement minus the contact time displacement is the maximum longitudinal displacement.

[0014] According to the surface tension of the surface of the culture medium and the characteristic that the surface tension suddenly disappears when the surface is ruptured, the resistance received by the line planting pen inoculation ring is detected by the force gauge, the time when the inoculation ring contacts the surface of the culture medium and the time when the surface of the culture medium is ruptured are identified according to the change of the resistance, and then the total displacement at different planting depths in the longitudinal direction is obtained according to the displacement of the line pen at the corresponding time. The change degree of the change characteristic is fully corresponding to the displacement of the line pen at different planting depths of the surface of the culture medium, so as to avoid planting lines under invalid displacement (longitudinal displacement without contacting the culture medium) of the line pen and planting lines under excessive displacement (longitudinal displacement breaking the surface of the culture medium) of the line pen, or invalid change degree of the change characteristic of the line pattern (the displacement of the line pen corresponding to the change degree is excessive displacement or invalid displacement), which also wastes the resolution of the change degree of the change characteristic.

[0015] Further, the maximum longitudinal displacement is determined by the following way: the line planting pen is lowered from a position away from the surface of the culture medium to be planted towards the surface of the culture medium, the resistance during the lowering of the line planting pen is detected, when the line planting pen contacts the surface of the culture medium, the displacement of the line pen at this time is recorded as the contact time displacement according to the change of the value of the force gauge; the line planting pen continues to lower until the surface of the culture medium is ruptured and the value of the force gauge changes suddenly, the displacement of the line pen at this time is recorded as the piercing time displacement, the piercing time displacement minus the contact time displacement is the maximum longitudinal displacement.

[0016] By using artificial intelligence machine learning, the image of the inoculation ring contacting the surface of the culture medium is recognized by the image recognition algorithm to obtain the corresponding contact time, and the time when the surface of the culture medium is ruptured is also recognized by the image recognition algorithm, and the total displacement at all planting depths in the longitudinal direction without rupturing the surface of the culture medium is obtained by the displacement difference of the inoculation pen at the corresponding time. Or use a person as an operator to replace artificial intelligence, manually mark the contact time when the operator observes that the inoculation ring contacts the surface of the culture medium, and manually mark the rupture time when the surface is observed to be ruptured.

[0017] Further, the set corresponding relationship is a linear relationship.

[0018] Further, the set corresponding relationship is a corresponding relationship obtained according to the elastic change characteristic of the surface spring.

[0019] The size of the surface tension of the culture medium and the displacement distance of the marking pen are in a nonlinear relationship, and due to the characteristics of the surface spring (plane spring or flat spring) on the surface of the culture medium, the corresponding relationship between the change degree and the displacement is obtained according to the elastic change characteristics of the surface spring, so that the marking force changes linearly with the change degree of the change characteristics of the line of the marking pattern during the marking planting process, which is beneficial to more uniform and more controllable planting.

[0020] Further, the change characteristics are one of a gray value, a color value of a set color, a brightness value, and a line thickness.

[0021] The technical scheme of the gradient-based microbial marking planting system of the present application comprises a processor, which executes instructions to realize the gradient-based microbial marking planting method as described above. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a marking pattern diagram of automatic marking in the prior art;

[0023] Figure 2 is an algorithm flow of computer processing of the marking pattern of the present application. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present application more clear and explicit, the present application will be further described in detail below with reference to the drawings and examples.

[0025] Method examples:

[0026] When a marking pen with a head having an inoculation loop is used to perform microbial marking inoculation on a culture medium, a large colony may be contaminated by other microorganisms, which is not conducive to bacterial culture, and therefore more single colonies are desired. When manual marking inoculation is performed, a few lines are quickly drawn at the beginning to quickly consume the bacterial liquid in the inoculation loop, and at the end of marking, the marking pen is lifted and gently drawn, so that more single colonies can be obtained at the end.

[0027] However, the existing automatic marking inoculation cannot control the inoculation force, and it is difficult to effectively obtain more single bacterial colonies. For example, as shown in the marking pattern of Figure 1 , the marking trajectories for three different bacterial species are shown, which are marking 1, marking 2, and marking 3, Figure 1 , the marking pattern shown in the drawing only has plane position information without force (depth) information, and cannot realize controllable inoculation force, cannot guarantee the amount of single colony production, and cannot customize the force of the marking pattern.

[0028] The gradient-based microbial streaking method of the present application represents the streaking pattern through a BMP or JPG image file, represents the streaking strength of the corresponding streaking position through the gray scale in the BMP or JPG image format, controls the streaking position by using the line position of the designed streaking pattern, controls the streaking strength or the streaking depth of the corresponding position by using the gray scale of the streaking pattern, and automatically controls the streaking inoculation.

[0029] The streaking pattern of the present application is drawn by, for example, a pressure-sensitive pen, the drawing strength is increased or decreased as needed during the drawing process, the drawing strength collected by the pressure-sensitive pen is converted into the gray scale value of the streaking pattern of the corresponding position, thereby obtaining the streaking pattern with different gray scale values, and the strength signal during the streaking is included in the gray scale value of the streaking pattern.

[0030] Still as Figure 1 shown in the streaking pattern (the gray scale change of the streaking pattern is not shown in the figure), in order to obtain more single-species colonies, the present application can design the early streaking of the streaking 1, the streaking 2 and the streaking 3 to be deeper, or the strength of the streaking to be larger, and design the end of the streaking to be lighter and shallower, according to the experience during manual streaking, to effectively control to obtain more single colonies.

[0031] For different microorganisms, there are also different optimal streaking depths during inoculation, and the present application can also design different streaking depths according to the different types of inoculated microorganisms.

[0032] It should be understood by those skilled in the art that in the present embodiment, the picture format capable of representing the gray scale is utilized to reflect the streaking depth through the gray scale, and therefore the present application does not limit the format of the image file, and other image formats capable of embodying or reflecting the gray scale can also be used.

[0033] Specifically, the method of the present application includes the following contents.

[0034] Firstly, the streaking pattern for inoculation is defined:

[0035] 1. The image is black (f000000);

[0036] 2. The track is a gray line;

[0037] 3. The gray scale value of the track represents the strength;

[0038] 4. The gray scale value is 0-255.

[0039] The process of the streaking inoculation is that the inoculation pen is controlled by the three-axis moving mechanism to move along the track line of the streaking pattern, and the inoculation pen is moved on the surface of the culture medium. The inoculation force is reflected by the displacement of the inoculation pen in the longitudinal direction: moving towards the culture medium in the longitudinal direction, that is, the inoculation force is large; moving away from the surface of the culture medium in the longitudinal direction, that is, the inoculation force is reduced. Therefore, in the process of moving along the track of the streaking pattern, the displacement of the inoculation pen in the longitudinal direction is also controlled according to the gray value of the corresponding position of the track, so as to realize the control of the streaking force.

[0040] Therefore, when the streaking force is initialized, the displacement range of the streaking pen in the longitudinal direction is first determined, and the process of inoculating the culture medium should avoid the surface of the culture medium being pierced, and the maximum longitudinal displacement of the surface of the culture medium being pierced is different for different culture media. Therefore, the maximum longitudinal displacement of the streaking pen in the process from contacting the surface of the culture medium to the surface of the culture medium being pierced is first determined. After the maximum longitudinal displacement is obtained, the maximum displacement is evenly divided into 255 parts, and one part is a unit displacement. Therefore, the streaking pen moves one unit displacement in the longitudinal direction for each change of 1 unit of gray value of the gray value. Specifically, when the gray value increases by 1 unit, the streaking pen moves down (i.e., close to the surface of the culture medium) by 1 unit displacement in the longitudinal direction, that is, the streaking force increases; when the gray value decreases by 1 unit, the streaking pen moves up (i.e., away from the surface of the culture medium) by 1 unit displacement in the longitudinal direction, that is, the streaking force decreases. When the gray value is 0, the inoculation ring of the streaking pen is away from the surface of the culture medium.

[0041] The gray value of 0-255 is obtained according to the range of gray values that can be recognized by the computer. As other embodiments, different ranges of gray values can also be set, and the range of gray values determines the control accuracy of the longitudinal movement of the streaking pen, or determines the resolution of the control of the streaking force. The finer the range of the gray value is divided, the higher the control accuracy and the resolution are.

[0042] The above unit displacement can be the maximum longitudinal displacement of the streaking pen divided by the number of divisions of the numerical range, for example, 255 in the above example. In this way, the change degree of the gray value of the streaking pattern line is linearly corresponding to the longitudinal displacement in the longitudinal displacement range of the streaking pen. Since the tension change caused by the deformation of the surface of the culture medium is similar to the elastic change characteristics of the surface spring, such a corresponding relationship can also be other functional relationships other than linear, for example, combined with the elastic change characteristics of the surface spring.

[0043] The maximum longitudinal displacement of the marker pen during the process from contacting the surface of the culture medium to the surface of the culture medium being punctured can be determined by the following method. The resistance during the descent of the marker pen is detected by a high-precision micro-force meter. When the marker pen contacts the surface of the culture medium, the surface tension generated by the deformation of the surface of the culture medium acts on the marker pen. According to the change in the value of the force meter (the start of the rising of the force value), the time when the marker pen contacts the surface of the culture medium is recorded, and the displacement of the marker pen at this time (the displacement at the time of contact) is recorded. The marker pen continues to descend, the deformation of the surface of the culture medium gradually increases, and the surface tension gradually increases. The value detected by the force meter changes linearly until the surface of the culture medium is punctured, and the value of the force meter suddenly changes. The middle time of the sudden change of the value of the force meter is taken as the time when the surface of the culture medium is punctured, and the displacement of the marker pen at this time (the displacement at the time of puncture) is recorded. The maximum longitudinal displacement of the marker pen during the process from contacting the surface of the culture medium to the surface of the culture medium being punctured is obtained by subtracting the displacement at the time of contact from the displacement at the time of puncture.

[0044] As other embodiments, artificial intelligence technology can also be used to collect images of the inoculation ring position of the marker pen, and to obtain the time when the marker pen contacts the surface of the culture medium and the displacement of the marker pen at the time of contact through image recognition. The time when the surface of the culture medium is punctured and the displacement of the marker pen at the time of puncture are also obtained. The maximum longitudinal displacement of the marker pen during the process from contacting the surface of the culture medium to the surface of the culture medium being punctured is obtained by subtracting the displacement at the time of contact from the displacement at the time of puncture.

[0045] According to the definition of the above-mentioned marking pattern, the marking pattern is generated or automatically generated by an algorithm according to the needs and technical requirements of actual marking inoculation. The marking pattern includes the trajectory of the line on the pattern reflecting the trajectory of the inoculation ring of the marker pen on the surface of the culture medium, and the gray value of the line on the pattern reflecting the longitudinal displacement of the inoculation ring of the marker pen, i.e., reflecting the size of the marking force.

[0046] After obtaining the marking pattern, the marking pattern is input into the control computer of the marker pen. The control computer obtains the trajectory of the line by image recognition to control the movement of the marker pen in the horizontal direction. At the same time, the gray value of the line is read by image recognition to control the longitudinal displacement of the marker pen at the corresponding horizontal position on the surface of the culture medium.

[0047] Specifically, the algorithm flow of the control computer processing the marking pattern is as follows: Figure 2As shown, the process includes the following. The computer loads a picture recording a line pattern, converts the picture size to 1024x1024. Then identify the position of the line center line, get the image coordinates of the continuous center line position with a certain step or control accuracy; and identify the line gray scale information at different center line coordinate positions. Define a three-dimensional array M (X=1024, Y=1024, Z=256), where X and Y are the horizontal coordinate axes, and Z is the vertical coordinate axis; and perform array assignment. Define the center line point coordinate array Point, which is a two-dimensional array, each row being a continuous curve. Calculate the gradient in the X, Y plane of adjacent points, when the gradient change rate is greater than 10% and the gray scale gradient change rate is less than 10%, record the point information, then loop to calculate the gradient in the X, Y plane of adjacent points until the data traversal is completed; after the data traversal is completed, the gray value is converted to the displacement of the Z axis, then the array for line pen control and the parameter space point coordinate linked list are returned, and the algorithm flow of the line pattern processing is completed.

[0048] The idea of the present application is that, when designing a line pattern, the lightness and darkness of the line drawing process are reflected through the visual changes of the readable and identifiable image, and when drawing, the changes of the designed line pattern are identified to adjust the lightness and darkness of the line drawing, so as to realize controllable line drawing force. Based on this idea, as other embodiments, the present application can also reflect the depth of line drawing through the brightness of the line color (i.e. the brightness of the line), or the color value of different color lines, or realize the identification of the lightness and darkness of line drawing through the thickness of the line pattern line. The corresponding relationship between the color depth, brightness change and line thickness and the line drawing lightness (the vertical displacement of the line pen inoculation ring between contacting the culture medium surface and piercing the culture medium surface) is pre-set.

[0049] System embodiment:

[0050] A gradient-based microbial line drawing planting system of the present application includes a control computer for controlling the action of a line pen to draw lines on the surface of a culture medium. The control computer can load a picture recording a line pattern, and process it, then control the movement of the line pen according to the trajectory and gray scale of the line on the pattern in two dimensions on the plane and the third dimension of force, to complete microbial planting with gradient control of force. The specific control method has been described in the planting embodiment and is clear enough, and will not be repeated here.

Claims

1. A gradient-based microbial streaking method, comprising: The reading scribe pattern includes lines reflecting the moving track of the scribe planting pen on the surface of the culture medium, and the lines also have a computer-readable varying feature in the extending direction, which is one of the following: gray value, color value, brightness value, line thickness; in the automatic scribe process, the inoculation pen is controlled by a three-axis moving mechanism to move along the track line of the scribe pattern and scribe on the surface of the culture medium, and the scribe strength is adjusted at the position corresponding to the planting track according to the varying feature; the scribe strength is reflected by the displacement of the scribe planting pen in the longitudinal direction, and the scribe strength decreases when the scribe planting pen is away from the surface of the culture medium and increases when the scribe planting pen approaches the surface of the culture medium; the scribe strength is reflected by the scribe depth, and the scribe pattern is designed to be deeper at the beginning and shallower at the end, so that the scribe strength is lighter and the line thickness is thinner at the end of the scribe to obtain more single colonies; The scribe pattern also includes a scribe strength initialization process before being generated, and the scribe strength initialization includes: the varying feature and the longitudinal displacement in the longitudinal displacement range of the scribe planting pen have a set corresponding relationship; the longitudinal displacement range is: the maximum longitudinal displacement of the scribe pen from contacting the surface of the culture medium to be planted to the process of piercing the surface of the culture medium to be planted.

2. The gradient-based microbial streaking method of claim 1, wherein, The scribe pattern is generated according to the needs and technical requirements of the actual scribe inoculation.

3. The gradient-based microbial streaking method of claim 1, wherein, The maximum longitudinal displacement is determined by the following method: the scribe planting pen is lowered from a position away from the surface of the culture medium to be planted towards the surface of the culture medium, the resistance in the lowering process of the scribe planting pen is detected, when the scribe planting pen contacts the surface of the culture medium, the displacement of the scribe pen at this time is recorded as the contact time displacement according to the change of the value of the dynamometer; the scribe planting pen continues to descend until the culture medium surface is broken and the value of the dynamometer changes suddenly, and the displacement of the scribe pen at this time is recorded as the piercing time displacement, and the piercing time displacement minus the contact time displacement is the maximum longitudinal displacement.

4. The gradient-based microbial streaking method of claim 1, wherein, The maximum longitudinal displacement is determined by the following method: the image of the position of the scribe planting pen inoculation ring is collected, the contact time displacement when the scribe pen contacts the surface of the culture medium is obtained through image recognition; at the same time, the piercing time displacement when the surface of the culture medium is pierced is obtained through image recognition, and the piercing time displacement minus the contact time displacement is the maximum longitudinal displacement.

5. The gradient-based microbial streaking method of claim 1, wherein, The set corresponding relationship is a linear relationship.

6. The gradient-based microbial streaking method of claim 1, wherein, The set corresponding relationship is obtained according to the elastic variation characteristics of the surface spring.

7. The gradient-based microbial streaking method according to any one of claims 1 to 6, characterized in that, The varying feature is the gray value.

8. The gradient-based microbial streaking method of claim 7, wherein, The scribe pattern is drawn by a pressure-sensitive pen, and the drawing strength is reduced during the drawing process according to the needs of the microbial scribe inoculation, and the drawing strength collected by the pressure-sensitive pen is converted into the gray value of the scribe pattern at the corresponding position.

9. A gradient-based microbial streaking system, comprising: The processor executes instructions to implement the gradient-based microbial scribe planting method according to any one of claims 1-8.

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