CN107680129B - Portable bacterial colony automatic counting method based on smart phone - Google Patents
Portable bacterial colony automatic counting method based on smart phone Download PDFInfo
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- CN107680129B CN107680129B CN201710813545.8A CN201710813545A CN107680129B CN 107680129 B CN107680129 B CN 107680129B CN 201710813545 A CN201710813545 A CN 201710813545A CN 107680129 B CN107680129 B CN 107680129B
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Abstract
The invention discloses a portable automatic bacterial colony counting device and method based on a smart phone. The illumination and control circuit board is arranged above the height adjusting board, the light source diffusion board is arranged above the illumination and control circuit board, the culture dish limiting plate is arranged above the light source diffusion board, and the culture dish limiting plate is provided with a bracket shell; the culture dish limiting plate is provided with a through hole, the bacterial culture dish is arranged in the through hole and is arranged on the light source diffusion plate, the support shell is provided with a smart phone placing position and places a smart phone, the smart phone placing position is provided with a mobile phone camera placing hole, and the camera is arranged in the mobile phone camera placing hole; the bacterial culture dish is provided with backlight illumination through the illumination and control circuit board so that the bacterial culture liquid is stable, and the camera shoots and collects images to obtain a counting result through image processing of a specific algorithm. The invention realizes the common colony counting operation in the biochemical sample detection, realizes higher accuracy and complete automation with lower cost, and is convenient for the field use in laboratories and in the field.
Description
Technical Field
The invention relates to the field of biochemical sample detection instruments, in particular to a portable automatic bacterial colony counting method based on a smart phone.
Background
Colony detection is one of important methods for quality detection in industries such as agriculture, food, medicine and health, quarantine inspection and the like, wherein the determination of the number of colonies is one of basic daily works. Currently, for the determination of the number of colonies, the national standard detection method, i.e. the ordinary nutrient agar pour plate method, is the so-called gold standard method with the highest confidence for inoculating and counting the colonies. After completion of colony culture, colonies on the culture dish need to be counted. The traditional counting is realized manually, so that the requirement on the experimenter is high, and even for an experienced experimenter, large human errors are still inevitable, especially for a high-density culture dish. Secondly, with the development of the laboratory, the workload of counting is also larger and larger, and the excessive time and energy of the laboratory staff are occupied, so that the experiment progress is influenced.
With the rapid development of computer image processing technology, in combination with industrial cameras and computers, the research on automated counting of bacterial colonies by using a machine vision method is increasing, related enterprises at home and abroad are also greatly put into research and development, and many mature instruments are also pushed to the market. For example, Spiral Biotech company, Germany Schuett company, Hangzhou county science and technology in China, Shanghai volkong, all successively provided their products, and also have applications in many laboratories at home and abroad. However, these commercial automatic colony counting devices are designed for laboratory use, are bulky, expensive, and not suitable for use in the field and in resource-poor areas.
In conclusion, the conventional colony counting device has low automation degree and high labor intensity, and commercial products can realize higher automation degree, but are often expensive, large in size and difficult to popularize and apply in a large range, and on the other hand, the device is difficult to adapt to field detection.
Disclosure of Invention
The invention aims to provide a portable automatic colony counting device and method based on a smart phone aiming at the defects of the prior art, which are based on a machine vision method, and the smart phone is used as a detection platform to automatically count colonies, so that the automatic colony counting with high accuracy is realized at lower cost, the overhead burden of a laboratory is reduced, and the portable automatic colony counting device and method are convenient for field detection.
In order to achieve the above purpose, the technical scheme adopted by the invention mainly comprises:
the utility model provides a portable bacterial colony automatic counting assembly based on smart mobile phone:
the device comprises a height adjusting plate, an illumination and control circuit board, a light source diffusion plate and a culture dish limiting plate, wherein the illumination and control circuit board is arranged above the height adjusting plate, the light source diffusion plate is arranged above the illumination and control circuit board and is fixedly connected with four corners of the illumination and control circuit board through connecting copper column bolts, the culture dish limiting plate is arranged above the light source diffusion plate, and a support shell is arranged on the culture dish limiting plate;
the culture dish limiting plate is provided with a through hole which is used as a placing position of the bacterial culture dish, the circle center of the through hole is superposed with the geometric center of the culture dish limiting plate, the bacterial culture dish is placed in the placing position of the bacterial culture dish in a penetrating way, the bottom of the bacterial culture dish is placed on the light source diffusion plate,
support shell top surface is equipped with the smart mobile phone and places the position, places the smart mobile phone that has the camera in the smart mobile phone places the position, and the smart mobile phone is placed the position and is equipped with the cell-phone camera and places the hole, and the cell-phone camera that the smart mobile phone has corresponds and places the hole in the cell-phone camera, and the cell-phone camera is placed the hole and is located the through-hole centre of a circle that runs through of bacteria culture dish placement position directly over.
The lighting and control circuit board is provided with a power module, a lighting module, a main control module and a communication module; the power supply module is electrically connected with the illumination module, the master control module and the communication module, the master control module is electrically connected with the illumination module and the communication module respectively, and the communication module is wirelessly connected with the smart phone; the lighting module is arranged in the middle of the top surface of the lighting and control circuit board.
The bacteria culture dish is internally provided with an object to be detected of a bacteria culture solution.
The intelligent mobile phone also comprises wireless communication functions such as Bluetooth and Wi-Fi, the intelligent mobile phone sends a control signal to the communication module in a wireless communication mode, the communication module transmits the received illumination control signal to the main control module, and the main control module controls the illumination module to start illumination according to the illumination control signal to provide backlight illumination for the bacteria culture dish placed in place; make the bacterial culture liquid in the bacteria culture dish stable after the illumination, host system passes through again communication module to smart mobile phone sends the acquisition control signal, smart mobile phone is receiving behind the acquisition control signal, control camera on the smart mobile phone shoots, gathers the image of bacteria culture dish.
After the app is controlled, a series of specific algorithms such as graying and Hough transformation are adopted to carry out noise filtering, segmentation and counting on the acquired image of the bacteria culture dish, counting results are obtained through calculation, and the counting results are displayed on a screen of the smart phone.
The illumination module of the illumination and control circuit mainly comprises a plurality of LEDs which are uniformly distributed and have variable colors, and light rays emitted during working penetrate through the light source diffusion plate to form uniform parallel backlight so as to provide illumination for the bacteria culture dish; under the control of the main control module, the lighting module changes different luminous intensities and luminous colors according to different shapes, colors and the like of bacteria in the bacteria culture dish, and the luminous colors are switched among red, green, blue and white.
The interior of the LED with variable colors in the illumination module consists of three primary colors of red, blue and green LEDs, and the main control module respectively controls the luminous intensity of the three primary colors of red, green and blue LEDs by outputting PWM signals with different duty ratios and finally mixes the signals to enable the illumination module to generate different luminous colors; after the luminous color is determined, the main control module outputs PWM signals with equal proportion change to control the luminous intensity of the red, green and blue three-primary-color LEDs and change the luminous intensity output by the illumination module.
The thickness of the height adjusting plate is fixed, different thicknesses can be selected according to the size of the bacteria culture dish and the focal length of a camera carried by the smart phone, so that the integrity of the image of the bacteria culture dish can be acquired by the camera of the smart phone, and meanwhile, the bacteria culture dish can occupy a large area in the acquired image.
The position size is placed to the bacterial culture dish in the culture dish limiting plate fixed, can select not unidimensional according to the culture dish of different models, guarantees the bacterial culture dish can place and keep motionless after the position is placed to the bacterial culture dish, and the centre of a circle position of placing basically with the centre of a circle of bacterial culture dish place the position and basically coincide.
And the bracket shell is covered by a layer of shading material, so that the influence of the change of environmental illumination conditions on the image quality acquired by the smart phone is reduced.
One side of the bracket shell can be opened and used for taking out, putting in and adjusting the height adjusting plate, the lighting and control circuit board, the light source diffusion plate, the culture dish limiting plate and the position of the bacteria culture dish.
The top of the support shell is provided with an inwards concave station serving as a mobile phone placing position, and meanwhile, the mobile phone placing position is provided with a through small hole which is a mobile phone camera placing hole and is used for placing a camera of the smart phone.
Secondly, a portable bacterial colony automatic counting method based on a smart phone comprises the following processes:
(1) the smart phone is provided with a control app of the automatic counting device, the control app sends a control signal to a communication module in the lighting and control circuit board in a wireless communication mode, the communication module transmits the received lighting control signal to the main control module, and the main control module controls the lighting module to start different lighting modes according to the lighting control signal to provide backlight illumination for the bacteria culture dish placed in place;
(2) after illumination, stabilizing a bacteria culture solution in a bacteria culture dish, sending an acquisition control signal to the control app by the main control module through the communication module, and controlling a camera of the smart phone to take a picture by the control app after receiving the acquisition control signal so as to acquire an image of the bacteria culture dish;
(3) and then, the steps of noise filtering, segmenting and counting are carried out on the acquired image of the bacteria culture dish through a specific algorithm in the control app, and a counting result is obtained through calculation.
The specific process of the step (3) is as follows:
(3.1) region of interest acquisition
Firstly, converting an original picture into a gray-scale image, then carrying out median and Gaussian filtering processing to filter out interference noise, and then detecting the region range of the outer wall of the bacterial culture dish by using Hough circle transformation, wherein specifically, the size of the outer wall of the bacterial culture dish is used as a limit in the original picture, and a circle with the same size is fitted as the circle of the outer wall of the bacterial culture dish;
finally, dividing the original picture by using the detected region range to obtain a picture only having complete bacteria culture solution and the wall thickness of the bacteria culture dish;
(3.2) threshold segmentation
Traversing pixel points in the picture obtained in the step (3.1), converting all the pixel points with the same color as the backlight color into white, graying the picture, performing threshold segmentation by using a maximum inter-class variance method to obtain a binary picture, dividing the area of the complete bacteria culture solution into areas with colony growth and without colony growth, setting the area without colony growth as white as a background, and setting the area with colony growth as black as a target;
(3.3) correction of colony count
To the colony that links to each other and is judged as belonging to same connected domain with bacteria culture dish wall, handle this connected domain alone. Detecting the area range where the wall thickness of the bacterial culture dish is located by using Hough circle transformation and deleting the area range, specifically, using the size of the inner wall of the bacterial culture dish as a limited picture and fitting circles with the same size as the circle where the inner wall of the bacterial culture dish is located;
(3.4) preliminary enumeration of colonies
Performing multiple opening operation on the picture obtained in the step (3.3), filling the holes by using a flooding filling method, traversing pixel points in the filled picture by using a seed filling method, acquiring all connected domains, recording coordinates of all pixel points contained in each connected domain, and simultaneously recording the total number of the connected domains;
the holes refer to the non-connected domain parts inside each obtained connected domain, namely the surrounded white background parts inside the target black area with colony growth.
(3.5) calculating the pixel area of each connected domain, and deleting the connected domains with the areas smaller than the preset area value by using the preset area value of the bacterial colony;
(3.6) then, calculating the external minimum convex hull of each connected domain, calculating the area proportion of the connected domain to the external minimum convex hull corresponding to the connected domain, and when the area proportion is larger than a preset proportion threshold value, considering that the connected domain only has one bacterial colony, otherwise, considering that the connected domain is adhered by a plurality of bacterial colonies;
(3.7) carrying out special treatment on the connected domain with a plurality of adhered bacterial colonies to obtain the total number of the bacterial colonies, and recording the total number as Y; counting the number of connected domains of only one colony as the total number of single colonies, and recording as X; the final calculation results in the total colony number S ═ X + Y.
In the step (3.7), the connected domain with a plurality of colonies adhered is specially processed to obtain the number of the multiple colonies, and specifically, the following three methods are respectively used for processing to obtain Y1, Y2 and Y3:
a. expanding the connected domain of the background once and recording the number of the expanded connected domains of the target by adopting an expansion maximum value searching method, repeating the steps for expansion and recording until the area of the connected domain of the target becomes 0, and recording the maximum value of the number of the connected domains of the target in the whole process as Y1;
b. performing the connected domain of each target by using a watershed segmentation method, and recording the number of segmented results in the connected domain of each target after segmentation as Y2;
c. calculating and drawing a minimum external rectangle of a target connected domain by adopting a distance conversion method, then calculating the Euclidean distance from each pixel point of the target connected domain to a background along the width direction of the minimum external rectangle aiming at each row of the minimum external rectangle, and taking the maximum value, traversing each row of the minimum external rectangle by taking one pixel as the stepping length along the length direction of the minimum external rectangle, drawing a variation curve of the maximum Euclidean distance of each row, and recording the number of valleys on the variation curve, wherein the sum of the number of the valleys is Y3;
finally, the total number of colonies obtained was calculated using the following formula:
Y＝aY1+bY2+cY3
wherein a, b and c are respectively a first coefficient, a second coefficient and a third coefficient, and a, b and c are all preset constants.
The invention has the beneficial effects that:
1. the invention realizes the automation of colony counting in biochemical quarantine. The common smart phone is used as a detection platform for interaction with a user, the operation is simple and visual, and the counting accuracy is improved while the labor intensity is reduced.
2. According to the invention, the camera of the smart phone is used as an image acquisition device, and the independently developed app on the smart phone is used as a control program to process the acquired image, so that the whole process from image acquisition to image processing to result acquisition is directly completed at the mobile phone end, the whole detection process is rapid and quick, and the instrument cost is greatly reduced.
3. The invention uses the color-changeable LED as the lighting source, and has lower power consumption and longer service life. Meanwhile, the intensity and the color of the light source are variable, and can be adjusted and changed according to the shape and the color of the bacteria to be detected, so that the accuracy of the counting result is improved.
4. The shell of the invention is covered by the shading material, thereby ensuring the controllability of the image acquisition condition of the bacteria culture dish, greatly reducing the interference of natural light, reducing the complexity of the algorithm and improving the accuracy.
5. The invention has simple structure, small volume and high portability, and is suitable for field use.
Drawings
FIG. 1 is a side view schematic of the present invention.
Fig. 2 is a schematic top view of the present invention.
Fig. 3 is a schematic diagram of the lighting and control circuit board of the present invention.
FIG. 4 is a top view of the plate for limiting culture plates in the present invention.
Fig. 5 is a schematic view of a method for adjusting the field of view of a camera of a mobile phone according to the present invention.
FIG. 6 is a detection flow diagram of the present invention.
In the figure: height adjustment board 1, illumination and control circuit board 2 connect copper post 3, light source diffuser plate 4, culture dish limiting plate 5, bacteria culture dish 6, and hole 7 is placed to the cell-phone camera, and the position 8 is placed to the smart mobile phone, power module 21, lighting module 22, main control module 23, communication module 24, and the position 51 is placed to the bacteria culture dish.
Detailed Description
The present invention will be further described with reference to the drawings attached to the specification, but the present invention is not limited to the following examples.
As shown in fig. 1 and 2, the invention comprises a height adjusting plate 1, a lighting and control circuit board 2, a connecting copper column 3, a light source diffusion plate 4 and a culture dish limiting plate 5, a mobile phone camera placing hole 7 with an inner concave block on the top and a shell of a mobile phone placing position 8. The height adjusting plate 1 with the horizontal surface is horizontally placed on a base of the whole device and is not fixedly connected with the base. The lighting and control circuit board 2 is fixedly connected with the light source diffusion plate 4 through the connecting copper column 3, and the whole body is horizontally placed above the height adjusting plate 1, and the light source diffusion plate 4 is arranged above the lighting and control circuit board 2. The culture dish limiting plate 5 is placed above the light source diffusion plate 4, and the bacterial culture dish 6 is placed in the through round hole in the center of the culture dish limiting plate 5. There is the cell-phone of indent to place position 8 that the shell top of whole device is used for placing the testing platform smart mobile phone that needs to use when detecting, and the cell-phone places hole 7 is then the position of placing the camera that the cell-phone was taken to the round hole cell-phone camera that runs through in position 8. The placing positions of the culture dish limiting plate 5, the round holes penetrating through the culture dish 6 and the mobile phone camera placing holes 7 are as concentric as possible in the vertical direction.
As shown in fig. 3, the lighting and control circuit board 2 is composed of a power module 21, a lighting module 22, a main control module 23 and a communication module 24. The power module 21 is electrically connected with the lighting module 22, the main control module 23 and the communication module 24, the main control module 23 is connected with the lighting module 22 and the communication module 24, and the communication module 24 is connected with the used smart phone in a wireless communication mode. The lighting module 22 is composed of a plurality of uniformly arranged color-changeable LEDs, and each LED is internally provided with red, green and blue three-primary-color LEDs. According to different PWM control signals, the red, green and blue three-primary-color LEDs respectively emit light with different light intensities, and the light with different colors and different intensities is finally emitted after mixing. The main control module 23 is used as a control core of the lighting and control circuit board 2, and drives the communication module 24 to communicate with the smart phone in a wireless mode, receive a control signal of a control end of the smart phone, and drive the lighting module 22 to light up or close and emit lighting light with required color and light intensity; and meanwhile, the feedback information is sent to the mobile phone control terminal through the communication module 24. In practical use, the communication module 24 may adopt a bluetooth module or a Wi-Fi module, and the main control module 23 may use an STM32 series single chip microcomputer, but is not limited thereto.
As shown in FIG. 4, the plate 5 is a rectangular plate having a circular hole at the center, i.e., a position 51 for placing a bacteria culture dish, and the surface thereof is covered with a black light-shielding material. To common 60mm diameter, the circular bacterial culture dish 6 of 90mm diameter and 150mm diameter, can use the bacterial culture dish to place position 51 diameter and be 65mm, 95mm and 155 mm's culture dish limiting plate 5, guarantee that whole bacterial culture dish 6 can put into the bacterial culture dish and place position 51 in, make bacterial culture dish 6 all can be lighted by lighting module 22, remaining activity space is little simultaneously, avoid bacterial culture dish 6 because the good unable complete image of being gathered by the cell-phone camera of locating position.
As shown in fig. 5, in specific implementation, the height adjustment plate 1 is used for aiming at different sizes of the bacteria culture dish 6 and different focal lengths of the mobile phone camera, and by adjusting the distance between the bacteria culture dish 6 and the mobile phone camera, it is ensured that the complete image of the bacteria culture dish 6 can be acquired by the mobile phone camera, and meanwhile, it is ensured that the bacteria culture dish 6 in the acquired image can occupy a larger area.
Taking a lens with a focal length of 28mm, namely a visual angle of 76 degrees, which is widely used in a mobile phone camera, as an example, the length-width ratio of the acquired photo is common 4:3, and the main body image of the bacteria culture dish 6 is appointed to be 10mm away from the edge of the photo. For the 150mm diameter bacteria culture dish, as shown in the top view of fig. 5(a), the rectangular ABCD is the final acquired image range with the aspect ratio of 4:3 by the mobile phone camera, the circle outside the rectangular ABCD passing through the four points of the ABCD is the theoretical acquired image range by the mobile phone camera lens, and the circle inside the rectangular ABCD is the top view of the 150mm diameter bacteria culture dish. According to convention the main body image of the bacteria culture dish 6 is 10mm from the edge of the photograph and the rectangular ABCD has an aspect ratio of 4:3 and a readily available OC length of (150/2+10)/3 x 4, i.e. 141.67 mm. FIG. 5(b) is a front view, wherein COA is the plane of the bacteria culture dish, and point P is the position of the mobile phone camera.
According to the geometrical relationship, the length of the readily available PO, i.e. the distance between the cell phone camera and the 150mm bacteria culture dish, should be 141.67mm/tan 38 °, i.e. 181.32 mm. Therefore, when a 150mm diameter dish is used for the test, the height adjustment plate 1 is required to adjust the distance between the dish 6 and the camera of the cell phone to 181.32 mm. Using a similar method, the thickness of the height adjustment plate 1 that needs to be used when changing to another size of the bacteria culture dish 6 can be calculated.
In FIGS. 5(c) to (d), the distance between the cell phone camera and the petri dish 6 used when a 90mm diameter petri dish was used was 117.33 mm.
FIGS. 5(e) to (f) show distances between the petri dish 6 and the mobile phone camera, which are 85.33mm, when a 60mm diameter petri dish is used.
As shown in fig. 6, the specific implementation process of the present invention is as follows:
1. according to the specification of the used bacteria culture dish and the focal length of the used mobile phone camera, the height adjusting plate 1 with the proper thickness and the culture dish limiting plate 5 with the proper size are selected.
2. Open counting assembly side door, from the bottom up put into in proper order height adjustment board 1, have used the illumination and the control circuit 2 and light source diffuser plate 4, the culture dish limiting plate 5 of connecting 3 fixed connection of copper post, place position 51 to the bacterial culture dish that waits that count 6 put into culture dish limiting plate 5 to guarantee as far as possible that bacterial culture dish 6 and bacterial culture dish place the centre of a circle coincidence of position 51 and place, close the side door. The smart phone is placed in the mobile phone placing position 8, the camera carried by the mobile phone is placed in the mobile phone camera placing hole 7, the control app installed on the mobile phone is opened, and meanwhile, the power supply of the automatic counting device is turned on.
3. The experimenter operates the control app on the mobile phone, sends a control signal to the lighting and control circuit board 2 through wireless communication modes such as Bluetooth and the like, the main control module 23 controls the lighting module 22 to send lighting light with corresponding color and intensity according to the received control signal, and after the control signal is stabilized for 1 minute, the main control module 23 sends a detection ready signal to the mobile phone through the communication module 24;
4. after the smart phone receives the ready signal, the app is controlled to open the mobile phone camera, the experimenter adjusts relevant photographing parameters such as aperture, shutter time, light sensitivity and focusing distance, the app is controlled to drive the mobile phone camera to take a picture, the image of the bacteria culture dish 6 is collected, and the image is stored in the mobile phone memory to be processed later.
5. After the image of the bacteria culture dish 6 is obtained, the app is controlled to sequentially execute the steps of obtaining the region of interest, segmenting the threshold value, initially counting the bacterial colonies, correcting the number of the bacterial colonies and the like, the number of the bacterial colonies in the target region is automatically calculated, and the final counting result is recorded, displayed and stored.
6. Open the side door of the counting device, change to the next culture dish, or end the test.
The foregoing detailed description is intended to be illustrative of the invention and is not to be construed as limiting, since any modifications and variations of the invention are possible within the spirit and scope of the invention as defined in the appended claims.
Claims (1)
1. A portable bacterial colony automatic counting method based on a smart phone is characterized by comprising the following steps: the method adopts the following device, the device comprises a height adjusting plate (1), an illumination and control circuit board (2), a light source diffusion plate (4) and a culture dish limiting plate (5), the illumination and control circuit board (2) is arranged above the height adjusting plate (1), the light source diffusion plate (4) is positioned above the illumination and control circuit board (2) and is fixedly connected with four corners of the illumination and control circuit board (2) through connecting copper columns (3), the culture dish limiting plate (5) is arranged above the light source diffusion plate (4), and a support shell is arranged on the culture dish limiting plate (5); the culture dish limiting plate (5) is provided with a through hole serving as a bacteria culture dish placing position (51), the circle center of the through hole coincides with the geometric center of the culture dish limiting plate (5), a bacteria culture dish (6) is placed in the bacteria culture dish placing position (51), the bottom of the bacteria culture dish is placed on the light source diffusion plate (4), the top surface of a support shell is provided with a smart phone placing position (8), a smart phone with a camera is placed in the smart phone placing position (8), the smart phone placing position (8) is provided with a mobile phone camera placing hole (7), the camera carried by the smart phone is correspondingly placed in the mobile phone camera placing hole (7), and the mobile phone camera placing hole (7) is located right above the circle center of the through hole of the bacteria culture dish placing position (51);
the method comprises the following steps:
(1) the control app of the automatic counting device is installed on the smart phone, the control app sends a lighting control signal to a communication module (24) in the lighting and control circuit board (2) in a wireless communication mode, the communication module (24) transmits the received lighting control signal to a main control module (23), and the main control module (23) controls a lighting module (22) to start different lighting modes according to the lighting control signal to provide backlight lighting for the bacteria culture dish (6) placed in place;
(2) after illumination, stabilizing the bacteria culture solution in the bacteria culture dish (6), sending an acquisition control signal to the control app by the main control module (23) through the communication module, and controlling a camera of the smart phone to take a picture by the control app after receiving the acquisition control signal so as to acquire an image of the bacteria culture dish (6);
(3) then, in the control app, the collected images of the bacteria culture dish (6) are subjected to noise filtering, segmentation and counting through a specific algorithm, and a counting result is obtained through calculation;
the specific process of the step (3) is as follows:
(3.1) region of interest acquisition
Converting an original picture into a gray-scale image, then performing median and Gaussian filtering processing, and detecting the region range of the outer wall of the bacterial culture dish by using Hough circle transformation;
finally, dividing the original picture by using the detected region range to obtain a picture only having complete bacteria culture solution and the wall thickness of the bacteria culture dish;
(3.2) threshold segmentation
Traversing pixel points in the picture obtained in the step (3.1), converting all the pixel points with the same color as the backlight color into white, graying the picture, performing threshold segmentation by using a maximum inter-class variance method to obtain a binary picture, dividing the area of the complete bacteria culture solution into areas with colony growth and without colony growth, setting the area without colony growth as white as a background, and setting the area with colony growth as black as a target;
(3.3) correction of colony count
Detecting the range of the region where the wall thickness of the bacterial culture dish is located by using Hough circle transformation and deleting the range;
(3.4) preliminary enumeration of colonies
Performing opening operation on the picture obtained in the step (3.3), filling the holes by using a flooding filling method, traversing pixel points in the filled picture by using a seed filling method, acquiring all connected domains, and recording the total number of the connected domains;
(3.5) deleting connected domains with the areas smaller than the preset area value by using the preset area value of the bacterial colony;
(3.6) then, calculating the circumscribed minimum convex hull of each connected domain, calculating the area proportion of the connected domain to the corresponding circumscribed minimum convex hull, and when the area proportion is larger than a preset proportion threshold value, considering that the connected domain only has one bacterial colony, otherwise, considering that the connected domain is adhered by a plurality of bacterial colonies;
(3.7) processing the connected domain with a plurality of colonies adhered to obtain the total number of the plurality of colonies, and recording the total number as Y;
counting the number of connected domains of only one colony as the total number of single colonies, and recording as X;
finally, calculating to obtain the total number S of the colonies which is X + Y;
in the step (3.7), the connected domain with a plurality of colonies adhered is processed to obtain the number of the multiple colonies, specifically, Y1, Y2 and Y3 are obtained by respectively processing the connected domain with the three methods,
a. expanding the connected domain of the background once and recording the number of the expanded connected domains of the target, repeating the steps for expanding and recording until the area of the connected domain of the target becomes 0, and recording the maximum value of the number of the connected domains of the target in the whole process as Y1;
b. dividing the connected domain of each target by using a watershed division method, and recording the number of divided results in the connected domain of each target as Y2;
c. firstly, calculating and drawing a minimum external rectangle of a target connected domain, then aiming at each row of the minimum external rectangle, calculating the Euclidean distance from each pixel point of the target connected domain to the background along the width direction of the minimum external rectangle and taking the maximum value, traversing each row of the minimum external rectangle by taking one pixel as the stepping length along the length direction of the minimum external rectangle, drawing a variation curve of the maximum Euclidean distance of each row, recording the number of valleys on the variation curve, and marking the number of the valleys plus one as Y3;
finally, the total number of colonies obtained was calculated using the following formula:
Y＝aY1+bY2+cY3
wherein a, b and c are respectively a first coefficient, a second coefficient and a third coefficient.
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| CN108801995A (en) * | 2018-06-05 | 2018-11-13 | 浙江大学 | A kind of the high pass amount detecting device and method of incorporating quantum point fluorescence and multispectral camera |
| CN108531385A (en) * | 2018-07-18 | 2018-09-14 | 贵州省流通环节食品安全检验中心 | A kind of microbe colony robot scaler |
| CN109001150A (en) * | 2018-09-11 | 2018-12-14 | 西北农林科技大学 | A kind of method and apparatus for realizing that bacterium colony is counted based on near-infrared image technology |
| CN109741357A (en) * | 2018-12-19 | 2019-05-10 | 浙江大学 | A kind of brassica napus inflorescence method of counting and device based on image procossing |
| CN109919863B (en) * | 2019-02-15 | 2023-06-20 | 佛山市博朋生物科技有限公司 | Full-automatic colony counter, system and colony counting method thereof |
| CN109948544B (en) * | 2019-03-20 | 2021-04-30 | 南京师范大学 | Automatic positioning and identifying method for target bacterial colony |
| CN112080416A (en) * | 2020-08-26 | 2020-12-15 | 浙江泰林医学工程有限公司 | Intelligent microbial culture counting system |
| CN112686857A (en) * | 2020-12-29 | 2021-04-20 | 中国科学院苏州生物医学工程技术研究所 | High-throughput multi-species colony counting method |
| JP7393836B1 (en) | 2023-04-25 | 2023-12-07 | 株式会社写真化学 | Bacterial body observation device |
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