A method of counting plankton

Abstract

The present application relates to the technical field of plankton counting, and particularly relates to a plankton counting method, which is used for sampling at a fixed flow rate in an actual environment; when any aerosol particle is detected by a plankton detector, the particle size of the aerosol particle is determined according to the corresponding scattered light of the aerosol particle, the number of layers of the plate to which the particle size belongs in an Anderson sampler is determined by looking up a table, the fluorescence of the aerosol particle is detected, the fluorescence intensity / scattered light intensity ratio of the aerosol particle is calculated, and the probability of the aerosol particle being plankton is calculated by using a probability calculation function corresponding to the range to which the particle size of the aerosol particle belongs, until the sum of the probabilities of each aerosol particle in the inhaled volume being plankton is calculated, and the total number of plankton in the inhaled volume is calculated; the method can improve the reliability and accuracy of plankton counting of the plankton detector in a fixed scene.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plankton counting, in particular to a plankton counting method. BACKGROUND

[0002] At present, the total number of bacteria in the indoor environment is regulated in the national standard GB / T18883-2020, which is 1500 CFU / m 3 The corresponding sampling method is the impact culture method. The impact method is to use an impact air microorganism sampler to make air pass through a slit or a small hole to generate a high-speed airflow, collect microorganisms suspended in the air onto a nutrient agar plate, and then determine the number of bacteria colonies after 48 hours of culture at 36℃±1℃. The commonly used sampler for this method is a six-stage sieve impact microorganism sampler, which distributes the particles of plankton from large to small on the nutrient agar plates from top to bottom. This method is the gold standard for detecting the number of plankton, but it is difficult to continuously monitor the number of plankton in the environment on a large scale because it requires the use of sterile culture medium and specific temperature culture in a sterile environment for 48 hours.

[0003] In order to meet the demand for continuous monitoring of the number of plankton in the environment, a plankton monitoring device based on ultraviolet-induced fluorescence has been developed. Since plankton with life activity has metabolic action, the fluorescence of the particles can be judged by tracking the substances in this process. Nicotinamide adenine dinucleotide (NAD) is a coenzyme that transfers protons, and it appears in many metabolic reactions in cells, such as glycolysis, gluconeogenesis, tricarboxylic acid cycle, and respiratory chain. The intermediate product will deliver the removed hydrogen to NAD to make it reduced NADH. NADH has a clear spectral absorption at 340nm and produces a strong fluorescence emission spectrum with a center position of 450nm. Therefore, when a focused light spot is irradiated on a certain particle to produce a fluorescence signal of this wavelength, the particle may be a plankton. In addition, when the particle is irradiated by light, it will produce scattering light in different directions, and the light intensity is based on the particle size. Therefore, by setting 1-2 laser beams and corresponding scattering light detectors and fluorescence detectors, it can be determined whether a certain particle is a plankton particle when the fluorescence intensity is greater than a certain threshold.

[0004] However, this monitoring method has the problem of weak anti-interference ability. Although it can respond to plankton, there are often many non-living particles with different fluorescence levels in the environment, and it is difficult to exclude interference while accurately counting plankton by simply judging whether it exceeds a certain fluorescence threshold. SUMMARY

[0005] The technical problem to be solved by the present invention is to provide a method for counting airborne bacteria, which addresses the shortcomings of the prior art.

[0006] The technical solution of the airborne bacteria counting method of the present invention is as follows:

[0007] In a real-world environment, the airborne bacteria detector is used to sample at a fixed flow rate;

[0008] When the airborne bacteria detector detects any aerosol particle, it determines the particle size of the aerosol particle based on the scattered light corresponding to the aerosol particle, and determines the number of plates in the Anderson sampler to which the particle size belongs by referring to a table. At the same time, it detects the fluorescence of the aerosol particle, calculates the ratio of the fluorescence intensity to the scattered light intensity of the aerosol particle, and uses the probability calculation function corresponding to the particle size range of the aerosol particle to calculate the probability that the aerosol particle is an airborne bacterium. This process is repeated until the sum of the probabilities that each aerosol particle in the inhalation volume is an airborne bacterium is obtained, and the total number of airborne bacteria in the inhalation volume is calculated.

[0009] The beneficial effects of the airborne bacteria counting method of the present invention are as follows:

[0010] This method can improve the reliability and accuracy of airborne bacteria counting. The fluorescence intensity of airborne bacteria is affected by their particle size; therefore, the probability that particles with the same level of fluorescence are airborne bacteria is influenced by particle size. Traditional methods use fluorescence thresholds to determine whether a particle is airborne, which may fail to count bacteria with low activity while counting highly fluorescent interfering particles. By using existing probability calculation functions, weaker fluorescent particles can contribute to the total count, while high-fluorescence interference can be excluded. Since there are many particles in the air, the expected airborne bacteria count in the environment can be calculated by calculating each aerosol individually.

[0011] Based on the above scheme, the airborne bacteria counting method of the present invention can be further improved as follows.

[0012] Furthermore, the process of defining the nth layer as the number of the plate to which any aerosol particle belongs in the Anderson sampler, and calculating the probability that any aerosol particle is a planktonic bacterium, includes:

[0013] Using an M-layer Anderson sampler, airborne bacteria are sampled at a preset location and cultured for a first preset time. The bacterial plaques of airborne bacteria cultured on the nth plate in the M-layer Anderson sampler are collected and dispersed in a liquid medium to obtain a suspension. An aerosol generator is used to generate the suspension to obtain a first aerosol containing airborne bacteria, where M and n are both positive integers.

[0014] The fluorescence light intensity / scattering light intensity ratio of each aerosol particle inhaled from the first aerosol is collected by using the plankton detector, and is recorded as a first fluorescence light intensity / scattering light intensity ratio;

[0015] A second aerosol without plankton is generated by using a liquid medium, the fluorescence light intensity / scattering light intensity ratio of each aerosol particle inhaled from the second aerosol is collected by using the plankton detector, and is recorded as a second fluorescence light intensity / scattering light intensity ratio;

[0016] The first fluorescence light intensity / scattering light intensity ratio and the second fluorescence light intensity / scattering light intensity ratio are both divided into multiple intervals according to the same preset standard, in each interval, the difference between the first fluorescence light intensity / scattering light intensity ratio and the second fluorescence light intensity / scattering light intensity ratio is used to obtain a fluorescence light intensity / scattering light intensity ratio distribution generated by plankton, and a normal function fitting is performed to calculate a standard deviation σ n , a mean μ n and a cultivable coefficient ρ n corresponding to the nth layer plate.

[0017] The probability formula is used to calculate the probability that any aerosol particle is plankton The probability formula is: F x represents the fluorescence light intensity of the any aerosol particle, and S x represents the scattering light intensity of the any aerosol particle.

[0018] Further, the total number of plankton in the inhaled volume is calculated, including:

[0019] The total number of plankton N in the inhaled volume is calculated by using a plankton total number calculation formula, and the plankton total number calculation formula is: Nm represents the total number of aerosol particles of the nth layer plate of the Anderson sampler corresponding to the particle size at the time of detection.

[0020] Further, the concentration range of the bacterial solution of the plankton in the first aerosol is 10 4 -10 6 CFU / m 3 .

[0021] Further, the method further comprises:

[0022] The first aerosol is re-sampled by using single-layer or multi-layer impact plankton sampling and cultured for a second preset time period, and finally the total number of bacterial colonies is recorded.

[0023] The beneficial effect of the above further scheme is that: by establishing the relationship between the actual number of cultivable planktonic bacteria and the sum of the difference between the first fluorescence light intensity / scattered light intensity ratio and the second fluorescence light intensity / scattered light intensity ratio, the cultivable coefficient ρ can be calculated n , so that the result of the planktonic bacteria total number calculation formula is closer to the result obtained by the culture method.

[0024] Further, it further comprises:

[0025] The concentration of planktonic bacteria in the suction volume is calculated as N / V, where V represents the suction volume and N represents the total number of planktonic bacteria in the suction volume.

[0026] Further, the number of intervals is not less than 6. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A flowchart of a planktonic bacteria counting method according to an embodiment of the present application;

[0028] Figure 2 A structural diagram of a planktonic bacteria detector according to an embodiment of the present application. DETAILED DESCRIPTION

[0029] As shown in the figure, a planktonic bacteria counting method according to an embodiment of the present application comprises the following steps: Figure 1

[0030] S1, in an actual environment, the planktonic bacteria detector is sampled at a fixed flow rate;

[0031] S2, when the planktonic bacteria detector detects any aerosol particle, the particle size of any aerosol particle is determined according to the corresponding scattered light of any aerosol particle, and the number of layers of the flat plate to which the particle size belongs in the Anderson sampler is determined by looking up the table, while the fluorescence of any aerosol particle is detected, the fluorescence light intensity / scattered light intensity ratio of any aerosol particle is calculated, and the probability that any aerosol particle is planktonic bacteria is calculated using the probability calculation function corresponding to the range to which the particle size belongs, until the sum of the probabilities that each aerosol particle in the suction volume is planktonic bacteria is calculated, and the total number of planktonic bacteria in the suction volume is calculated.

[0032] As shown in the figure, the planktonic bacteria detector comprises a highly directional light beam 1, a scattered light detector 2, a fluorescence detector 3, a scattered light filter 4, a fluorescence condenser 5 and a scattered light condenser 6, the highly directional light beam is focused on the particle passing position, the wavelength is ≤400nm, and can be used to excite the fluorescence of NADH, Figure 2 The solid line in the figure is the scattered light path, and the dashed line is the fluorescence light path. Figure 2

[0033] ​​Optionally, in the above technical solution, the number of layers of the flat plate in the Anderson sampler to which any aerosol particle belongs is defined as the nth layer, and the process of calculating the probability of any aerosol particle being plankton includes:

[0034] S20, using an M-layer Anderson sampler to sample plankton at a preset position and culture for a first preset time, collecting the bacterial plaque of the plankton cultured on the nth layer flat plate in the M-layer Anderson sampler and dispersing it in a liquid medium to obtain a suspension, and using an aerosol generating device to generate the suspension to obtain a first aerosol with plankton, wherein M and n are positive integers, and M is generally equal to 6 or 8;

[0035] Wherein, the M-layer impact type plankton sampling is realized by a six-stage impact type microbial sampler, for example, each layer of the six-stage impact type microbial sampler is provided with a culture flat plate, and a bacterial plaque can be generated after the plankton impacts and cultures on the culture flat plate. The number of bacterial plaques is observed by naked eye, and the volume is calculated combined with the sampling time and flow rate. The number / volume is CFU / m 3 For the 6-layer impact type microbial sampler, when the sampling flow rate is fixed at 28.3 L / min, the upper limit particle size is 7.0 μm, 4.7 μm, 3.3 μm, 2.1 μm, 1.1 μm and 0.65 μm from top to bottom. The reason for this difference is that the size of each layer of the hole plate is different, and it decreases from top to bottom.

[0036] S21, using a plankton detector to collect the fluorescence light intensity / scattering light intensity ratio of each aerosol particle inhaled from the first aerosol, denoted as the first fluorescence light intensity / scattering light intensity ratio;

[0037] S22, using a liquid medium to generate a second aerosol without plankton, and using a plankton detector to collect the fluorescence light intensity / scattering light intensity ratio of each aerosol particle inhaled from the second aerosol, denoted as the second fluorescence light intensity / scattering light intensity ratio, wherein the liquid medium is physiological saline or phosphate buffer solution PBS, etc.

[0038] S23, according to the same preset standard, the first fluorescence light intensity / scattering light intensity ratio and the second fluorescence light intensity / scattering light intensity ratio are divided into multiple intervals, in each interval, the difference between the first fluorescence light intensity / scattering light intensity ratio and the second fluorescence light intensity / scattering light intensity ratio is used to obtain the fluorescence light intensity / scattering light intensity ratio distribution generated by the plankton, and normal function fitting is performed to calculate the standard deviation σ n , the mean μ n and the cultivable coefficient ρ n of the nth layer flat plate.

[0039] Optionally, the preset standard for dividing the intervals needs to consider the maximum and minimum values of the first fluorescent light intensity / scattering light intensity ratio, divide the difference between the two by the number of intervals to be divided, and obtain the range of each interval, so that the number of data points in the interval containing the maximum value of the first fluorescent light intensity / scattering light intensity ratio does not exceed 2.5% of the total number of data points.

[0040] S24, calculate the probability of any aerosol particle being plankton by using a probability formula The probability formula is: F x represents the fluorescent light intensity of any aerosol particle, S x represents the scattering light intensity of any aerosol particle.

[0041] wherein the concentration range of the plankton in the first aerosol is 10 4 -10 6 CFU / m 3 ;

[0042] Optionally, in the above technical solution, in S2, the total number of plankton in the inhaled volume is calculated, including:

[0043] S24, calculate the total number of plankton N in the inhaled volume by using a plankton total number calculation formula, and the plankton total number calculation formula is: Nm represents the total number of aerosol particles in the nth layer plate of the Anderson sampler corresponding to the particle size at the time of detection.

[0044] Optionally, in the above technical solution, it further includes:

[0045] S3, re-performing single-layer or multi-layer impact type plankton sampling and collecting on the first aerosol and culturing for a second preset time period, and finally recording the total plaque number.

[0046] wherein the first preset time period and the second preset time period are both 48 hours, and can also be set according to actual conditions.

[0047] Optionally, in the above technical solution, it further includes:

[0048] S4, calculate the concentration of plankton in the inhaled volume as N / V, wherein V represents the inhaled volume, and N represents the total number of plankton in the inhaled volume.

[0049] Optionally, in the above technical solution, the number of intervals is not less than 6.

[0050] In another embodiment, it includes:

[0051] S101, perform M-layer impact type plankton sampling on a place or similar place where a biological aerosol detector needs to be continuously and long-term placed, and culture the culture plates of each layer.

[0052] S102, collect and re-disperse the bacteria plaque of each layer of flat plate culture in liquid medium (such as physiological saline, phosphate buffer PBS), and use an aerosol generating device to generate an aerosol with planktonic bacteria, so that the concentration of the bacterial solution is about 10 4 -10 6 CFU / m 3 , the generated aerosol is re-sampled by single or multi-layer impact planktonic bacteria and cultured, and the total plaque number N is finally recorded i At the same time, the biological aerosol detector to be calibrated is used to collect all the aerosols and record the scattering light intensity F / scattering light intensity S value of each aerosol particle. Finally, an aerosol is generated using the same concentration of dispersion liquid (without bacteria), and the biological aerosol detector to be calibrated is used to collect the aerosol and record the F / S value of each aerosol particle. F / S is divided into n intervals (generally n≥6) according to intensity, and the F / S value of each particle of the above two experimental results is counted according to the interval and the statistical result of the bacteria is subtracted from the blank statistical result, to obtain the F / S value distribution of the planktonic bacteria.

[0053] S103, the F / S value (average value of each interval) distribution of the aerosol particles generated by the original environment sampling of each layer of plate culture is fitted with a normal function, and the total number is N c , and σ, μ and the culturable coefficient ρ of each particle are obtained by fitting the normal function according to the data, and the specific formula is as follows.

[0054]

[0055] S104, when sampling in the actual environment sampling at a fixed flow rate, when a particle x is detected, its particle size is inferred (which can be realized by calibrated microspheres with known particle size), the particle size range of the corresponding planktonic bacteria is judged according to the scattering light S x , and the data σ n , μ n , ρ n of the layer are used to calculate the probability that it is a culturable planktonic bacteria For a specific inhalation volume V, the total number N of culturable planktonic bacteria is the sum of the probabilities of all particles, and the number in the mth layer range according to the particle size distribution is N m , then The concentration of planktonic bacteria is N / V.

[0056] The implementation principle and beneficial effects of the present application are as follows:

[0057] 1)NADH in each planktonic bacteria limited content, so if any particle if planktonic bacteria, its fluorescence intensity will not be very strong, only meet certain requirements of fluorescence intensity may be planktonic bacteria.

[0058] 2) using fluorescence F / scattering light S to calculate the approximate fluorescence efficiency of the particle, but still affected by particle size, so the different volume of bacteria stratification processing, each layer of bacteria fluorescence F / scattering light S should meet the distribution rule.

[0059] 3) due to the existence of fixed fluorescence threshold, will lead to division is very difficult, too absolute, so consider each particle, calculate the probability of each particle is bacteria, and then find out all the measured particles are bacteria expectations, so as to avoid the fixed threshold adjustment for bacteria count is particularly large problem. Can cleverly solve the false positive problem of high fluorescence particles, also can make low fluorescence particles produce certain contribution.

[0060] 4) using the method of sampling, culture, calibration of the scene, can make the parameters more suitable for the same similar environment, help to improve the reliability of the data of planktonic bacteria detector in fixed scene.

[0061] 5) using a certain probability function to convert the fluorescence value F or scattering light intensity F / scattering light intensity S value, and then calculate the probability expectation of planktonic bacteria in all particles, rather than by setting the fluorescence value F threshold to calculate the number of particles exceeding the threshold.

[0062] 6) through the stratified statistical method of multi-layer impact sampler, the parameters of bacteria in a certain particle size range are calculated, which reduces the problem of inconsistent fluorescence characteristics of bacteria of different particle sizes, and makes the overall result more accurate.

[0063] In the above embodiments, although the steps are numbered S1, S2, etc., it is only a specific embodiment given by the present application, and those skilled in the art can adjust the execution order of S1, S2, etc. according to the actual situation, which is also within the protection scope of the present application. It can be understood that in some embodiments, some or all of the above embodiments can be included.

[0064] Those skilled in the art know that the present application can be implemented as a system, a method or a computer program product.

[0065] Therefore, the present disclosure can be embodied in the form of hardware only, software only (including firmware, resident software, micro-code, etc.), or a combination thereof, which can be referred to as a "circuit", "module" or "system" hereinafter. Furthermore, in some embodiments, the present disclosure can also be embodied in the form of a computer program product on one or more computer readable medium(s) containing computer readable program codes.

[0066] Any combination of one or more computer readable medium(s) can be employed. The computer readable medium can be a computer readable signal medium or a computer readable storage medium. A computer readable storage medium can be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any suitable combination of the foregoing. More specific examples (a non-exhaustive list) of the computer readable storage medium include: an electrical connection having one or more wires, a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the foregoing. In the present document, a computer readable storage medium can be any tangible medium that contains or stores a program for use by or in connection with an instruction execution system, apparatus, or device.

[0067] Although the embodiments of the present disclosure have been shown and described above, it should be understood that the above-described embodiments are exemplary, and should not be construed as limiting the present disclosure, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present disclosure.

Claims

1. A method for counting airborne bacteria, characterized in that, include: In a real-world environment, the airborne bacteria detector is used to sample at a fixed flow rate; When the airborne bacteria detector detects any aerosol particle, it determines the particle size of the aerosol particle based on the scattered light corresponding to the aerosol particle, and determines the number of plates in the Anderson sampler to which the particle size belongs by referring to a table. At the same time, it detects the fluorescence of the aerosol particle, calculates the ratio of the fluorescence intensity to the scattered light intensity of the aerosol particle, and uses the probability calculation function corresponding to the particle size range of the aerosol particle to calculate the probability that the aerosol particle is an airborne bacterium. This process is repeated until the sum of the probabilities that each aerosol particle in the inhalation volume is an airborne bacterium is obtained, and the total number of airborne bacteria in the inhalation volume is calculated. The process of defining the nth layer as the number of the plate to which any aerosol particle belongs in the Anderson sampler, and calculating the probability that any aerosol particle is a planktonic bacterium, includes: Using an M-layer Anderson sampler, airborne bacteria are sampled at a preset location and cultured for a first preset time. The bacterial plaques of airborne bacteria cultured on the nth plate in the M-layer Anderson sampler are collected and dispersed in a liquid medium to obtain a suspension. An aerosol generator is used to generate the suspension to obtain a first aerosol containing airborne bacteria, where M and n are both positive integers. The fluorescent light intensity / scattered light intensity ratio of each aerosol particle inhaled from the first aerosol is collected using the airborne bacteria detector and recorded as the first fluorescent light intensity / scattered light intensity ratio. A second aerosol without airborne bacteria is generated using a liquid medium. The airborne bacteria detector is used to collect the ratio of fluorescence intensity to scattered light intensity of each aerosol particle inhaled from the second aerosol, which is recorded as the second fluorescence intensity to scattered light intensity ratio. According to the same preset standard, the ratios of the first fluorescence intensity to the scattered light intensity and the second fluorescence intensity to the scattered light intensity are both divided into multiple intervals. Within each interval, the distribution of the fluorescence intensity to the scattered light intensity ratio produced by the planktonic bacteria is obtained by using the difference between the first fluorescence intensity to the scattered light intensity ratio and the second fluorescence intensity to the scattered light intensity ratio. A normal function is then fitted to calculate the standard deviation σ corresponding to the nth plate. n Mean μ n and culturability coefficient ρ n ; The probability that any aerosol particle is a planktonic bacterium can be calculated using the aforementioned probability formula. The probability formula is: F x S represents the fluorescence intensity of any of the aerosol particles. x This represents the intensity of the scattered light from any of the aerosol particles. The total number of airborne bacteria in the inspiratory volume was calculated, including: The total number of airborne bacteria N in the inhalation volume is calculated using the formula for calculating the total number of airborne bacteria. N m This represents the total number of aerosol particles on the nth plate of the Anderson sampler corresponding to the particle size during detection.

2. The method for counting airborne bacteria according to claim 1, characterized in that, The bacterial concentration range of the airborne bacteria in the first aerosol is 10. 4 -10 6 CFU / m 3 .

3. The method for counting airborne bacteria according to claim 1, characterized in that, Also includes: The first aerosol is resampled using a single-layer or multi-layer impactor-type airborne bacteria sampling method and cultured for a second preset time. The total number of bacterial plaques is then recorded.

4. The method for counting airborne bacteria according to claim 1, further comprising: The concentration of airborne bacteria in the inhalation volume is calculated as N / V, where V represents the inhalation volume and N represents the total number of airborne bacteria in the inhalation volume.

5. The method for counting airborne bacteria according to claim 1, characterized in that, The number of intervals is no less than 6.

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