阅读说明：本技术 一种检测细菌数量的方法 (Method for detecting bacterial quantity ) 是由 贺令娟 程菊红 伍凤祥 于 2019-11-12 设计创作，主要内容包括：本发明公开了一种检测细菌数量的方法,涉及微生物检测技术领域。所述检测细菌数量的方法,在观察计数前先对样品进行一系列的处理,再采用扫描电子显微镜进行成像计数,在一系列的处理中,固定处理避免了计数过程中细菌运动导致计数不准确的问题,同时固定处理还可以避免由于气压和电子束轰击等导致细菌结构易变形的问题；利用扫描电子显微镜成像计数,可以直接利用样品表面材料的物质性能进行微观成像,对细菌浓度没有要求,且可以放大几十到上百万倍,基本上对细菌大小没有限制,同时扫描电子显微镜自带测量尺寸的功能,可以快捷地测量细菌的尺寸,且识别率高,能够区分微生物与微小杂物,在计数时将微小杂物排除,提高了计数的准确度。(The invention discloses a method for detecting the number of bacteria, and relates to the technical field of microbial detection. According to the method for detecting the number of bacteria, a series of treatments are carried out on a sample before observation and counting, then an imaging counting is carried out by adopting a scanning electron microscope, in the series of treatments, the problem of inaccurate counting caused by bacterial movement in the counting process is avoided by fixing treatment, and meanwhile, the problem of easy deformation of a bacterial structure caused by air pressure, electron beam bombardment and the like can also be avoided by fixing treatment; utilize scanning electron microscope imaging count, can directly utilize the material performance of sample surface material to carry out the microcosmic formation of image, do not have the requirement to bacterium concentration, and can enlarge dozens to millions of times, do not have the restriction to the bacterium size basically, scanning electron microscope is from taking the function of measuring size simultaneously, can swiftly measure the size of bacterium, and the recognition rate is high, can distinguish microorganism and small debris, gets rid of small debris when the count, has improved the degree of accuracy of count.)

1. A method for detecting bacterial numbers, comprising the steps of:

step 1: weighing a proper amount of sample, dissolving and diluting the sample, centrifuging the sample again, and removing supernatant;

step 2: fixing the sample solution treated in the step (1), centrifuging again, and removing the supernatant;

and step 3: rinsing the sample solution treated in the step 2, centrifuging again, and removing supernatant;

and 4, step 4: dehydrating the sample solution treated in the step (3), centrifuging again, and removing the supernatant;

and 5: carrying out ultrasonic dispersion treatment on the sample liquid treated in the step (4), and carrying out gold spraying treatment on a proper amount of sample to obtain a sample to be observed;

step 6: and (3) putting a sample to be observed into a sample chamber of a field emission scanning electron microscope, and performing imaging counting to obtain the number of bacteria in a unit sample.

2. The method for detecting the number of bacteria according to claim 1, wherein in the step 1, the sample is subjected to a lysis dilution treatment using a phosphate buffer.

3. The method for detecting the number of bacteria according to claim 2, wherein the phosphate buffer has a pH of 7.4.

4. The method for detecting the number of bacteria according to claim 3, wherein the phosphate buffer is prepared by: 79ml of 0.1mol/L sodium hydroxide solution is added into 1.36g of monopotassium phosphate, and the mixture is diluted to 200ml by water, so that the phosphate buffer solution with the pH value of 7.4 is obtained.

5. The method for detecting the number of bacteria according to claim 1, wherein in the step 2, 2.5% phosphoric acid buffered glutaraldehyde fixing solution is used for fixing the sample, and the specific fixing treatment steps are as follows:

2.1, injecting 2.5% phosphoric acid buffer glutaraldehyde stationary liquid into a sample, fully and uniformly sealing, wherein the cold storage time is 1-2 hours, and the cold storage temperature is 0-5 ℃;

2.2, carrying out ultrasonic dispersion treatment on the refrigerated sample, and then continuously refrigerating for 1-2 hours at the refrigerating temperature of 0-5 ℃.

6. The method for detecting the number of bacteria according to claim 5, wherein the 2.5% phosphate buffered glutaraldehyde fixing solution is prepared from 25% glutaraldehyde, sterile water and a phosphate buffer solution in a volume ratio of 1:4: 5.

7. The method for detecting the number of bacteria according to claim 1, wherein in the step 4, the ethanol with the mass percentage concentration of 30%, 50%, 70%, 80%, 90%, 95% is sequentially adopted for gradient centrifugal dehydration, and the centrifugal dehydration time of each gradient is 8-15 min.

8. The method for detecting the number of bacteria according to claim 1, wherein the specific operation process of the step 5 is as follows: and (4) adding 100% ethanol in percentage by mass into the sample liquid treated in the step (4), performing ultrasonic dispersion for 5-10 min, placing a proper amount of sample on a sample stage of a Scanning Electron Microscope (SEM) adhered with conductive adhesive, performing vacuum-pumping drying treatment, and performing gold spraying treatment.

9. The method for detecting the number of bacteria according to claim 1, wherein in the step 6, the number of bacteria per unit sample is calculated by the formula:

wherein the content of the first and second substances,Uthe number of bacteria per sample, in CFU/g,Xthe total count of bacteria in a scanning electron microscope, in CFU,ais the dilution factor of the sample, in ml,mis the sample size in step 1, in g,lthe sample size in ml is taken in step 5.

Technical Field

The invention belongs to the technical field of microbial detection, and particularly relates to a method for detecting the number of bacteria by using field emission scanning electron microscope imaging.

Background

At present, microorganism counting methods are classified into direct counting methods and indirect counting methods, and the microorganism microscope direct counting method is a method of dropping microorganism homogenized bacteria on a clean blood counting chamber, then placing the blood counting chamber under a microscope, amplifying the microorganisms to a proper multiple, and observing the microorganisms with naked eyes to count the microorganisms.

The direct counting method of the microorganism microscope has the following defects that ① is not suitable for counting the moving bacteria, ② needs relatively high bacteria concentration, ③ small bacteria are difficult to observe under the microscope and are only suitable for thalli or spores with larger thalli, the process of measuring the size of ④ thalli is troublesome, ⑤ tiny impurities can be counted to cause a higher result, ⑥ can not distinguish live bacteria from dead bacteria to cause a higher result of counting the live bacteria, and the defects cause the direct counting method of the microorganism microscope to have certain limitation.

Disclosure of Invention

Aiming at the defects of the direct counting method of the microbial microscope in the prior art, the invention provides a method for detecting the number of bacteria, which aims to solve the defects of the direct counting method of the microbial microscope in the background technology.

The invention solves the technical problems through the following technical scheme: a method for detecting bacterial numbers comprising the steps of:

step 1: weighing a proper amount of sample, dissolving and diluting the sample, centrifuging the sample again, and removing supernatant;

step 2: fixing the sample solution treated in the step (1), centrifuging again, and removing the supernatant;

and step 3: rinsing the sample solution treated in the step 2, centrifuging again, and removing supernatant;

and 4, step 4: dehydrating the sample solution treated in the step (3), centrifuging again, and removing the supernatant;

and 5: carrying out ultrasonic dispersion treatment on the sample liquid treated in the step (4), and carrying out gold spraying treatment on a proper amount of sample to obtain a sample to be observed;

step 6: and (3) putting a sample to be observed into a sample chamber of a field emission scanning electron microscope, and performing imaging counting to obtain the number of bacteria in a unit sample.

According to the method, a series of treatments are carried out on the sample before imaging counting, then the scanning electron microscope is adopted for imaging counting, in the series of treatments, the problem of inaccurate counting caused by bacterial movement in the counting process is avoided through fixing treatment, and meanwhile, the problem that the bacterial structure is easy to deform due to air pressure, electron beam bombardment and the like can also be avoided through fixing treatment; the scanning electron microscope is used for imaging counting, the material performance of the surface material of a sample can be directly used for microscopic imaging, the requirement on the concentration of bacteria is avoided, the amplification is dozens to millions of times, the size of the bacteria is basically not limited, meanwhile, the scanning electron microscope has the function of size measurement, the size of the bacteria can be rapidly measured, the recognition rate is high, microorganisms and tiny impurities can be distinguished, the tiny impurities are removed during counting, and the counting accuracy is improved; in steps 1 to 4, the supernatant is discarded after centrifugation to increase the concentration of bacteria, so that the bacteria fill the sample chamber during observation, and the detection precision of the number of bacteria is improved.

Further, in the step 1, the sample is subjected to a lysis dilution treatment using a phosphate buffer, and the pH of the bacteria is adjusted to maintain the bacteria at a constant pH, so that the bacteria can be observed in an optimal state.

Further, the pH of the phosphate buffer was 7.4.

Further, the preparation method of the phosphate buffer solution comprises the following steps: 79ml of 0.1mol/L sodium hydroxide solution is added into 1.36g of monopotassium phosphate, and the mixture is diluted to 200ml by water, so that the phosphate buffer solution with the pH value of 7.4 is obtained.

Further, in the step 2, 2.5% phosphoric acid buffered glutaraldehyde fixing solution is used for fixing the sample, and the specific fixing treatment steps are as follows:

2.1, injecting 2.5% phosphoric acid buffer glutaraldehyde stationary liquid into a sample, fully and uniformly sealing, wherein the cold storage time is 1-2 hours, and the cold storage temperature is 0-5 ℃;

2.2, carrying out ultrasonic dispersion treatment on the refrigerated sample, and then continuously refrigerating for 1-2 hours at the refrigerating temperature of 0-5 ℃.

Further, the 2.5% phosphoric acid buffered glutaraldehyde stationary liquid is prepared from 25% glutaraldehyde, sterile water and a phosphate buffer solution according to a volume ratio of 1:4: 5.

Further, in the step 4, gradient centrifugal dehydration is sequentially performed by adopting 30%, 50%, 70%, 80%, 90% and 95% ethanol in mass percentage concentration, and the centrifugal dehydration time of each gradient is 8-15 min.

Further, the specific operation process of step 5 is as follows: and (4) adding 100% ethanol in percentage by mass into the sample liquid treated in the step (4), performing ultrasonic dispersion for 5-10 min, placing a proper amount of sample on a sample stage of a Scanning Electron Microscope (SEM) adhered with conductive adhesive, performing vacuum-pumping drying treatment, and performing gold spraying treatment for observation.

Further, in step 6, the calculation formula of the number of bacteria per unit sample is:

wherein the content of the first and second substances,Uthe number of bacteria per sample, in CFU/g,Xthe total count of bacteria in a scanning electron microscope, in CFU,ais the dilution factor of the sample, in ml,mis the sample size in step 1, in g,lthe sample size in ml is taken in step 5.

Advantageous effects

Compared with the prior art, the method for detecting the number of bacteria provided by the invention has the advantages that a series of treatments are firstly carried out on a sample before observation and counting, then the scanning electron microscope is adopted for imaging and counting, in the series of treatments, the problem of inaccurate counting caused by the movement of bacteria in the counting process is avoided by fixing treatment, and meanwhile, the problem of easy deformation of a bacteria structure caused by air pressure, electron beam bombardment and the like can also be avoided by fixing treatment; utilize scanning electron microscope imaging count, can directly utilize the material performance of sample surface material to carry out the microcosmic formation of image, do not have the requirement to bacterium concentration, and can enlarge dozens to millions of times, do not have the restriction to the bacterium size basically, scanning electron microscope is from taking the function of measuring size simultaneously, can swiftly measure the size of bacterium, and the recognition rate is high, can distinguish microorganism and small debris, gets rid of small debris when the count, has improved the degree of accuracy of count.

Detailed Description

The technical solutions in the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Taking commercial probiotics (2 g/per lactobacillus, marked by 200 hundred million units of lactic acid bacteria, namely 100 hundred million CFU/g) as an example, the method for detecting the bacterial number provided by the invention comprises the following steps:

1. 0.01g of probiotic bacteria was weighed, dissolved and diluted to an appropriate solubility (which is determined by the number of bacteria on the sample stage to which the conductive gel was adhered) with a phosphate buffer solution having a pH of 7.4, and then centrifuged.

The specific preparation method of the phosphate buffer solution with the pH value of 7.4 comprises the following steps: 79ml of 0.1mol/L sodium hydroxide solution is added into 1.36g of monopotassium phosphate, and the mixture is diluted to 200ml by water, so that the phosphate buffer solution with the pH value of 7.4 is obtained. The probiotics in the sample can be kept at a constant pH value by adopting phosphate buffer solution with the pH value of 7.4 for dissolving and diluting so as to observe under the optimal state of bacteria. In order to reduce the usage amount of phosphate buffer solution, the specific dissolving and diluting steps are as follows: diluting 0.01g of sample to 10ml by using a phosphate buffer solution, and shaking up to obtain a first diluted sample solution; adding 1ml of the first diluted sample solution into 9ml of phosphate buffer solution, and shaking up to obtain a second diluted sample solution; adding 1ml of the second diluted sample solution into 9ml of phosphate buffer solution, and shaking up to obtain a third diluted sample solution; adding 1ml of the third diluted sample solution into 9ml of phosphate buffer solution, and shaking up to obtain a fourth diluted sample solution; and adding 1ml of fourth diluted sample liquid into 9ml of phosphate buffer solution, shaking up to obtain fifth diluted sample liquid, and centrifuging the fifth diluted sample liquid.

The container used for dissolving and diluting is a sterilized centrifugal tube with 10ml scales, and in the subsequent processing steps (steps 1 to 5), the sample liquid is correspondingly processed in the centrifugal tube, so that the pollution and the error caused by the transfer of the sample liquid in the processing process are avoided.

And (4) centrifuging for 8min at the rotating speed of 1500r/min by using a commercial centrifuge, discarding supernatant after precipitation, and taking a sample solution after precipitation so as to carry out the next operation.

2. And (3) fixing the sample liquid after the centrifugal treatment in the step (1), and then, removing the supernatant after the centrifugal treatment.

Under appropriate environmental conditions, biological samples are typically observed in a scanning electron microscope without fixation. However, biological samples with high water content such as bacteria have a long observation time under a scanning electron microscope due to air pressure, electron beam bombardment, and the like, and the structures thereof are easily deformed. The chemical substances are adopted for fixing, so that the tissue and cell components of the bacteria can generate a cross-linking effect to form a relatively stable bracket, and the bracket is not easy to deform. Therefore, the bacterial sample is considered to be fixed with a chemical substance and then observed in a scanning electron microscope.

The sample is fixed by adopting 2.5 percent phosphoric acid buffer glutaraldehyde fixing solution, and the specific fixing treatment steps are as follows:

2.1, injecting 1ml of 2.5% phosphoric acid buffer glutaraldehyde stationary liquid into a centrifuge tube filled with the sample liquid treated in the step 1 by using a straw, fully shaking up to ensure that thalli are fully contacted with the stationary liquid, covering a cover for sealing, and placing the centrifuge tube into a refrigerator refrigerating chamber, wherein the refrigerating time is 1 hour, and the refrigerating temperature is 3 ℃;

2.2, in order to prevent bacteria from depositing in the static refrigeration process and not completely contacting with 2.5% phosphoric acid buffering glutaraldehyde stationary liquid, taking out the centrifugal tube, putting the centrifugal tube into a small beaker filled with water (the tube opening of the centrifugal tube is upward, the beaker does not fall in an ultrasonic cleaning instrument during the water in the beaker is subjected to ultrasound and the water does not enter the centrifugal tube), putting the beaker with the centrifugal tube into a water tank of a 25kHZ ultrasonic cleaning instrument, performing ultrasound for 10min, and then continuously putting the beaker into a refrigeration chamber for refrigeration, wherein the refrigeration time is 1 hour, and the refrigeration temperature is 3 ℃;

and 2.3, taking out the centrifugal tube, carrying out centrifugal treatment for 10min at the rotating speed of 1500r/min, removing supernatant after thalli are precipitated, and taking a sample solution after precipitation so as to carry out the next operation.

The 2.5% phosphoric acid buffer glutaraldehyde stationary liquid has good preservation effect on bacterial structures, avoids easy deformation of the bacterial structures, and is prepared from 25% glutaraldehyde, sterile water and a phosphate buffer solution in a volume ratio of 1:4: 5. Sterile water is obtained by adding 100mL of water to 200mL or 250mL triangular flask, adding tampon, bandaging, and autoclaving.

3. And (3) rinsing the sample liquid after the centrifugal treatment in the step (2), and then, removing the supernatant after the centrifugal treatment.

The purpose of the rinsing treatment is to avoid that the residual fixing solution attached to the sample reacts with the dehydrating agent used in the step 4 to damage the real appearance of the surface of the sample, and the rinsing method comprises the following steps: and (3) injecting a phosphate buffer solution into the centrifuge tube filled with the sample, covering a cover, repeatedly vibrating for 3-5 min, centrifuging for 8min at 1500r/min, removing supernatant after precipitation, and repeating the step for 3 times to obtain the sample solution processed in the step 3 so as to carry out the next operation.

4. And (4) dehydrating the sample liquid treated in the step (3), centrifuging again, and then removing the supernatant.

And (3) sequentially and respectively taking 10ml of 30%, 50%, 70%, 80%, 90% and 95% ethanol in mass percentage concentration to perform gradient centrifugal dehydration on the sample liquid treated in the step (3), wherein the centrifugal dehydration time of each gradient is 10 min. After gradient centrifugal dehydration, centrifuging at the rotating speed of 1500r/min for 8min, discarding supernatant after precipitation, and taking precipitated sample liquid for further operation. The gradient centrifugal dehydration treatment ensures that the ordered structure of bacteria in the sample is not damaged, thereby being convenient for the subsequent effective observation.

5. Adding 100% ethanol to 10ml into the sample solution treated in the step 4, performing ultrasonic dispersion for 5min in a 25kHZ ultrasonic cleaning instrument, taking 0.05ml (50 microliter, 50 CFU) of sample by using a microsyringe, placing the sample on a sample table of a Scanning Electron Microscope (SEM) adhered with conductive adhesive, placing the sample table into a gold spraying instrument, performing vacuum drying treatment (the drying time is 5 min), and performing gold spraying treatment for observation. The gold spraying treatment ensures that the bacteria in the sample have good conductivity, thereby facilitating the stability of subsequent imaging. The number of bacteria is small, the counting error is large, the number of bacteria is large, and the bacteria are difficult to count, so that the number of the bacteria on the sample table adhered with the conductive adhesive is preferably 30-100 CFU.

6. Putting a sample to be observed into a sample chamber of a field emission scanning electron microscope, selecting 15kV accelerating voltage, carrying out imaging counting after focusing and stigmation elimination, and counting the total amount of bacteria on the whole conductive gelXObtaining the number of bacteria in the unit sample and the number of bacteria in the unit sampleUComprises the following steps:

，Xtotal count of bacteria in a scanning electron microscope in CFU, 10⁵Is the dilution of the sample in ml, 0.01 is the sample size in g for step 1, and 0.05 is the sample size in ml for step 5. The field emission scanning electron microscope is an existing device and is sold in the market.

According to the method for detecting the number of bacteria, a series of treatments are carried out on a sample before imaging counting, then the scanning electron microscope is adopted for imaging counting, in the series of treatments, the problem of inaccurate counting caused by bacterial movement in the counting process is avoided through fixing treatment, and meanwhile, the problem that the bacterial structure is easy to deform due to air pressure, electron beam bombardment and the like can also be avoided through fixing treatment; the scanning electron microscope is used for imaging counting, the material performance of the surface material of a sample can be directly used for microscopic imaging, the requirement on the concentration of bacteria is avoided, the amplification is dozens to millions of times, the size of the bacteria is basically not limited, meanwhile, the scanning electron microscope has the function of size measurement, the size of the bacteria can be rapidly measured, the recognition rate is high, microorganisms and tiny impurities can be distinguished, the tiny impurities are removed during counting, and the counting accuracy is improved; because the concentration of the sample liquid is reduced by dilution, fixation, rinsing and dehydration treatment, the number of bacteria in a unit sample is reduced, therefore, the supernatant is discarded after the centrifugation treatment is carried out in the steps 1 to 4 to improve the number of bacteria in the unit sample, so that the bacteria fill the sample chamber during observation, and the detection precision of the number of the bacteria is improved.

The above disclosure is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of changes or modifications within the technical scope of the present invention, and shall be covered by the scope of the present invention.