WO2017107333A1 - 一种细菌计数方法 - Google Patents

一种细菌计数方法 Download PDF

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WO2017107333A1
WO2017107333A1 PCT/CN2016/077794 CN2016077794W WO2017107333A1 WO 2017107333 A1 WO2017107333 A1 WO 2017107333A1 CN 2016077794 W CN2016077794 W CN 2016077794W WO 2017107333 A1 WO2017107333 A1 WO 2017107333A1
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bacterial
bacteria
polyaniline
composite film
glassy carbon
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French (fr)
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邹小波
李志华
石吉勇
黄晓玮
周煦成
胡雪桃
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Jiangsu University
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Jiangsu University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0656—Investigating concentration of particle suspensions using electric, e.g. electrostatic methods or magnetic methods
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1024—Counting particles by non-optical means

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  • the invention belongs to the field of material science and microbiology, and in particular relates to a method for counting bacteria based on a polyaniline/bacteria composite film.
  • the detection of bacterial cell concentration is of great significance in the fields of microbial fermentation, environmental monitoring, food quality testing and so on.
  • the commonly used bacterial counting method is a culture method, including a plate counting method, an MPN method, and the like.
  • the plate counting method is a national standard test method for detecting the total number of colonies (see GB/T 4789.2-2010 Food Hygiene Microbiology Test Colony Total Determination [S]), which is a standard method for judging the accuracy of other counting methods.
  • S Food Hygiene Microbiology Test Colony Total Determination
  • MPN is a dilution culture method: it is suitable for the detection of foods with a large number of competing bacteria and their raw materials and untreated foods containing a small amount of Staphylococcus aureus.
  • the MPN method is a common indirect counting method, but it has certain limitations.
  • the fluorescence microscope counting method and the flow cytometry counting method are relatively reliable and widely recognized, and the fluorescence microscope (Fluorescence microscope) counting method: the fluorescence microscope is an ultraviolet light source for illuminating the object to be fluoresced. Then, observe the shape, location and number of objects under the microscope.
  • Flow cytometry is the counting of particles passing through a laser beam by a laser that refracts and reflects light as it passes through the laser beam. This refracted and reflected signal is recorded by the detector. Each time a cell passes, a peak is generated and the number of peaks is recorded.
  • the purpose of the present invention is to solve the deficiencies in the prior art, and to provide an operation that is simple, time-consuming, and cost-effective.
  • Low bacterial counting method enables rapid and accurate detection of bacterial cell concentration.
  • the technical solution of the present invention is: a method for counting bacteria, comprising the following steps:
  • step S1 specifically includes the following steps:
  • the cyclic voltammetry curve is measured until the oxidation-reduction peak potential difference falls within 80 mV, and the glassy carbon electrode is dried; the standard suspension suspension is taken to the glassy carbon electrode. After the surface, the bacteria are fixed on the surface of the glassy carbon electrode by drying;
  • the above-mentioned bacteria-immobilized glassy carbon electrode was placed in a sulfuric acid solution containing aniline and scanned by cyclic voltammetry.
  • the aniline was polymerized on the surface of the glassy carbon electrode to obtain a polyaniline/bacterial composite film.
  • the sulfuric acid solution is 0.5M
  • the aniline is 0.1M
  • the cyclic voltammetry scan is 1 to 20 cycles
  • the scanning rate is 5 to 100 mV/s
  • the voltage lower limit is -0.6 to 0 V
  • the upper limit of voltage is 0.75 to 1.2. V.
  • the number of scanning turns is 10 turns, the scanning rate is 50 mV/s, and the scanning voltage ranges from -0.2 to 0.9V.
  • step S2 specifically includes the following steps:
  • step S1 The standard bacterial suspension obtained in step S1 is sequentially diluted by gradient to obtain different concentrations of the bacterial suspension;
  • step S3 specifically includes the following steps:
  • step S1 1) preparing a polyaniline/bacterial composite film on the surface of the glassy carbon electrode by using the suspension of the test sample according to step S1, and measuring the cyclic voltammetry curve according to step S2;
  • the present invention utilizes electropolymerization to prepare a polyaniline/bacterial composite film on the surface of a glassy carbon electrode, and the bacteria fixed on the surface of the glassy carbon electrode have an inhibitory effect on the polymerization of the aniline on the surface of the electrode, thereby preparing the polyaniline/bacterial composite film. It exhibits different electrochemical properties and enables rapid and accurate detection of bacterial cell concentration.
  • the aniline is the main reagent of the invention, and the consumption of the liquid to be tested is small, so the detection cost is low and the equipment is simple.
  • the method of the invention has the advantages of good repeatability and wide detection linear range for the same bacterial cell concentration measurement.
  • Figure 1 is a schematic view showing the overall preparation process of a polyaniline/bacterial composite film.
  • Fig. 2(a) is a graph showing the change of the cyclic volt-ampere curve of the electrode during the preparation of the polyaniline-bacterial composite film by cyclic voltammetry.
  • Fig. 2(b) is a comparison diagram of the morphology of the prepared polyaniline/bacterial composite film and the pure polyaniline film.
  • Fig. 3(a) is a cyclic voltammetry graph of a polyaniline/bacterial composite membrane modified glassy carbon electrode prepared by using different concentrations of Bacillus subtilis suspension in a 0.1 M H 2 SO 4 solution.
  • Fig. 3(b) is a standard curve of the peak current of the cyclic voltammetry curve of the polyaniline/Bacillus subtilis film-modified electrode at 0.2V.
  • Fig. 4 is a comparison diagram of the results of detection of different concentrations of Bacillus subtilis suspension samples by plate colony counting method and polyaniline/bacterial film counting method according to the present invention.
  • Figure 5 is a standard curve of the peak current of the cyclic voltammetry curve of the polyaniline/E. coli film modified electrode at 0.2V.
  • Fig. 6 is a standard curve of the peak current of the cyclic voltammetry curve of the polyaniline/S. thermophilus film-modified electrode at 0.2V.
  • aniline, sulfuric acid, yeast extract and trypsin used in the present invention are all purchased from Sinopharm Chemical Co., Ltd. Company, aniline is used after distillation under reduced pressure.
  • the electrochemical measurement was performed using CHI660D electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.). This example is described by taking Bacillus subtilis as an example, and the strain is purchased from the China Industrial Microbial Culture Collection.
  • the bacterial counting method comprises the following steps:
  • Figure 1 shows the overall preparation process of a polyaniline/bacterial composite film. The specific steps are as follows:
  • the glassy carbon electrode was sequentially polished with metallographic sandpaper, 0.3 ⁇ m and 0.05 ⁇ m Al 2 O 3 powder, and then ultrasonically washed with ethanol and water, respectively, and the glassy carbon electrode was measured in a 1 mM K 3 [Fe(CN) 6 ] solution. Cyclic volt-ampere (CV) curve until the oxidation-reduction peak potential difference falls within 80mV, and air-dry to ensure the initial state of the electrode is consistent; take 10 ⁇ L of standard Bacillus subtilis suspension to the surface of the glassy carbon electrode, and pass the oven at 50 °C for 15min. Drying can fix bacteria on the surface of the glassy carbon electrode to obtain a bacteria/glassy carbon electrode;
  • the above-mentioned bacteria-immobilized glassy carbon electrode was placed in a sulfuric acid (0.5 M) solution containing 0.1 M aniline by a cyclic voltammetry using a platinum wire electrode as a counter electrode and a silver/silver chloride electrode as a reference electrode.
  • scan 10 turns scan rate is 50 mV/s, voltage lower limit is -0.2 V, and upper voltage limit is 0.9 V to obtain a polyaniline/Bacillus subtilis composite film.
  • Fig. 2(a) shows the cyclic volt-ampere curve of the electrode during the preparation of polyaniline/bacterial composite film by cyclic voltammetry.
  • Fig. 2(b) is a comparison of the morphology of the polyaniline/Bacillus subtilis composite film and the pure polyaniline film prepared at a bacterial concentration of 5.33 ⁇ 10 5 CFU ⁇ mL -1 , and the two films are obviously different in morphology.
  • the immobilization of the bacteria has a great influence on the deposition of polyaniline on the surface of the electrode, so that it exhibits different electrochemical characteristics, which provides a basis for quantitative detection of the concentration of the cells.
  • the standard bacterial suspension having the concentration of 5.33 ⁇ 10 8 CFU ⁇ mL -1 obtained in the step S1 was sequentially diluted to 1.066 ⁇ 10 8 CFU ⁇ mL -1 , 5.33 ⁇ 10 7 CFU ⁇ mL -1 , 5.33 ⁇ 10 6 CFU ⁇ mL. -1 , 1.066 ⁇ 10 6 CFU ⁇ mL -1 , 5.33 ⁇ 10 5 CFU ⁇ mL -1 , 5.33 ⁇ 10 4 CFU ⁇ mL -1 .
  • the polyaniline/Bacillus subtilis composite film was prepared on the surface of the glassy carbon electrode by using the obtained different concentrations of the bacterial suspension according to the step S1, and the cyclic voltammetry curve was measured in a 0.1 M H 2 SO 4 solution after rinsing with distilled water.
  • the scan range voltage is –0.2 to 0.9V and the scan rate is 50mV/s.
  • the polyaniline/Bacillus subtilis composite membrane was prepared according to the step S1 using the suspension of the test bacteria, and its cyclic voltammetry curve in 0.1 M H 2 SO 4 solution was determined according to step S2, according to its peak at 0.2 V.
  • the current was calculated from the standard curve of the polyaniline/Bacillus subtilis composite film modified electrode obtained in the step S2.
  • Table 1 and FIG. 4 are comparisons between the results of the polyaniline/bacterial composite film counting method and the plate colony technique, and the results show that the mean concentration of the bacterial suspension measured by the method of the present invention is determined by the plate colony counting method.
  • the results are basically the same, but the relative standard deviation is significantly lower than the plate colony counting method, indicating that it has better stability.
  • the polyaniline/bacteria composite film counting method of the present invention is superior to the traditional plate colony counting method.
  • This embodiment is the same as the method used in the embodiment 1, except that the present embodiment is described by taking Escherichia coli as an example, and the strain is purchased from the China Industrial Microbial Culture Collection.
  • a standard E. coli suspension having a cell concentration of 9.26 ⁇ 10 8 CFU ⁇ mL -1 was obtained by the same method as that described in the step S1 of Example 1, and sequentially diluted to 9.26 ⁇ 10 7 CFU ⁇ mL -1 , 9.26 ⁇ 10 6 CFU ⁇ mL -1 , 9.26 ⁇ 10 5 CFU ⁇ mL -1 , 9.26 ⁇ 10 4 CFU ⁇ mL -1 , 9.26 ⁇ 10 3 CFU ⁇ mL -1 .
  • the sample of the Escherichia coli sample to be tested is determined according to the step S3 in the embodiment 1.
  • Table 2 is a comparison of the results of the E. coli sample determination based on the polyaniline/bacterial composite film counting method and the plate colony technique, and the results show that the present invention
  • the average concentration of the bacterial suspension measured by the method is basically the same as that of the plate colony counting method, but the relative standard deviation is significantly lower than the plate colony counting method, indicating that it has better stability, and the polyaniline of the present invention.
  • Bacterial composite film counting method is also applicable to Escherichia coli samples.
  • This example is the same as the method used in Examples 1 and 2, except that this example is described by using Streptococcus thermophilus as an example, and the strain is purchased from the China Industrial Microbial Culture Collection.
  • the standard suspension was sequentially diluted to 6.72 ⁇ 10 8 CFU ⁇ mL -1 , 6.72 ⁇ 10 7 CFU ⁇ mL -1 , 6.72 ⁇ 10 6 CFU ⁇ mL -1 , 6.72 ⁇ 10 5 CFU ⁇ mL -1 , 6.72 ⁇ 10 4 CFU ⁇ mL -1 .
  • the sample of Streptococcus thermophilus was measured as described in S1 of Example 1, and Table 3 is a comparison of the results of the S. thermophilus sample based on the polyaniline/bacterial composite film counting method and the plate colony technique.
  • the average concentration of the bacterial suspension measured by the method of the present invention is basically the same as that of the plate colony counting method, but the relative standard deviation is significantly lower than the plate colony counting method, indicating that it has better stability, and the present invention
  • the polyaniline/bacterial composite film counting method is also applicable to the S. thermophilus sample.

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Abstract

一种细菌计数方法，包括以下步骤，S1：利用电聚合法在玻碳电极表面制备聚苯胺/细菌复合薄膜；S2：绘制所述聚苯胺/细菌复合薄膜修饰电极标准曲线；S3：根据所述步骤S2所得标准曲线测定待测菌液样本的细菌浓度。本方法实施过程中无需对细菌进行培养，因此耗时短、操作简便；本方法以苯胺为主要试剂，待测液消耗量少，因此检测费用低、设备简单；本方法具有重复性好、检测线性范围宽的优点；是一种操作简便、耗时短、成本低的细菌计数方法，本方法是基于聚苯胺/细菌复合薄膜的细菌计数方法，能实现细菌菌体浓度的快速准确检测。

Description

一种细菌计数方法 技术领域
本发明属于材料科学、微生物学领域，尤其涉及一种基于聚苯胺/细菌复合薄膜的细菌计数方法。
背景技术
细菌菌体浓度的检测在微生物发酵、环境监测、食品质量检测等领域都具有重要意义。目前，常用的细菌计数方法为培养法，包括平板计数法、MPN法等。
平板计数法是检测菌落总数的国家标准检验法(参考文献见：GB/T 4789.2-2010食品卫生微生物学检验菌落总数测定[S])，是判断其他计数方法准确性的标准方法，该方法对于平皿中的单菌落计数较准确，但由于培养皿中菌落疏密不均匀地排列，依靠人工观察粘连菌落并计数具有一定的主观性且计数不准确，整体来说该方法操作繁琐、耗时长、误差大、效率低。
MPN是稀释培养测数法：适用于检测带有大量竞争菌的食品及其原料和未经处理的含少量金黄色葡萄球菌的食品。MPN法是一种常见的间接计数法，但其存在一定的局限性。
这些培养法存在耗时较长，无菌操作要求严格，手续繁琐，劳动强度大，受培养条件影响较大等不足。
为克服培养法的不足，多种计数方法被广泛采用。其中相对可靠并被广泛认可的有荧光显微镜计数法和流式细胞仪计数法，其中荧光显微镜(Fluorescence microscope)计数法：荧光显微镜是以紫外线为光源，用以照射被检物体，使之发出荧光，然后在显微镜下观察物体的形状、所在位置及其数量。流式细胞仪计数法是通过激光对通过激光束的颗粒进行计数，当颗粒或者细胞通过激光束的时候，会对光线产生折射和反射。这个折射和反射的信号被探测器记录下来。每通过一个细胞，就会产生一个峰值，最后记录峰值的个数。
虽然这两种方法具有快速、准确的特点，但存在设备昂贵，使用和维护成本高等不足。因此，寻求一种操作简便、快速，成本低的细菌计数方法具有重要价值。
发明内容
本发明的目的是为了解决现有技术中的不足，提出了一种操作简便、耗时短、成本 低的细菌计数方法，能实现细菌菌体浓度的快速准确检测。
本发明的技术方案是：一种细菌计数方法，包括以下步骤:
S1：利用电聚合法在玻碳电极表面制备聚苯胺/细菌复合薄膜；
S2：绘制所述聚苯胺/细菌复合薄膜修饰电极标准曲线；
S3：根据所述步骤S2所得标准曲线测定待测菌液样本的细菌浓度。
上述方案中，所述步骤S1具体包括以下步骤:
1)标准菌悬液准备：
配制细菌培养液，高压灭菌后接种适量菌种进行培养，将培养后所获得菌液离心清洗，便得到标准菌悬液；
2)细菌在玻碳电极表面的固定：
所述玻碳电极进行预处理后，测定其循环伏安曲线，直至氧化-还原峰电位差降至80mV以内，将所述玻碳电极晾干；取所述标准菌悬液滴至玻碳电极表面后，经烘干即可将细菌固定在所述玻碳电极表面；
3)苯胺在电极表面的聚合：
将上述固定有细菌的玻碳电极置于含有苯胺的硫酸溶液中采用循环伏安法扫描，苯胺在玻碳电极表面聚合得到聚苯胺/细菌复合薄膜。
上述方案中，所述硫酸溶液为0.5M，苯胺为0.1M，循环伏安法扫描1～20圈，扫描速率为5～100mV/s，电压下限为–0.6～0V，电压上限为0.75～1.2V。
优选的，所述扫描圈数为10圈，所述扫描速率为50mV/s，所述扫描的电压范围为-0.2～0.9V。
上述方案中，所述步骤S2具体包括以下步骤：
1)将步骤S1所得的标准菌悬液依次按梯度稀释获得不同浓度的菌悬液；
2)利用所获得的不同浓度的菌悬液分别按照步骤S1所述在玻碳电极表面制备聚苯胺/细菌复合薄膜，蒸馏水漂洗后于0.1M H2SO4溶液中测定其循环伏安曲线，扫描速率为50mV/s，扫描的电压范围为–0.2～0.9V；
3)以细菌浓度对数为横坐标，以2)中所得循环伏安曲线最高峰所对应的峰电流为纵坐标，绘制所述聚苯胺/细菌复合薄膜修饰电极标准曲线。
上术方案中，所述步骤S3具体包括以下步骤：
1)利用待测菌悬液按照步骤S1所述在玻碳电极表面制备聚苯胺/细菌复合薄膜，按照步骤S2所述测定其循环伏安曲线；
2)确定循环伏安曲线的峰电流，根据步骤S2中所得聚苯胺/细菌复合薄膜修饰电极标准曲线计算其细菌浓度。
现有技术相比本发明的有益效果：
1、本发明利用电聚合法在玻碳电极表面制备聚苯胺/细菌复合薄膜，由于固定于玻碳电极表面的细菌对苯胺在电极表面聚合具有阻碍作用，从而使得所制备聚苯胺/细菌复合薄膜表现出不同的电化学特性，能实现细菌菌体浓度的快速准确检测。
2、本发明实施过程中无需像传统平板计数法等对细菌进行培养后再统计，因此耗时短、操作简便。
3、本发明以苯胺为主要试剂，待测液消耗量少，因此检测费用低、设备简单。
4、本发明所述方法对同一细菌菌体浓度测量具有重复性好、检测线性范围宽的优点。
附图说明
图1为聚苯胺/细菌复合薄膜的整体制备过程示意图。
图2(a)为利用循环伏安法制备聚苯胺-细菌复合薄膜过程中电极循环伏安曲线变化情况图。
图2(b)为所制备聚苯胺/细菌复合薄膜与纯聚苯胺膜的形貌对比图。
图3(a)为采用不同浓度枯草芽孢杆菌菌悬液所制备聚苯胺/细菌复合膜修饰玻碳电极在0.1M H2SO4溶液中的循环伏安曲线图。
图3(b)为聚苯胺/枯草芽孢杆菌薄膜修饰电极的循环伏安曲线在0.2V处峰电流的标准曲线图。
图4为不同浓度枯草芽孢杆菌菌悬液样本采用平板菌落计数法和本发明所述聚苯胺/细菌薄膜计数法检测结果对比图。
图5为聚苯胺/大肠杆菌薄膜修饰电极的循环伏安曲线在0.2V处峰电流的标准曲线图。
图6为聚苯胺/嗜热链球菌薄膜修饰电极的循环伏安曲线在0.2V处峰电流的标准曲线图。
具体实施方式
下面分别以枯草芽孢杆菌、埃希氏大肠杆菌和嗜热链球菌为具体实施方式并结合附图对本发明作进一步详细说明，但本发明的保护范围并不限于此。
实施例1
本发明使用的苯胺、硫酸、酵母提取物、胰化蛋白胨均购自国药集团化学试剂有限 公司，苯胺经过减压蒸馏后使用。电化学测定采用CHI660D电化学工作站(上海辰华仪器有限公司)。本实施例以枯草芽孢杆菌为例进行说明，菌种购自中国工业微生物菌种保藏中心。
所述细菌计数方法包括以下步骤：
S1、利用电聚合法在玻碳电极表面制备聚苯胺/细菌复合薄膜；
图1显示了聚苯胺/细菌复合薄膜的整体制备过程，具体步骤如下：
(1)标准菌悬液准备
取酵母提取物5g，胰化蛋白胨10g，氯化钠10g，加水配制成1L培养液，利用NaOH调节pH至7.0，121℃，20min，高压灭菌后接种适量枯草芽孢杆菌菌种，37℃恒温培养箱培养20h。将所获得菌液在3500ɡ离心力下4℃离心10min清洗3次，得到标准枯草芽孢杆菌悬浮液，采用平板菌落计数法测定其菌体浓度为5.33×108CFU·mL-1；
(2)细菌在玻碳电极表面的固定
玻碳电极依次用金相砂纸、0.3μm和0.05μm的Al2O3粉末抛光，再分别用乙醇、水超声清洗后，在1mM的K3[Fe(CN)6]溶液中测定玻碳电极的循环伏安(CV)曲线，直至氧化-还原峰电位差降至80mV以内，晾干，确保电极初始状态一致；取10μL标准枯草芽孢杆菌悬液滴至玻碳电极表面，经50℃烘箱15min烘干即可将细菌固定在所述玻碳电极表面，得到细菌/玻碳电极；
(3)苯胺在电极表面的聚合
将上述固定有细菌的玻碳电极置于含有0.1M苯胺的硫酸(0.5M)溶液中采用循环伏安法以铂丝电极为对电极，银/氯化银电极为参比电极扫描。为了获取较高的检测灵敏度，优选地，扫描10圈，扫描速率为50mV/s，电压下限为–0.2V，电压上限为0.9V，得到聚苯胺/枯草芽孢杆菌复合薄膜。图2(a)为利用循环伏安法制备聚苯胺/细菌复合薄膜过程中电极循环伏安曲线变化情况，其中出现了3对典型的氧化还原峰：Ιa/Ic，IIa/IIc，Шa/Шc，说明了苯胺在电极表面的成功聚合；此外，扫描电流随着扫描圈数的增加逐渐增大，这印证了聚苯胺聚合时的自催化作用。图2(b)是细菌浓度为5.33×105CFU·mL-1时所制备聚苯胺/枯草芽孢杆菌复合薄膜与纯聚苯胺膜的形貌对比，两种薄膜在形貌上明显不同，说明菌体的固定对聚苯胺在电极表面的沉积具有较大影响，从而使其表现出不同的电化学特性，为菌体浓度的定量检测提供基础。
S2.绘制所述聚苯胺/细菌复合薄膜修饰电极标准曲线；
将步骤S1所得浓度为5.33×108CFU·mL-1的标准菌悬液依次稀释为1.066×108 CFU·mL-1，5.33×107CFU·mL-1，5.33×106CFU·mL-1，1.066×106CFU·mL-1，5.33×105CFU·mL-1，5.33×104CFU·mL-1。利用所获得的不同浓度的菌悬液按照步骤S1所述在玻碳电极表面制备聚苯胺/枯草芽孢杆菌复合薄膜，蒸馏水漂洗后于0.1M H2SO4溶液中测定其循环伏安曲线。扫描范围电压为–0.2～0.9V，扫描速率为50mV/s。
以细菌浓度对数为横坐标X，以所得循环伏安曲线在0.2V处的峰电流为纵坐标Y，绘制聚苯胺/枯草芽孢杆菌复合薄膜修饰电极标准曲线。图3(a)为不同浓度菌悬液所对应的循环伏安曲线，图3(b)其峰电流标准曲线，由图3可知电极在0.2V处的峰电流与细菌浓度对数呈现良好的线性关系：Y＝-30.413X+272.560,R2＝0.982。
S3.测定待测菌液样本的细菌浓度。
利用待测菌悬液按照步骤S1所述制备聚苯胺/枯草芽孢杆菌复合膜，按照步骤S2所述测定其在0.1M H2SO4溶液中的循环伏安曲线，根据其在0.2V处的峰电流由步骤S2中所得聚苯胺/枯草芽孢杆菌复合薄膜修饰电极标准曲线计算其菌体浓度。表1和图4为基于所述聚苯胺/细菌复合薄膜计数法和平板菌落技术法的测定结果对比，结果表明，本发明所述方法5次测定的菌悬液浓度均值与平板菌落计数法测定结果基本一致，但其相对标准偏差明显低于平板菌落计数法，说明其具有更好的稳定性，本发明所述聚苯胺/细菌复合薄膜计数法计数结果优于传统的平板菌落计数法。
表1基于聚苯胺/细菌复合薄膜计数法和平板菌落技术法的测定枯草芽孢杆菌样本结果对
Figure PCTCN2016077794-appb-000001
比
实施例2
本实施例与实施例1采用的方法相同，不同之处在于本实施例以埃希氏大肠杆菌为例进行说明，菌种购自中国工业微生物菌种保藏中心。
采用与实施例1中步骤S1所述相同的方法获得菌体浓度为9.26×108CFU·mL-1的标准大肠杆菌悬液，依次稀释为9.26×107CFU·mL-1，9.26×106CFU·mL-1，9.26×105CFU·mL-1， 9.26×104CFU·mL-1,9.26×103CFU·mL-1。
按照实施例1中步骤S2所述方法以大肠杆菌浓度对数为横坐标X，以所得循环伏安曲线在0.2V处的峰电流为纵坐标Y，绘制聚苯胺/大肠杆菌复合薄膜修饰电极标准曲线。由图5可知电极在0.2V处的峰电流与大肠杆菌浓度对数呈现良好的线性关系：Y＝-24.249X+217.33,R2＝0.996。
按照实施例1中步骤S3所述测定待测大肠杆菌菌液样本，表2为基于所述聚苯胺/细菌复合薄膜计数法和平板菌落技术法的测定大肠杆菌样本结果对比，结果表明，本发明所述方法5次测定的菌悬液浓度均值与平板菌落计数法测定结果基本一致，但其相对标准偏差明显低于平板菌落计数法，说明其具有更好的稳定性，本发明所述聚苯胺/细菌复合薄膜计数法同样适用于埃希氏大肠杆菌样本。
表2基于所述聚苯胺/细菌复合薄膜计数法和平板菌落技术法的测定大肠杆菌样本结果对比
Figure PCTCN2016077794-appb-000002
实施例3
本实施例与实施例1和2采用的方法相同，不同之处在于本实施例以嗜热链球菌为例进行说明，菌种购自中国工业微生物菌种保藏中心。
称取MRS肉汤培养基(购自青岛海博生物技术有限公司)52.4g，加热溶解于1L蒸馏水中，118℃高压灭菌15min，冷却后接种适量嗜热链球菌菌种，37℃恒温培养箱培养20h。将所获得菌液在3500ɡ离心力下4℃离心10min清洗3次，得到标准嗜热链球菌悬浮液，采用平板菌落计数法测定其菌体浓度为6.72×109CFU·mL-1。将该标准悬浮液依次稀释为6.72×108CFU·mL-1，6.72×107CFU·mL-1，6.72×106CFU·mL-1，6.72×105CFU·mL-1，6.72×104CFU·mL-1。按照实施例1中步骤S2所述方法以细菌浓度对数为横坐标X，以所得循环伏安曲线在0.2V处的峰电流为纵坐标Y，绘制聚苯胺/嗜热链球菌复合 薄膜修饰电极标准曲线。由图6可知电极在0.2V处的峰电流与细菌浓度对数呈现良好的线性关系：Y＝-28.601X+278.430，R2＝0.988。
按照实施例1中S3所述测定待测嗜热链球菌菌液样本，表3为基于所述聚苯胺/细菌复合薄膜计数法和平板菌落技术法的测定嗜热链球菌样本结果对比，结果表明，本发明所述方法5次测定的菌悬液浓度均值与平板菌落计数法测定结果基本一致，但其相对标准偏差明显低于平板菌落计数法，说明其具有更好的稳定性，本发明所述聚苯胺/细菌复合薄膜计数法同样适用于嗜热链球菌样本。
表3基于所述聚苯胺/细菌复合薄膜计数法和平板菌落技术法的测定嗜热链球菌样本结果对比
Figure PCTCN2016077794-appb-000003
所述实施例为本发明的优选的实施方式，但本发明并不限于上述实施方式，在不背离本发明的实质内容的情况下，本领域技术人员能够做出的任何显而易见的改进、替换或变型均属于本发明的保护范围。

Claims (6)

  1. 一种细菌计数方法，其特征在于，包括以下步骤:
    S1：利用电聚合法在玻碳电极表面制备聚苯胺/细菌复合薄膜；
    S2：绘制所述聚苯胺/细菌复合薄膜修饰电极标准曲线；
    S3：根据所述步骤S2所得标准曲线测定待测菌液样本的细菌浓度。
  2. 根据权利要求1所述的细菌计数方法，其特征在于，所述步骤S1具体包括以下步骤:
    1)标准菌悬液准备：
    配制细菌培养液，高压灭菌后接种适量菌种进行培养，将培养后所获得菌液离心清洗，便得到标准菌悬液；
    2)细菌在玻碳电极表面的固定：
    所述玻碳电极进行预处理后，测定其循环伏安曲线，直至氧化-还原峰电位差降至80mV以内，将所述玻碳电极晾干；取所述标准菌悬液滴至玻碳电极表面后，经烘干即可将细菌固定在所述玻碳电极表面；
    3)苯胺在电极表面的聚合：
    将上述固定有细菌的玻碳电极置于含有苯胺的硫酸溶液中采用循环伏安法扫描，苯胺在玻碳电极表面聚合得到聚苯胺/细菌复合薄膜。
  3. 根据权利要求2所述的细菌计数方法，其特征在于，所述硫酸溶液为0.5M，苯胺为0.1M，循环伏安法扫描1～20圈，扫描速率为5～100mV/s，电压下限为–0.6～0V，电压上限为0.75～1.2V。
  4. 根据权利要求3所述的细菌计数方法，其特征在于，所述扫描圈数为10圈，所述扫描速率为50mV/s，所述扫描的电压范围为-0.2～0.9V。
  5. 根据权利要求1所述的细菌计数方法，其特征在于，所述步骤S2具体包括以下步骤：
    1)将步骤S1所得的标准菌悬液依次按梯度稀释获得不同浓度的菌悬液；
    2)利用所获得的不同浓度的菌悬液分别按照步骤S1所述在玻碳电极表面制备聚苯胺/细菌复合薄膜，蒸馏水漂洗后于0.1M H2SO4溶液中测定其循环伏安曲线，扫描速率为50mV/s，扫描的电压范围为–0.2～0.9V；
    3)以细菌浓度对数为横坐标，以2)中所得循环伏安曲线最高峰所对应的峰电流为纵坐标，绘制所述聚苯胺/细菌复合薄膜修饰电极标准曲线。
  6. 根据权利要求1所述的细菌计数方法，其特征在于，所述步骤S3具体包括以下步骤：
    1)利用待测菌悬液按照步骤S1所述在玻碳电极表面制备聚苯胺/细菌复合薄膜，按照步骤S2所述测定其循环伏安曲线；
    2)确定循环伏安曲线的峰电流，根据步骤S2中所得聚苯胺/细菌复合薄膜修饰电极标准曲线计算其细菌浓度。
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US10883135B2 (en) 2015-08-25 2021-01-05 Avails Medical, Inc. Devices, systems and methods for detecting viable infectious agents in a fluid sample
US11021732B2 (en) 2016-05-31 2021-06-01 Avails Medical, Inc. Devices, systems and methods to detect viable infectious agents in a fluid sample and susceptibility of infectious agents to anti-infectives
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4528270A (en) * 1982-11-02 1985-07-09 Kabushiki Kaisya Advance Kaihatsu Kenkyujo Electrochemical method for detection and classification of microbial cell
US20040175780A1 (en) * 2002-08-06 2004-09-09 Yanbin Li Rapid and automated electrochemical method for detection of viable microbial pathogens
CN101003830A (zh) * 2006-12-31 2007-07-25 华东师范大学 快速计数水体中的大肠杆菌的方法
CN101216449A (zh) * 2008-01-10 2008-07-09 沈阳师范大学 识别死-活细胞的聚苯胺修饰电极的制备方法
CN104198558A (zh) * 2014-09-05 2014-12-10 天津工业大学 一种新型大肠杆菌电化学传感器的制备方法
CN104407031A (zh) * 2014-11-05 2015-03-11 宁波大学 PBP-1A亲和型β-内酰胺类抗生素电化学生物传感器及其制备方法和应用
CN105067694A (zh) * 2015-08-03 2015-11-18 河北科技大学 用于快速检测阪崎肠杆菌的纳米免疫传感器的制备方法及其检测方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005160334A (ja) * 2003-12-01 2005-06-23 Daikin Ind Ltd 細菌数測定方法
US8005526B2 (en) * 2005-08-31 2011-08-23 The Regents Of The University Of Michigan Biologically integrated electrode devices
CN102375009B (zh) * 2011-09-14 2013-09-18 东南大学 应用电化学方法快速检测细菌的方法
CN102392069B (zh) * 2011-10-25 2013-07-10 常熟理工学院 基于功能化纳米金电极的快速检测菌落总数的方法
CN103645237A (zh) * 2013-12-16 2014-03-19 江苏大学 检测雾滴中多菌灵含量的电化学传感器及制备和使用方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4528270A (en) * 1982-11-02 1985-07-09 Kabushiki Kaisya Advance Kaihatsu Kenkyujo Electrochemical method for detection and classification of microbial cell
US20040175780A1 (en) * 2002-08-06 2004-09-09 Yanbin Li Rapid and automated electrochemical method for detection of viable microbial pathogens
CN101003830A (zh) * 2006-12-31 2007-07-25 华东师范大学 快速计数水体中的大肠杆菌的方法
CN101216449A (zh) * 2008-01-10 2008-07-09 沈阳师范大学 识别死-活细胞的聚苯胺修饰电极的制备方法
CN104198558A (zh) * 2014-09-05 2014-12-10 天津工业大学 一种新型大肠杆菌电化学传感器的制备方法
CN104407031A (zh) * 2014-11-05 2015-03-11 宁波大学 PBP-1A亲和型β-内酰胺类抗生素电化学生物传感器及其制备方法和应用
CN105067694A (zh) * 2015-08-03 2015-11-18 河北科技大学 用于快速检测阪崎肠杆菌的纳米免疫传感器的制备方法及其检测方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GAO HONGYAN ET AL.: "On bacteria biosensor based on E.coli modified carbon paste electrode by cyclic voltammetry", JOURNAL OF SHENYANG NORMAL UNIVERSITY (NATURAL SCIENCE EDITION), vol. 29, no. 2, pages 305 - 308 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10883135B2 (en) 2015-08-25 2021-01-05 Avails Medical, Inc. Devices, systems and methods for detecting viable infectious agents in a fluid sample
US11021732B2 (en) 2016-05-31 2021-06-01 Avails Medical, Inc. Devices, systems and methods to detect viable infectious agents in a fluid sample and susceptibility of infectious agents to anti-infectives
US11913058B2 (en) 2016-05-31 2024-02-27 Avails Medical, Inc. Devices, systems and methods to detect viable infectious agents in a fluid sample and susceptibility of infectious agents to anti-infectives
US12275982B2 (en) 2016-05-31 2025-04-15 Avails Medical, Inc. Devices, systems and methods to detect viable infectious agents in a fluid sample and susceptibility of infectious agents to anti-infectives
US12352714B2 (en) 2016-12-13 2025-07-08 Avails Medical, Inc. Devices, systems and methods to detect the presence of ß-lactam antibiotic hydrolyzing bacteria in a sample
US11385200B2 (en) 2017-06-27 2022-07-12 Avails Medical, Inc. Apparatus, systems, and methods for determining susceptibility of microorganisms to anti-infectives
US12276634B2 (en) 2017-06-27 2025-04-15 Avails Medical, Inc. Apparatus, systems, and methods for determining susceptibility of microorganisms to anti-infectives
WO2019070739A1 (en) * 2017-10-03 2019-04-11 Avails Medical, Inc. APPARATUSES, SYSTEMS AND METHODS FOR DETERMINING THE CONCENTRATION OF MICROORGANISMS AND THE SENSITIVITY OF MICROORGANISMS TO ANTI-INFECTIOUS, BASED ON OXIDOREDUCTION REACTIONS
US11655494B2 (en) 2017-10-03 2023-05-23 Avails Medical, Inc. Apparatus, systems, and methods for determining the concentration of microorganisms and the susceptibility of microorganisms to anti-infectives based on redox reactions
US12275981B2 (en) 2017-10-03 2025-04-15 Avails Medical, Inc. Apparatus, systems, and methods for determining the concentration of microorganisms and the susceptibility of microorganisms to anti-infectives based on redox reactions
US12422343B2 (en) 2017-12-05 2025-09-23 Avails Medical, Inc. Apparatus, systems, and methods for preparing an output sample comprising a defined concentration of an infectious agent for downstream testing
US12379373B2 (en) 2018-12-03 2025-08-05 Avails Medical, Inc. Apparatus, systems, and methods for quantifying infectious agents

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