Method for detecting yeast morphology and sprouting ratio based on electrical impedance anti-flow type

A technology of flow detection and electrical impedance, which is applied in the direction of biochemical equipment and methods, bioreactor/fermenter combination, specific-purpose bioreactor/fermenter, etc., which can solve the complex and cumbersome analysis method without further analysis of yeast Problems such as the detection of budding ratio, etc., achieve the effect of high throughput, fast calculation of budding ratio, and simple method

Inactive Publication Date: 2019-09-20
INST OF MEDICAL SUPPORT TECH OF ACAD OF SYST ENG OF ACAD OF MILITARY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires precise image and impedance matching, and it can only be realized by building a relatively complex system, and the analysis method is also complicated and cumbersome.
This article also did not further analyze the detection of the proportion of a large number of yeast buds

Method used

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  • Method for detecting yeast morphology and sprouting ratio based on electrical impedance anti-flow type
  • Method for detecting yeast morphology and sprouting ratio based on electrical impedance anti-flow type
  • Method for detecting yeast morphology and sprouting ratio based on electrical impedance anti-flow type

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Using the pulse width histogram to identify yeast buds and calculate the percentage of buds

[0027] Follow the steps below to implement:

[0028] Step 101, cultivating yeast. Inoculate the yeast into the YPD medium under sterile conditions, and culture at 30°C for 24 hours to recover the strains. Under sterile conditions, take 100 μL of the resuscitated strain culture solution and add it into 100 mL of YPD liquid medium, and cultivate at 30°C and 180 r / min for 6-24 hours.

[0029] Step 102, take a small amount of yeast samples, dilute the cultured yeast cell liquid with phosphate buffered saline (PBS), so that the concentration of yeast cells is 10 5 / mL or so;

[0030] Step 103, collecting the impedance signal of the yeast by using an electrical impedance flow cytometry system. The system is required to allow the rod-shaped particles to pass through the detection part one by one with the long axis parallel to the flow direction of the liquid in the cent...

Embodiment 2

[0051] Embodiment 2: use the pulse height, R value scatter plot to identify yeast buds and calculate the bud ratio according to the following steps:

[0052] Steps 101-104 are the same as in Embodiment 1.

[0053] Step 105, determination of budding yeast identification threshold. Skip this step if thresholds have already been determined.

[0054] Specifically include the following steps:

[0055] a, according to the aforementioned steps 101-104, collect the pulse height and R value of 100 typical budding live yeast cells for statistics;

[0056] b, the pulse height and R value of 100 typical ungerminated single live yeast cells were also collected for statistics;

[0057] c, Comparing the numerical distribution of the scatter plots of the pulse statistics results of typical budding yeast and typical unbudding live yeast cells, and determining the parameter boundary threshold of budding yeast and non-budding yeast. All the collected 200 yeast signals can be arranged in asce...

Embodiment 3

[0060] Embodiment 3: Using the measured budding ratio to guide the cultivation of yeast

[0061] The budding ratio detected can be used to guide the cultivation of yeast and fermentation industry. As shown in the following experimental results, the budding ratio of yeast at different culture stages has different values, which reflects the proliferation ability and vitality of yeast, and also indicates whether the culture environment it lives in is suitable for rapid reproduction.

[0062] Step 1, cultivating yeast. Inoculate the yeast into the YPD medium under sterile conditions, and culture at 30°C for 24 hours to recover the strains. Under sterile conditions, take 100 μL of the resuscitated strain culture solution and add it into 100 mL of YPD liquid medium, and culture it at 30 °C and 180 r / min for 48 h.

[0063] Step 2. Starting from injecting the resuscitated bacteria liquid into the medium, select 0, 6, 12, 18, 24, 30, and 48 hours as the detection time points, and det...

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Abstract

The invention discloses a method for distinguishing yeast cell morphology and a sprouting ratio based on microfluidic electrical impedance detection. The method uses an electrical impedance anti-flow detection system to collect a yeast impedance signal. The system ensures that the yeast cells pass through a detection site with a long axis parallel to a flow direction of the central liquid in a pipe. According to the invention, the statistical results of the parameters such as the width, height, width / height ratio R of typical sprouted and non-sprouted live yeast cells of the same number are numerically compared to determine a parameter boundary threshold of the sprouted yeast and the non-sprouted yeast; in the formal test, the cell morphology of a yeast sample to-be-tested is distinguished according to a threshold value, and the value of the ratio of the sprouted yeast is calculated. The method is simple and has high flux, and can realize rapid and automatic detection of yeast morphology and budding ratio calculation, and can well reflect the activity and proliferation ability of yeast.

Description

technical field [0001] The invention relates to a microfluidic microorganism detection method, in particular to a method for detecting yeast morphology and budding ratio based on electrical impedance flow. Background technique [0002] The budding ratio of yeast is an important parameter to reflect the proliferation ability of yeast. When the yeast buds, a raised bud grows on the approximately spherical cell and gradually grows, the cell shape gradually becomes two spheres connected together, which is closer to a rod-shaped morphology. Morphology analysis is an important method in the research of screening excellent yeast. The traditional method needs to rely on human eye observation. This method is labor-intensive, lacks quantitative analysis methods, and relies on manual experience to count and is prone to errors, resulting in low efficiency. Microfluidic electrical impedance detection technology is a relatively common method in the field of single cell detection. This m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/34
CPCC12M41/36
Inventor 谢新武徐新喜杜耀华田丰张志伟程智吴金辉
Owner INST OF MEDICAL SUPPORT TECH OF ACAD OF SYST ENG OF ACAD OF MILITARY SCI
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