CN103352068A - Method for detecting activities of cells in fermenting process through adopting flow cytometry (FCM) - Google Patents

Abstract

The invention relates to a method for detecting the activities of cells in a fermenting process through adopting FCM. In the invention, the activities of microbial cells (yeast or moulds) cultured through Rh123/PI double staining are detected in a shaking bottle under normal fermentation or high salt stress through the FCM to obtain an FCM map, an appropriate cross door is adjusted, and a reliable ratio of dead cells to living cells to apoptotic cells can be obtained through quadrant analysis. The method is a simple method for detecting the activities of the cells in the industrial production.

Description

A kind of method that adopts cytoactive in the Flow cytometry fermenting process

Technical field

A kind of method that adopts cytoactive in the Flow cytometry fermenting process belongs to bioengineering field, and particularly this method is convenient and reliable in suitability for industrialized production.

Background technology

Biological fermentation process have pollute little, generally take glucose as substrate, low cost and other advantages, be an extremely promising method, therefore people focus on sight in the research of biological fermentation process in recent years.Yet the multiple pressure factors such as temperature, pH, hunger can make activity decreased even the self-dissolving of cell, affect output.Therefore, set up easy, quick, reliable cytoactive detection method fermentative production is had practical significance.

The ordinary method that detects at present the microorganism cells activity has colony counting method, methylene blue method, than oxygen consumption rate detection method etc., the shortcoming such as these methods all have complicated operation, detection sensitivity is poor and consuming time.In recent years, flow cytometry (flowcytometry, FCM) usually is used for substituting ordinary method as a kind of effective and valuable method and detects microorganism active.Several thousand cells of flow cytometer in can disposable detection cell colony make the researchist set up the multidimensional presentation of individual cells in colony, and level of automation is high, and each the detection can obtain very large quantity of information.Yet this technology during the fermentation report of cytoactive context of detection is also fewer, is in conceptual phase.

Summary of the invention

The objective of the invention is to provide in the fermenting process a kind of easy, quick, reliable cytoactive detection method.

Technical scheme of the present invention:

One, cell cultures and pre-treatment

The check of cytoactive standard when 1, PI singly dyes

1. fermentation culture is got 1ml fermented liquid totally 3 pipes approximately to exponential phase cells;

2. get wherein 1 pipe and place in the boiling water ice bath behind the heating 8-10min, be numbered 1, two pipes are put at room temperature in addition, are numbered respectively 2 and 3;

3. from 1 and No. 2 pipe, get in 500ul fermented liquid to 4 pipe mixing;

4. a certain size aperture grid filters 1,3,4 pipe fermented liquids;

5. fermented liquid is centrifugal, abandons supernatant, and collecting cell is with PBS re-suspended cell gently;

6. get 10 ⁵-10 ⁶Cell centrifugation is abandoned supernatant, adds PBS, gently re-suspended cell;

7. add finite concentration propidium iodide staining fluid, mixing gently, ice bath behind the room temperature lucifuge reaction 5-10min.Detect with flow cytometer.

2, the cytoactive under the high-salt stress

Cytoactive when 1) PI singly dyes under the high salt

Experimental technique is with reference to 1.

2) cytoactive that Rh123/PI is two under the high salt when dying

1. fermentation culture is got the 1ml fermented liquid approximately to exponential phase cells

2. fermented liquid is centrifugal, abandons supernatant, and collecting cell is with PBS re-suspended cell gently;

3. get 10 ⁵-10 ⁶Cell centrifugation is abandoned supernatant, adds PBS, gently re-suspended cell;

4. add 450 μ L PBS and 50 μ L Rh123 working fluids, mixing, room temperature lucifuge reaction 10min, centrifugal and collection thalline;

5. twice rear PI dyeing of PBS washed cell, mixing, room temperature lucifuge reaction 5min carries out machine testing on the streaming immediately.

2, flow cytometer detects

The sample that dyeing is good is put into the loading pipe, with FL1 and FL3 passage fluorescence intensity signal, mixes up voltage, obtains the FCM collection of illustrative plates.Expression dead cell in R1 district in the collection of illustrative plates, R2 district expression viable cell, R3 district expression apoptotic cell.

Description of drawings

FCM figure (A: untreated cell sample when Fig. 1 PI singly dyes; B: the cell sample that heating causes death; C: each A of 50% and the cell sample of B)

FCM figure when PI singly dyes and dyes with Rh123/PI is two under the high salt of Fig. 2

Embodiment

Flow cytometer detects the embodiment of cytoactive when below being the torulopsis glabrata shake flask fermentation.

Torulopsis glabrata (T.glabrata) CCTCC M202019, VitB1, nicotinic acid, vitamin H and pyridoxol quadruple vitamin defective type, and pyruvic carboxylase Active components type reduces, be this research department's seed selection, be mainly used to fermentation production of acetone acid, this bacterial classification has been applied for Chinese patent, and application number is: 02113142.2, and publication number is: CN1392246A.

Seed culture medium (g/L):

Glucose 30, soy peptone 10, KH ₂PO ₄1, MgSO ₄7H ₂O 0.5, and regulating the initial pH of substratum with 6mol/L HCl is 5.5.The tap water constant volume.

Medium of shaking flask fermentation (g/L):

Glucose 100, urea 7, CH ₃COONa 3, KH ₂PO ₄3, MgSO ₄7H ₂O 0.8, liquid microelement 10mL, and VITAMIN liquid 10mL, pH 5.5.The tap water constant volume.During high-salt stress, add 70g/L NaCl in the fermention medium.

Liquid microelement: MnCl ₂4H ₂O 0.2g, FeSO ₄7H ₂O 2g, CaCl ₂2H ₂O 2g, CuSO ₄5H ₂O 0.05g, ZnCl ₂0.5g, be settled to 1L after the dissolving.

VITAMIN liquid: vitamin 1.5mg, Bio 0.004g, pyridoxine hydrochloride 0.04g, nicotinic acid 0.8g is settled to 1L after the 6mol/LHCl dissolving.

Embodiment 1

Torulopsis glabrata activates through seed culture medium, in the inoculation Medium of shaking flask fermentation, is cultured to exponential phase cells 24h, respectively gets 1ml fermented liquid 3 pipes.Get wherein 1 pipe and place in the boiling water ice bath behind the heating 8-10min, be numbered 1, two pipes are put at room temperature in addition, are numbered respectively 2 and 3.From 1 and No. 2 pipe, respectively get mixing in 500ul fermented liquid to 4 pipe.The aperture grid of 40um filters the fermented liquid of dilution certain multiple in 1,3,4 pipes, and the centrifugal 5min of 3000g abandons supernatant, and collecting cell use the PBS washed cell, centrifugal after, adding 500ul PBS, gently re-suspended cell.Add 5ul100ug/ml propidium iodide staining fluid, mixing gently, ice bath behind the room temperature lucifuge reaction 5min detects with flow cytometer.

Embodiment 2

Torulopsis glabrata activates through seed culture medium, and inoculation advances to have added in the 70g/LNaCl fermention medium, is cultured to exponential phase cells 24h, gets 1ml fermented liquid 2 pipes.The aperture grid of 40um filters the fermented liquid of dilution certain multiple, and the centrifugal 5min of 5000g abandons supernatant, and collecting cell use the PBS washed cell, centrifugal after, adding 500ul PBS, gently re-suspended cell.Wherein a pipe adds 5ul100ug/ml propidium iodide staining fluid, mixing gently, ice bath behind the room temperature lucifuge reaction 5min.Another pipe adds 450 μ L PBS and 50 μ LRh123 working fluids, mixing, room temperature lucifuge reaction 10min, centrifugal and collection thalline, twice rear PI dyeing of PBS washed cell, mixing, room temperature lucifuge reaction 5min.Detect with flow cytometer immediately at last.

Embodiment 3

The sample that dyeing is good is put into the loading pipe, with FL1 and FL3 passage fluorescence intensity signal, mix up detection voltage, obtain the FCM collection of illustrative plates, mix up suitable " cross door ", Rh123 and PI dye liquor can be divided into cell mass three districts, and quadrant analysis obtains reliable dead cell, apoptotic cell and viable cell ratio.

Claims (2)

1. utilize the two microorganism cellss (yeast or mould) of cultivating that dye of Rh123/PI, when the Flow cytometry shaking flask is normally fermented and the cytoactive under the high-salt stress, obtain the FCM collection of illustrative plates, mix up suitable " cross door ", quadrant analysis obtains the ratio of reliable dead cell, viable cell and apoptotic cell.

2. method according to claim 1, take torulopsis glabrata (Torulopsisglabrata) the CCTCC M202019 of multiple vitamin defective type as producing bacterial strain, carry out fermentation culture in the 500ml shaking flask, the employing Flow cytometry is analyzed, and can obtain easy, reliably the ratio of dead cell, viable cell and apoptotic cell.

Medium of shaking flask fermentation (g/L):

Glucose 100, urea 7, CH ₃COONa 3, KH ₂PO ₄3, MgSO ₄7H ₂O 0.8, liquid microelement 10mL, and VITAMIN liquid 10mL, pH 5.5.The tap water constant volume.During high-salt stress, add 70g/LNaCl in the fermention medium.

Liquid microelement: MnCl ₂4H ₂O 0.2g, FeSO ₄7H ₂O 2g, CaCl ₂2H ₂O 2g, CuSO ₄5H ₂O 0.05g, ZnCl ₂0.5g, be settled to 1L after the dissolving.

VITAMIN liquid: vitamin 1.5mg, Bio 0.004g, pyridoxine hydrochloride 0.04g, nicotinic acid 0.8g is settled to 1L after the 6mol/LHCl dissolving.

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