CN116200270A - Method for high-throughput separation, culture and identification of microorganisms - Google Patents

Abstract

The invention provides a method for high-throughput separation, culture and identification of microorganisms, comprising the following steps: pretreating a sample of the mixed microorganism, adding chitosan oligosaccharide or polyethylene glycol, and vibrating to disperse the microorganism to form a single-cell microorganism suspension; adding glycerol PBS buffer solution, mixing, and freezing for preservation; randomly sampling, dripping a liquid culture medium containing CCK8 for gradient dilution, pre-culturing for 2-7 days to obtain the density of the culturable microorganisms, thawing the sample according to the dilution of the bacterial growth rate of 10-35%, adding the liquid culture medium containing CCK-8, uniformly mixing, separating liquid, culturing for 3-7 days, collecting liquid in holes with color change and obvious turbidity, and identifying strains. According to the technical scheme, the microorganisms can be better dispersed, more single-hole pure-bred microorganisms are obtained, the later-period microorganism recovery success rate is high, the operation is simple and convenient, batch completion can be realized, and the comprehensive cost is lower.

Description

Method for high-throughput separation, culture and identification of microorganisms

Technical Field

The invention relates to the technical field of microorganisms, in particular to a method for separating, culturing and identifying microorganisms with high flux.

Background

The microorganism has important application potential in the fields of medicine, environmental protection, chemical industry, materials and the like. In recent years, intestinal microorganisms have been shown to have important probiotic effects on animals and humans. Microorganisms derived from extreme environments have also been shown to assist in remediation of contaminated soil and water. Furthermore, natural chemical products of microbial origin are often developed into pharmaceuticals, and specific compounds can be biosynthesized using specific microorganisms. Therefore, microorganisms are important industrial development resources.

The research, development and utilization of microorganisms firstly need to separate and culture single microorganisms from environment, soil and feces to obtain a pure culture, but as the individual microorganisms are very small and various microorganisms are mixed in the environment, how to separate and culture the microorganisms by pure culture is always a difficult problem in the process of researching the microorganisms. The traditional method generally comprises the steps of preparing an environmental sample into suspension, diluting the suspension to a sufficient degree, coating or streaking the suspension on a solid flat-plate culture medium, culturing for a period of time, observing cultured colonies by naked eyes, and repeatedly streaking and separating the colonies to finally obtain pure culture strains. The method is complex in operation and has the following defects which are difficult to overcome. First, during plate culture, a variety of microorganisms grow together, including fast growing slow growing microorganisms, which typically grow into a larger strain, potentially masking slow growing microorganisms; and fast growing microorganisms consume nutrients in the medium rapidly, resulting in slow growing microorganisms failing to grow due to lack of moisture or nutrition. Second, the samples typically contain a large amount of fungal spores that are difficult to remove during plate separation, where the hyphae of the mold grow at a very rapid rate and significantly inhibit the growth of bacteria, resulting in a failure of the bacteria to survive and separation failure. Thirdly, after isolated culture, colonies need to be collected, DNA is extracted, and sequencing and identification of strains are performed. After the plate separation, a large amount of colonies are manually picked, liquid culture is performed to harvest thalli for DNA extraction, and the operation is very complicated. For the above reasons, the plate separation method is difficult to be applied to the separation culture of microorganisms in large scale and in batches, and is unfavorable for the development of microbial strain resources.

Another common method for separating and culturing microorganisms is a liquid dilution culture method, namely, after a sample is oscillated or ground, preparing a suspension, carrying out gradient dilution on the suspension, redistributing 96-well plates into different wells for liquid culture, theoretically, after dilution to a certain extent, each well contains at most one microorganism cell, and after culture, pure microorganisms can be obtained in batches. This method is theoretically possible and can be used for large-scale isolated culture. However, in the practical process, it is often difficult for researchers to obtain a large number of strains efficiently by this method. First, microorganisms such as bacteria in a sample are not present in the sample in a single cell dispersed form, and the microorganisms are closely adhered to each other or to soil particles. Conventional grinding, shaking, etc. do not disperse the microorganisms well into individual suspensions, which results in that some microorganisms cannot be separated or a plurality of microorganisms are mixed in one well, resulting in impurity of the harvested microorganisms. Second, as the microorganism is growing or dying, the concentration of the culturable microorganism is changing for the same suspension sample. The existing method is to distribute suspensions with different dilution concentrations into 96-well plates in a large quantity through gradient dilution, and bacteria grow in all holes due to insufficient dilution caused by improper dilution, and various bacteria exist in the same hole, so that the suspensions are impure. Too high a dilution will occur and there will be no bacterial growth in most wells, resulting in increased separation effort. Thus, there is a need to develop a viable method for determining the density of culturable microorganisms. Third, the liquid culture is to judge whether or not bacterial cells are present by visually observing whether or not the culture solution is turbid. The growth rates of different species of microorganisms vary widely. At normal room temperature, some microorganisms can become turbid within one day, and some require up to 2 weeks. In the batch culture process, if whether microorganisms are growing is still judged by whether the bacterial liquid is turbid, all the wells need to be cultured for 2 weeks or more. For fast growing bacteria, after 1-2 weeks of culture, the bacteria die due to nutrition depletion or the activity of the bacteria is reduced, so that the bacteria are difficult to recover after subsequent cryopreservation. For the above reasons, it is difficult to perform large-scale and high-throughput isolated culture of microorganisms in a practical operation using a liquid dilution culture method.

Disclosure of Invention

Aiming at the technical problems, the invention discloses a method for separating, culturing and identifying microorganisms with high flux, which can separate and culture microorganisms with high flux from a sample, and can harvest as many single-hole pure culture as possible in a rapid and batch manner.

In this regard, the invention adopts the following technical scheme:

a method for high throughput isolation, cultivation and identification of microorganisms comprising the steps of:

step S1, pretreating a sample of the mixed microorganism, adding PBS buffer solution containing glycerol, chitosan oligosaccharide or polyethylene glycol, uniformly mixing to disperse the microorganism to form single-cell microorganism suspension, standing, taking supernatant as the sample to be separated, uniformly mixing, sub-packaging into small tubes, and then placing in a freezing environment for preservation;

step S2, randomly sampling the sample to be separated obtained in the step S1, carrying out gradient dilution on the sampled sample by using a liquid culture medium containing CCK8, then carrying out preculture for 2-7 days in an environment of 20-37 ℃, and counting the bacterial growth rate to obtain the density of the culturable microorganisms;

and S3, obtaining dilution with the bacterial growth rate of 10-35% according to the density of the obtained culturable microorganisms, adding a liquid culture medium containing CCK-8 after thawing the sample according to the dilution, uniformly mixing, separating the liquid, culturing for 3-7 days in an environment of 20-37 ℃, collecting liquid in holes with color change and obvious turbidity, and freezing to keep the bacterial strain or carrying out bacterial strain identification. Further, for freezing and preserving the surviving strain, adding 20% glycerol into the collected liquid, uniformly mixing, and freezing and preserving; for strain identification, the supernatant is removed by centrifugation, and then strain identification is performed on the liquid.

The technical scheme provides a method for separating and culturing bacteria from a sample mixed with various microorganisms and carrying out sequencing identification, and the method can be used for better dispersing the microorganisms to form single-cell suspension; the method comprises the steps of carrying out gradient dilution and then carrying out pre-culture to determine the density of the culturable microorganisms in the sample, and sensitively indicating the growth of the microorganisms by adding CCK8, thereby shortening the culture time, reducing the culture cost, and more importantly avoiding the problem that the bacteria cannot be recovered after being frozen for storage due to aging of the strain cultured for a long time. By improving the methods of freezing, preserving and separating the samples, the density of the microorganisms which can be cultured in the same sample is ensured to be unchanged when the same sample is subjected to separation culture at different times, and the repeatability of the batch separation culture is high. In general, the method can be used for separating, culturing and identifying single-pore pure-breeding microorganisms in a large scale and in a batch mode, and the method has the advantages of more single-pore pure-breeding microorganisms, simpler and more convenient operation, lower comprehensive cost and higher later-stage microorganism recovery power.

As a further improvement of the invention, in the step S1, after the water sample is centrifuged to remove most of supernatant, chitosan oligosaccharide with the final concentration of 0.2-0.8g/L or polyethylene glycol with the final concentration of 1-5g/L is added into the residual liquid, and vortex oscillation is carried out for more than 5min, so as to obtain single-cell microorganism suspension;

for solid samples such as feces, soil, sediment and the like, 1-5g of the sample is taken, 5-20 times of PBS buffer solution containing 0.2-0.8g/L chitosan oligosaccharide or 1-5g/L polyethylene glycol and 20% (volume ratio) glycerol is added, vortex shaking is carried out for 5min to form dispersion suspension, natural sedimentation is carried out for 10-20min, supernatant is taken, or a filter membrane with a pore diameter of more than 1.6 mu m is used for filtering and removing large particulate matters to obtain single-cell microorganism suspension.

As a further improvement of the invention, the polyethylene glycol has a molecular weight of 200-2000.

As a further improvement of the invention, in the step S1, 20% glycerol PBS buffer solution is added into the obtained microorganism suspension, and after vortex mixing, the microorganism suspension is packaged into a preservation tube and is preserved in an environment of minus 80 ℃. All the preservation pipes should be preserved in the refrigerator for at least 12 hours, then follow-up experiments are carried out, a new sample is taken out of the refrigerator when each experiment is carried out, and the unused sample is directly discarded and cannot be replaced in the refrigerator. The aim of the above operation is to ensure that each sample tube is only freeze-thawed once, ensuring consistent reproducibility of the subsequent separation operations performed at different times.

As a further improvement of the invention, in the step S2, 1ml of the sample is taken and added into 9ml of liquid culture medium containing 1X CCK8 for gradient dilution; wherein the 1X CCK8 is solution containing 300 mg/L2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt and 6 mg/L1-methoxy-5-methylphenazine dimethyl sulfate.

As a further improvement of the present invention, the liquid medium is used after 5-10-fold dilution at a conventional concentration.

As a further improvement of the invention, the liquid culture medium is R2A or 2216E.

As a further improvement of the invention, the solution obtained by gradient dilution is distributed into 96-well deep-well plates, 10-20 wells are distributed for each dilution gradient, each well contains 0.2-0.4ml of culture medium, and 10 bacteria-free culture mediums are distributed as controls to detect whether the culture medium and the operation process are polluted or not;

for anaerobic bacteria, operating in an anaerobic workstation, adding 150-300 mu L of sterilized liquid paraffin on the surface of liquid, and attaching an impermeable film; for aerobic bacteria, attaching a ventilated membrane on the surface of a 96-well plate, and placing the 96-well plate on a shaking table with the rotating speed of 100-200rpm for culturing; culturing the 96-well plate in 20-37deg.C for 2-7 days;

each well was observed daily during the incubation period as per Kong Bianse, indicating the presence of microbial growth in the well; after the preculture is finished, counting the ratio of the number of the holes grown by each dilution to the total separation hole number, namely the growth rate, calculating the dilution with the growth rate of 10-35% as the dilution of the mass culture, and selecting the dilution to carry out the next mass separation culture.

As a further improvement of the invention, in the step S3, according to the dilution degree corresponding to the 10-35% of the long bacteria rate obtained in the step S2, adding a liquid culture medium which is precooled at 4 ℃ and contains CCK-8 into a thawed sample for dilution, magnetically stirring to ensure that bacteria are in a uniform state in a culture solution, and simultaneously maintaining the culture medium at the environment of 4 ℃;

distributing the diluted culture medium into 96-well plates, distributing 94 wells per plate, leaving 2 wells for adding blank culture medium as control, adding 0.2-0.4ml culture medium per well, and culturing for 3-7 days according to the pre-culture step in step S2;

after the culture is finished, collecting liquid in the holes with color change or obvious turbidity, adding one part of the collected liquid into 20% glycerol, uniformly mixing, freezing to keep the viable bacteria strain, and carrying out strain identification on the other part.

As a further improvement of the invention, transferring the collected liquid into 2 new 96-well plates, adding 20% glycerol into one part of the liquid, uniformly mixing, freezing to keep the viable bacteria, centrifuging the other part of the liquid to remove the supernatant, and using the supernatant in DNA extraction, PCR amplification and sequencing for bacteria identification; the bacterial liquid of the same hole corresponds to the position of the two 96-well plates, and the sequencing result corresponds to the strain. 94 strains were collected on each 96-well plate, and 2 additional wells were used for negative controls for subsequent DNA extraction and PCR amplification.

As a further improvement of the invention, the DNA extraction adopts a commercial nucleic acid extractor and a bacterial genome DNA extraction reagent by a magnetic bead method.

As a further improvement of the invention, according to different types of the separated microorganisms, 16S, 18S or ITS partial sequences are selected for PCR amplification and sequencing, and a barcode region of 6-8 bases is arranged outside the 5' end of the universal forward and reverse primers to obtain a series of primer groups with different barcodes, and after the forward and reverse primers are combined again, different primer combinations are obtained;

when PCR amplification is carried out, one hole uses one primer combination, different holes use different primer combinations, and the barcode sequence outside the primer corresponding to the hole is recorded; after PCR amplification, agarose gel electrophoresis is carried out, PCR products with correct amplified bands are collected, and the PCR products with different holes are mixed together to be used as a PCR product mixed sample;

and (3) purifying, constructing a library and sequencing a PCR product mixed sample in high throughput, filtering sequencing data to remove unqualified data, then using the sequencing data for sequence analysis and strain annotation, and determining the corresponding 96-well plate and the position on the 96-well plate according to the information of the barcode at the two sides of the sequence, and corresponding to the cryopreserved viable bacteria.

Compared with the prior art, the invention has the beneficial effects that:

by adopting the technical scheme of the invention, the microorganism can be better dispersed, the density of the culturable microorganism in the sample can be determined, the microorganism growth is sensitively indicated, more single-hole pure microorganism is obtained, the later-stage microorganism recovery success rate is high, the operation is simpler and more convenient, the separation culture and the sequencing identification of the strain can be completed in batches, and the workload and the comprehensive cost of the sequencing identification of the strain are greatly reduced.

Drawings

FIG. 1 shows the growth of well plates on day 3 after CCK-8 addition in example 1 of the present invention.

FIG. 2 is a gel electrophoresis chart of the PCR product of example 1 of the present invention, wherein a) is the front 48 wells of plate 6, b) is the rear 48 wells of plate 6, c) is the front 48 wells of plate 12, d) is the rear 48 wells of plate 12.

FIG. 3 shows the gram stain results for a sample suspension without chitosan or polyethylene glycol added in example 2 of the present invention.

FIG. 4 shows the results of gram staining of sample suspensions of three pretreatment modes (A), (B) and (C) in example 2 of the present invention; wherein (a), (B) and (C) correspond to the samples of the three pretreatment modes (A), (B) and (C), respectively.

FIG. 5 shows the deep-well plate pictures of the three pretreatment samples (A), (B) and (C) and the blank group according to the embodiment 2 of the present invention, wherein (a), (B) and (C) correspond to the three pretreatment methods (A), (B) and (C), respectively, and (d) is a blank group sample after 5 days of incubation.

FIG. 6 is a photograph of a deep-well plate after 2 days of incubation of the sample in example 3 of the present invention.

FIG. 7 is a photograph of a deep well plate after 5 days of culture of the sample in example 3 of the present invention.

Detailed Description

Preferred embodiments of the present invention are described in further detail below.

A method for high-throughput separation, culture and identification of microorganisms, which adopts a liquid dilution mode to separate and culture mixed microorganisms, and further performs DNA extraction, amplification, purification and sequencing on the separated bacteria, comprises the following specific steps:

(1) Pretreatment of the sample:

(1) and (3) water sample: and (3) enriching microorganisms in the sample by centrifugation at 5000rpm for 15min, removing most of supernatant, adding chitosan oligosaccharide with the final concentration of 0.2-0.8g/L or polyethylene glycol with the final concentration of 1-5g/L (molecular weight of 200-2000) into the residual liquid, and vortex-vibrating for 5min. The chitosan oligosaccharide and the polyethylene glycol have positive charges, and are combined with negative charges on the surfaces of bacteria, so that interactions between bacteria or between bacteria and other matrixes are destroyed, and the bacteria are promoted to disperse to form single-cell suspension.

(2) For samples such as feces, soil, sediment and the like, 1-5g of the sample is taken, 5-20 times of PBS buffer solution containing 0.2-0.8g/L chitosan oligosaccharide or 1-5g/L polyethylene glycol (molecular weight of 200-2000) is added, and vortex shaking is carried out for 5min to help microorganisms form dispersion suspension. Naturally settling for 10-20min, collecting supernatant, or filtering with a filter membrane with pore diameter of more than 1.6 μm to remove large particulate matters to obtain microorganism suspension.

(3) Then adding 10 times of 20% glycerol PBS buffer solution into the microorganism suspension, fully vortex mixing, split charging into 1.5-2ml tubes, filling 1ml liquid into each tube, and storing in a refrigerator at-80 ℃. All the preservation pipes should be preserved in the refrigerator at the temperature of minus 80 ℃ for at least 12 hours, then subsequent experiments are carried out, a new sample is taken out of the refrigerator when each experiment is carried out, and the unused sample is directly discarded and cannot be replaced into the refrigerator again. The aim of the above operation is to ensure that each sample tube is only freeze-thawed once, ensuring consistent reproducibility of the subsequent separation operations performed at different times.

(2) Densitometry of the culturable microorganisms in the sample:

taking 2-3 tubes of the frozen samples, adding 1ml of the frozen samples into 9ml of liquid culture medium containing 1X CCK8 for 10-time dilution, and carrying out 9-12 rounds of dilution so as to obtain 10-time gradient diluted samples. CCK8 at 1X concentration means that the solution contains 300 mg/L2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt and 6 mg/L1-methoxy-5-methylphenoxazine sulfate dimethyl ester. The liquid medium is selected according to the target strain or sample source, including but not limited to R2A,2216E, etc.; the culture medium is diluted 5-10 times at conventional concentration to avoid partial oligotrophic microorganism incapable of growing due to too high concentration of nutrient substances.

The solution obtained by the gradient dilution is distributed into 96-well deep well plates, and 10-20 wells are distributed for each dilution gradient, and each well contains 0.2-0.4ml of culture medium. 10 bacteria-free media were simultaneously dispensed as controls to detect contamination of the media and the process. For aerobic bacteria, a gas permeable membrane is attached to the surface of a 96-well plate. For anaerobic bacteria, the above operation should be performed in an anaerobic workstation, 150 to 300. Mu.L of sterilized liquid paraffin is added to the liquid surface, and an impermeable film is attached. Depending on the source of the sample, the 96-well plate was incubated at 20-37℃for 2-7 days. If the bacteria are aerobic bacteria, the 96-well plate is placed on a shaking table with the rotating speed of 100-200rpm during the culture, and dissolved oxygen is increased. Each well was observed daily during the incubation period. If the well turns red, it indicates that there is microbial growth within the well. After the end of the preculture, the ratio of the number of wells grown by each dilution to the total number of isolated wells (growth rate) was counted. On this basis, the dilution with a growth rate of 10% -35% was calculated as the dilution for the bulk culture, and the bulk isolated culture was selected at this dilution.

(3) Mass isolation culture of microorganisms

According to the dilution degree corresponding to the 10-35% of the long bacterial rate, a new sample is taken out from the refrigerator, 1-2L of culture medium which is precooled at 4 ℃ and contains CCK-8 is prepared after thawing and placed on a magnetic stirrer, bacteria are kept in a uniformly mixed state in the culture solution, and meanwhile, the culture medium is kept in the environment at 4 ℃ so as to avoid the change of the density of the culturable microorganisms in the culture solution caused by bacterial division and propagation in a large number of separation processes.

The above media was dispensed into 96-well plates using an automatic liquid separator, 94 wells were allocated per plate, leaving 2 wells for addition of blank media as a control. 0.2-0.4ml of culture medium is added to each well. Culturing for 3-7 days according to the conditions in the "Density determination of culturable microorganisms in sample" step (2).

If anaerobic bacteria are present, the above-mentioned process should be carried out in an anaerobic workstation.

After the culture is finished, collecting liquid in a hole with color change and obvious turbidity, transferring the collected liquid into 2 new 96-well plates, adding 20% glycerol into one part of the liquid to mix uniformly, freezing to keep the viable bacteria strain, and centrifuging the other part of the liquid to remove the supernatant, and then using the liquid in DNA extraction, 16S amplification and sequencing for strain identification. The position of the bacterial liquid in the same hole on two 96-well plates should be consistent, and the sequencing result corresponds to the strain. 94 strains were collected on each 96-well plate, and 2 additional wells were used for negative controls for subsequent DNA extraction and PCR amplification.

(4) DNA extraction, PCR amplification, high throughput sequencing and strain identification

DNA extraction was performed using a commercial nucleic acid extractor in combination with a bacterial genomic DNA extraction reagent by the magnetic bead method.

Conventional microbial 16S sequencing identification 16S was sequenced using a generation sanger sequencing. However, the method has small flux and is not suitable for large-scale bacterial separation and culture. The invention adopts a high-throughput sequencing method to sequence and identify a large number of strains, so as to simplify the operation process and reduce the comprehensive cost.

The extracted DNA was PCR amplified using a self-designed primer combination and Taq Mix kit. Depending on the type of microorganism isolated, 16S, 18S or ITS partial sequences are selected for PCR amplification and sequencing. The 5' end outer sides of the universal forward and reverse primers are respectively provided with a barcode region with 6-8 bases, the barcode sequence region can be changed continuously, a series of primer groups with different barcodes are generated, and after the forward and reverse primers are combined again, tens of thousands of primer combinations can be finally generated.

In performing PCR amplification, one well uses one primer combination, different wells use different primer combinations, and the barcode sequence outside the primer corresponding to the well is recorded. After PCR amplification according to the conventional method, agarose gel electrophoresis is performed, PCR products with correct amplified bands are collected, and PCR products of different wells are mixed together to serve as one sample.

The PCR product mix samples described above were purified, library constructed and high throughput sequenced according to the instructions of the Illumina sequencer and related reagents. The amount of data collected is the total number of wells multiplied by 500.

The sequencing data are filtered to remove unqualified data and then used for sequence analysis and strain annotation. According to the information of the barcode at two sides of the sequence, the corresponding 96-well plate and the position on the 96-well plate can be determined, and then the corresponding cryopreserved viable bacteria can be found for subsequent scientific research and industrial development.

The following description is made with reference to specific examples.

Example 1

Isolation and purification of bacteria in fresh water samples (R2A group):

(1) Sample pretreatment: 5g of fresh water sediment sample is placed into a 50ml sterile centrifuge tube, 30ml of PBS solution containing 5g/L polyethylene glycol 400 and 20% glycerol (volume ratio) is added, shaking and standing are carried out fully, 3 tubes are prepared repeatedly to reduce sampling errors, when solid-liquid delamination is obvious, 15ml of supernatant is taken out of each tube on an ultra clean bench and is combined into the 50ml sterile centrifuge tube. Mixing, packaging into 45 systems of 1ml, and freeze preserving in-80deg.C refrigerator.

(2) Pre-experiment of samples: a10 XCCK-8 solution (composed of 3g/L of WST-8 reagent and 60mg/L of PMS reagent) was prepared and sterilized by filtration. Added to sterilized 1/10R2A medium, and CCK-8 was added at a final concentration of 1X. The following operations are performed in the ultra clean bench: 12 sterile, 15ml, sterile, centrifuge tubes were taken and 9ml of the above medium was added to each tube. One branch was taken for blank control, and the other 11 branches were numbered-1 to-11. 1ml of the sample was added to the-1 tube using a 1ml pipette, vortexed on the mixer for 10s, 1ml was aspirated from the-1 tube after changing the tip and added to the-2 tube, and the above procedure was repeated until-11. I.e. diluting the pretreatment liquid to a concentration of from 10 ^-1 To 10 ^-11 Total 11 dilutions. Tubes-4 to-11 were selected, the first 11 wells of a row of 96-well plates were used for each dilution, 400 μl of the diluted sample of the corresponding dilution was added dropwise to the 11 wells using a 1ml pipette, and the same volume of medium was added dropwise to the last 1 well of each row as a blank. Incremental dilution by degree, i.e. 10 ^-4 To 10 ^-11 The well plates were added in the order 96 well plates a to H. The last hole of each row is added with a blank culture medium, an anti-pollution breathable film is attached, and the culture is carried out by placing the culture medium on a shaking table at 28 ℃ and 120rpm after the cover is covered. Observations were made daily.

(3) FIG. 1 is a photograph of a culture day 3, according to which 10 ^-6 Only 1 out of 11 wells at dilution had bacterial growth. Thus, 2×10 is selected ^-6 (extensive isolation culture at this dilution was predicted, about 20% of wells will be bacterial growth)As the optimal concentration, a large number of separation cultures were performed using an atomic single channel dispenser, totaling 50 96 well plates.

(4) When the culture is carried out for 7 days, the growth is found to meet the requirement, no new Kong Changjun exists, bacterial liquid in 50 96-well plates is collected into the new 96-well plates, and the A1 and A2 wells are reserved as blank controls. Sucking 100 mu L of the collected bacterial liquid into a new sterilized PCR plate, adding 50 mu L of 60% glycerol PBS buffer solution into each hole, whipping and mixing the bacterial liquid, sticking an aluminum film sealing film on the PCR plate, boxing, and storing in a refrigerator at-80 ℃, wherein the glycerol strain is obtained.

(5) The remainder of the 96-well plate was subjected to DNA extraction according to the magaBio bacterial genomic DNA purification kit III. The method comprises the following specific steps:

(1) the remaining bacterial liquid in the 96-well plate was centrifuged at 4000r/min for 10min in a centrifuge. The supernatant was sucked as far as possible in the super clean bench without sucking off the sediment.

(2) After all of the self-contained lysozymes of the kit were dissolved in TET Buffer, 200 μl was added to each well of the 96-well plate and the bacteria were sufficiently dispersed and resuspended by shaking.

(3) The temperature bath was carried out at 37℃for 60 minutes.

(4) 200. Mu.L of Lysis Buffer and 20. Mu.L of PK were added and mixed with vigorous shaking for 20 seconds.

(5) The incubation was carried out at 70℃for 10 minutes, and then the 96-well plate was removed from the incubation at 70 ℃.

(6) All the liquid in the 96-well plate is transferred to the Binding Buffer pre-allocation plate, so that cross contamination is avoided.

(7) Putting the 96-well plate into an NPA-96 nucleic acid extraction and purification instrument, wherein the arrangement sequence of the 96 Kong Shiji plates from left to right is as follows: binding Buffer Reagent board, magaBio Reagent board, WB1 Buffer Reagent board, wash Buffer Reagent board, and ElutionBuffer Reagent board are equipped with magnetic bar sheath.

(8) An automated extraction experiment was performed on a nucleic acid extractor following the procedure of table 1 below:

TABLE 1

(9) After the automatic program is finished, the Elution Buffer pre-assembled plate is taken down and stuck with SF-520 film, if not used immediately, the plate is preserved at the temperature of minus 20 ℃.

The extracted DNA was subjected to PCR amplification according to the BioReady PCR MIX (with dye) kit instructions. The method comprises the following specific steps:

(1) After removal of 2X TaqMix (with dye), the mixture was gently inverted and mixed up and down after sufficient thawing, and centrifuged briefly before preparation.

(2) The reaction solution components of the following Table 2 were added to a PCR plate or a PCR reaction tube on ice. Wherein the primer is a 16S V3V4 universal primer with a barcode sequence. After the sealing film of the PCR plate is attached, the PCR plate is centrifuged briefly, so that all reagents are concentrated at the bottom of the container, and the PCR amplification is performed after bubbles are completely removed.

TABLE 2

(3) The PCR plate was placed in a PCR apparatus and amplified according to the procedure of Table 3 below.

TABLE 3 Table 3

And (3) performing gel electrophoresis on the PCR product, and observing and photographing and preserving the PCR product by a full-automatic chemiluminescence imager. Electrophoresis pictures of the PCR products of plates 6 and 12 were attached as shown in FIG. 2.

(6) The PCR products were collected according to the electrophoresis results, and the collected products were purified using a general DNA product purification kit. The method comprises the following specific steps:

(1) the PCR products were collected into 1 EP tube according to the brightness of the bands after electrophoresis, and the bright bands were judged to be bright and slightly dark, 4. Mu.L was collected, 8. Mu.L was collected slightly dark, and no bands were collected. The collected product was mixed into a tube as one sample.

(2) 500. Mu.L of balance liquid BL is added into the adsorption column CB2 (the adsorption column is placed in the collecting pipe), the mixture is centrifuged at 12000rpm for 1min, waste liquid in the collecting pipe is poured out, and the adsorption column CB2 is replaced in the collecting pipe.

(3) To the collected PCR reaction solution, 5 times of the volume of the binding solution PB was added, followed by thoroughly mixing. Adding into an adsorption column CB2 (the adsorption column is placed in a collecting pipe), standing at room temperature for 2min, centrifuging at 12000rpm (about 13400 Xg) for 60s, pouring out waste liquid in the collecting pipe, and placing the adsorption column CB2 into the collecting pipe.

(4) 600. Mu.L of rinse PW (absolute ethanol should be added before use) was added to the adsorption column CB2, the mixture was centrifuged at 12000rpm for 60s, the waste liquid in the collection tube was poured out, and the adsorption column CB2 was placed in the collection tube. This step was repeated 1 time.

(5) The adsorption column CB2 was returned to the collection tube, and centrifuged at 12000rpm (. About.13400 Xg) for 2 minutes to remove the rinse solution as much as possible. The adsorption column CB2 is left at room temperature for a few minutes and is thoroughly dried to prevent the residual rinsing liquid from affecting the next experiment.

(6) Placing the adsorption column CB2 into a clean 1.5mL centrifuge tube, and suspending and dripping 50 μl of eluent ddH into the middle of the adsorption film ₂ O, standing at room temperature for 5min. Centrifuge at 12000rpm for 2min, discard CB2 column, cover the centrifuge tube lid.

(7) Taking the purified product in a 3 mu L centrifuge tube to carry out agarose gel electrophoresis, and sending the qualified strip to a commissioner to carry out second-generation high-throughput sequencing.

(7) And (3) performing quality control on a sequencing result, removing unqualified reads, performing internal self-alignment after sequence splicing, merging repeated sequences, performing blast alignment on the obtained non-repeated sequences and Genbank, and determining the strain name corresponding to each sequence. Within the same well, it was determined whether it was a single fungus. The final result shows that the current mass separation and co-culture is carried out on 550 holes, wherein 125 holes are subjected to bacterial growth, 27 bacteria with different species are obtained after sequencing, and the holes with 85% bacterial growth only contain one kind of bacteria, which are regarded as pure bacterial cultures, and can be directly used for subsequent researches.

Example 2

Based on the example 1, 6 parts of the same fresh water sediment sample are taken for each 5g part, wherein 3 parts of the fresh water sediment sample are pretreated as follows:

(A) 30ml of PBS solution containing 20% glycerol and 5g/L polyethylene glycol 400 was added,

(B) 30ml of PBS solution containing 20% glycerol and 5g/L polyethylene glycol 2000 was added,

(C) 30ml of PBS solution containing 20% glycerol and 0.8g/L chitosan oligosaccharide was added.

A control group was prepared for each treatment group simultaneously, i.e., 30ml of PBS containing only 20% glycerol was added. Shaking, standing for 10min, and taking supernatant as a sample to be separated for later use.

The supernatant was subjected to 10-fold gradient dilution with 1/10R2A medium (without affecting gram stain, without adding CCK-8), from 10 ^-3 Samples were taken from dilutions to make smears and bacterial morphology was observed after gram staining (see figures 3 and 4). FIG. 3 shows that no polyethylene glycol or chitosan oligosaccharide is added, and the prepared bacterial suspension has obvious microorganism aggregation phenomenon, which is obviously unfavorable for the separation of microorganisms, and the situation that a plurality of microorganisms exist in the same hole later. FIG. 4 shows the results of the three pretreatment samples (A), (B) and (C), and the microorganisms are in a single dispersion state, so that the dispersion effect is better than that of the control group, and the subsequent microorganism separation is facilitated.

In addition, 0.4ml was added to the deep well plate from each dilution, 11 duplicate wells per dilution, respectively. Then pasting an anti-pollution breathable film on the surface of the deep hole plate, placing the deep hole plate on a shaking table at 28 ℃ for 120rpm for shaking culture for 5 days, and observing the growth condition of bacteria in the hole plate. After 5 days of incubation, the deep-well plate was photographed, as shown in FIG. 5, and 5 (a), 5 (B), 5 (C), and 5 (d) were each pretreated with 5g/L polyethylene glycol 400 (i.e., pretreated with (A)), 5g/L polyethylene glycol 2000 (i.e., pretreated with (B)), 0.8g/L chitosan oligosaccharide (i.e., pretreated with (C)), and control groups (without polyethylene glycol or chitosan oligosaccharide). In FIG. 5, each deep well plate is shown at 10 from top to bottom for each row ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 、10 ^-8 Dilution. Comparative example 10 ^-5 The dilutions of FIGS. 5 (a), 5 (b), 5 (c) and 5 (d) were 7, 4, respectively,The existence of microorganism growth in 5 and 1 holes further indicates that the addition of polyethylene glycol and chitosan can obviously provide the dispersing effect of microorganisms in suspension, thereby being more beneficial to the separation and culture of microorganisms.

Example 3

This example is mainly to compare the effects played by CCK-8.

(1) Sample pretreatment: taking 5g of a fresh water sediment sample, adding 30ml of PBS solution containing 5g/L polyethylene glycol and 20% glycerol, shaking uniformly, standing for 10 minutes, and taking supernatant as a sample to be separated for later use. This step is the same as the pretreatment step of the example.

(2) The testing method comprises the following steps: a10 XCCK-8 solution (composed of 3g/L of WST-8 reagent and 60mg/L of PMS reagent) was prepared and sterilized by filtration. The above supernatant was subjected to 10-fold gradient dilution using 1/10R2A medium containing 1 XCCK-8, and 0.4ml of each dilution was added to a deep well plate, and 11 wells per dilution were used as replicates. Then pasting an anti-pollution breathable film on the surface of the deep hole plate, and placing the deep hole plate on a shaking table at 28 ℃ for shaking culture at 120 rpm. The wells were observed daily for discoloration and growth.

FIG. 6 is a photograph of a deep well plate after 2 days incubation of a sample with CCK-8. Obviously, part of the wells had turned orange, indicating that there was bacterial growth, but no apparent precipitation in the wells.

FIG. 7 is a photograph of day 5 of incubation, with a darker color compared to FIG. 6. First row of holes (10 times dilution), second row of holes (10 dilution) ² Multiple) of which a distinct cloudy precipitate (centered in each well) was present, but a third row of wells (dilution 10 ³ Multiple) of the discoloured pores still had no significant precipitation. The above results demonstrate that the color response of CCK-8 is significantly earlier than the time at which precipitation occurs. Without CCK-8, researchers can only determine if there is bacterial growth by observing if there is precipitation, which would take 5 days or more to confirm if there is bacterial growth in the wells. After adding CKK-8, the color change can be observed after 2 days of culture, and whether bacteria grow or not can be confirmed. Obviously, the holes with the growth of bacteria can be determined earlier by adding CKK-8, the observation time is obviously shortened, and the bacteria collected earlier can be recovered more easily after being stored in a liquid.

Example 4

The effect of measuring the density of the culturable microorganisms was compared with the method of example 1 using a flow cytometer.

The experimental method comprises the following steps: taking 5g of fresh water sediment in a 50ml centrifuge tube, adding 30ml of PBS solution containing 20% glycerol and 5g/L polyethylene glycol, vibrating and dispersing, standing and taking supernatant for later use. Different from the pretreatment of example 1.

The microorganism density was measured and mass-isolated culture was performed in the following two ways.

(1) A1 ml sample of the liquid was placed in an EP tube, SYBR Green I and propidium iodide dye were added, and the microorganisms in the sample were counted using a flow cytometer. The final measured density of total microorganisms in the sample was 6.06X10 ⁷ ±2.20×10 ⁷ . According to the density, the samples are diluted by using a culture medium, the theoretical microorganism density after dilution is 0.5 bacteria in each milliliter of liquid, 10 96-well plates are separated and cultured according to 0.4ml of each well, and shake culture is carried out for 5 days at 28 ℃. In theory there should be 20% of the growth of bacteria in the wells, but practical statistics have found that only no more than 3% of the wells per plate grow, well below the theoretical density.

(2) Samples obtained by pretreatment were sub-packed into 1ml volumes, frozen at-80℃for 1 day, and 10-fold gradient dilutions were performed in 1 tube, each dilution separating 11 wells, and 0.4ml of liquid per well. After two days of cultivation, the microorganism density was calculated to be 1.36×10 ⁶ And each ml. According to the density, a dilution with a bacterial growth rate of 20% is obtained, according to the dilution, another tube of unfrozen bacterial liquid is taken, the bacterial liquid is diluted by a culture medium, the theoretical microbial density after dilution is 0.5 bacteria per milliliter of liquid, 10 96-well plates are carried out according to 0.4ml per well, and shake culture is carried out for 5 days at 28 ℃. In theory, 20% of the cells should have bacterial growth, and actual statistics show that 28% of the cells per plate have bacterial growth, which is close to the theoretical density.

The comparison of the results shows that the microorganism density measured by using a flow cytometer is higher, but most of bacteria cannot be cultivated, the obtained result has no reference significance for a large amount of separation cultivation, serious waste of pore plates can be caused, the workload of separation is increased, the efficiency of effective bacteria collection is low, and the method is not suitable for high-flux microorganism separation cultivation.

The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.

Claims (10)

1. A method for high throughput separation, cultivation and identification of microorganisms, characterized by: the method comprises the following steps:

step S1, pretreating a sample of the mixed microorganism, adding PBS buffer solution containing glycerol, chitosan oligosaccharide or polyethylene glycol, uniformly mixing to disperse the microorganism to form single-cell microorganism suspension, standing, and taking supernatant as a sample to be separated for later use; the method comprises the steps of carrying out a first treatment on the surface of the

Step S2, randomly sampling the sample to be separated obtained in the step S1, dropwise adding a liquid culture medium containing CCK8 into the sampled sample for gradient dilution, then pre-culturing for 2-7 days in an environment of 20-37 ℃, and counting the bacterial growth rate to obtain the density of the culturable microorganisms;

and S3, obtaining dilution with the bacterial growth rate of 10-35% according to the density of the obtained culturable microorganisms, adding a liquid culture medium containing CCK-8 after thawing the sample according to the dilution, uniformly mixing, separating the liquid, culturing for 3-7 days in an environment of 20-37 ℃, and collecting liquid in holes with color change and obvious turbidity for freezing and preserving the bacterial species and identifying the bacterial species.

2. The method for high throughput separation, cultivation and identification of microorganisms according to claim 1, wherein: in the step S1, for a water sample, after most of supernatant is removed by centrifugation, chitosan oligosaccharide with the final concentration of 0.2-0.8g/L or polyethylene glycol with the final concentration of 1-5g/L is added into the residual liquid, and vortex oscillation is carried out for more than 5min, so as to obtain single-cell microorganism suspension;

for solid samples, 1-5g of samples are taken, 5-20 times of PBS buffer solution containing 0.2-0.8g/L chitosan oligosaccharide or 1-5g/L polyethylene glycol is added, vortex oscillation is carried out for 5min to form dispersion suspension, natural sedimentation is carried out for 10-20min, and supernatant is taken, or a filter membrane with the aperture of more than 1.6 mu m is used for filtering and removing large particulate matters to obtain single-cell microorganism suspension.

3. The method for high throughput separation, cultivation and identification of microorganisms according to claim 2, wherein: the molecular weight of the polyethylene glycol is 200-2000.

4. The method for high throughput separation, cultivation and identification of microorganisms according to claim 2, wherein: in the step S1, PBS buffer solution containing 20% of glycerol by volume is added into the obtained microorganism suspension, and after vortex mixing, the mixture is packaged into a preservation tube and preserved in an environment of-80 ℃.

5. The method for high throughput separation, cultivation and identification of microorganisms according to claim 1, wherein: in the step S2, 1ml of sample is taken and added into 9ml of liquid culture medium containing 1X CCK8 for gradient dilution;

wherein the 1X CCK8 is solution containing 300 mg/L2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt and 6 mg/L1-methoxy-5-methylphenazine dimethyl sulfate.

6. The method for high throughput separation, cultivation and identification of microorganisms according to claim 5, wherein: the liquid medium is used after 5-10-fold dilution at a conventional concentration.

7. The method for high throughput separation, cultivation and identification of microorganisms according to claim 5, wherein: distributing the solution obtained by gradient dilution into 96-well deep hole plates, distributing 10-20 wells in each dilution gradient, wherein each well contains 0.2-0.4ml of culture medium, and simultaneously distributing 10-well culture medium without bacteria as a control to detect whether the culture medium and the operation process are polluted or not;

for anaerobic bacteria, operating in an anaerobic workstation, adding 150-300 mu L of sterilized liquid paraffin on the surface of liquid, and attaching an impermeable film; for aerobic bacteria, attaching a ventilated membrane on the surface of a 96-well plate, and placing the 96-well plate on a shaking table with the rotating speed of 100-200rpm for culturing; culturing the 96-well plate in 20-37deg.C for 2-7 days;

each well was observed daily during the incubation period as per Kong Bianse, indicating the presence of microbial growth in the well; after the preculture is finished, counting the ratio of the number of the holes grown by each dilution to the total separation hole number, namely the growth rate, calculating the dilution with the growth rate of 10-35% as the dilution of the mass culture, and selecting the dilution to carry out the next mass separation culture.

8. The method for high throughput separation, cultivation and identification of microorganisms according to claim 5, wherein: in the step S3, according to the dilution degree corresponding to the 10-35% of the long bacteria rate obtained in the step S2, adding a liquid culture medium which is precooled at 4 ℃ and contains CCK-8 into a thawed sample for dilution, magnetically stirring to ensure that bacteria are in a uniform mixing state in a culture solution, and simultaneously maintaining the culture medium at the environment of 4 ℃;

distributing the diluted culture medium into 96-well plates, distributing 94 wells per plate, leaving 2 wells for adding blank culture medium as control, adding 0.2-0.4ml culture medium per well, and culturing for 3-7 days according to the pre-culture step in step S2;

after the culture is finished, collecting liquid in the holes with color change or obvious turbidity, adding one part of the collected liquid into 20% glycerol, uniformly mixing, freezing to keep the viable bacteria strain, and carrying out strain identification on the other part.

9. The method for high throughput separation, cultivation and identification of microorganisms of claim 8, wherein: transferring the collected liquid into 2 new 96-well plates, adding 20% glycerol into one part, mixing, freezing to keep alive strain, centrifuging to remove supernatant, and performing DNA extraction, PCR amplification, sequencing for strain identification; the bacterial solutions of the same well correspond to the positions on two 96-well plates, 94 strains are collected on each 96-well plate, and the other 2 wells are used for negative control for subsequent DNA extraction and PCR amplification.

10. The method for high throughput separation, cultivation and identification of microorganisms according to claim 9, wherein: the DNA extraction adopts a commercial nucleic acid extractor matched with a bacterial genome DNA extraction reagent by a magnetic bead method;

according to different types of the separated microorganisms, selecting 16S, 18S or ITS partial sequences for PCR amplification and sequencing, and setting a barcode region of 6-8 bases outside the 5' end of the universal forward and reverse primers to obtain a series of primer groups with different barcodes, and combining the forward and reverse primers to obtain different primer combinations;

when PCR amplification is carried out, one hole uses one primer combination, different holes use different primer combinations, and the barcode sequence outside the primer corresponding to the hole is recorded; after PCR amplification, agarose gel electrophoresis is carried out, PCR products with correct amplified bands are collected, and the PCR products with different holes are mixed together to be used as a PCR product mixed sample;

and (3) purifying, constructing a library and sequencing a PCR product mixed sample in high throughput, filtering sequencing data to remove unqualified data, then using the sequencing data for sequence analysis and strain annotation, and determining the corresponding 96-well plate and the position on the 96-well plate according to the information of the barcode at the two sides of the sequence, and corresponding to the cryopreserved viable bacteria.

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