Bacterial apoptosis evaluation method based on cell membrane phosphatidylserine fluorescence staining
A technology of phosphatidylserine and cell membrane, which is applied in the field of cell staining, can solve the problems of low dyeing efficiency and achieve obvious staining effect, strong practicability, and simple operation
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Embodiment 1
[0059] Phosphatidylserine eversion staining on the cell membrane of a Gram-negative bacterium Ochrobactrum sp. that can degrade phenol
[0060] Take the Ochrobactrum sp. culture solution 1.5mL (10 8cells / mL), add 30mL ethanol-water solution with a volume percentage of 70%, the volume ratio of the ethanol solution to the liquid medium containing the bacteria to be detected is 20:1, mix well, fix for 2h, no fixed bacterial cells Mix with the same volume of sterile water and let stand for 2h;
[0061] Centrifuge the fixed bacterial cells under the centrifugal force of 5000rpm for 5min, retain the bacterial cells, and remove excess ethanol-water solution;
[0062] Prepare EDTA-Na with sterile water 2 Solution 30mL (200mg / L) to wash the bacterial cell precipitate, centrifuge again at 5000g for 5min, retain the bacterial cell, and remove excess liquid;
[0063] Resuspend the bacteria to a volume of 27 mL with sterile phosphate buffered saline (PBS).
[0064] Prepare 3mL (2000mg / ...
Embodiment 2
[0071] Phosphatidylserine outward staining on the cell membrane of two phenol-degrading bacteria under different treatments (lysozyme, ethanol + lysozyme, high temperature sterilization, high temperature sterilization + lysozyme).
[0072] Prepare LB medium, sterilize at 121°C, inoculate Ochrobactrum sp. and Micrococcus sp. respectively, and place them on a 180rpm shaker at 30°C for 24 hours. When both bacteria are in the logarithmic growth phase, samples are taken at the same time, and different experimental groups and Control treatment.
[0073] The bacterial cells of Ochrobactrum sp. and Micrococcus sp. treated with lysozyme were only subjected to wall-lysis treatment, and lysozyme with a final concentration of 200 mg / L was added to the bacterial culture solution diluted 20 times with PBS, kept at 37°C for 1 hour in a water bath, and discarded after centrifugation. clear;
[0074] In the experimental control group of Ochrobactrum sp. and Micrococcus sp. treated with ethano...
Embodiment 3
[0078] The relationship between the fluorescence intensity of two strains of phenol-degrading bacteria under the starvation environment pressure and the starvation time of bacterial cells under the two staining methods was verified.
[0079] Two strains of bacteria were screened into the sludge of the coking wastewater treatment reactor, including a Gram-negative strain and a Gram-positive strain, both of which had good phenol degradation effects. Control the inoculum size at 2%, carry out the degradation experiment with phenol as the only carbon source, find that in 0-60h, two strains of bacteria can realize the complete degradation of 750mg / L phenol ( Figure 4 ). Figure 4 A in A is Ochrobactrum sp., B is Micrococcus sp. Subsequently, through the full-length sequencing of the 16S rRNA of the two phenol-degrading bacteria, they were compared with the BLAST tool of NCBI and found that the two strains belonged to the genera Ochrobactrum and Micrococcus respectively.
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