Application of Pseudomonas ias03
A technology for Pseudomonas and mold, applied in the field of microorganisms, can solve problems such as residual risk, malachite green is not easy to degrade, and mutagenic
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Embodiment 1
[0025] Example 1 Isolation of Pseudomonas strain IAS03.
[0026] Test material: pond bottom mud.
[0027] Preparation of medium:
[0028] Beef extract peptone solid medium: peptone 10.0g, beef extract 3.0g, sodium chloride 5.0g, agar powder 15.0-20.0g, distilled water 1000mL, pH 7.2, sterilized at 121°C for 20min (beef extract peptone liquid medium is not Add agar powder).
[0029] KB solid medium: peptone 20.0g, glycerol 10.0mL, K 2 HPO 4 1.5g, MgSO 4 .7H 2 O1.5g, agar powder 15.0g-20.0g, deionized water 1000mL, pH 7.2, high temperature sterilization at 121°C for 20min.
[0030] experiment method:
[0031] (1) Weigh 10g of pond sediment into an Erlenmeyer flask, add 90mL of sterilized water, culture at 28°C, 160r / min constant temperature shaking for 30min, let stand for 5min, take the supernatant, and use the gradient dilution method to dilute the supernatant to 10 3 times, take 0.15mL of the diluted solution and spread it evenly on the beef extract peptone solid me...
Embodiment 2
[0035] The 16S rDNA species identification of embodiment 2 Pseudomonas bacterial strain IAS03
[0036] Test materials: bacterial universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGTTACCTTGTTACGACTT-3'), synthesized by Guangzhou Huada Gene Company.
[0037] experiment method:
[0038] (1) Amplification of 16S rDNA. The preparation of the seed solution of Pseudomonas IAS03 strain is the same as that in Example 1. The seed liquid of the strain was taken for colony PCR, and the primers were universal primers (concentration 10 μM): 27F and 1492R; the reaction system was 25 μL, including ddH 2 O 15.00 μL, KOD-Plus buffer 2.5 μL, dNTP mix (200 mmol / L) 2.5 μL, primer 27F 1.0 μL, primer 1492R 1.0 μL, bacterial solution 1.0 μL, KOD-Plus enzyme 0.5 μL, MgSO 4 (25mmol / L) 1.5μL. The reaction process is as follows: pre-denaturation: 95°C, 5min; denaturation: 94°C, 1min; annealing: 54°C, 1min; extension: 72°C, 90s; re-extension: 72°C, 10min. The PCR product was subjected...
Embodiment 3
[0042] The toxicity experiment of embodiment 3 bacterial strains
[0043] Experimental materials: 105 grass carp (body weight 30.0±5.5g, body length 12±2.5cm), 0.9% sterile saline.
[0044] Preparation of medium: the preparation of beef extract peptone medium is the same as in Example 1.
[0045] Preparation of injection bacterial solution:
[0046] Take the Pseudomonas IAS03 strain, and use an inoculation loop to streak and activate it on the beef extract peptone solid medium. After cultivating at a constant temperature at 28°C for 16 hours, use an inoculation loop to pick a single colony and inoculate it in the beef extract peptone liquid medium, at 28°C , 160r / min constant temperature shaking culture for 12h, then centrifuged at 4°C at 7500r / min for 15min, washed three times with 0.9% sterile normal saline, resuspended with 0.9% sterile normal saline, and finally passed the ultraviolet spectrum Photometer to measure OD 600 value, the concentration of the bacterial soluti...
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