Halobacterium halobium containing extreme halophiles halocin and enrichment culturing method and usage thereof
A technology of halophilic archaea and enrichment culture, applied in the field of microorganisms, can solve the problems of late start of halophilic research, few discovered species, few application reports, etc., and achieves the effects of stable properties, low cost and simple preparation process.
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Embodiment 1
[0008] Example 1: Culture and biological characteristics of halophilic bacteria Natrinema pallidum CGMCC No.1873
[0009] The halophilic bacteria (Natrinema pallidum) CGMCC No.1873 was inoculated in the complex medium, and the medium composition was as follows: sodium chloride 200g / l, hydrated magnesium sulfate 20g / l, potassium chloride 2g / l, trisodium citrate 3g / l , Yeast paste 10g / l, casein amino acid 7.5g / l, pH 7.0. 1.5% agar was added to the solid medium. Sterilize at 121°C for 30 minutes. Shake culture at 42°C and 150rpm on a shaker and static culture at a constant temperature in an incubator at 42°C. The bacteria has the following characteristics: (1) Colony morphology: the colony is round, with smooth surface, flat edges, raised, and reddish due to the presence of bacteriocin. (2) Cell morphology: Gram-negative bacteria, showing motility under the cell microscope (Olympus, BX40), the morphology is polymorphic, mostly rod-shaped (3-5μm×0.8-1.2μm), and some cells are s...
Embodiment 2
[0010] Example 2: PCR amplification and sequence determination of 16S rRNA gene of halophilic bacteria Natrinema pallidum CGMCC No. 1873
[0011] Natrinema pallidum CGMCC No.1873 was inoculated into the complex medium, and a ring of cells was directly picked from the slant, added with 400 μL of sterile water, mixed well, water bathed at 70°C for 5 minutes, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was directly used for PCR. A pair of universal primers for amplifying the 16S rRNA gene are as follows: forward primer: 5'-ATTCCGGTTGATCCTGC-3', reverse primer: 5'-AGGAGGTGATCCAGCCGCAG-3'. They correspond to bases 6-22 and 1540-1521 of the 16S rRNA gene of E. coli, respectively. The PCR reaction system (50 μL) was: 5 μL of 10×buffer, 1 μL of 10 mM dNTPs, 1 μL of 10 mM primers, 42 μL of ddH2O, 0.5 μL of Taq enzyme, and 0.5 μL of template DNA. The PCR reaction conditions were: denaturation at 94°C for 45s; annealing at 55°C for 45s; extension at 72°C for 90s, follow...
Embodiment 3
[0013] Example 3: Halophilic bacteria Natrinema pallidum CGMCC No.1873 halophilic extraction and screening of sensitive strains
[0014] Natrinema pallidum CGMCC No. 1873 CGMCC No.1873 cultured to the end of logarithmic growth phase was centrifuged at 4000rpm for 20min, the precipitate was discarded, the supernatant was dropped on a double-layer plate coated with indicator bacteria, and cultured at 37°C for 7-14d to observe whether a bacteriostatic zone was formed. Indicator strains used include: Natrinema altunense AJ2 T , Nnm.sp.AB53, Nnm.pellirubrum JCM 10476 T , Nnm.pallidum JCM 8980 T , Haloarcula sp.AJ4, Halobiforma lacisalsi AJ5 T , Hbf.haloterrestris JCM 11672 T , Hbf.sp.AB1, Halorubrum sp.AJ201, Hrr.saccharovorum NCIMB 2081 T , Hrr.coriense ch2 T , Hrr.distributum JCM 9100 T , Haloterrigena thermotolerans JCM 11050 T , Htg.saccharevitans AB14 T , Htg.sp.AJ233, Haloferax sp.YT226, Hfx.sp.YT228, Hfx.gibbonsii.CGMCC 1.2148 T , Hfx.volacnii CGMCC AS1.2150 T , Na...
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