# US 2014/0155273 A1 · Method for Detecting the Genus of Bacillus in Samples from Oil Reservoirs > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0155273 A1 - Document ID: US20140155273A1 - Status: Published application - Kind: A1 - Application number: 13/868,561 - Filed: 2013-04-23 - Published: 2014-06-05 - Earliest claimed: 2012-11-30 - Assignees: China University of Geosciences (Beijing) - Applicants: China University of Geosciences, Beijing - Inventors: Fan Zhang; Yuehui She; Dujie Hou; Lujun Chai - Web page: https://patentgrep.com/patent/US20140155273A1 ## CPC - C12Q 1/689: for bacteria - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The invention provides a quick method for detecting the genus of Bacillus in samples from oil reservoirs, which comprise: collecting samples from oil reservoirs; inactivating microbes except for spore-forming Bacillus in the samples through high temperature; collecting spores from the samples; incubating the collected spores in a medium, and stimulating to resurrect Bacillus under in situ temperatures of the oil reservoirs; etecting the resurrected Bacillus in the cultures using molecular biological techniques. The present invention provides an effective method to detect the genus of Bacillus in samples from oil reservoirs, which is conducive to the discovery of the functional Bacillus for microbial enhanced oil recovery (MEOR) and to revealing the ecosystem of Bacillus in oil reservoirs. ## Claims ### Claim 1 A quick method for detecting the genus Bacillus in samples from oil reservoirs, which comprises: (a) collecting samples from oil reservoirs; (b) inactivating microbes except for spore-forming Bacillus in the samples through high temperature; (c) collecting spores from the samples; (d) incubating the collected spores in an appropriate medium, and stimulating to resurrect the Bacillus under the in situ temperatures of the oil reservoirs; (e) detecting the resurrected Bacillus in the cultures using molecular biological techniques. ### Claim 2 (depends on 1) A quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 1, wherein said reservoirs are various oil reservoirs with different geological conditions and exploitation methods. ### Claim 3 (depends on 2) A quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 2, wherein said samples are selected from oil-water samples collected directly from the wellheads of injection wells, or production wells, or formation water samples, or flowing-back sample of the oil wells, or core samples from oil reservoirs. ### Claim 4 (depends on 3) A quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 3, wherein said step (a) of collecting samples comprising: oil-water samples are collected and put into sterile plastic bottles, full-filled, sealed and record the in situ temperature of the oil reservoirs of the collected samples. ### Claim 5 (depends on 4) A quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 4, wherein said step (b) of inactivating microbes except for spore-forming Bacillus in the samples through high temperature comprising: a 500 ml of the oil-water sample is transferred into a sterile flask in a sterile condition, and sealed with a sterile ventilate membrane; and put the flask on an electric heater and keep the oil-water sample boiling for 10 minutes, cool down at room temperature. ### Claim 6 (depends on 5) A quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 5, wherein said step (c) of collecting spores from the inactivating samples comprising: after cool down, the inactivating oil-water sample is transferred into 50 ml sterile centrifuge tubes in sterile condition and centrifuge at 10,000×g for 15 minutes, remove the supernatant to a sterile container for medium preparation; resuspend the pellets of precipitation in each centrifuge tube with 1 ml sterile water, and combine the suspension from all the centrifuge tubes into a 10 ml mixture; the mixture contains spores. ### Claim 7 (depends on 6) A quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 6, wherein said step (d) of incubating the collected spores in a medium, and stimulating to resurrect the Bacillus under the in situ temperatures of the oil reservoirs comprising: the 10 ml mixture collected in step (c) is incubated into a 100 ml flask with 30 ml sterilized medium, seal the cover of the flask, cultivate to resurrect the Bacillus at in situ temperature of the oil reservoir 10° C. to 110° C., from which the sample was collected, for 7-15 days at 150 rmp to obtain the resurrection medium; wherein said medium is compatible with the environments from which samples were collected, and can provide nutrients for stimulating spores, the medium (100 ml) includes: 2 g molasses, 0.16 g (NH4)2HPO4, 0.2 g KNO3, constant the volume with the supernatant collected in step 3 to 100 ml, and add 2 g crude oil; autoclave at 121° C. for 20 minutes, the crude oil is from the oil reservoirs where the sample was collected. ### Claim 8 (depends on 7) A quick method for detecting the genus Bacillus in samples from oil reservoirs according to claim 7, wherein said step (e) of detecting the resurrected Bacillus in the cultures using molecular biological techniques comprising: a 5 ml of the stimulating medium in the step (d) is transferred into a sterile centrifuge tube in a sterile condition, centrifuged at 10,000×g for 10 minutes for cell collection, remove the supernatant and add 1 ml distilled water to resuspend the cells sedimentation; DNA in the resuspension is extracted following the manufacturer's protocol of the FastDNA Spin Kit for Soil, and the extracted DNA is amplified by PCR using specific 16S RNA gene primers for bacteria to construct clone library, the representative clones are sequenced and are analysed to determine their phylogenetic affiliations.