Method for isolating and culturing unculturable microorganisms
First Claim
1. A method for isolating and culturing a previously unculturable microorganism, which comprises:
- (i) collecting a sample from an environmental source;
(ii) counting/estimating the number of microorganisms in the sample;
(iii) diluting the sample in an appropriate medium;
(iv) adding a gelating agent such as to entrap one or more microorganisms within a sphere of the gelating agent;
(v) coating the spheres containing the entrapped microorganism(s) with a natural or synthetic polymer to form a polymeric membrane;
(vi) incubating the coated spheres in the original environment for an appropriate time;
(vii) cutting the spheres and scanning for microorganisms colonies; and
(viii) isolating the microorganisms, and repeating steps (iii) to (vii) until a pure clone of said previously unculturable microorganism is obtained.
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Accused Products
Abstract
The invention provides a method for isolating and culturing a previously unculturable microorganism, which comprises: (i) collecting a sample from an environmental source; (ii) counting/estimating the number of microorganisms in the sample; (iii) diluting the sample in an appropriate medium; (iv) adding a gelating agent such as to entrap one or more microorganisms within a sphere of the gelating agent; (v) coating the spheres containing the entrapped microorganism(s) with a natural or synthetic polymer to form a polymeric membrane; (vi) incubating the coated spheres in the original environment for an appropriate time; (vii) cutting the spheres and scanning for microorganisms colonies; and (viii) isolating the microorganisms, and repeating steps (iii) to (vii) until a pure clone of said previously unculturable microorganism is obtained.
121 Citations
19 Claims
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1. A method for isolating and culturing a previously unculturable microorganism, which comprises:
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(i) collecting a sample from an environmental source;
(ii) counting/estimating the number of microorganisms in the sample;
(iii) diluting the sample in an appropriate medium;
(iv) adding a gelating agent such as to entrap one or more microorganisms within a sphere of the gelating agent;
(v) coating the spheres containing the entrapped microorganism(s) with a natural or synthetic polymer to form a polymeric membrane;
(vi) incubating the coated spheres in the original environment for an appropriate time;
(vii) cutting the spheres and scanning for microorganisms colonies; and
(viii) isolating the microorganisms, and repeating steps (iii) to (vii) until a pure clone of said previously unculturable microorganism is obtained. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 18, 19)
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10. A method for genomic characterization of previously unculturable microorganisms, which comprises:
- (i) collecting a sample from an environmental source;
(ii) counting/estimating the number of microorganisms in the sample;
(iii) diluting the sample in an appropriate medium;
(iv) adding a gelating agent such as to entrap one or more microorganisms within a sphere of the gelating agent;
(v) coating the spheres containing the entrapped microorganism(s) with a natural or synthetic polymer to form a polymeric membrane;
(vi) incubating the coated spheres in the original environment for an appropriate time;
(vii) cutting the spheres and extracting the microorganisms by chemical lysis using an agent for extraction of genomic DNA;
(viii) processing the total genomic DNA to establish the restriction fragment length polymorphism (RFLP) pattern of the microorganisms;
(ix) analyzing the RFLP patterns to identify unique clones that are submitted to sequence analysis; and
(x) identifying the microorganisms by comparison of these sequences with sequences available at the GenBank database. - View Dependent Claims (11, 12, 13, 14, 15, 16)
- (i) collecting a sample from an environmental source;
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17. (canceled)
Specification