Claims
- 1. A method for isolating a microorganism from an environmental sample, said method comprising the steps of:
a) providing a sample from an environment to be tested; b) providing a growth chamber, said growth chamber when sealed being permeable to diffusion of components from said environment but not permeable to cells of microorganisms; c) mixing an inoculum from said sample with a semi-solid support medium inside said growth chamber; d) sealing said chamber; e) incubating said sealed chamber under growth conditions such that components from said environment diffuse into said chamber and growth of microorganisms occurs; f) opening said chamber and examining said support medium for the presence of colonies of microorganisms; g) isolating cells from one of said colonies; and h) identifying said isolated microorganism.
- 2. The method of claim 1, further comprising the step of comparing characteristics of said newly isolated microorganism with a database of characteristics of known microorganisms to determine novelty.
- 3. The method of claim 1, wherein said sampled environment is selected from the group consisting of fresh water, seawater, sediments and soils.
- 4. The method of claim 3, wherein said soils comprise forest soil, farmland, tundra, alpine soil or landfill.
- 5. The method of claim 1, wherein said sampled environment is an area in a building and said sample is taken from a site selected from the group consisting of ventilation system, bathroom wall surface and hospital room surface.
- 6. The method of claim 1, wherein said growth chamber is sealable by a semi-permeable membrane, said membrane being permeable to diffusion of components of said environment but not permeable to cells of microorganisms.
- 7. The method of claim 1, wherein, in said mixing step, said support medium is soft agar.
- 8. The method of claim 7, wherein said soft agar is 0.7% agar.
- 9. The method of claim 7, wherein, in said mixing step, said inoculum and said soft agar are further mixed with autoclaved sand or mud.
- 10. The method of claim 1, wherein, in said opening and examining step, said surface is examined microscopically for the presence of microcolonies of microorganisms, and wherein, further, in said isolating step, cells are isolated from one of said microcolonies.
- 11. The method of claim 1, wherein, in said isolating step, cells are isolated from an indwelling colony in said support medium.
- 12. A method for isolating a microorganism from an environmental sample, said method comprising the steps of:
a) providing a sample from an environment to be tested; b) providing a growth chamber, said growth chamber when sealed being permeable to diffusion of components from said environment but not permeable to cells of microorganisms; c) mixing an inoculum from said sample with a support medium; d) processing said inoculum and said support medium to form a gel microdrop matrix; e) depositing said gel microdrop matrix comprising said inoculum in said growth chamber; f) seedling said chamber; g) incubating said sealed chamber under growth conditions such that components from said environment diffuse into said chamber and growth of microorganisms occurs; h) opening said chamber and examining said support medium in said gel microdrops for the presence of colonies of microorganisms; i) isolating cells from one of said colonies; and j) identifying said isolated microorganism.
- 13. A method for isolating a novel microorganism from an environmental sample, wherein said novel microorganism is capable of growing in artificial media only in co-culture with a helper microorganism, said method comprising the steps of:
a) providing a sample from an environment to be tested; b) combining a representative amount from said environmental sample with a helper microorganism to form an inoculum, wherein, in said inoculum, the number of microorganisms from said sample is in excess over the number of helper microorganisms; c) inoculating a first nutrient support medium with said inoculum; d) incubating said inoculated first nutrient support medium under growth conditions such that growth of colonies of microorganisms occurs; e) examining said colonies growing on said first nutrient support medium for the presence of clusters of colonies surrounding a central colony; f) isolating cells from one of said colonies from a cluster; g) inoculating a second nutrient support medium with said cells, in the absence of a helper microorganism; h) incubating said inoculated second nutrient support medium under said growth conditions, whereby growth of colonies of microorganisms would occur if said second nutrient support medium were capable of supporting growth of said cells without said helper microorganism; and i) designating any of said cells that grow on nutrient support medium in the presence of said helper microorganism but not in the absence of said helper microorganism as said novel microorganism capable of growing in artificial media only in co-culture with a helper microorganism.
- 14. The method of claim 13, wherein, in said inoculating steps, said inoculum is mixed with said nutrient support medium.
- 15. A method for isolating a novel microorganism from an environmental sample, wherein said novel microorganism is capable of growing in artificial media only in co-culture with a helper microorganism, said method comprising the steps of:
a) providing a sample from an environment to be tested; b) inoculating a first nutrient support medium with an inoculum from said sample; c) incubating said inoculated first nutrient support medium under growth conditions such that growth of colonies of microorganisms occurs; d) examining said first nutrient support medium for colonies; e) isolating cells from two or more of said colonies growing on said first nutrient support medium; f) placing said cells from two or more of said colonies growing on said first nutrient support medium adjacent to each other on a second nutrient support medium; g) incubating said cells on said second nutrient support medium under said growth conditions; h) examining said second nutrient support medium for colonies; i) isolating cells from one or more of said colonies growing on said second nutrient support medium; j) inoculating said cells from said one or more of said colonies growing on said second nutrient support medium onto separate nutrient support media; k) incubating said separate nutrient support media with said cells under said growth conditions; l) examining said separate nutrient support media for colonies; m) designating as a novel microorganism capable of growing in artificial media only in co-culture with a helper microorganism one that is seen to grow in step (h) but not in step (l); and n) designating as a helper microorganism capable of growing alone in artificial media and capable of supporting growth in co-culture of said novel microorganism one that is seen to grow in step (h) and also in step (l).
- 16. A method for isolating a novel microorganism from marine sediment, said method comprising the steps of:
a) providing a sample of marine sediment; b) providing a growth chamber, said growth chamber being sealable by a semi-permeable membrane, wherein said membrane is permeable to diffusion of growth components of seawater but is not permeable to cells of microorganisms; c) mixing an inoculum from said sample with a semi-solid support medium inside said growth chamber; d) sealing said chamber with said semi-permeable membrane; e) incubating said sealed chamber in a marine environment wherein components of seawater from said environment diffuse into said chamber and growth of microorganisms occurs; f) opening said chamber and examining said support medium microscopically for the presence of microcolonies of microorganisms; g) isolating cells from one of said microcolonies; and h) comparing characteristics of said newly isolated cells with a database of characteristics of known microorganisms to determine novelty.
- 17. The method of claim 16, wherein, in said mixing step, said support medium is soft agar.
- 18. The method of claim 17, wherein, in said mixing step, said inoculum and said soft agar are further mixed with autoclaved sand or mud.
- 19. The method of claim 17, wherein said soft agar is 0.7% agar.
- 20. A microorganism isolated according to the method of any one of claim 13 or claim 15.
- 21. A method of cultivating a microorganism according to claim 20, said method comprising the steps of:
a) providing a sample of a microorganism according to claim 20;b) combining a representative amount from said sample with a a helper microorganism to form an inoculum; c) inoculating a nutrient culture medium with said inoculum; and d) incubating said inoculated culture medium under growth conditions such that growth of microorganisms occurs.
- 22. A method of cultivating a microorganism according to claim 20, said method comprising the steps of:
a) providing a sample of a microorganism according to claim 20;b) providing an extract of or supernatant from cultivation of a helper microorganism; c) combining said sample with said extract or said supernatant to form an inoculum; d) inoculating a nutrient culture medium with said inoculum; and e) incubating said inoculated culture medium under growth conditions such that growth of microorganisms occurs.
- 23. The method of claim 22, wherein step (b) is carried out by providing an extract of or supernatant from microorganisms cultivated according to the method of claim 21.
- 24. A method of isolating a signaling compound for triggering growth of a microorganism capable of growing in artificial media only in co-culture with a helper microorganism, said method comprising the steps of:
a) providing an extract of or supernatant from cultivation of a helper microorganism; b) fractionating said extract or said supernatant; c) carrying out the method of claim 22, wherein, in step (b), fractions of said extract or said supernatant are used; d) identifying the active fraction from step(b); and e) continuing to purify the active compound in said active fraction to obtain said signaling compound.
- 25. The method of claim 24, wherein step (a) is carried out by providing an extract of or supernatant from microorganisms cultivated according to the method of claim 21.
- 26. A signaling compound isolated by the method of claim 24.
- 27. A method of domesticating a microorganism according to claim 20, said method comprising the steps of:
a) providing said microorganism; b) inoculating a nutrient support medium with said sample; c) incubating said inoculated nutrient support medium under growth conditions known to support growth of colonies of said microorganism only in the presence of a helper microorganism; d) examining said nutrient support medium for colonies; and e) isolating cells from a colony growing independently on said nutrient support medium as a domesticated said microorganism.
- 28. The method of claim 27, wherein, between step (a) and step (b), said microorganism is exposed to a mutagen.
- 29. An isolated culture of a microorganism, said microorganism comprising a 16S rRNA nucleic acid sequence having GenBank Accession No. AY062176 and ATCC Deposit No. ______, said organism being denominated MSC1.
- 30. An isolated culture of a microorganism, said microorganism comprising a 16S rRNA nucleic acid sequence having GenBank Accession No. AY062177 and ATCC Deposit No. , said organism being denominated MSC2.
- 31. A method of identifying a novel biologically active compound, comprising:
carrying out the method of claim 13 or claim 15;isolating a novel microorganism identified by said method; isolating a biologically active compound from said novel microorganism; determining the molecular structure of said biologically active compound; and comparing characteristics of said newly isolate biologically active compound with a database of characteristics of known compounds.
- 32. A biologically active compound identified by the method of claim 31.
- 33. A method of drug development, comprising:
carrying out the method of claim 31;preparing a new compound having a structure related to the molecular structure of said biologically active compound identified by the method of claim 31; and testing said new compound in an assay for biological activity.
- 34. A biologically active compound identified by the method of claim 33.
- 35. A method for manufacturing a pharmaceutical preparation, comprising:
carrying out the method of claim 31; and formulating the biologically active compound as a pharmaceutical preparation suitable for administration to a patient.
- 36. A method for manufacturing a pharmaceutical preparation, comprising:
carrying out the method of claim 33; and formulating the biologically active compound as a pharmaceutical preparation suitable for adminstration to a patient.
- 37. A pharmaceutical preparation comprising the compound of claim 32 formulated for administration to a patient.
- 38. A pharmaceutical preparation comprising the compound of claim 34 formulated for adminstration to a patient.
- 39. A method for treating a medical condition comprising:
carrying out the method of claim 35; and administering to a patient said pharmaceutical preparation manufactured by said method.
- 40. A method for treating a medical condition comprising:
carrying out the method of claim 36; and administering to a patient said pharmaceutical preparation manufactured by said method.
- 41. A method for treating a medical condition comprising:
administering to a patient the pharmaceutical preparation of claim 37.
- 42. A method for treating a medical condition comprising:
administering to a patient the pharmaceutical preparation of claim 38.
- 43. A method for isolating a microorganism from an environmental sample, said method comprising the steps of:
providing a sample from an environment to be tested; providing a growth chamber, said growth chamber when sealed being permeable to diffusion of components from said environment but not permeable to cells of microorganisms; mixing an inoculum from said sample with a support medium inside said growth chamber; sealing said chamber; incubating said sealed chamber under growth conditions such that components from said environment diffuse into said chamber and growth of microorganisms occurs; opening said chamber and examining said support medium for the presence of colonies of microorganisms; isolating cells from one of said colonies; and identifying said isolated microorganism.
- 44. A method of domesticating a microorganism isolated according to claim 1, said method comprising the steps of:
a) providing said microorganism isolated from a growth chamber; b) inoculating an artificial nutrient support medium with said microorganism; c) incubating said inoculated nutrient support medium; d) examining said nutrient support medium for colonies; and e) isolating cells from a colony growing independently on said nutrient support medium as a domesticated said microorganism.
- 45. The method of claim 44, wherein, between step (a) and step (b), said microorganism is exposed to a mutagen.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of U.S. Provisional Application No. 60/325,052 filed Sep. 26, 2001, entitled METHOD AND DEVICE FOR CULTIVATION AND ISOLATION OF MICROORGANISMS FROM NATURAL ENVIRONMENTS, and of U.S. Non-Provisional Application No. ______ filed May 1, 2002, entitled ISOLATION AND CULTIVATION OF MICROORGANISMS FROM NATURAL ENVIRONMENTS AND DRUG DISCOVERY BASED THEREON, the whole of which are hereby incorporated by reference herein.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Part of the work leading to this invention was carried out with United States Government support provided under a grant from the National Science Foundation, Grant No. OCE0102248. Therefore, the U.S. Government has certain rights in this invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60325052 |
Sep 2001 |
US |