Method for isolating and culturing unculturable microorganisms

a microorganism and uncultivable technology, applied in the field of isolating and culturing novel “ uncultivable” microorganisms, can solve the problems of small percentage (less than 1%) of viable bacteria in soil and restricted cell movemen

Inactive Publication Date: 2006-10-26
BEN GURION UNIVERSITY OF THE NEGEV
View PDF6 Cites 122 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010] In one preferred embodiment of the invention, the sample is collected from an environmental source and diluted in an appropriate medium after counting / estimating the number of microorganisms in the sample. A gelating agent is then added such as to entrap one or more microorganisms within a sphere of the gelating agent. The spheres containing the entrapped microorganism(s) are then coated with a natural or synthetic polymer to form a polymeric membrane. The coated spheres with the entrapped microorganisms are incubated in the original environment and, after an appropriate time, are cut and scanned for microorganisms colonies. The microorganisms are isolated and subjected one or more times to the steps of dilution in an appropriate medium, entrapping in a gelating agent, coating of the spheres containing the entrapped microorganisms, incubation of the coated spheres, cutting the spheres and scanning for microorganisms colonies, until a pure clone of said previously unculturable microorganism is obtained.

Problems solved by technology

However, only a small percentage (less than 1%) of the viable bacteria in soil can be cultured on known nutrient media using current techniques such as petri dishes (Handelsman et al.
The membranes allowed exchange of chemicals between the chamber and the environment, but restricted movement of cells.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for isolating and culturing unculturable microorganisms
  • Method for isolating and culturing unculturable microorganisms

Examples

Experimental program
Comparison scheme
Effect test

example 1

Laboratory Scale Wastewater Bioreactor

[0054] An environmental sample was obtained from laboratory scale wastewater bioreactor (waste water from Ramat Hovav Toxic Waste Dumping Site, Negev, Israel) and estimated for microorganism number by DAPI-staining and microscope direct counting (108-109 cell / mL). The sample was diluted 8- and 9-fold with water in order to entrap approximately one microorganism in one agar sphere.

[0055] The diluted samples were mixed with warm (50° C.) autoclaved agar (DIFCO) (900 μl agar per 100 μl diluted sample, final concentration 1.5% agar). Agar spheres of approximately 1-2 mm in diameter containing the entrapped microorganism(s) were formed by dripping droplets of the mixture into cold mineral oil.

[0056] A solution of 10% polysulfone of m.w. ca. 35,000 (Sigma-Aldrich, Product No. 42,830-2) in DMF was prepared and used to coat the dried agar spheres containing the entrapped microorganism(s). For this purpose, the agar spheres were introduced into the po...

example 2

Microbial Communities Inhabiting Coral Mucus

[0059] Mucus of coral heads from the Red Sea was sampled by collecting ca. 1 ml−1 in a sterile disposable 50-ml polypropylene centrifuge tube. Mucus bacteria were counted directly under phase microscopy. Mucus was diluted by sterile seawater and placed on marine agar for culturable colony forming units (CFU). Several dilutions (10−7, 10−6, 10−5 and 10−4) were mixed with 2% marine agar in ratio 1 to 1 and agar spheres were coated with polysulphone and incubated in a seawater aquarium containing corals for several weeks. Agar spheres of approximately 1-2 mm in diameter containing the entrapped microorganism(s) were formed by dripping droplets of the mixture into cold mineral oil.

[0060] A solution of 10% polysulfone of m.w. ca. 35,000 (Sigma-Aldrich, Product No. 42,830-2) in DMF was prepared and used to coat the dried agar spheres containing the entrapped microorganism(s). For this purpose, the agar spheres were introduced into the polymer ...

example 3

Microbial Communities Inhabiting Soil

[0069] A sample was collected from the soil of the Halutza region, Negev, Israel, and processed as described in Example 2. The medium was sterile tapwater and the incubation of the polysulfone-coated agar spheres was carried out in pots filled with soil material from the Halutza region. A clone library constructed from agar spheres (10 colonies) inoculated with the soil bacteria was incubated for 3 weeks, analyzed as described above and revealed two different patterns by RFLP by ratio 9 to 1. DNA from two clones were partially sequenced by using direct (8F) and reversed (1512R) primers and microorganism identification was based on comparison of these sequences with the GenBank database.

[0070] The most abundant sequence (90%) of this sample of soil bacteria is characterized by partial 16S rDNA sequences of SEQ ID NO:9 and SEQ ID NO:10, that represent Proteobacteria (phylum), Betaproteobacteria (class), Burkholderiales (order), Alcaligenaceae (fa...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
sizeaaaaaaaaaa
sizeaaaaaaaaaa
thicknessaaaaaaaaaa
Login to view more

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.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for isolating and culturing novel “uncultivable” microorganisms. BACKGROUND OF THE INVENTION [0002] Cultured microorganisms are the most common source of antibiotics and other medicinal agents. However, only a small percentage (less than 1%) of the viable bacteria in soil can be cultured on known nutrient media using current techniques such as petri dishes (Handelsman et al. 1998; Amann et al. 1995). The other 99% of uncultured / uncultivable microorganisms, with their genetic and biochemical diversity, may emerge as a major source of new natural chemical structures that may be useful for humans, for example as drugs. [0003] The exploration of previously uncultured microorganisms for the discovery of new useful natural products is now being carried out in several laboratories. The main approach involves genomics techniques such as the approach designated metagenomics for the analysis of the collective genomes of t...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/04C12N1/20C12N1/00C12P21/04
CPCC12N1/00
Inventor KUSHMARO, ARIELGERESH, SHIMONAGERESH, SHAUL
Owner BEN GURION UNIVERSITY OF THE NEGEV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap