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Isolation of Microorganisms

Jrgen Frhlich, Helmut Knig and Dietmar Kahle
Jrgen Frhlich and Helmut Knig; Institute for Microbiology and Wine Research,
Johannes-Gutenberg-University; Mainz, Germany
Dietmar Kahle, Eppendorf AG Problem The isolation of microorganisms (such as bacteria or yeast) from complex mixed cultures and their cultivation in a pure culture is an essential prerequisite for their precise identification and characterization.

Several different processes are used for this purpose, including the classic platecasting process invented by Robert Koch, as well as dilution methods or selective enrichment.

However, it can prove to be very difficult to isolate microbes when the conditions for their growth are not known or when the microbes grow in a heterogeneous mixed culture in very small numbers only.

Such experiments are now possible thanks to new techniques which have been developed recently (e.g., optical laser tweezers).

This article outlines a novel method which, with the aid of a micromanipulation workstation, enables the isolation of individual bacteria from a mixed culture.

These cells can then be cultivated further or analyzed via single cell PCR*.

Isolation of individual bacteria Material The workstation consists of an inverse research microscope with up to 100-fold magnification (phase contrast), an Eppendorf Micromanipulator 5171 or TransferMan device as well as the Eppendorf CellTram Oil hydraulic piston pump.

Isolation was carried out with special sterile glass capillaries. The dimensions of these microcapillaries depend on the size of the cells to be isolated. For typical experiments, capillaries with an inner diameter of 5 m and a tip angle of 45 were used. These capillaries were designed in accordance with our specifications (Eppendorf).

Dried bacteria cultures were resuspended using suitable sterile nutrient solutions. The isolated cells were then transferred into the appropriate micro test tubes for further processing.

Advantages
  • This process can be used for cloning and isolating microorganisms (such as bacteria, archaea, protozoa and fungi) as well as eukaryotic organelles and cell nuclei.
  • The risk of contamination during the identification of non-cultivatable microorganisms from mixed cultures by means of single cell PCR is greatly reduced.
  • Microorganisms present in low numbers can also be examined.
  • In addition to aerobic microorganisms, it is also possible to isolate anaerobic microorganisms (with the appropriate accessories).
  • The electronic control of the micro-manipulator and the high-resolution piston pump guarantee rapid and simple isolation.
Conclusion Microorganisms which were previously difficult to access can now be isolated and characterized rapidly using this simple and relatively inexpensive method.

J. Frhlich and H. Knig (1999) Rapid isolation of single microbial cells from mixed natural and laboratory populations with the aid of a micromanipulator. System. Appl. Microbiol. 22, 249-257.


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