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
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
These cells can then be cultivated further or analyzed via single cell
Isolation of individual bacteria
The workstation consists of an inverse research microscope with up to
100-fold magnification (phase contrast), an Eppendorf Micromanipulator 5171
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.
- 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
- 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.
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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