Symbiotic Fungi: Principles and Practice (Soil Biology)

7 In Vitro Compartmented Systems to Study Transport 117
be conducted by the measurement of the relative abundance of isotopes if stable
isotopes are used as tracers or of radioactivity in the case of radioactive tracers.
Instruments used to detect stable isotopes include principally mass spectrographs
and nuclear magnetic resonance (NMR) spectrometers. Instruments used to detect
radiation may include liquid scintillation counters, gamma counters and (micro)autoradiographs.
The isotopic tracer should be sterilized before addition to the Petri plate. Filtersterilization
using a 0.2 mm filter is recommended.
7.6.1 Stable or Radio-Isotopic Tracer?
The choice between stable and radioactive isotopic labeling depends on:
l The purpose of the experiment. If the goal is to demonstrate the presence of an
element, both stable and radioactive isotopes can be used. However, if the goal is
to study a metabolic process, a stable isotope should be used in combination with
NMR spectrometry or mass spectrometry (MS).
l The availability of the stable and suitability of the radioactive isotope. Some
elements have no stable isotope (i.e. technetium, tungsten and promethium and
all elements with an atomic number greater than 82). Therefore, all tracers used
to investigate these elements must be their radioactive forms. Conversely,
some elements have only radioactive isotopes with a very short half-life (i.e.
inferior to a few days). Therefore, they cannot, or only with difficulty, be used
for transport studies. In this case the tracer must be a low-abundance stable
isotope prepared in enriched form. Also, not all stable isotopes can be used
with NMR spectrometry (i.e. isotopes with a zero nuclear spin) or mass
spectrometry (i.e. isobars). Note that isobars and isobar molecules can be
distinguished using Accelerator Mass spectrometry (AMS) or Resonance-
Ionization Mass Spectrometry (RIMS), but these instruments are only available
in some highly specialized laboratories.
l The detection limit of the instruments. The choice between stable and radioactive
isotopes can be determined by the so-called ‘‘dilution factor’’, which is
a measure of the concentration of tracer required for detection. Generally,
radioactive tracers may be detected in much lower quantities than stable tracers.
For example, a pure 13 C tracer would be detectable after being diluted 100,000 to
1 million times with natural 12 C. Radioactive 14 C, however, can be detected after
being diluted 25 billion times with 12,13 C.
For many studies, it is possible to choose between stable-isotope tracers
and radioactive-isotope tracers. For example, it is possible to choose between 13 C
(stable) and 14 C (radioactive) or 31 P (stable) and 32,33 P (radioactive) isotopes.