By carefully designing the medium to contain selective agents, and
diagnostic compounds which will give a strong signal when they are
metabolized, it is possible to use electrical methods to estimate the
activity (and hence, by calibration, numbers) of specific groups of
organisms. Thus the incorporation of trimethylamine oxide (TMAO)
into a selective enrichment broth can be used as a pre-screening for the
presence ofSalmonella. The presence of the enzyme TMAO reductase
converts this neutral molecule into the strongly charged trimethylam-
monium ion which has a considerable effect on the conductance of the
medium. Absence of a detection time in control sample cells inoculated
with a Salmonella-specific phage act as further confirmation of the
presence ofSalmonella.
10.5.3 ATP Determination
Adenosine triphosphate is found in all living cells and is the universal
agent for the transfer of free energy from catabolic processes to anabolic
processes. A number of quite different living organisms have evolved a
mechanism for producing light by the activity of enzymes known as
luciferases on substrates known as luciferins. These reactions require the
presence of ATP and magnesium ions and produce one photon of light at
the expense of the hydrolysis of one molecule of ATP through a series of
intermediates. An ATP molecule facilitates the formation of an enzyme–
substrate complex which is oxidized by molecular oxygen to an electron-
ically excited state. The excited state of the molecule returns to the lower
energy ground state with the release of a photon of light and dissociates
to release the enzyme luciferase again. These reactions are summarized
below:-
Because instruments are now available which can accurately measure low
levels of light emission and pure luciferin and luciferase from, for
example fireflies, can be manufactured, the reaction can be used as a
very sensitive assay for ATP. Sensitive instruments using photomultiplier
386 Methods for the Microbiological Examination of Foods