Spectroscopy WORLD OF MICROBIOLOGY AND IMMUNOLOGY524•
absorbance of a control. Typically, a bacterial control is
uninoculated growth medium, so the absorbance should be
zero. In typical growth curve studies, the bacterial culture can
be grown in a special flask called a side-arm flask. The side
arm is a test tube that can be inserted directly into a spec-
trophotometer.
Double-beam spectrophotometers are also available and
are the norm now in research microbiology laboratories. In
these instruments the light beam is split into two beams by
means of mirrors. One light path goes through the sample
chamber and the other light beam passes through what is
referred to as the reference cell or chamber. The ration of the
absorbance between the two chambers is computed and is
used to determine sample concentration.
Depending on the spectrophotometer, absorbance can
be taken at a single wavelength, or scanned through a spec-
trum of wavelengths. The latter can be a useful means of iden-
tifying components of the sample, based on their preferential
absorption of certain wavelengths of light.See alsoLaboratory techniques in microbiologySSpectroscopyPECTROSCOPY
Because organisms present unique spectroscopic patterns,
spectroscopic examination (e.g., Raman spectroscopy) of
microorganisms(e.g., microbial cells) can help to differentiate
between species and strains of microbes. Spectroscopic exam-
ination can also aid in the identification and measurement of
subcellular processes (e.g., CO 2 production) that facilitate the
understanding of cell growth, response to environmental stim-
uli, and drug actions.
The measurement of the absorption, emission, or scat-
tering of electromagnetic radiation by atoms or molecules is
referred to as spectroscopy. A transition from a lower energy
level to a higher level with transfer of electromagnetic energy
to the atom or molecule is called absorption; a transition from
a higher energy level to a lower level results in the emission of
a photon if energy is transferred to the electromagnetic field;
and the redirection of light as a result of its interaction with
matter is called scattering.
When atoms or molecules absorb electromagnetic
energy, the incoming energy transfers the quantized atomic or
molecular system to a higher energy level. Electrons are pro-
moted to higher orbitals by ultraviolet or visible light; vibra-
tions are excited by infrared light, and rotations are excited by
microwaves. Atomic-absorption spectroscopy measures the
concentration of an element in a sample, whereas atomic-
emission spectroscopy aims at measuring the concentration of
elements in samples. UV-VIS absorption spectroscopy is used
to obtain qualitative information from the electronic absorp-
tion spectrum, or to measure the concentration of an analyte
molecule in solution. Molecular fluorescence spectroscopy is
a technique for obtaining qualitative information from the
electronic fluorescence spectrum, or, again, for measuring the
concentration of an analyte in solution.Infrared spectroscopy has been widely used in the study
of surfaces. The most frequently used portion of the infrared
spectrum is the region where molecular vibrational frequen-
cies occur. This technique was first applied around the turn of
the twentieth century in an attempt to distinguish water of
crystallization from water of constitution in solids.
Ultraviolet spectroscopy takes advantage of the selec-
tive absorbance of ultraviolet radiation by various substances.
The technique is especially useful in investigating biologically
active substances such as compounds in body fluids, and drugs
and narcotics either in the living body (in vivo) or outside it
(in vitro). Ultraviolet instruments have also been used to mon-
itor air and water pollution, to analyze dyestuffs, to study car-
cinogens, to identify food additives, to analyze petroleum
fractions, and to analyze pesticide residues. Ultraviolet photo-
electron spectroscopy, a technique that is analogous to x-ray
photoelectron spectroscopy, has been used to study valence
electrons in gases.
Microwave spectroscopy, or molecular rotational reso-
nance spectroscopy, addresses the microwave region and the
absorption of energy by molecules as they undergo transitions
between rotational energy levels. From these spectra, it is pos-
sible to obtain information about molecular structure, including
bond distances and bond angles. One example of the applica-
tion of this technique is in the distinction of trans and gauche
rotational isomers. It is also possible to determine dipole
moments and molecular collision rates from these spectra.
In nuclear magnetic resonance (NMR), resonant energy
is transferred between a radio-frequency alternating magnetic
field and a nucleusplaced in a field sufficiently strong to
decouple the nuclear spin from the influence of atomic elec-
trons. Transitions induced between substrates correspond to
different quantized orientations of the nuclear spin relative to
the direction of the magnetic field. Nuclear magnetic reso-
nance spectroscopy has two subfields: broadline NMR and
high resolution NMR. High resolution NMR has been used in
inorganic and organic chemistry to measure subtle electronic
effects, to determine structure, to study chemical reactions,
and to follow the motion of molecules or groups of atoms
within molecules.
Electron paramagnetic resonance is a spectroscopic
technique similar to nuclear magnetic resonance except that
microwave radiation is employed instead of radio frequencies.
Electron paramagnetic resonance has been used extensively to
study paramagnetic species present on various solid surfaces.
These species may be metal ions, surface defects, or adsorbed
molecules or ions with one or more unpaired electrons. This
technique also provides a basis for determining the bonding
characteristics and orientation of a surface complex. Because
the technique can be used with low concentrations of active
sites, it has proven valuable in studies of oxidation states.
Atoms or molecules that have been excited to high
energy levels can decay to lower levels by emitting radiation.
For atoms excited by light energy, the emission is referred to as
atomic fluorescence; for atoms excited by higher energies, the
emission is called atomic or optical emission. In the case of
molecules, the emission is called fluorescence if the transitionwomi_S 5/7/03 8:20 AM Page 524