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The cell-by-cell instrumental analysis of a bacterial population is new and advantageous. Traditional biochemical tests to characterize bacteria are often performed on isolated colonies grown from a single cell. Because these colonies are grown outside the fermentation vat, the conditions may be different, yielding a nonrepresentative sample. In addition, some methods of on-line analysis give average composition data over the entire population. Single cell analysis by MALDI MS has been performed on much larger eukaryotic cells, but the point-and-shoot ease of confocal Raman microspectroscopy is attractive. Also attractive is the cost. Schuster is using a
K. CHRISTIAN SCHUSTER
ance. In the November 15 issue of Analytical Chemistry (pp 5529–5534), Bernhard Lendl, K. Christian Schuster, and colleagues at the Vienna University of Technology (Austria) and Jobin-Yvon/ Dilor GmbH (Germany) reveal a unique way to study a nonhomogeneous population of Clostridium beijerinckii as it grows and changes: Raman microspectroscopy of the individual cell members. Cells within a bioprocess population will, at times, differ in properties and physiological status. In the case of C. beijerinckii, which is of interest because of its ability to convert agricultural waste into basic chemicals and fuels, the early growth and division stages produce butyrate and acetate. But in a later phase, cells differentiate into spore-forming cells or into large forms for storing starchlike granulose. Solvents such as acetone and butanol are produced in this stage, too. The performance of this fermentation and the related changes in metabolic pathways of the organism are not completely understood. Confocal Raman microspectroscopy enables single cells to be chosen visually, focused upon manually with the laser beam (which is at an attenuated power level to avoid damaging the cells), and finally analyzed. When focused, the beam illuminates about the same area as the cell (~1 µm2 for C. beijerinckii). “For many organisms, the bulk composition of [a] biomass is known or can be analyzed,” perhaps by FTIR spectroscopy, says Schuster. “[And] this data can potentially be used for ‘calibrating’ single cell spectra.”
The Raman microscope with (left) bacterial cells as seen through the light microscope attachment (actual size: 65 3 43 µm).
commercially available system that costs $60,000–70,000, which is significantly less than a MALDI mass spectrometer. Lendl and his co-workers point out that Raman microspectroscopy has advantages over the other techniques for single cell analysis. IR microspectroscopy has a spatial resolution of only ~10 µm, which
is too poor to single out smaller bacterial cells. Flow cytometry and microscopy with image analysis can zero in on a single bacterium, but these techniques introduce chemical stains and see only one, or a few, components. Raman microspectroscopy, on the other hand, does not introduce interfering substances; it uses only distilled water for washing and suspending the cells. And several different compounds might be detected from Raman spectral information. Nevertheless, there were some challenges in optimizing the system. To achieve a high enough sensitivity, and thus avoid more complicated methods like UV-excited resonance Raman or surface-enhanced Raman spectroscopy, the researchers chose a HeNe laser at 632.8 nm for excitation. They also had to direct their attention to finding the optimal carrier material. Sodium glass— from which microscope slides are made— had a high fluorescent background at the desired excitation wavelength. Quartz glass produced a lower noise level. But calcium fluoride carriers were best, producing little noise and a very flat baseline with only one sharp peak at 322 cm21. Schuster says that they will soon perform experiments to identify granulose and other suitable markers in the life cycle of C. beijerinckii. When asked if he has pointed his Raman microspectroscopy apparatus at other varieties of bacteria, he notes, “Not extensively, but experiments are on the way.” He cautions, “Other applications for different bioprocesses are feasible, but have to be studied in detail from case to case.” Gerald Keller
MEETINGS Frederick Conference on CE—Britt Erickson reports from Frederick, MD. Microbes meet CE It’s not too often that words such as prokaryotes, fungi, and protists show up in Analytical Chemistry, and if you are
like most of our readers, you probably don’t remember exactly what they mean. It may be time to pull out those dusty
biology books. On the basis of a group of talks presented at the 11th Frederick Conference on CE in October, it is
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which of these bacteria is causing the evident that separation scientists have begun bridging the gap between analyt- problem, directly from a urine sample. In another example, Armstrong used ical chemistry and microbiology. CE to quantify the active ingredient (a Daniel Armstrong of Iowa State microorganism) in acidophilus pills. University set the tone with a presentation on the challenges of separating inMicrobes in the tact bacteria. A big problem, he noted, environment is that there are few, if any, pure standards for microbes. Another problem, Characterizing microbes in the environhe pointed out, is that microbes tend to ment is another area in which CE has a aggregate, which can clog separation lot to offer, as demonstrated by Patrick channels. Although sonication helps Gillevet and co-workers at George prevent clumping, it is not foolproof. Mason University and SpectruMedix Microorganisms also tend to secrete Corp. The researchers have been substances that can surveying prokaryotes (bacteria), cause havoc with a fungi, and protists (algae and proseparation, and many tozoa) to establish a baseline inpolymer buffers comventory for microorganisms in esmonly used in CE are tuarine coastal environments. not compatible with According to Gillevet,
