Chapter 18 : Chemical Analysis

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The methods selected for this chapter are most commonly used in research relating to microbial components and metabolism, but most are also suited to advanced undergraduate and graduate teaching laboratories. The incorporation of radioactively labeled compounds into bacterial cells is used as a means of estimating the rates and amounts of synthesis as well as the distribution of the main small molecule and macromolecule fractions of the cells. Macromolecules are precipitated by cold dilute solutions of trichloroacetic acid. The precipitate fraction of macromolecules is sequentially extracted with organic solvents for lipids, alkali for RNAs, and hot trichloroacetic acid for DNAs; the precipitate remaining after these extractions contains the cell proteins and peptidoglycan. Some macromolecules contain both carbohydrate and noncarbohydrate components (lipopolysaccharides, peptidoglycans, teichoic acids, lipoteichoic acids, teichuronic acids, nucleic acids, glycoproteins). In some cases gas-liquid chromatography can be useful when coupled with mass spectrometry. For nitrite analysis, the most widely accepted method involves modification of the diazotization and coupling reactions. The methods described in this section differ mainly in how the DNA is purified and prepared. A number of short-chain organic acids and alcohols are formed in bacterial cells as intermediates or end products of the citric acid, glycolytic, and other metabolic pathways.

Citation: Daniels L, Hanson R, Phillips J. 2007. Chemical Analysis, p 462-503. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch18
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Flow diagram of fractionation procedure.

Citation: Daniels L, Hanson R, Phillips J. 2007. Chemical Analysis, p 462-503. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch18
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Generic image for table

Comparison of analytical techniques for analysis of nonprotein nitrogen compounds

For further information and sensitivity, see analysis listed under appropriate nitrogen class.

Citation: Daniels L, Hanson R, Phillips J. 2007. Chemical Analysis, p 462-503. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch18
Generic image for table

Comparison of analytical techniques available for protein assay

Sensitivity is given in micrograms per ml of solution in the assay. This can be converted easily to micrograms per ml of original solution by accounting for the dilution factors. When a range is given, both the extrasensitive microassay and the routine, less sensitive assays are described. Interfering substances may reduce this sensitivity but still allow accurate assays in an appropriate range.

DTT, dithiothreitol.

Glucose, Tris buffer, and ammonium sulfate can cause interference but can be corrected with proper blanks or pH adjustment.

Citation: Daniels L, Hanson R, Phillips J. 2007. Chemical Analysis, p 462-503. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch18

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