Growth Measurement

Arthur L. Koch

doi:10.1128/9781555817497.ch9

Chapter 9 : Growth Measurement

Author: Arthur L. Koch

Content Type: Reference

Publication Year: 2007

Category: Microbial Genetics and Molecular Biology

Book DOI: 10.1128/9781555817497

Chapter DOI: 10.1128/9781555817497.ch9

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Abstract:

This chapter talks about four classes of pitfalls in bacterial growth measurement. The change of enteric bacteria from large RNA-rich forms in the exponential phase to small RNA-poor forms in the stationary phase has many of the aspects of differentiation. Evidence of the difficulties involved is the fact that many of the people who helped develop the technique for measuring distribution of cell volumes no longer use it. This article, therefore, only presents the principles, mentions the difficulties and the attempted solutions to these difficulties, and directs the reader to published literature. Three colony count methods are discussed. The first is the spread plate, in which all colonies are surface colonies. The second method is the layered plate. The third method is the pour plate. Flow cytometry has become an extremely powerful method for the studies of many aspects of the biology of eucaryotes, but the methods are only now coming into their own in the study of the biology of procaryotes. The concentration of viable cells can be roughly estimated by the most-probable-number (MPN) method. For growth measurements, the time can be precisely defined, and the major error is in the measurement of the number of cells or other indices of biomass.

Citation: Koch A. 2007. Growth Measurement, p 172-199. 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.ch9

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Growth Measurement

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FIGURE 1

Spreaders. The one at the top is bent from a paper clip and fitted into a length of Teflon tubing. The one at the bottom is made by fusing the end of a Pasteur pipette and then bending it sharply in two places by using a Bunsen burner with a wing tip to define the spreading region. The handles of both are bent to the convenience of the user. Both spreaders have low heat capacity. The spreader at the top absorbs fewer bacteria because of its Teflon construction.

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FIGURE 2

Errors that arise in using the MPN method. The CV for a single level of dilution is shown as a function of the percentage of sterile tubes (bottom abscissa) and the mean number of cells per tube (top abscissa). The respective optima occur at 20.8% and 1.59 mean cells per tube for a varying number of tubes (n) prepared at a single dilution level.

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FIGURE 2

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FIGURE 3

Typical water sorption isotherm curve for bacterial cells.

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FIGURE 3

Typical water sorption isotherm curve for bacterial cells.

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FIGURE 4

Light-scattering patterns from randomly oriented bacteria showing the angular distribution of light scattered by a beam traversing from left to right through a suspension of cells with the size and physical properties of Escherichia coli grown in minimal medium. The upper pattern shows the distribution if the cells are ellipsoidal with an axial ratio of 4:1. The lower pattern shows the distribution for spherical cells.

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FIGURE 5

Schematic designs for filter-type “colorimeter” instruments. (A) Narrow-beam instrument. Almost all the scattered light deviates enough to escape falling on the photodetector. (B) Wide-beam instrument. A variable fraction of the scattered light falls on the detector, and thus the sensitivity is lowered.

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FIGURE 6

Absorbance (at 420 nm) as a function of bacterial cell concentration. The dashed line shows the theoretical relationship. The solid line shows an actual result obtained with 15 dilutions of a bacterial culture, whose concentrations were measured by dry-weight determinations and whose plot was obtained by the best-fitting quadratic curve that was forced to pass through the origin.

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FIGURE 7

Growth curve plotted on semilogarithmic paper. See the text for conditions. The horizontal arrows are drawn to the curve at values of bacterial concentrations that are convenient powers of 2 apart, which facilitates estimations of the doubling time.

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Tables

Growth Measurement

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TABLE 1

Sample data for exponential growth by graphical estimation or pocket calculator computation

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TABLE 1

Sample data for exponential growth by graphical estimation or pocket calculator computation

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SAMPLE DATA FOR MPN COMPUTATION

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SAMPLE DATA FOR MPN COMPUTATION

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TABLE A1

Sample data and output, E. coli ML308, 28°C, M9 Med, 10/25/2003, Run B

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TABLE A1

Sample data and output, E. coli ML308, 28°C, M9 Med, 10/25/2003, Run B