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Determination of Cell Mass
Appendix
13 Determination of Cell Mass
Growth of cultures can be monitored by spectrophotometry. The number of photons scattered is proportional to the mass of the cells in a sample (except for very concentrated cultures, as discussed in the next paragraph) or, for particular growth conditions, to the cell concentration. However, the geometry of each spectrophotometer determines how much scattered light falls onto a photodetector. Therefore, a calibration curve that relates cell concentration and absorbance must be made for each spectrophotometer. This is done by measuring the absorbance of suspensions of cells at different concentrations and determining the cell concentration of each suspension by direct counting or by plating on agar and measuring the number of colonies formed. It is important to note that the absorbance is a measure of cell mass rather than cell number. Cell size varies with growth phase, so it is best to calibrate the spectrophotometer with exponentially growing cells because such cells are used in most experiments. Cell size also varies from one growth medium to the next, decreasing as the medium becomes poorer, so a calibration curve is needed for each growth medium, and often for each strain. As a rough guide, for many Escherichia coli strains, A6oo = 1.0 for ~-8 x 10 8 cells/ml and for Saccharomyces cerevisiae, A6oo = 1.0 for ---3 x 10 7 cells/ml. If the cell density is too high, a photon may be deflected away from the photodetector by one cell and then back again by a second cell. This effect causes the absorbance to be lower than if multiple 219
220
Appendix 13
scattering were not occurring; it becomes important at values of A6oo above 0.7. Thus, when the concentration of a dense culture is to be determined by spectrophotometry, the culture is diluted prior to reading. The measured value is then corrected by the dilution factor. Wavelengths other than 600 nm can be employed in determining cell density and, in fact, the sensitivity increases as the wavelength decreases. Wavelengths as low as 400 nm may be used, but not with all rich media. These media usually absorb short-wavelength light significantly and the absorption complicates the measurements.
13 Determination of Cell Mass
Growth of cultures can be monitored by spectrophotometry. The number of photons scattered is proportional to the mass of the cells in a sample (except for very concentrated cultures, as discussed in the next paragraph) or, for particular growth conditions, to the cell concentration. However, the geometry of each spectrophotometer determines how much scattered light falls onto a photodetector. Therefore, a calibration curve that relates cell concentration and absorbance must be made for each spectrophotometer. This is done by measuring the absorbance of suspensions of cells at different concentrations and determining the cell concentration of each suspension by direct counting or by plating on agar and measuring the number of colonies formed. It is important to note that the absorbance is a measure of cell mass rather than cell number. Cell size varies with growth phase, so it is best to calibrate the spectrophotometer with exponentially growing cells because such cells are used in most experiments. Cell size also varies from one growth medium to the next, decreasing as the medium becomes poorer, so a calibration curve is needed for each growth medium, and often for each strain. As a rough guide, for many Escherichia coli strains, A6oo = 1.0 for ~-8 x 10 8 cells/ml and for Saccharomyces cerevisiae, A6oo = 1.0 for ---3 x 10 7 cells/ml. If the cell density is too high, a photon may be deflected away from the photodetector by one cell and then back again by a second cell. This effect causes the absorbance to be lower than if multiple 219
220
Appendix 13
scattering were not occurring; it becomes important at values of A6oo above 0.7. Thus, when the concentration of a dense culture is to be determined by spectrophotometry, the culture is diluted prior to reading. The measured value is then corrected by the dilution factor. Wavelengths other than 600 nm can be employed in determining cell density and, in fact, the sensitivity increases as the wavelength decreases. Wavelengths as low as 400 nm may be used, but not with all rich media. These media usually absorb short-wavelength light significantly and the absorption complicates the measurements.