Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The Amino Acid Sequence Of Ribitol Dehydrogenase-f, A Mutant Enzyme With Improved Xylitol Dehydrogenase Activity

A mutant ribitol dehydrogenase (RDH-F) was purified from Klebsiella aerogenes strain F which evolved from the wild-type strain A under selective pressure to improve growth on xylitol, a poor substrate used as sole carbon source. The ratio of activities on xylitol (500 mM) and ribitol (50 mM) was 0.154 for RDH-F compared to 0.033 for the wild-type (RDH-A) enzyme. The complete amino acid sequence of RDH-F showed the mutations. Q60 for E60 and V215 for L215 in the single polypeptide chain of 249 amino acid residues. Structural modeling based on homologies with two other microbial dehydrogenases suggests that E60 → Q60 is a neutral mutation, since it lies in a region far from the catalytic site and should not cause structural perturbations. In contrast, L215 → V215 lies in variable region II and would shift a loop that interacts with the NADH cofactor. Another improved ribitol dehydrogenase, RDH-D, contains an Al96 → P196 mutation that would disrupt a surface α-helix in region II. Hence conformational changes in this region appear to be responsible for the improved xylitol specificity. © 1999 Plenum Publishing Corporation.184489495Burleigh, B.D., Rigby, P.W.J., Hartley, B.S., (1974) Biochem. J., 143, pp. 341-352Butler, P.J.G., Hartley, B.S., (1972) Methods in Enzymology, 25, pp. 191-199. , (Hirs, C. H., and Timasheff, S. N., eds:), Academic Press, New YorkCintra, A.C.O., Vieira, C.A., Giglio, J.R., (1990) J. Protein Chem., 9, pp. 221-227Cintra, A.C.O., Marangoni, S., Oliveira, B., Giglio, J.R., (1993) J. Protein Chem., 12, pp. 57-64Dothie, J.M., Giglio, J.R., Moore, C.B., Taylor, S.S., Hartley, B.S., (1985) Biochem, J., 230, pp. 569-578Ghosh, D., Weeks, C.M., Grochulski, P., Duax, W.L., Erman, M., Rimsay, R.L., Orr, J.C., (1991) Proc. Natl. Acad. Sci. USA, 88, pp. 10064-10068Giglio, J.R., (1977) Anal. Biochem., 82, pp. 262-264Gray, W.R., (1972) Methods in Enzymology, 25, pp. 333-344. , (Hirs, C. H., and Timasheff, S. N., eds.), Academic Press, New YorkGuex, N., Peitsch, M.C., (1997) Electrophoresis, 18, pp. 2714-2723Hartley, B.S., (1984) Microorganisms As Model Systems for Studying Evolution, pp. 23-54. , (Mortlock, R. P., ed.), Plenum Press, New YorkHartley, B.S., (1984) Microorganisms As Model Systems for Studying Evolution, pp. 55-108. , (Mortlock, R. P., ed.), Plenum Press, New YorkHartley, B.S., Altosaar, I., Dothie, J.W., Neuberger, M.S., (1976) Structure-Function Relationship of Proteins, pp. 191-200. , (Markham, R., and Horn, R. W., eds.), Elsevier/North-Holland, AmsterdamHulsmeyer, M., Hecht, H.J., Niefeld, K., Hofer, B., Eltis, L.D., Timmis, K.N., Schomberg, D., (1998) Protein Sci., 7, pp. 1286-1293Itzaki, R.F., Gill, D.M., (1964) Analyt. Biochem., 9, pp. 401-410Laemmli, U.K., (1970) Nature, 227, pp. 680-685Lerner, S.A., Wu, T.T., Lin, E.C.C., (1964) Science, 146, pp. 1313-1315Loviny, T., Norton, P.M., Hartley, B.S., (1985) Biochem. J., 230, pp. 579-585Marangoni, S., Ghiso, J., Sampaio, S.V., Arantes, E.C., Giglio, J.R., Oliveira, B., Frangione, B., (1990) J. Protein Chem., 9, pp. 595-601Mortlock, R.P., Fossit, D.D., Wood, W.A., (1965) Proc. Natl. Acad. Sci. USA, 54, pp. 572-579Offord, R.E., (1966) Nature, 211, pp. 591-593Rigby, W.J., Burleigh, B.D., Hartley, B.S., (1974) Nature, 251, pp. 200-204Ryle, A.P., Sanger, F., Smith, L.F., Kitai, R., (1955) Biochem. J., 60, pp. 541-556Skoog, B., Wichman, A., (1986) Trends Anal. Chem., 5, pp. 82-93Taylor, S.S., Rigby, P.W.J., Hartley, B.S., (1974) Biochem. J., 141, pp. 693-700Wu, T.T., Lin, E.C.C., Tanaka, S., (1968) J. Bact., 96, pp. 447-45