One part is measured at nm using a photometer, while the other part of the sample is subjected to serial dilution. A defined volume of each dilution is then spread onto a separate agar plate and incubated. If the values are plotted against the concentration, a calibration curve will result that will serve as the basis for subsequent measurements of the same strain using the same instrument. The following points should be considered when measuring OD : Since a culture constitutes a suspension, homogeneity of the sample is critical.
For this reason, the culture should be mixed well prior to withdrawal of samples and, if applicable, mixing should be repeated just prior to measurement. The same fresh culture medium that is used for the culture should serve as the blank. If, however, higher accuracy is required and if the medium changes color over the course of the incubation, it may be advantageous to use the culture supernatant as the blank.
If the measured optical density exceeds the linear range, the sample should be diluted accordingly using culture medium. Further possible causes for varying measured values are listed in White Paper 27 [3]. Microbiological turbidimetry using standard photometers. In practice however, it has been shown that despite of the absence of absorbance, the Beer-Lambert can be used for low density cultures.
In other words, the OD value can be directly related to the number of microorganisms in very low-density suspensions. For higher density cultures, the OD measurement results in a rather parabolic curve. The number of organisms in such a culture can only be calculated after a calibration of OD values to a count of organisms 1. Cuvette measurements are recommended to monitor production processes with huge volumes. At regular intervals e.
The method requires approximately 1. Alternatively, microplates can be used to continuously monitor microbial growth. As there are typically 96 wells in a microplate it is perfectly suited to measure multiple samples in replicates and to compare different media conditions or strains. For mid- to high-throughput applications, processes can be automated. An example is shown in the video below. The measurement of light scattering by particles using the absorbance mode may give different results from one instrument to another.
The reason is that different instruments use different light beams and the detector is positioned at different distances from the sample. Think of a light beam being scattered by a microorganism: a nearby detector still captures the light, while a detector positioned further away does not Figure 3.
Therefore, choosing one instrument series for microbial growth measurements by OD is recommended. Clearly, the decision for cuvettes or microplates narrows the choice as either a cuvette spectrophotometer or a microplate reader is required. Instruments that perform both, cuvette and microplate measurements, such as the SPECTROstar Nano are an alternative that equips you for all future needs.
For the measurement, microorganism suspensions with known microorganism concentrations are prepared and OD is measured at the instrument of choice using the same volume as used in later experiments. The measured values can then be used to generate a calibration curve. OD values of unknown samples can later be related to this curve and the concentration can be back calculated.
As the light-scattering changes with the size and shape of particles it is recommended to repeat calibration for each organism of interest.
Likewise, a calibration needs to be performed when changing the analysis instrument or the sample volume. The measurement of microbial growth by OD in microplates is increasingly used in combination with detection modes such as luminescence or fluorescence.
The measurement of microbial growth serves as normalization for another functional assay. A beta-galactosidase reporter was measured using a fluorescence readout and the signal indicating enzyme activity was normalized to OD Another example monitored L. The nephelometric method to measure microbial growth is also based on the light scattering of microorganisms.
Contrary to OD measurements where the loss of transmission due to scattering is measured, nephelometry directly detects the scattered light. Compared to the absorbance-based method, nephelometry is more sensitive and detects lower amounts of particles, therefore being suitable to suspensions with very low microorganism numbers.
A fluorophore such as GFP or RFP stably expressed by the microorganism of interest is capable of reporting on microbial growth. Fluorescence intensity is linear to the fluorophore concentration. Thus, it reports on an increase in fluorescent microorganisms. OD is difficult to use for the detection of a mycoplasma contamination , as they mycoplasma are very small, slowly proliferating and usually to be detected in combined cultures with eukaryotic cells.
A substantial deviation between OD and C is visible. During the initial part of the log phase OD and C show the same time dependency. This increase in OD for constant N is the result of an increase in cell mass as cells filament and increase in size , rather than increasing N Fig.
There can be a substantial difference between the expected OD and C relationship for scatterers of a fixed size and those whose size changes during growth. For a constant cell size Supplementary Fig. Supplementary Figure 6 shows the difference between growth rates obtained from non-calibrated OD and C measurements for the same cell culture.
The value and time point at which maximum growth rate is reached in Supplementary Figure 6A corresponding to Fig. Additionally, Supplementary Figure 6B shows that the two values differ significantly, by a factor of two. Apart from the size of the scatterer, changes in the difference between refractive index of the growth media n m and the refractive index of the scatterer n p can have a significant effect on the OD calibration curve.
As the relationship decreases, the OD of a fixed N is similarly reduced. The effect is small for beads as the relative difference between n p and n m is large, but will be more pronounced for biological samples like bacteria, which have a smaller n Supplementary Table 2. Finally, we investigated the effect of bacterial lysis and intracellular matter leaking into the media on n m , which can occur during growth under antibiotics.
We measured the refractive index of LB media with different concentrations of lysed E. The intracellular material Supplementary Table 3 and Supplementary Fig. The increase is substantially lower than n m variations caused by the introduction of even low concentrations of sucrose to ddH 2 O Supplementary Fig. Thus, cell lysis as a result of growth under different antibiotics will unlikely change n m sufficiently to alter the OD versus C calibration curve.
Nevertheless, growth at high sugar concentrations, such as in the food industry, will. We have presented potential issues and the calibration protocols needed for quantitative measurements of microbial growth rates based on OD measurements. We show that different spectrophotometers and microplate readers need to be cross-calibrated to compare the OD readings as an absolute number.
Furthermore, variations in diameter D and refractive index of the cell or of the media need to be considered and calibrated to avoid substantially over- or underestimating the number of cells present in the sample. Therefore, we recommend first determining if considerable changes in cell size are expected during growth of the culture. If not, and size is expected to remain constant, calibration of OD against N needs to be performed once for each D and index of refraction, and ideally reported in publications.
Changes in refractive index can be particularly relevant during growth in media with high sugar concentrations such as those in food sciences 23 , 24 , 25 , 26 and drinks with high osmolarity like beer. We have shown that changes in n m due to lysis induced leakage of cell material, for example when grown in the presence of antibiotics, are small.
However, we note that the effect of changing media index of refraction is likely to be more pronounced in bioreactor experiments where C is far larger. If cell size is expected to change substantially during the course of growth of the microbial culture for example: growth under antibiotics or various other stresses, growth of shape-inducing mutants, growth of over-expression strains, and growth of strains that induce chains or clumps , OD measurements are no longer suitable and direct counting of N should be performed, using, for example, microscopy.
All bacterial cell culture studies were conducted using E. All experiments were conducted in LB media except where explicitly stated. MM9 medium contained 0. MM9 Modified M9 is of the same composition as M9 28 except sodium phosphate buffer only was used. Yeast studies used three Sc-derived strains: BY, BY 29 and the cln3 homozygous deletion derived from the Saccharomyces genome deletion project Colloidal bead cultures were created using dilutions of polystyrene beads of known diameter D 0.
For all samples C was experimentally determined by counting the number of beads in 10 of the dilution in a microscope tunnel slide The cells were then diluted in increments to provide a range of OD readings.
A single dilution of cells for each series was then imaged in the brightfield microscope as above for the polystyrene beads. All measurements in the main text were reported using the BMG with correction values, which is given as the measured OD multiplied by 1. Yeast cell counts were performed using a Neubauer improved bright-line haemocytometer Marienfeld. Calibration between the two spectrophotometers was performed using E. The relative difference in measurements was then calculated and used to correct the data gathered for yeast.
The calibration is shown in Supplementary Figure 1B. Stacks of images through each sample were acquired every 0. True values of C were experimentally determined by counting N present in the known stack volume determined by the field of view of the microscope For each each bead preparation in Fig.
Similarly, for each cell preparation in Fig. In Fig. For each osmolarity, C was determined by counting N using brightfield microscopy as above.
Osmolalities were measured using a freezing point depression osmometer Camlab. Fitting of the data presented in Fig. Data was first trimmed to remove points where the spectrophotometer had saturated then fitted as a 2nd degree polynomial with robust fitting using the bisquare method. The polynomials found are presented in Supplementary Table 4.
When an increase in OD above the baseline was observed sampling for C began, with each new sample taken from a separate well. For all growth curves at least 8 wells were left untouched to provide a complete growth curve for comparison. In the rare cases where the growth curve deviated significantly from the average, those traces were excluded from all measurements. Samples were then imaged under brightfield illumination and N counted manually to determine C for each time point.
For Fig. For Supplementary Figure 5 solid and dashed fits were produced using 1st and 2nd degree polynomials respectively. The fitted region for each was selected by expanding sequentially from zero until the fit quality started to drop. This cell lysate was diluted to 0. The original cell extract was counted as above to determine the concentration of cells before lysing.
Refractive indices of solutions were measured in a manual refractometer Bellingham and Stanley, London. Sucrose data was obtained from a standard brix index How to cite this article: Stevenson, K. General calibration of microbial growth in microplate readers. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Neidhardt, F. Klumpp, S. Cell , — Article Google Scholar. Scott, M. Science , — Andrews, J. Determination of minimum inhibitory concentrations.
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