In life and other experimental sciences, serial dilution of substances is a routine procedure. A process that is reproducible and accurate is critically important for generating trustworthy results. The user-friendly software Andrew Lab, combined with the pipetting robot Andrew, simplifies volume calculation and the design of the experiment.
This combination also generates serial dilutions that are, on average, five times, but can be up to ten times more accurate and reproducible than an average operator.
Serial Dilutions Enable Production of Samples with Known Concentration
Changing the concentration of samples, buffers and reagents is a daily reality in science. It is much more accurate to make several smaller stepwise dilutions to reach a final concentration when the required reduction in concentration is large. Certain experiments even need each intermediate dilution step to be available for use as standard curves for quantitative assays or for testing different concentrations of a reagent.
Serial dilutions can also be used in other situations, to reduce initial sample viscosity and dilute out substances or inhibitors in the samples that may interfere with analytics downstream. Regular stepwise dilutions are made in cascade in a classical serial dilution. This results in an exponential reduction in the concentration. These kinds of experiments can be easily designed using the Andrew Lab software. The software automatically calculates the required concentrations and volumes and Andrew, the pipetting robot, handles the actual pipetting accurately.
Clearly, accurate pipetting during preparation of serial dilutions is critical, because any deviation will propagate to all of the subsequent steps. Furthermore, the precision and accuracy of serial dilutions depend on thorough mixing at each step. This is critical to achieving a homogenous mixture that has a constant concentration over the sample volume. Any aliquot can randomly have a lower or higher concentration than expected if proper mixing does not occur (Figure 1). If this happens, the sampled aliquot can dramatically affect the downstream concentrations when it is transferred to the next step.
Figure 1. An example of what the distribution of a sample inside a tube could look like with and without good mixing.
Andrew Guarantees Homogenous and Reproducible Mixing at Each Dilution Step
As well as the correct calculation and definition of serial dilution volumes, it is necessary to include reproducible and precise mixing methods after each dilution step. Ideally, at least two thirds of the total volume of a solution should be used when mixing by up-and-down pipetting. This is especially true when working with solutions that are viscous. Unfortunately, this process often requires pipettes or volume settings to be changed at each step and this increases the number of repetitive motions dramatically.
This makes such experiments cumbersome, variable and prone to errors. In a 10-fold dilution with the targets final volume of 200 µl, for example, the intermediate steps would be a buffer volume of 180 µl and an immediately upstream sample volume of 20 µl. In this example, the minimum volume that should be used to ensure proper mixing would be 100 µl, since the final volume in each dilution is 200 µl.
However, since the previous pipetting step uses a volume of 20 µl, a change of pipette and volume setting to at least 100 µl is required to ensure proper mixing. A proper and perfectly reproducible experimental workflow can be designed with the Andrew Lab software, according to user preference and excluding any ambiguity (Figure 2). For instance, variations in the level of mixing at each step can be avoided by adapting the number of pipette aspirations and how they are dispensed for mixing. This avoids experimental differences. Human error can be eliminated with the automatic execution of the experimental flow using Andrew. This will further ensure the accuracy and reproducibility of every experiment.
Figure 2. An example of a 1:10 serial dilution requiring the incorporation of a mixing step (2) with a higher volume for a proper mixing. Incorporation of this critical step in serial dilutions made by hand is cumbersome and renders the experiment prone to error and variation. The use of the Andrew pipetting robot not only facilitates the performance of serial dilutions but also allows more accurate and reproducible results.
Testing Quality of Serial Dilutions Made by Hand or with Andrew
Two-fold, eight-step serial dilutions of Ponceau S (700 mg/L) in water were undertaken both using Andrew and manually. It is important to note that distinct mixing steps were included at each dilation step and the same pipettes were used in both cases. A mixing volume of 135 µl was chosen because the total volume at the mixing step was 200 µl.
The mixing volume chosen corresponds to two-thirds of the total volume, for a final volume of 100 µl. A standard high accuracy spectrophotometer was used to measure dye absorbance. Five of the eight serial dilution points (175, 87.5, 43,75, 21.9 and 10.9 mg/L) fall within the measurable dynamic range of the device. Twelve replicates, over several days, were used to test the precision and accuracy of these serial dilutions. The absorbance of the first and last two dilution steps (350, 5.47 and 2.73 mg/L) were outside the range of the plate reader and, as such, were not included in the analysis. In order to avoid dependence on the initial sample concentration, the first readable concentration was defined as 100%. All other data points of the dilution series were compared in relation to this reference point.
Andrew can Perform Reproducible Serial Dilutions
In order to evaluate the variability of serial dilutions performed by a highly trained human operator and by Andrew, the mean Optical Density (OD) of 12 individual serial dilutions was calculated for each method (Manual and Andrew). The standard deviation of the mean was calculated and then the mean ± 3 SD was displayed in a bar chart (Figure 3).
Serial dilutions performed by Andrew are more reproducible and consistent and this is demonstrated by the smaller error bars. This was further demonstrated by calculating the coefficient of variation of each dilution (Table 1). The maximum co-efficient of variation (CV) for the dilution steps performed by Andrew was 1.29%. In all manually done measurement steps this was consistently higher, with values from 1.56% up to 9.54%.
This CV represents the random error, encompassing the random error of the manual pipette (of 0.15% per maximum Gilson permissible errors), Andrew, and the sensitivity of the plate reader. The statistical error of the plate reader contributes to 1% of this variability. The constant performance across many replicates demonstrates without a doubt that, compared to a human operator, the pipetting robot Andrew can achieve serial dilution in a more reproducible and accurate way.
Table 1. Concentrations measured at each dilution step as Percentage of the Initial 100% and calculated CVs (n=12).
||CV observed with Andrew
||CV observed with Manual
|1 (175 mg/L)
|2 (87.5 mg/L)
|3 (43.75 mg/L)
|4 (21.88 mg/L)
|5 (10.94 mg/L)
Serial Dilutions with Andrew are also More Accurate
A target dilution curve of dye concentrations at each of the dilution steps was calculated for the above serial dilutions, in order to assess the systematic error. These calculations were based on the assumption that a perfect dilution was possible. In order to assess how closely the target concentrations were achieved with both methods, the standard curve was compared with the average serial dilution curves generated either manually or from Andrew.
The final dilution step achieved by Andrew differed from the target dilution by only 0.05%, and all the intermediate steps deviated by less than 0.2% of their target dilutions (Figure 4). At every step, the difference from the target dilution falls within the range of the CV. This indicates that the systemic error observed in serial dilutions made by Andrew, is negligible.
Conversely, observed deviation in the manually performed serial dilutions was consistently higher at every dilution step. Deviation was between 0.26 and 0.85% of the target dilution values (Figure 4).
These results strongly support the premise that the pipetting robot Andrew can perform serial dilutions more accurately than those achieved manually. The main difference between the serial dilutions made manually and by Andrew is the consistency of the separate mixing step. Typically, this is accomplished by a carefully controlled and consistent pipetting technique and this can only be achieved with the use of automated solutions such as Andrew.
Furthermore, the precision of the serial dilution may also be affected by other biases such as pipette verticality, variations in the pipette volume setting hysteresis, thumb speed, tip insertion depth, first-stop detection force and tip insertion force. All of these are eliminated by using Andrew.
Figure 3. Serial dilutions made by Andrew are consistently more reproducible than their manually performed counterparts. Mean OD measured at each dilution step and ±3 SD from the mean are plotted.
Figure 4. Serial dilutions made by Andrew are more accurate than their manually performed counterparts. A standard curve for the ideal target diluted concentration at each dilution point was calculated using the dilution factor of 2. This was compared with serial dilution curves produced either by hand or by Andrew. The relative deviation from the target OD value observed for both curves at each dilution step is depicted.
Programming a Serial Dilution in Andrew
Andrew Lab includes an extremely user-friendly tool for making serial dilutions. In a few clicks, the amount and concentration of the final sample, or the dilution factor, can be indicated. The concentrations and volumes required at each dilution step are calculated by Andrew Lab, allowing the mixing methods to be defined accurately. When designing a serial dilution, the dilution factor and buffer volume to be dispensed in each dilution step can be indicated (Figure 5a).
Alternatively, the concentration of any of the serial dilution points can be indicated. This means that Andrew Lab can calculate the initial volume of reagent/sample required, and also the volume to be moved from well to well during dilution. In addition, a different volume for the mixing step can be set up. This is done by introducing the numeric value in the entry box designated for that end in the mixing panel of options (Figure 5b). At least two-thirds of the total volume should be used when mixing difficult samples using up-and-down pipetting.
Figure 5. Example steps to easily create a serial dilution protocol in Andrew Lab 1.4. (A) Determine the Dilution Factor and Buffer volume to be dispensed in each dilution step. (B) Determine the optimal mixing volume in the designated entry box at the “reagent destination” conditions.
For accurate and reproducible serial dilution experiments, precise pipetting and mixing is compulsory. However, when doing repetitively serial dilutions manually this is often overlooked as this is a time-consuming and repetitive operation. A serial dilution can be described easily and in a few seconds in Andrew Lab. Andrew can then deliver, unattended, results that are on average five times, and up to ten times more reproducible, and ten times more precise than an average operator.
About Andrew Alliance S.A.
Andrew Alliance is an independent, privately financed company, based in Geneva, Boston and Paris. The company was created in March 2011.
Andrew Alliance is dedicated to advance science by working with scientists to create a new class of easy-to-use robots and connected devices that take repeatability, performance, and efficiency of laboratory experiments to the level required by 21st-century biology.
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