Serial Dilution with the Agilent Bravo
Automated Liquid Handling Platform
Technical Overview

Summary• Accurate serial dilution with a fullchannel head
• Efficient mixing with as few as three
mix cycles• No tip changes necessary• No wash steps necessary

Introduction
Many laboratory protocols require the serial
dilution of reagents or compounds. IC50
assays are commonly used to evaluate drug
efficacy, and assay development procedures
as well as standard curve generation involvethe serial dilution of proteins, compounds,
or other detection agents. These processes
can be streamlined with automated liquidhandling equipment with serial dilution
capabilities.
The Bravo Automated Liquid Handling
Platform from Agilent Automation Solutions
can be utilized to perform serial dilutions. It
has nine standard deck positions that canhold any SBS standard microplate. Thesedeck positions can also be configured forheating, cooling, shaking, and tip washing.
The design of the head, along with various
tip and head options, allows serial dilutions
to be performed by column or row, from
300 nL to 200 μL dilutions.
Operators can easily write protocols with theincluded Agilent VWorks software package.
One key feature is the Serial Dilution
Wizard. The Serial Dilution Wizard lets theoperator easily program the instrument for

The Agilent Bravo has nine deck positions and can beconfigured with interchangeable 8-, 16-, 96-, or 384-fixedand disposable tip heads.

making serial dilutions. The wizard walks
the operator through the steps of the serial
dilution task: tip changes, mix parameters,and volume transfers. The operator caneither determine the transfer based on initialvolumes (transfer by volume) or by a concentration gradient (transfer by concentration).
This technical overview explores the parameters that lead to an efficient, precise, andaccurate serial dilution protocol. The goal isto produce good precision and accuracy foreach dilution in the least amount of time.
The mix parameters are essential for arobust serial dilution task. The parameters
explored include:••••

The number of mix cycles
Mix height
Liquid class settings
Tip retraction/extension

Materials Used
• Bravo with a 96-Channel LT Disposable
Tip Head• Agilent 96LT 200 μL tips
(Now replaced with 250 μL tips,
product no. 19477-002)• Agilent 96-well manual fill reservoirs(product no. G5498B/G#049)• 96-well polystyrene, black flat clear
bottom plates (Greiner 655087)• Fluorescein solution(3 μM in 50 mM Tris-HCl, pH 8.0)
• SPECTRAFluor Fluorescence
Spectrophotometer (Tecan)

Method
A reservoir containing 60 mL of fluorescein
solution is placed on position 2 of theAgilent Bravo (fluorescein reservoir for serial
dilution by Row is placed on position 7). Areservoir containing 100 mL of 50 mM Tris-HCl
is placed on position 3. A 96-well polystyrene
plate is placed on position 5. 200 μL tip
boxes are placed on positions 6 and 9. AnAgilent Works liquid class for 51-200 μL
dispense is utilized.
Figure 1. Bravo Deck Layout
for Serial Dilution by Column.

1. Tips are pressed onto the head fromposition 9.
2. 150 μL Tris-HCl solution is transferredfrom position 3 to position 5. Aspirate
parameters are 6 mm from the bottom ofthe reservoir with a 2 μL pre-aspirate
volume. Dispense parameters are 2 mmfrom the bottom of the plate, with a 2 μL
blowout volume.
3. Tips are unloaded back to position 9.
4. Eight tips are pressed onto the last columnof the head from position 6.
5. Fluorescein solution is mixed at position
2. Mix parameters are 150 μL, 3 mix
cycles 6 mm from the bottom of theplate, with a 2 μL air gap.
6. 150 μL fluorescein solution is transferredfrom position 2 to Column 1 of the plate
at position 5. Aspirate parameters are
6 mm from the bottom of the reservoir
with a 2 μL pre-aspirate volume.
Dispense parameters are 2 mm fromthe bottom of the plate, with a 2 μL
blowout volume.

Results
Number of mix cycles
The first experiment was to determine thenumber of mixes required to produce aprecise and accurate 1:2 serial dilution.
The volume of the mix was just under the# of Mixes

wavelength with a gain of 63, 5 flash
cycles, and a 40 ms integration time.

7. 190 μL was mixed in Column 1 of the96-well plate. Mix parameters vary oneach experiment performed throughout
the study as described below.
8. A 1:2 serial dilution (150 μL) was performed from Column 1-10. All aspirations
have a 2 μL pre-aspirate volume at 2 mmfrom the bottom of the plate. All dispenses occur 2 mm from the bottom ofthe plate, with a 2 μL blowout volume.
Mix parameters vary on each experiment
performed throughout the study as
described below.
9. 150 μL of excess volume Column 10 istransferred to waste.
10. Tips are unloaded back to position 6.
11. Plates are centrifuged at 1800 rpm for
60 seconds to ensure consistentwell menisci.

A similar serial dilution method by row isperformed, with the exception that thefluorescein reservoir is placed on position 7.
Fluorescence measurements from each well
are used to determine the precision of thetransfer. Coefficient of Variance (CV) calculations are made by dividing the standard
deviation by the mean for each column/
row. Accuracy is calculated based on anequation derived from a fluorescein/Tris-HCl
calibration curve consisting of data points at
2, 1.5, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01 and
0.005 μM, compared to the actual fluorescence value in each well.

12. Fluorescence absorption is read at
485 nm excitation and 535 nm emission

Results may vary, depending on individual
experimental methods and liquid class optimization.

maximum volume of the tip, and the numberof mixes was varied from 3-20 cycles. Table
1 shows the resulting CV of each column foreach mix cycle protocol. The average precision (averaging CVs for columns 1-10) showsthat the precision improves asymptoticallyas the number of mix cycles is increased(Figure 2). Three mixes yielded an average CVof 11.8%, while 20 mixes had a considerably
better CV of 1.7%. The precision in all cases
worsens as the serial dilution proceeds
across the plate; this is expected as the error
in the earlier columns is propagated witheach transfer.

CV/Column561234

3

0.8%

6.1%

6.1%

3.9%

9.7%

11.5%

50.4%

2.0%

4.1%

3.9%

2.6%

4.7%

10

0.6%

1.2%

1.4%

1.6%

2.2%

20

0.5%

0.6%

1.0%

1.3%

1.5%

78

Avg. Precision

Time (sec/plate)Acc. Ratio

22.4%

11.8%

340

1:1.85

5.9%

4.2%

440

1:1.92

5.0%

5.3%

2.7%

690

1:1.97

2.4%

3.4%

1.7%

1200

1:2.0191020.1% 18.7%

19.2%

4.5%

6.3%

8.1%

3.1%

3.2%

3.6%

1.7%

2.4%

2.6%

Table 1. Precision, accuracy, and serial dilution time with varying number of mix cycles.

2Figure 2. Average precision improves as the number of mix cycles increases.

In addition, the accuracy ratio improves as
the number of mix cycles increases (Figure3). The accuracy ratio is an average of theconcentration of the diluted column compared to the previous column—a perfectserial dilution would have an accuracy ratioof 1:2.00 across the entire plate. The accuracy ratio of the plate improves with moremix cycles, varying from 1:1.85 to 1:2.01.

Figure 3. As the number of mix cycles increases, the observed concentrations
approach the idealized 1:2 serial dilution.

This is expected as the fluorescein would be
more equally distributed in the well themore the well contents are mixed, ensuringthat transfers are made from a uniformly
mixed solution.
While the precision and accuracy with 20
mix cycles is very close to a perfect serial
dilution, it also undoubtedly takes too much

Mix Height
The mix height was adjusted in order todetermine the effect of distributing the liquid
at a different location in the well. As the mix
height was increased, the average precision
improved. Figure 4 shows that at a height of
3 mm, the average precision is 3.9%, significantly lower than 15% at a height of 0.1 mm.
Following the same trend as previously
observed, accuracy tracks with precision, so
the higher mix height also improves our
accuracy ratio to 1.95 (Table 2). This trend isobserved because the higher dispense
height at this dispense speed ensures that
more of the sample is circulated by the mix
cycle: in a mix far from the bottom of thewell, dispensed liquid is forced closer to thebottom while dispensing, and aspirated liquid
is pulled from the top of the well. If the mix
occurs closer to the bottom of the plate, thedispensed liquid is pulled back into the tip
during the aspiration.
Mix Heighttime for an automated process. The 20 mix
cycle protocol takes 20 minutes to perform,
while a 3 mix cycle protocol takes less thansix minutes. Efforts were then focused on
the factors that can improve the 3 mix cycle
protocol to produce accuracy and precision
results that were closer to the achieved 20mix cycle protocol.

Figure 4. Average precision improves as mix height increases.

123

4CV/Column
5678

9

10

Avg. PrecisionAcc. Ratio

0.1

7.9%

8.5%

11.2%

11.5%

11.8%

19.8%

15.8%

17.5%

25.1%

0.5

6.6%

4.7%

6.9%

16.3%

6.9%

12.3%

13.5%

21.9%

22.7%

21.1%

15.0%

1:1.78

20.0%

13.2%

1

6.3%

4.3%

6.3%

8.8%

14.2%

9.7%

12.1%

15.0%

1:1.74

11.1%

11.4%

9.9%

1:1.82

2

1.8%

4.3%

7.5%

6.8%

5.2%

4.6%

10.4%

13.8%

16.6%

14.1%

8.5%

1:1.86

2.5

2.2%

2.1%

3.5%

8.2%

8.5%

8.7%

11.1%

18.9%

15.2%

11.6%

9.0%

1:1.90

2.75

3.7%

4.1%

4.9%

3.8%

3.8%

5.2%

6.2%

9.2%

4.7%

7.4%

5.3%

1:1.92

3

1.8%

2.5%

2.8%

2.4%

2.1%

3.1%

4.2%

6.4%

5.8%

7.9%

3.9%

1:1.95

Table 2. Precision and accuracy with varying mix heights.

3

The original liquid class speed for the mix
was 100 μL/s velocity and 500 μL/s2 acceleration. Based on the average precision ofthe data (Table 3), mixing became more
effective as the mix speed increased.
Accuracy is also improved as the mix speed
increases, because of consistency of theMix Liquid Class Setting
The next parameter explored was the mix
liquid class settings. Each liquid class has a
set of velocities and accelerations for theaspirate and dispense. This experiment was
to determine if increasing the aspirate anddispense speeds of the mix would result in
improved serial dilution results.
Liquid Class SpeedVel.
Acceler.
(μL/s)(μL/s2)

1

2

3

4

CV/Column56mixes across the plate. Under these conditions, a mix liquid class setting of a velocity
300 μL/s and an acceleration 1000 μL/s2
was utilized. There was no appreciable difference in time or performance by increasing the velocity from 300 μL/s to 500 μL/s.

Avg. Precision
78910Acc. Ratio

100

500

5.6%

3.7%

5.6%

10.3%

10.1%

19.0%

21.1%

17.8%

14.5%

15.7%

12.4%

1:1.90

300

1000

2.1%

1.9%

1.9%

1.9%

1.6%

1.8%

1.7%

2.9%

3.0%

6.7%

2.6%

1:1.98

500

1000

2.0%

1.3%

1.4%

2.6%

2.2%

2.3%

3.6%

4.0%

4.9%

5.1%

2.9%

1:1.97

Table 3. Precision and accuracy with varying mix liquid class settings.

Dynamic Tip Retraction/Extension
To further explore improving precision andaccuracy, dynamic tip retraction/extension
was used on the mix parameter. Utilizing
this particular parameter in the mix cycle,
the tip moves into the well during each
aspiration and retracts during each dispense.
The following retraction/extension test
heights were calculated based on the optimal
mix height on experiment 2 to give an effective mixing. Mix area was also consideredon this test (Figure 5). This will showwhether retracting/extending tips during
the mixing gives a noticeable change in theCV and amount transferred.

Dynamic Tip
Retraction/
Extension (mm/μL)
no retraction/
extension
0.02
0.01
0.01

Mix
Mix
HeightArea(mm)2
no retraction/extension
0.1
whole0.1
bottom2
top

In this experiment, the average precision
improved slightly as mix area increased(Table 4). Mixing in half the well was marginally worse than mixing in the entire well,and mixing in the lower half of the well was
marginally worse than mixing in the top half.
In fact, mixing in half the well was worse
than not moving the tips during the mix at all.
There was little to no variation in accuracy as
the tip retraction changed, suggesting that
this parameter did not have an effect,
and/or the mix parameters were optimized tothe best degree possible.
Figure 5. Mix area schematic.

CV/Column
45

1

2

3

2.1%

1.9%

1.9%

1.9%

2.4%
1.4%
2.6%

1.9%
1.6%
1.8%

1.7%
2.5%
2.8%

2.2%
2.5%
2.4%

Table 4. Precision and accuracy with varying mix tip retraction/extension.

4Avg. Acc. Ratio
Precision

678910

1.6%

1.8%

1.7%

2.9%

3.0%

6.7%

2.6%

1:1.98

1.3%
2.4%
2.3%

1.4%
3.5%
2.8%

1.4%
3.8%
3.3%

1.9%
4.6%
2.8%

2.6%
5.1%
3.5%

6.1%
6.4%
5.3%

2.3%
3.4%
3.0%

1:1.97
1:1.98
1:1.98

Number of mix cycles with the improved
mix parameters
Based on the experiments above, the conclusion was that the best mix parametersshould have the following characteristics:• Three mix cycles• 2 mm mix height• 300 μL/s velocity and1000 μL/s2 acceleration• 0.02 mm/μL tip retraction

Figure 6. Average precisionof improved vs. original
parameters.

To verify this conclusion, the first experiment(varying the number of mix cycles) wasrepeated with the improved mix parameters.
Table 5 compares the old mix parameterswith the improved mix parameters. The
improved mix parameters (Figure 6) provides

increased precision and accuracy because
the mix was more effective. Additionally,
the improved mix parameters decreased thetime required to run a serial dilution protocol

by nearly a minute. Most of this improvement
was due to increased liquid class speeds.
Accuracy also improved in comparison tothe original mix settings.

Mix Parameters
Number of mix cycles Average Precision
35
1020

2.8%
2.9%
2.2%
2.0%

Improved
Serial Dilution Time(seconds/plate)
285
365
545
900

Accuracy Ratio

Average Precision

1:2.01
1:2.021:2.041:2.04

11.9%
4.2%
2.7%
1.7%

Original
Serial Dilution Time(seconds/plate)
340
440
690
1200

Accuracy Ratio1:1.85
1:1.92
1:1.971:2.01

Table 5. Precision, accuracy, and serial dilution time utilizing improved vs. original mix parameters.

Serial Dilution by Row
The Agilent Bravo can perform serial dilutions by column and by row. While all of theprevious experiments were serial dilution bycolumn, it was necessary to confirm that theserial dilution by row would work equally
well. Table 6 shows that by applying theimproved mix parameters, high precision
and accuracy were possible in another serial
dilution method. Average precision wasimproved mostly due to the fact that there
were seven dilutions instead of 10; error
was propagated over a fewer number ofdilutions. With fewer dilutions, the overall
serial dilution time decreased as well.

Plate Number

CV/Row
4
1.1%
1.4%
1.6%
1.3%

12
34

1
1.4%
1.0%
0.6%
0.9%

2
1.6%
1.3%
1.1%
1.7%

3
0.9%
1.0%
1.6%
1.2%

5
1.1%
1.5%
1.7%
1.4%

6
2.2%
1.8%
2.2%
1.6%

Table 6. Precision and accuracy of serial dilution by row.

5

7
2.5%
1.3%
2.8%
2.8%

Average
Precision
1.5%
1.3%
1.6%
1.5%

Serial Dilution
Time (sec/plate)
225
225
225
225

AccuracyRatio
1:1.97
1:1.97
1:1.95
1:1.95

Conclusion
This study shows that the Agilent Bravo canquickly and reliably serial dilute acrosscolumns or rows of a plate. Based on theseexperiments, the following settings gave us
low CV and an accurate overall dispense. It is
possible to achieve accurate and precise serial
dilution results in a short process time with:• No tip changes• A minimum number of mix cycles• No wash steps
The ability of the Bravo to do serial dilution
with a 96- or 384-channel disposable tip headmeans that serial dilution can be performed
with one instrument with one head type. A
head swap is not necessary. A parametersetting in the protocol allowed a change fromtransferring liquid with the entire head totransferring with a single row or column.
Serial dilution experiments can be optimized
through a number of different parameterchanges, and based on the results shown
here, the keys to obtaining accurate andprecise mixing are the following, in order ofdecreasing contribution to overall mixing
efficiency:• Maximize volume of mixing• Maximize mixing speed• Maximize the area traversed by tip
retraction/extension.
Please contact your sales representative orAgilent Applications Support if you haveparticular questions regarding your specificapplication. Supplemental information(protocol files and data analysis spreadsheets) are also available upon request.
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© Agilent Technologies, Inc. 2014, 2015
Published in the U.S.A. September 4, 2015
5990-3647EN