Wash Protocols with theAgilent Open Bath Wash Station
Technical Overview

Summary

3. Wash step. Wash parameters vary oneach experiment performed throughout
the study as described below.

Wash station effectively washes tips withlow carryover achievable.

Introduction
Figure 1. Agilent Open Wash Station.

Laboratory automation systems can run
cycles of liquid handling processes withdisposable tips. There are two options for
disposable tip processes: to change disposable tips every cycle or wash the disposable
tips. Washing disposable tips can potentiallyreduce running costs, but it requires aneffective and efficient wash process without
wasting time and wash solution.
This technical overview evaluates thewashing capability of the Agilent Open
Wash Station (product no. G5498B/G#048).
This wash station is a white polypropylene
open wash bath with a 250 mL capacity. It
has 5 input/output channels (see Figure 1)
designed to perform different methods ofwashing, such as “fill & empty” and “continuous flow” wash methods. The goal of thiswork was to create a wash process thatwould have the lowest possible carryover
and also minimize wash volume andprocess time.

Materials• Agilent Bravo Automated Liquid
Handling Platform with a 384-channel
ST Disposable Tip Head
• Agilent 384ST 30 μL disposable tips
(product no.11484-202)• Open Wash Station(product no. G5498B/G#048)• Agilent Pump Module• Agilent 384-well manual fill reservoir(product no. G5498B/G#050)• 384-well polystyrene, black flat bottomplates (Costar 3710)• 10 mM Fluorescein dissolved in DMSO
• 50 mM Tris-HCl pH 8.0• Fluorescence Spectrophotometer(Tecan SPECTRAFluor)

MethodA wash station plumbed with water isplaced on position 1. 60 mL of fluorescein
solution was poured into a manual fill reservoir and placed on position 2 of the Bravo. A
384-well polystyrene plate pre-filled with 60 μL
of 50 mM Tris-HCl is placed on location 4(the “external” carryover plate). An empty
384-well polystyrene plate is placed on
position 5 (the “internal” carryover plate).
A 30 μL disposable tip box is placed on
position 3. An Agilent VWorks liquid classfor 11-50 μL dispense was utilized.
1. Tips on are pressed onto the head.
2. Contamination step. 20 μL of 10 mM
fluorescein solution is mixed from themanual fill reservoir. Mix parameters are
3 mix cycles, 6 mm from the bottom ofthe plate, with a 6 μL pre-aspirate
volume and a 2 μL blowout volume.

Fill empty method: wash station is filledwith wash fluid [water]. Tips are mixed avarying number of times in the bath. The
wash station is emptied and refilled.
Continuous flow method: wash station isfilled and emptied with wash fluid continuously as the tips are mixed for avarying number of times in the bath.
4. Steps 2 and 3 are repeated five times.
5. After every 5th round of contamination
and washing, 30 μL is transferred from an‘external’ carryover plate to the ‘internal’
carryover plate.
6. Process from steps 2-5 is repeatedfour times to replicate 20 plate/contamination cycles.
Plates are centrifuged at 1800 rpm for 60
seconds to ensure consistent well menisci.
Fluorescence was read at 485 nm excitationand 535 nm emission wavelength.
Fluorescence values in each well were used
to determine the effectiveness of the wash
protocol. Carryover was calculated based onan equation derived from a Fluorescein inDMSO calibration curve consisting of data
points from 0 to 1000 part per million, compared to the actual fluorescence value ineach well.
Individual results may vary, and are expected
to be highly dependent on contaminating
solvent and the wash solution chosen. It ishighly recommended to do analogous experiments based on your application prior toadopting a wash protocol.

Results
Fill & Empty Wash Method
In the “fill & empty” method, the wash station is configured as shown in Figure 2. In
this configuration the wash bath is filledfrom both ends but is drained via the bottom
outlet. In this method the bath can be filled
and emptied one or more times between
each wash cycle.
The first experiments were to determine
how to minimize carryover in the shortest
process time with the lowest consumptionof wash buffer. The tips were mixed at fullvolume three times per fill & empty cycle.
Based on the results in Table 1, two fill &empty cycles are sufficient to eliminate
carryover. As process time and wash buffer
volume are also critical in determining anoptimal wash protocol, those parameters
were the next explored.
Number of Mix Cycles
Based on the results in Table 1, two fill &empty cycle were sufficient to reduce carryover to undetectable levels. As this firstexperiment was performed with three mix
cycles per fill and empty cycle, we next
determined if we could reduce the process
time by reducing the number of mix cycles.
Table 2 shows the result of these experiments. Two mixes per fill and empty cycle
was sufficient to reduce carryover below
detectable levels.
Volume Exchange
After optimizing this method, the next step
was to determine if there was a chance oflong term contaminant build-up based on
this protocol. The protocol was run for 20
cycles of contamination and tip washing,and on every 5th plate a liquid transfer wasperformed between an “external” carryoverplate (the aspirate step will leave contaminating fluid from the outside of the tips) andan “internal” carryover plate (the dispense
will leave contaminating fluid from theinside of the tips).
Since process time and wash buffer volumes are critical in considering an optimized wash protocol, we also included anexperiment on ‘used’ wash buffer solution,in which case the wash station is still filled

Figure 2. Configuration for a fill & empty wash method.

Number of Fill &Empty Cycles

Number of wells withcarryover
1-10ppm>10ppmTime (sec/plate)

Volume(mL)

1161190250

200180500

300270750

Table 1. Number of carryover, process time and wash volume consumption on varying number of fill/empty cycles.

Number of Mixes
1

Number of wells with carryover
1-10ppm>10ppm206
1

Time (sec/plate)

Volume (mL)

150500500

200165300180500

500210500

Table 2. Number of wells with carryover, process time and volume of wash solution consumed by varying the number ofmix cycles per fill & empty cycle.

Wash solution for aFill & Empty wash method

Process Time (sec)Volume of wash solution (mL)Fresh

180500Used

120250

Table 3. Time and volume of wash solution consumed when washing with ‘fresh’ or ‘used’ wash solution for twowash cycles.

from the previous wash cycle for the first fill& empty step [‘used’ wash solution]. A‘fresh’ wash solution is used for the secondwash. This technique would reduce process
time and consumption of wash solution.
This method was compared to a process
where the reservoir was drained and filledwith fresh wash solution prior to the firstwash cycle [‘fresh’ wash solution].
Washing with a fresh wash solution
increases the likelihood of achieving low
carryover on tips. Using a ‘fresh’ wash
solution for every wash cycle may consume2

more wash solution and have a longer
process time compared to washing with‘used’ wash solution (Table 3). Washingwith ‘used’ wash solution almost halves theprocess time and wash volume consumption.
Based on the results from Table 4, both
‘fresh’ and ‘used’ wash solution were about
99% effective in reducing carryover to undetectable levels over 20 processed plates.
Carryover occurred randomly in about 1% ofwells in a 384-well plate from random tips ofa 384-channel head. Contamination in nocase was greater than 30 ppm.

Continuous flow wash method
Another method of tip washing is the continuous flow wash method. Fresh wash
solution is continuously pumped into thewash station, overflows, and is removedwaste. An advantage of this method is thatthe process time is shorter compared to thefill & empty method, therefore improvingthroughput. This method does consume
a larger volume of wash solution. The
wash parameters of two washes and three
mix cycles per wash were also applied tothis method.

PlateNumber

The results of the continuous flow wash
method are shown in Table 5.
Approximately 99.5% of wells had nodetectable carryover. The 0.5% of wells that
had measurable carryover did not exceed 8
ppm carryover. This wash method does
indeed run faster (80 seconds, an improvement of 55%). The volume of wash solution
is larger in comparison (800 mL vs. 500 mL)
to the fill and empty method.

'Fresh' wash solution'Used' wash solution
Wells w/external Wells w/internal Wells w/external Wells w/internal
carryovercarryovercarryovercarryover
1-10ppm >10ppm 1-10ppm >10ppm 1-10ppm >10ppm 1-10ppm >10ppm

5th32

3

100

20

10th

000000

30

15th

000

100

40

20th

00

10

0

0

40

Table 4. Number of wells with carryover on plates processed with a “fill & empty” wash method with ‘fresh’ and ‘used’
wash solution.

The previous results demonstrate that thereis very low carryover from plate to plate. In
testing this method, we found that there
was no cross contamination from contaminated wells into uncontaminated wells, suggesting that the open bath wash station
would not increase the risk of cross contamination in comparison to chimney-styled
wash stations.

Conclusion
This technical overview demonstrates two
feasible methods of washing tips that arecapable of reducing carryover in disposable
tip usage cases with the Agilent Open
Wash Station. The reservoir is designed toallow multiple methods of cleaning tips.
Each of the methods studied in this technicaloverview has different strong characteristics.
The empty and fill method results in clean
tips with a minimal use of wash volume.
This may be advantageous in cases where
disposal costs are prohibitive, and it is anacceptable tradeoff to have a slightly longer
wash time in order to minimize waste.

Figure 3. Configuration for a continuous flow wash method.

Plate
NumberComparison to chimney wash stations
One concern in using a wash station of thisstyle is the possibility of cross contamination from one tip to the other. To test forthis possibility, we changed our carryoverplate to have alternating rows of fluorescein
contaminant and no contaminant at all. By
looking for carryover in the tips that had
never been exposed to fluorescein, wecould determine if there was a chance ofcross contamination.

External wells
Internal wellswith carryoverwith carryover
1-10ppm >10ppm 1-10ppm >10ppm

Process Time(sec)Volume of Wash
Solution (mL)

5th001080

800

10th001080

800

15th000080

800

20th000080

800

Table 5. Number of wells with carryover with the continuous flow wash method.

3The other method, the continuous flow
method, is useful in cases where throughput
times are paramount and the cost of disposing of waste fluid is comparatively lower.
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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Published in the U.S.A. April 7, 2016
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