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From Burettes to Pipettes to Robots: Master Liquid Handling Basics for Peak Performance

Congratulations! You've landed a great new job in a facility that has a tool that's new to you – an automated liquid handler. This tool has the potential to increase both your output and accuracy – if you know how to use it.

 

Researchers who are new to the use of automated liquid handling can quickly become well informed and proficient liquid handling problem-solvers by participating in the Liquid Handling Essentials Short Course being presented at SLAS2016 on Jan. 24 in San Diego, CA. This full-day program offers an interactive primer in fundamentals vital to the successful operation of automated liquid handling devices.

The lead instructor of the course, Dana Campbell, field applications specialist at Artel, says the Liquid Handling Essentials Short Course is intended to close a gap faced by many experimenters.

"A lot of scientists get thrown into this arena when they start their careers," he notes. "They may start in hand pipetting, and then need to learn how to run a robot, or operate a liquid handler. There's a big learning curve, and what our course is designed to do is bring fundamental knowledge to a lot of these users so that they can get the most out of their instruments."

Another instructor, Nat Hentz, Ph.D., technology training director at North Carolina State University, emphasizes that the SLAS2016 Short Course is designed for "more entry level people; to offer them a good foundation to make decisions in the lab to help build on their experiences. We are showing some critical features that are typically involved in a laboratory, things like mixing, discrete transfers versus multiple transfers from a single aspiration step, which are common operations across any liquid handler whether it's manual or automated, whoever the vendor happens to be."

A Short History

A liquid handling device transfers a selected quantity of liquid from one vessel to another. The simplest machine moves an allotted volume of liquid via a manual or motorized pipette or syringe. Currently, more sophisticated liquid transfer devices operate under microprocessor or computer control, and are generally referred to as robotic liquid handlers.

In the late 18th century, Francois Descroizilles, a French chemist and pharmacist, developed the burette and pipette, essentially a graduated cylinder capable of accurately drawing out, and injecting measured doses of experimental fluids. In a related vein, the invention of the syringe, (Martin Overlach) with its sealed piston that further facilitated the accurate transfer of liquids, added to the utility of the burette and pipette.

In the 1940s, the development of analytical devices capable of doing laboratory analysis on microliter-sized samples necessitated the development of pipettes capable of delivering smaller and more precise amounts of fluid. The further development of gas chromatography spurred further development of microliter syringes.

In the 1970s, the development of micro-scale dc motor and valve technology led to the introduction of highly accurate semi-automated motorized syringe-based pipetting devices. And, in the 1980s and 90s, the evolution of motor and microprocessor technology enabled the development of the first, true automated liquid handling workstations.

Nowadays, robots often eliminate the manual handling of pipettes, and mechanize more repetitive actions, says Lisa Knapp, field applications scientist in genomics automation at Agilent Technologies, and one of the instructors of the Liquid Handling Essentials Short Course.

"If you're ever in a lab pipetting manually, you notice as the day goes on that your hand gets tired, or you're mentally fatigued," she explains. "Automation takes that away from the assayist, so that you get consistent results time after time. The liquid handling course is trying to help optimize that, so researchers achieve optimal results."

A Bridge to Knowledge

Setting up a robotic liquid handler properly is a matter of great interest among those working in research labs these days, Campbell explains. "The goal of our course is not to teach people how to program automated liquid handlers, but to broaden researchers' understanding of how to make the most of what the robots do best.

"I want people to understand the importance of the liquid types they are dealing with on automated liquid handling platforms, but also the roots of manual pipetting as well," Hentz adds.

Hentz explains that most labs pipette fluids that are less than ideal including blood, serum, proteins, detergents, oil, organic solvents or salts. "By treating these fluid types the same as water, significant errors can be introduced to an assay process, depending on volume. In addition to viscosity, scientists need to also consider density, vapor pressure and surface tension. By successfully programming automated liquid handlers, improved precision and accuracy is often attained. For example, when pipetting solvents, which tend to have high vapor pressures, a person needs to constantly adjust the pressure on the plunger, change speed and move quickly between aspirate and dispense," says Hentz.

Knapp expects to share her expertise in discriminating between different fluids that respond well to the use of an automated liquid handler.

"Different liquids will behave very differently when pipetted," says Knapp. "For example, methanol will drip out of a tip as soon as you aspirate it so you must put an air gap after it to hold it in the tip until you get to the plate where you want to pipette it. Glycerol-based solutions are an example where the viscosity of the liquid is important. When you pipette glycerol-based solutions such as master mixes, you have to leave the tip in the liquid for a bit of time after the plunger in the pipettor finishes its movement before pulling the pipette tip out of the liquid. If you do not, you will not aspirate the full volume desired. You will likely have an air gap at the bottom of the tip. You will also want to look at the speed of aspiration and dispense. This is based upon viscosity and sometimes the volume you are pipetting. In the case of glycerol, you would pipette much more slowly than say water or ethanol to get accurate and precise pipetting."

Another thing to consider is when it's appropriate to take advantage of the capabilities of a robot. "If processes are not automated, time is often wasted in the overnight hours or early in the morning when the instruments are sitting idle," says Hentz. "Automation can be set up to run constantly, but scientists first need to understand where the inefficiencies are in their processes. If there are gaps in the process, then (partial or full) automation can improve the efficiency and repeatability. Sometimes these systems are just not appropriate and when you try to force a particular assay into an automation realm, errors pop up unexpectedly. Not that there is anything wrong with the design of the automation hardware, but it just may not be right for that particular application or the assay may need further validation. Other considerations are that automated liquid handlers usually require an expert user, depending on how sophisticated the system is and that assays that are used intermittently may not benefit from automated liquid handling. It's really about assay consistency and whether upfront investment in time and capital is justified for long-term gain."

Interactive and Wide-Ranging

Campbell says the SLAS2016 Liquid Handling Essentials Short Course has been designed to provide the participants some exposure to a selected variety of automated liquid handlers.

"There's a part of the room that is set up with robots from different manufacturers," he explains, "and we aim to have a total of six stations. At different points throughout the day, we break into groups and participants rotate among the stations and robots." The stations feature equipment from Agilent, Beckman Coulter, CyBio, Hamilton Company and Tecan; and representatives from each manufacturer are there to provide technical support to instructors and participants. One of the six stations demonstrates traditional handheld pipetting techniques to provide a baseline upon which to compare what's learned about the operation of the robots.

Campbell goes on to say that with three instructors, the goal is to offer course participants "multiple viewpoints from different people with different backgrounds."

When the Inevitable Happens

Another aspect of the Liquid Handling Essentials Short Course prepares participants for what to expect and how to respond when things go wrong.

"One of the key parts of our course is to discuss troubleshooting," Campbell explains, "because unexpected things happen all the time no matter how good a system is. To troubleshoot something, you can talk about it, but without actually seeing it, and seeing how things affect pipetting, it's hard to put it into practice. The interactive part of our course really helps with that."

In addition, the SLAS2016 Liquid Handling Essentials Short Course delves into quality control, according to Hentz.

"We talk about methods to measure the performance of the liquid handlers, including a number of in-house methods people use. We're going to make sure people are using the appropriate tools for the appropriate jobs."

At the end of the course, Hentz and his colleagues hope those in attendance leave with a more thorough and nuanced foundation for how to make robotic liquid handlers perform effectively in the lab.

"I'm hoping they'll understand the complexities of the fluid they'll be dealing with, whatever it is, whether they're in a traditional high-throughput screening lab or in a service laboratory conducting a lot of routine analysis," Hentz says. "I also want them to understand the limitations of their liquid handlers, and to measure that appropriately."

Campbell says this Short Course's emphasis on the basics is planned in direct response to feedback from participants in past SLAS conferences who requested that the needs of relative newcomers to the field of automated liquid handling be the priority focus.

"This course is for the beginner," Campbell says. "We are targeting people who have a stake and an interest in liquid handling and offer fundamental principles that are important for any beginner that is new to automation, or new to pipetting in general."

And, while this Short Course does not specifically address programming of liquid handlers and the design of experiments around their use, SLAS Director of Education Steve Hamilton says the SLAS2016 Short Course entitled Study Design and Statistical Analysis for High-Throughput Screening (HTS) Experiments addresses these areas of vital interest to more experienced practitioners. "It all begins with design," says Hamilton, "and good design involves a thorough examination of statistics."

Hamilton adds that, "Approximately one-third of the course covers basic study design principles (e.g., randomization, internal validity, avoiding confounding variables) and introductory statistical principles (e.g., systematic versus random error, data visualization, inferential versus exploratory data analysis, false positive versus false negative errors). Time is spent applying these principles to HTS experiments, covering both primary and secondary (validation) screens."

Information about all 19 SLAS2016 Short Courses is available on the SLAS2016 website. 

November 30, 2015