Picking apart your pipetting

It’s amazing how the commonly seen can so easily become the unseen, but when it comes to pipetting – you must beat this habit writes Craig Bush

Walk down the street and you don’t really notice the street lights, road and parking signs, railings and fences. Pressing the button for the Zebra crossing is automatic. In our daily lives the plethora of street furniture, no matter how sophisticated the technology contained within them, is not merely unobtrusive, it’s invisible.

Is your laboratory any different, or are we all in the habit of taking the furniture for granted here as well? The careful dispensing of precise volumes of liquids is an essential component of many scientists’ lives and the pipettes we use to do this are found in laboratories of almost every discipline. Sophisticated, yes, but so commonplace we can be inclined to keep on pushing the button automatically.

And, there are a lot of buttons to press. In a recent survey of over 8,000 laboratory scientists that we carried out, 91% used variable volume single channel pipettes routinely in their work. Six out of ten used 4-6 different pipettes while one in five used more than 10 different pipettes

The prevalence, number and variety of pipettes in use means that the safeguarding of accuracy and precision through a unified approach to routine checking procedures and calibration is essential. The importance of calibration is recognised by all responsible pipette manufacturers. However, a quick glance through the specification sheets for different makes of pipettes reveals a bewildering array of facts and figures presented regarding the maximum permissible errors of individual pipettes and volume ranges. And, this complexity is carried through to individual manufacturers’ calibration certificates supplied when the pipette was bought.

Fortunately, precision measuring instruments have norms and regulations describing the calibration and operating parameters to which they should conform. For pipettes, this is the seven-part international standard EN ISO 8655 that identifies and details:

• design requirements which are valid for pipettes manufacturers,
• maximal permissible errors which manufacturers can use as norms for their quality assurance programme, pipette operators can use as acceptance criteria and regular in-use testing criteria, and testing institutions can use as a basis for independent certification,
• environmental conditions in which the testing and calibration occurs,
• the gravimetric method, detailed in ISO 8655-6, normally used as the basis for testing. This relies on precise weighing of ultra-pure water dispensed from the pipette and back-calculating to the volume. The method is time consuming and requires specialist weighing equipment but is the most common calibration method.

What this means is that, as a pipette user, you can use one set of standardised specifications for all your pipettes, regardless of make or model and depending only on its type and nominal volume. You can also adopt this standard and write it into any SOPs (standard operating procedures) for your laboratory. You can use it to compare and contrast between rival performance claims when shopping for pipettes – comparing ‘apples to apples’ in your purchasing decision. And, you can ensure that testing and calibration throughout the pipette’s lifetime is carried out in a way that continues to guarantee its accuracy and precision in your experiments.

At Anachem, we have configured and equipped our new calibration laboratory to comply with the standards requirements.

For instance, the standard dictates that the test shall be carried out in a draught-free room with a stable environment. The test room shall have a relative humidity above 50% and a constant temperature between 15?C and 30?C, plus or minus 0.5?C. Prior to the test, the apparatus to be tested and the test water shall have stood in the room for a sufficient time, at least two hours, to reach equilibrium with the room conditions. At the start and end of the weighing procedure, the water temperature, barometric pressure and relative humidity should all be recorded.

The latitude allowed in the range of temperatures and barometric pressures is good news when it comes to doing your own testing and calibration checks in your laboratory – the standard goes through the calculations required to factor in this variance. More problematic is the requirement to keep whatever temperature you have constant, irrespective of the time of day, number of people at work and other equipment and instruments that may be running.

The standard is equally stringent when it comes to the minimum requirements for the weighing equipment to be used, which, for practical purposes, depends on the nominal volume of the pipette being tested (Table 1).

If your laboratory does not have access to precision balances, you would need to adjust for the uncertainty this brings to your own calibration measurements if you choose to perform regular checks on your pipettes.

Even with a dedicated, purpose-built calibration laboratory, error due to evaporation must be taken into consideration and this is especially important when dealing with small volumes of less than 50 µl. We have determined experimentally the error due to evaporation within our laboratory using our equipment and procedures, and this is taken into account in our overall error compensation.

But stable, draught-free conditions and sensitive balances can only go so far! ISO 8655 also says that the test cycle time, defined as the time required to complete the weighing of one dispensed volume, shall be kept to a minimum – ideally less than sixty seconds. Also, that it is consistent both within each cycle and between cycles so that a reliable calculation of error due to evaporation can be applied.

This brings us to the importance of operator training and technique. ISO 8655 is clear that the total pipetting system consists of three parts:

• the pipette
• the pipette tip
• the operator

In doing so, it recognises that the combination of pipette and pipette tip, even when not co-sourced from a single manufacturer, should conform to the maximum permissible errors set out in the standard and allows you as a user to choose a pipette tip for your individual applications based on design, quality and fit rather than brand. You can decide whether to have your pipettes calibrated either to the manufacturers specification (with the manufacturers pipette tip) or to the ISO standard using the tip of your choice.

The standard also recognises that the source of greatest variability is the operator – in other words, you and me! And, it sets out to address this at a very basic level by prescribing exactly how the pipetting of the test volume into the weighing vessel shall be carried out. At the same time as you are concentrating on being quick whilst smooth and consistent, you have to note down the weights, record the overall cycle time and take the temperature of the test water. If you only carry out this procedure quarterly or annually then knowing how to carry out the process will probably not make up for variability introduced through lack of practice.

It is common sense to look after your pipettes and keep them in good working condition. And, it is good practice to test and calibrate them regularly. As a general rule, a 20% annual failure rate can be expected if a pipette is not maintained or serviced regularly. Over 95% of these failures are attributable to preventable sealing system defects. So, daily pipette checks and routine basic servicing will reduce failures and lower the risk of experimental failure.

Alongside your in-house preventative maintenance, making sure your pipettes are professionally serviced and calibrated by an ISO 17025 compliant service centre is essential. Only pipette service companies holding this standard can guarantee the quality and traceability of the work being performed on your pipette.

And no, I am not advocating specialist training in ISO 8655 procedures for every person that picks up a pipette. But, I do encourage training in pipetting techniques for every person who is expected to use a pipette in the course of their work. And, for every pipette user to examine their work to see if their pipetting technique is influencing the results they achieve and how this may be corrected. As a guide, table 2 shows how to recognise possible sources of error and ways of correcting them.

At Anachem, we appreciate that ISO 8655 is designed around using pipettes in real laboratories, under real conditions, by real people rather than in an idealised environment in the manufacturer’s laboratory. Through our intense involvement in the requirements of the standard, we firmly believe that an optimised pipetting system is only as good as the people who use it. Good technique, and knowing which techniques to use for various liquids, affects precision, accuracy and the reproducibility of your results.

Our Good Pipetting Practice programme offers a systematic approach to maximising pipetting accuracy and precision through a package of risk analyses, educational materials, seminars and training. It may help you appreciate the commonplace pipette and see it in a new light.

Author: Craig Bush, Pipette Service Manager, Anachem Limited