Good Pipetting Practice

Pipette Tip Quality

& Certification

Pipette Calibration
& Technique

The Ergonomics
of Pipetting

The Influence of Pipetting

on Experimental Results
Content
Content

1. Introduction 5
2. Pipette Tip Quality & Certification  7
2.1.  Rainin BioClean Tips: Certified Contamination-free7
2.2.  Pipette Tip Quality: Influence on Experimental Results9
3. Pipette Calibration & Technique 13
4. The Ergonomics of Pipetting 16
5. Literature References 22

METTLER TOLEDO The Influence of Pipetting on Experimental Results 3

Introduction
 1. Introduction

Pipettes are the workhorses in the laboratory. Together with tips and the
operator, they form a system that influences the accuracy, precision and
repeatability of experiments.

While there are many publications on the influence of proper handling and good pipetting tech-
nique on the quality of results, the influence of pipette tips commonly receives lesser attention. We
will give an introduction into the topic and provide detailed advice on what to look for when choos-
ing a good pipette tip, i.e., a tip that does not influence the experiment’s results.

We will also look at the impact that service, e.g. pipette calibration as well as pipetting technique
of the operator have on experimental results. Last, we will look at pipette ergonomics, and how
these influence the operator directly, for instance in the form of Repetitive Strain Injuries (RSI).

METTLER TOLEDO The Influence of Pipetting on Experimental Results 5

Pipette Tip Quality & Certification
 2. Pipette Tip Quality & Certification

Pipetting accuracy is useful only if the tips are also guaranteed not to
contribute biological and chemical contaminants to the samples.

2.1. Rainin BioClean Tips: Certified Contamination-free

Pipetting accuracy with Rainin’s products is achieved through the use of certified pipetting instru-
mentation and tips, which have been designed and tested to work together as a system for the as-
piration and dispensing of pre-set fluid volumes. Consummate accuracy is required to ensure pre-
dictable reaction conditions and hence, data fidelity. However, it often goes unnoticed that reaction
conditions can also be compromised by the inadvertent release of miscellaneous contaminants
by the pipette tip, once it is brought into contact with the sample. These diverse contaminants can
exhibit adverse, unpredictable effects upon enzyme activity and longevity, molecular interaction,
spectrophotometric and chromatographic analysis, etc. Collectively, this diverse contaminant pool
is comprised of biological, organic and non-organic substances which may become associated
with the pipette tip during any stage of the tip molding and packaging process.

Releasing Agents
In the November 7th, 2008 issue of Science1, a paper was published which described how exog-
enous agents associated with certain brands of pipette tips and tubes adversely affected biolog-
ical reactions and data fidelity. Mass spectroscopy conducted on leachates from these tips and
tubes implicated two compounds from the manufacturing process as likely candidates respon-
sible for the adulteration of the reactions described above. The two contaminants are a detergent
(DiHEMDA) and a releasing agent (oleamide), Both are commonly used by other companies in
the injection molding of polypropylene products. After the release of this report, Rainin enlisted
the services of an independent analytical lab to conduct these same analyses on its products2.
The results indicated ZERO presence of either DiHEMDA or oleamide in Rainin’s products.

Rainin uses the purest virgin polypropylene resin in the form of CFR 21 for all tip molding. This
material is free of any additives or dyes and is extensively tested to ensure lot-to-lot purity.

Trace Metals and Trace Organics

Another independent laboratory described above was also contracted to perform analyses for
both metal metal and trace organic leachates in Rainin tips2. ICP-MS analyses was used for the
detection of any metals which were mobilized from Rainin tips by an exceptionally aggressive
treatment with concentrated nitric acid. As with the tests for releasing agents, a volume of nitric
acid was used to concentrate the heavy metal leachables from a total of ten tips. For the trace
organic analyses, groups of tips were washed with 1.5 ml of a 3:1 mixture of methanol and
toluene. The resulting organic leachate was tested using both GC-MS and LC-MS.

The results for trace metal analysis indicated that even under the harsh acidic conditions em-
ployed, only negligible amounts of various metals were detectable. Under normal laboratory
conditions in which aqueous buffers are used, it is likely that no detectable metals would be
present in the leachate. For trace organics analyses, the results indicated zero presence of con-
taminating organic species.

METTLER TOLEDO The Influence of Pipetting on Experimental Results 7

 Bisphenol A
Pipette Tip Quality & Certification
Known to be esterogenic since the mid 1930s, Bisphenol A (BPA) is used in the synthesis of
polycarbonate and epoxy resins and in other applications. As a result, it has been found in a
variety of plastic products. In 2010, a report by the FDA raised additional concerns regarding
the exposure of fetuses, infants and young children. Shortly thereafter, Canada declared BPA a
“toxic substance” because of its ability to mimic the effects of estrogens. Due to this potential
biological activity, studies were undertaken at Rainin to determine if BPA could be mobilized as
a leachable from its pipette tips3.

250 µl of an aqueous buffer was cycled through thirty pipette tips and the leachate subjected
to GC-MS analysis. The polypropylene homopolymer pellet material used by Rainin was also
subjected to this analysis. One gram of pellet material was first heated to 150 °C for 15 minutes.
The pellets were allowed to cool and then combined with 1 ml of aqueous buffer in a glass
scintillation vial and the sample shaken for 15 minutes. The pellet leachate was also subjected
to GC-MS analysis. No BPA was detected in either leachate with a Limit of Detection (LOD) of
0.06 µg/g (pellet sample) and 0.015 µg/g (per 30 tips).

PCR Inhibitors
Leachates from Rainin tips exhibit no demonstrable inhibition of PCR4. In addition to the tip, Rainin
employs sintered polyethylene filters to prevent aerosol-borne cross-contamination, particularly in
PCRs. Other companies use “self-sealing” pipette tip filters which contain an embedded additive
called “carboxy- methyl cellulose” (CMC). This additive is highly hydrophilic and will gel upon con-
tact with aqueous liquids. This feature allows such filters to act as plugs if liquid is aspirated into
the filter. The intention is to prevent the aspiration of liquid into the pipette shaft. Due to the manner
in which the CMC fibers are retained within the filter, they are often shed into the barrel of the tip
during handling, allowing them to come into contact with any aspirated liquid.

Biological Contaminants
Rainin’s BioClean pipette tips are manufactured under Class 100K clean room conditions with pro-
cesses that eliminate the opportunity for human contact with the product, starting with the polypro-
pylene pellet material and ending with the sealed racked tips. Rainin’s tips are quality-tested for the
presence of biological contaminants in both internal and external labs, to the most stringent quality
specifications in the industry (see table below). These rigorous quality testing procedures are de-
signed to detect vanishingly small amounts of different biological contaminants. For example, our
test for DNA contamination employs Quantitative PCR (qPCR) with a sensitivity level of less than
one copy number of human DNA. All testing regimens for biological contaminants are designed to
consolidate any contaminants found within a group of several tips. This method of testing ensures
both a high probability of detecting any contamination which may be unevenly distributed among
a sample set (product lot) and that even the slightest amounts of contamination will be detected.

Contaminant Limit of detection (LOD)

RNase < 10-9 Kunitz units/µl
DNase < 10-7 Kunitz units/µl
DNA < 1 copy human DNA
Pyrogens 0.001 EU/mlw
ATP <2 x 10-12 mg/ µl

8 METTLER TOLEDO The Influence of Pipetting on Experimental Results

 Sterility
BioClean filter and pre-sterilized tips are gamma-irradiated to guarantee the packaged product is
free of viable microorganisms.

In Conclusion
The stringent test specifications combined with the design of the testing procedures collectively
ensure the absolute purity of Rainin’s pipette tips. This means there is essentially zero possibility
for any Rainin tip to contribute a contaminant to the customer sample. Absolute pipetting accuracy
and absolute product purity mean absolute confidence in experimental integrity and data fidelity.

While there are many publications on the influence of proper handling and
good pipetting technique on the quality of results, the influence of pipette tips
commonly receives lesser attention. This article will give an introduction into
the topic and provide detailed advice on what to look for when choosing a
good pipette tip, i.e., a tip that does not influence the experiment’s results.

2.2. Pipette Tip Quality: Influence on Experimental Results

When you are surrounded by sophisticated laboratory instruments, pipetting just seems so simple.
In fact, finding the right pipette amongst the multitude that inhabit most labs often seems the most
difficult part of the job! Then, you just need to put a tip on the end and get on with the experiment,
right? And, the great thing is, all your attention can be reserved for the experimental details.

If this is the reality for most scientists working in a lab, it’s a testament to the exquisite engineer-
ing design and manufacturing excellence put into the humble pipette and pipette tip that they are
expected to perform as advertised no matter how little attention we pay them.

Unfortunately, even the best engineering cannot overcome inattention and inappropriate use. Al-
most obscured by our expectations that the pipetting system will continue to perform is a series
of potential problems that may completely change the experimental results that we obtain. And,
no matter whether you are just running routine clinical tests, starting out on your postgraduate
work, or developing the latest blockbuster drug, inconclusive or incorrect results mean only one
thing – waste. Wasted time, wasted money, wasted opportunity – we squander the chance of ac-
curate, precise and repeatable results at our peril.

Missing Tips
You don’t have to look very hard to find many papers full of ‘hints and tips’ about the impor-
tance of maintaining pipettes in good condition. It’s also easy to find guidelines on ‘correct
pipetting techniques’. All of these stress the contribution that the pipette can make to maximizing
the potential for success.

However, the bit that actually comes into contact with the liquid sample is almost always either
forgotten completely or relegated to a footnote. It’s a tip – a simple consumable that requires no
thought. You just use them once and throw them away. What could be easier?

METTLER TOLEDO The Influence of Pipetting on Experimental Results 9

Pipette Tip Quality & Certification
Unfortunately, this generalization hides some serious issues that all users should be aware of.
These issues should also be considered as part of the risk assessment when preparing to per-
form any experiments.

In a perfect world, an ideal tip would:

• Fit perfectly onto the pipette - there should be no need to tighten the tip onto the pipette to as-
sure good fit. An ideal tip should always fit correctly, whether you have a single or multichan-
nel pipette.
• Remain sealed – it may seem obvious but the pipette tip must maintain a seal throughout the
pipetting operation, as even the tiniest leak changes the volume accuracy.
• Come off easily – again, it may appear obvious but, when you are finished with the tip and
ejection comes around, the ideal tip will eject easily without requiring excessive force.
• Be free of defects - the shape and finish of the tip should show no blemishes or defects, such
as extra plastic hanging off the walls.
• Be absolutely pure – your tips should be manufactured from the purest of polypropylene and
not contain any special additives to aid the molding process and help the manufacturer pro-
duce more tips per hour. Experience has shown that these additives can leach from the walls
of the tip into the aspirated liquid, which may cause chemical inhibition or changes to experi-
mental reactions. Refer to the Additional Resources section to learn more about leachables
and extractables.
• Be clean – tips should be free of the threat of biological contamination, having been manu-
factured and packaged in an ultra-clean environment.
• Arrive with an assurance of their quality – tips from a reputable manufacturer will be pack-
aged with a certificate affirming which tests were performed on the products to verify that any
potential contamination was lower than a specific high sensitivity number.

Why Aim For Perfection?

If you have already begun to question the status of the tips you are currently using against these
seemingly simple criteria, perhaps it is worth exploring in more depth why these attributes are
important and what can happen if the less than ideal tip is used.

Tip fit and finish are very important characteristics controlled by the design, manufacturing and
packaging process. Any deviations in manufacturing reproducibility can directly affect the size
and shape of the critical tip sealing area. As an end-user, you’ll become aware of this through
having to apply variable forces to acquire the tips and ensure a consistent seal. In addition, any
changes to the tip finish can change the characteristics associated with aspiration and dispens-
ing and will ultimately affect the accuracy of the pipetted volume. Therefore, any manufacturer
seeking to consistently make the most reproducible tip must ensure that the mold for the tip
must be of the highest quality steel and finest precision design. Furthermore, the molds must be
routinely inspected and serviced in order to sustain the same dimensions of tip and the produc-
tion quality assurance procedures must routinely test the tips produced in the mold to check that
all dimensions are consistent and that there are no deviations to the finish. In particular, if the
dimensions of the seal vary, the fit to the pipette can change dramatically and this can increase
the amount of force required to put the tip onto the pipette, the effectiveness of the actual seal
and the amount of force required to eject the tip. Ergonomically this puts stress on both the user
and the tip ejection mechanism of the pipette which can result in increased repair costs for the
pipette. For those who do not apply enough force to ensure correct sealing, aspiration and dis-
pensing volumes can vary significantly, enough to change end results completely and require
repeat experiments.

10 METTLER TOLEDO The Influence of Pipetting on Experimental Results

For these reasons, many pipette manufacturers also manufacture pipette tips to control both
the pipette and tip dimensions and all pipette manufacturers specifically recommend tips that fit
their pipettes.

The quality of the tip molding process, or rather the lack of it, is also seen in the small frag-
ments of plastic material that may be found on many tips. This bad finish may range from obvi-
ous surface flaws and blemishes and ‘flashings’ around the tip end to microscopic imperfec-
tions in the tip finish. Whichever it is, variability in the finish of the tip itself can affect the correct
dispensing of liquid sufficiently to compromise volume accuracy enough to necessitate a repeat
of the experiment.

The material used to make the tips is also an important factor is producing the finest of tips
and, importantly, this choice of material is purely up to the manufacturer. Manufacturers who
want to make the lowest cost tip possible will use the lowest cost polypropylene, which may
contain impurities, and include additives, such as releasing agents, to increase the speed of the
manufacturing process. After all, the more tips that can be made per hour, the cheaper each tip
becomes to manufacture. Unfortunately, the use of low quality polypropylene and additives can
produce ‘leachables’ in the tips that can be removed by the passage of liquid into and out of the
tip. Some of these leachables are the organic molecules used as releasing agents to speed up
the manufacturing process while it has been shown that some metal ions can also leach from
the surface of the tip in sufficient concentrations to inhibit some sensitive enzyme based experi-
ments. Even though in most pipetting protocols the liquid only remains in contact with the tip for
a brief time, it is sufficient for some of the leachate material to go into solution and get carried
into your experiment. The result can be invalid results that necessitate a repeat of the entire ex-
periment, as witnessed by many scientific publications.

Finally, tips may be exposed to routine biological contaminants, such as DNA (from human
skin and hair) or pyrogens (from bacterial cell walls), during the manufacturing and packaging
process. Both these two contaminants can alter the experimental results if transferred from the
tip to the experimental solutions. Both are also difficult to remove and even autoclaving may not
remove or destroy them (see publication reference). Similarly, DNases and RNases are common
contaminants that can alter experimental results and are difficult to remove from the tips. Con-
sequently, many manufacturers routinely test for many of these contaminants and accompany
the pipette tips with a certificate indicating there is no contamination. However, even here care
must be taken, as some certificates are not sufficiently specific. For instance, it is not enough
for a certificate to simply claim the tips are ‘free’ of DNA. More correctly, the certificate should
detail the mechanism by which the product is tested so that the accuracy of the statement can
be evaluated. For instance, quoting a test result using a qPCR method rather than a less sensi-
tive end point PCR method would allow claims to be made that the DNA contamination is guar-
anteed to be less than a specific amount. This data provides the scientist with accurate detailed
information instead of the very vague „free of DNA“ with no testing data.

Tips to Rely On
In today’s labs, it is difficult to imagine how we would manage without the pipette – it is the un-
derstated workhorse for so many of our experimental procedures. But, next time you pick one up
and cast around for a few tips, I hope you’ll stop and think more carefully about the technologi-
cal achievement they represent and the value that should be attached to choosing and using the
right tip.

METTLER TOLEDO The Influence of Pipetting on Experimental Results 11

Pipette Tip Quality & Certification
Additional Resources
We suggest to read the following articles to learn more about leachables and extractables in
pipette tips and their influence on results.

[1]
McDonald et al: Human Monamine Oxidase Assay. Science 2008
[2]
McDonald et al: Benzodiazipine Binding to GABA-A Receptors. G.I.T. Laboratory Journal 2009
[3]
Belaiche et al: Mitochondrial Enzymatic Activities. G.I.T. Clinical Chemistry 2009
[4]
Watson et al: GCPR Assay. J Biomol Screen 2009
[5]
Reuhl et al: NMDA and nAChR Channel Assay. Brain Res Bull 2009
[6]
Glossmann et al: Ca Ion Channel Assays. PNAS 1993
[7]
Lewis et al: DNA/Protein Assays. Biotechniques 2010
[8]
Ocvirk et al: Radioimmunoassays of Progesterone. J Stereoid Biochemistry 2009

12 METTLER TOLEDO The Influence of Pipetting on Experimental Results

 3. Pipette Calibration & Technique

Pipette Calibration & Technique

The use of pipettes to transfer liquids is a daily activity in most life science
research labs. From academic labs involved in leading edge discoveries to
testing labs that follow routine standard operating procedures, the data gener-
ated can be greatly influenced by the performance of the pipette and technique
of the user.

Pipette performance is a function of many factors, including keeping the pipette well maintained
in order to achieve the desired performance and periodically checking to ensure that it meets the
desired specifications. The other major factor, technique, requires users to develop their pipet-
ting skill, such that maximum performance is routinely achieved and data is reliably produced.

When the two key criteria of routine maintenance and user technique are met, inaccuracies aris-
ing from these variables are significantly reduced and reliable results obtained, no matter what
the application.

A brief review of recent scientific literature indicates a constant stream of reminders that pipettes
and pipetting technique can play a major role in the success or failure of an experiment. Like-
wise, the outcome of ignoring guidance on technique can result in significant loss of time and
money, which are crucial for any lab.

The review of these papers can be subdivided into many classes of applications. This review fo-
cuses on the information from genomics and proteomics papers published in the last few years.

Pipette Calibration and Experiment Outcome

Many genomics experiments include a PCR or qPCR com-
ponent that requires the careful addition of reaction
components or preparation of a standard curve. Many
publications indicate that not only careful pipetting,
but also maintaining a calibrated pipette is essential
if the data to be generated is accurate. Pennington
and Edwards5, using qPCR for gene expression
studies in cultured cells and small tissue samples,
recommend avoiding pipetting less than 2 µL
since precise pipetting is vital to the success of
the qPCR experiment. Toh et al6, also using qPCR,
recommend that readers specifically set aside a set
of dedicated pipettes and have them calibrated on
a regular basis. Grgicak et al7 demonstrate that vari-
ability between standard curve dilutions has a significant
impact on calibration curve stability and that using a Part of the GPP - Good Pipetting Practice Series.
single calibrated pipette showed minimal error in com-
parison to using either two pipettes or an uncalibrated pipette.

METTLER TOLEDO The Influence of Pipetting on Experimental Results 13

 The underlying theme in this selection of papers is the importance of maintaining calibrated
Pipette Calibration & Technique
pipettes so that, at a minimum, the mechanical variability of the pipette is minimized as a result
of routine professional calibration. This process can be enhanced by regular verification that
the pipette in use meets the published specifications, a check that can be performed by using a
high performance balance.

Pipette Technique and Experiment Outcome

Separately, many of these papers also provide guidance and reminders about pipetting tech-
nique that, if not followed, can also lead to substantial errors. For example, in Morga et al8, the
ability to obtain highly reproducible measurements with qPCR experiments depends on a num-
ber of factors, including the ability to perform “skilled pipetting.”

In Vallania et al9, Allele quantification was shown to be affected by pipetting errors during the
process of DNA pooling. This was confirmed in further genome-wide association studies where
inaccurate pipetting was shown to be a primary source of error.

Venegas et al10, studying mitochondrial DNA with qPCR, indicated that inconsistencies with
intra-run results were due to errors in pipetting of reagents, DNA template or primers, and that
pipetting accuracy is very important.

Frendewey et al11, studying cell screening and mouse genotyping by qPCR, indicate that differ-
ences in value between duplicate samples reflect differences in pipetting accuracy and repro-
ducibility.

Life Technologies, a leading supplier of qPCR products, provides significant support to their plat-
forms, including guidance on optimizing and troubleshooting. The guidance given in their qPCR
protocols indicates that because low volume pipetting (<5 µL) negatively affects precision, they
do not recommend it unless using pipettes designed for such volumes. The consequences of
inaccurate pipetting of the test sample include high standard deviations and a number of errors
that can occur when preparing the standard curve. Most of these lead to the production of an in-
accurate standard curve, resulting in an artificially lower or higher amplification efficiency score,
depending on whether the error is due to excess or deficit pipetting. This in turn can violate MIQE
guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments).12

Simple calculations of pipetting error show the potential effects caused by these gross inac-
curacies in a qPCR experiment. For example, if a 10 µL pipette is being used down to 5 µL, the
mechanical accuracy is ± 0.075 µL. If the copy number in the 5 µL is 30,000, then the inherent
copy number variability for the pipette alone (excluding user technique) can range from 30,450
down to 29,550 copies of DNA. And this assumes a well-calibrated and maintained pipette.

Depending on user skill, technique can add a range of ± 2 to 7%. The consequence of this ad-
ditive error is a copy number range from 31,972 down to 28,072. The errors will accumulate
during a dilution series and this accumulation can make significant differences in a standard
curve and ultimately an assay.

Unlike genomics, which has a finite number of assay and detection techniques, proteomics has
many detection systems with highly varying needs for volume, format and purity of protein. The
analysis of the final sample in the detection system of choice results from a number of prepara-
tion steps involving pipetting, each step being capable of adding to the variance and inaccuracy
of the data that is generated.

14 METTLER TOLEDO The Influence of Pipetting on Experimental Results

In an Alzheimer’s disease study by Teunissen et al13 there is a review of an inter-laboratory study
that focuses on a specific biomarker assay. The clear outcome of the study is that even though
each lab received the same sample and performed the same assay with the same materials,
there was high variability in the results produced by the different labs. One of the areas of con-
cern involved pipetting techniques, indicating that differences in technique contributed to the
inter-lab variance.

An extension of the concern for technique includes making sure that the correct tips are securely
fitted to the pipette to obtain a sufficient seal. For example, in the chapter “Immunoassays in
Veterinary Plant-made Vaccines,” Guzman et al14 suggest that for their ELISA analysis not only is
good pipetting technique essential, but the reader is reminded to “Always inspect the pipette and
tips for correct seal, and ensure that consistent pipetting technique is used.”

Recommendations: “Self Check” and External Calibration

Not only is operator technique important, but the physical capability of the pipette should be
checked to verify that it meets the specification needs for the intended applications. In the work
by Alamooti et al15 using ELISA and flow cytometry studies, the authors state that the accuracy
and reproducibility of all pipettes and technicians were checked every month by the gravimetric
method. It is worth noting that only highly-trained and experienced service technicians are ca-
pable of performing truly accurate independent checks of individual pipettes.

Numerous organizations suggest variable frequencies for pipette calibration and checking. For
example, ORA-LAB. 5.5 from the FDA suggests that all volumetric delivery devices, such as
mechanical pipettes, be calibrated at a minimum of every six months.16 In the review by Ber-
termann, the recommendation is for pipettes to be “calibrated according to documented proce-
dures along with periodic checks to ensure proper ongoing performance.”

Individual labs and researchers should evaluate their need for routine checks based on the sen-
sitivity of their experiments to pipetting errors and to the risks they would assume if their data
were compromised. The Risk Check Tool at www.mt.com/gpp is a useful tool for ascertaining
pipetting risks.

METTLER TOLEDO The Influence of Pipetting on Experimental Results 15

 4. The Ergonomics of Pipetting
The Ergonomics of Pipetting

The process of pipetting is well understood by scientists around the world.

Pipettes are ubiquitous, providing a simple mechanism to complete workflow
requirements, from the starting liquid sample preparation to introducing the
sample into a final analysis system. Using many different pipettes throughout
the day is common and each unit may have its own unique challenges – high
volume, low volume or multichannel. The ergonomic factors associated with
a variety of pipettes and applications is an important consideration – poor
ergonomics can lead to fatigue, pain and the risk of injuries.

Several critical areas have been identified as key to degrading the comfort level of the pipette
and subsequent development of various painful ailments, generally called Repetitive Stress Inju-
ries (RSIs). This information has been published by ergonomics scientists in an effort to create
awareness of the usability issues associated with pipetting.17 18 19 20 21 22 A common theme among
all studies is that pipetting is a forceful and repetitive activity, and that there is a strong associa-
tion between pipetting and developing RSIs. In fact, pipetting just over an hour a day over the
course of a year is enough to put you at risk, and the chances increase exponentially with work-
load and age.17 18 19 22

A key reference point is the amount of force required to perform an activity, such as depressing
the plunger or ejecting tips. Most of these forces are transmitted through the thumb. Generally
speaking, the goal of pipette manufacturers has been to lower pipetting forces
as much as possible in order to improve their ergonomic impact.

Tip Loading and Tip Ejection

Loading a tip on the end of a pipette and subsequently
ejecting it at the end of the pipetting cycle are very closely
linked and constitute some of the heaviest forces associ-
ated with pipetting. With most pipetting systems the de-
sign of the distal end of the pipette is conical in shape.

The process of loading the tip on the pipette requires a

certain amount of force to ensure an adequate seal of the
system – too little force and there is a risk of leakage and
volumetric error. User technique for tip loading can vary as
there is no clear way to know when enough force had been
used to seal the system. Part of the GPP - Good Pipetting Practice Series.

Typically a user will adopt a technique that provides reasonable assurance that the tip has
sealed and will stay with that technique for most of their working life. Unfortunately, for tradi-
tional conical tip systems, there is a strong correlation between the amount of force needed to
load the tip and the amount of force required to eject it, so for those who adopt a technique that
uses high force to add the tip, a high force will be required to eject it (see Figure 1, red line).

16 METTLER TOLEDO The Influence of Pipetting on Experimental Results

 Tip Ejection Forces at Increasing Tip Loading Forces
Brand X Traditional Tip vs. Rainin LTS

4.0

Ejection Force (kg) 3.0

Brand X Tip, 250 µL
2.0
Rainin LTS Tip, 250 µL
1.0

0.0
0.0 5.0 10.0 15.0
Insertion Force (kg)

Figure 1
The relationship between tip loading and ejection forces – a traditional conical tip compared with a Rainin LTS tip.

In the paper by Lichty et al,21 a number of different manufacturer’s pipettes are reviewed and
force measurements compiled for plunger forces and tip ejection forces. It is clear that tip ejec-
tion forces are the most significant force associated with the many steps involved in pipetting,
but there is great variation in tip ejection forces between pipettes (See Figure 2).

2.1 kg - Female
Rainin
Pipet-Lite (LTS) 3.O kg - Male

Manuf. 3’s
Manual Pipette

Manuf. 2’s
Manual Pipette

Manuf. 4’s
Manual Pipette

0 1 2 3 4 5
kg Force
Data from Work, 39, (2011), 177-185. Lichty et al

Figure 2
Comparison of tip ejection forces from four popular pipettes. The maximum recommended thumb exertion force is
2.1 kgf for females and 3.0 kgf for males.23

Rainin solved this problem with LiteTouch System (LTS) of pipettes and tips (See Figure 3).
Here both the distal end of the pipette and the inside of the tip have a closely matched cylindri-
cal surface. LTS tips also have a sealing system close to the entry point and a hard stop end
point inside the tip. The end stop prevents the shaft from being pushed further into the tip with
increased force. This strictly limits the maximum sealing force, while still guaranteeing that there
will be no leakage. A strict limit on sealing force ensures a minimal ejection force (See Figure 1).
This unique yet simple design provides a tool that significantly reduces one of the major sources
of muscle fatigue and causes of RSI for pipettors.

METTLER TOLEDO The Influence of Pipetting on Experimental Results 17

The Ergonomics of Pipetting

Traditional LTS
Large seal Small seal
Conical Cylindrical
Friction fit Positive Stop

Figure 3
Mechanics of Rainin LTS tip sealing system compared to traditional conical tip sealing systems.

Plunger Forces
The plunger system of manual pipettes constitutes the second area of ergonomic interest to the
user where repetitive tasks can influence the outcome of RSI’s. After tip insertion, pressing the
plunger down to the first stop against the stroke spring (See Figure 4) prepares the pipette/tip
system for aspiration. A clear distinctive first stop is critical in that it defines the distance needed
to move the plunger and aspirate the desired volume.

“Home“ First Second

Position Stop Stop

Figure 4
Pressing manual pipette plunger to show home position (start dispense), first stop (full dispense)
and second stop (blowout).

Mechanically, this first stop is created by the plunger hitting a stiffer pre-loaded blowout spring
that has greater resistance than the stroke spring. After the plunger is pressed down to the
first stop, the tip is inserted into the solution and the plunger slowly released to aspirate. The
stroke spring performs the aspiration by pushing the plunger back up to the home position. The
sample is dispensed at the last step – pushing the plunger down against the force of the stroke
spring, through the first stop position and then against the force of the blow-out spring to re-
move all of the liquid. With all liquid dispensed, the plunger is released and returns back to the
home position.

18 METTLER TOLEDO The Influence of Pipetting on Experimental Results

In the paper by Lichty,21 blow-out forces are the second largest force in the process of pipetting (af-
ter tip ejection forces) and stroke spring forces the smallest.

An important aspect of the forces in this process is the in-

Piston
ternal piston seal system (Figure 5). The seal produces a
partial vacuum that enables liquid to be drawn into the tip Piston spring
as the piston moves up the shaft. The seal system must
be tight and strong enough to hold the vacuum required
for the entire operating range of the pipette. If the seal is Seal retainer
too tight the force required to push the plunger down and
then return it to the home position will be excessive. If the Seal
O-ring
seal is relatively loose, the forces required to move the
plunger back and forth will be less, but the potential for Figure 5
Sealing system in manual pipettes.
leakage increases.

For manufacturers of pipettes there is a balance between the seal design and the stroke spring
forces that must be considered in order to meet both the technical needs for accurate pipetting and
the individual user’s need for an ergonomic solution. This challenge is met differently by various
manufacturers, resulting in variable plunger forces (See Figure 6).

Manuf. 4’s
Manual Pipette

Manuf. 3’s
Manual Pipette

Manuf. 2’s
Manual Pipette

Rainin
Pipet-Lite* (LTS)

0 1 2 3 4 5 6 7
kg Force

Depress Hold Aspirate Dispense and Blowout

Figure 6
Comparison of plunger forces from four popular pipettes. The maximum recommended thumb exertion force is
2.1 kgf for females and 3.0 kgf for males.23 *New Pipet-Lite XLS reduces plunger forces by up to 43%.

Rainin developed high precision, low friction seals to help reduce stroke spring plunger forces.
These seals require minimal force to move while maintaining a vacuum, which allows for a
lighter stroke spring. A lighter stroke spring allows for a lighter blow-out spring, because the dif-
ference in spring forces defines the first stop. Thus blow-out forces are reduced as well.

Ultimately, the best solution for avoiding plunger forces is to use an electronic pipette, where a
microprocessor-controlled motor moves the plunger, resulting in near zero plunger forces. This
can be particularly helpful with multichannel pipettes, which have the highest plunger forces,
and when a researcher needs to perform intensive repetitive mixing of the liquid samples.

METTLER TOLEDO The Influence of Pipetting on Experimental Results 19

 Volume Setting – Changing the Aspiration Volume
The Ergonomics of Pipetting
Changing the volume of a manual pipette is often necessary to meet the requirements of the
experiment (e.g., changing the setting of a 200 µL pipette down to 50 µL for specific buffer
addition, and then back up to 200 µL for the next buffer). The process of changing the volume
setting screw – normally a small diameter button – requires several turns of the hand-fingers-
thumb combination. Experienced pipettors may even do this with one hand, both holding and
adjusting the pipette at the same time, while holding a liquid sample in the other hand.

A study by Asundi et al20 indicates that volume adjustment is an activity that requires high mus-
cle activity. In the four different thumb-related muscles that were tested, volume changes gene-
rated significant muscular response, indicating a potential for fatigue. Different pipette designs
can either reduce or increase the forces required for changing volume. A further study by Lichty
et al21 found a positive correlation between ease of volume adjustment and worker productivity,
probably because significant time is spent adjusting the volume setting during routine operation.

Rainin reduced the forces associated with volume adjustment by providing a large easy-grip
button and low-friction screw mechanism. An adjustable lock prevents the inadvertent changing
of the volume setting between pipetting steps. With this combination an operator can easily
unlock, adjust and lock the volume setting mechanism with one hand and with minimal force
requirements, (see Figure 7).

Figure 7
Setting the pipette volume with one hand.

Repetition
Pipetting once will not affect your hand, but pipetting hundreds of times over the course of sev-
eral hours certainly will. The chance of getting injured increases greatly with repetitive work.
Fortunately there are several straight-forward solutions.

The first kind of solution is to use products that do the work for you. Multichannel pipettes and
96-well pipetting platforms reduce repetition by pipetting multiple samples at once. Electronic
pipettes can semi-automate certain functions, such as dispensing multiple aliquots from a
single aspiration, automatically mixing, or automatically changing the programmed volumes in
a pre-determined sequence.

20 METTLER TOLEDO The Influence of Pipetting on Experimental Results

The second kind of solution is to modify pipetting habits to decrease the effects of repetitive work.
Try to take regular breaks, switch hands, stretch your limbs and maintain good posture (minimize
twisting of your shoulders, arms and wrists; manipulate objects at shoulder height or below).

Grip
Grip comfort has been positively associated with reducing hand and arm discomfort.21 Factors
important for grip comfort include the shape of the pipette body and fingerhook, and the di-
stance your thumb needs to reach to the top of the plunger. Try to grasp the pipette with as little
force as necessary to maintain control.

Conclusion and Recommendations

The ergonomics of pipetting are generally well understood. Higher pipetting forces and long
hours of pipetting are associated with developing RSIs. The following recommendations will as-
sist researchers in minimizing their chance of developing RSIs.

Look for Lower Plunger Forces

• Check spring forces associated with the plunger.
• Compare new pipettes with old ones.
• Use electronic pipettes when possible.
• Service your pipettes regularly to maintain optimal mechanics.
• Keeping the piston and seals clean will both lower friction, thus reducing pipetting force, and
maintain better accuracy and precision.

Look for lower tip ejection forces

Compare the tip ejection forces of different pipettes. Consider that a pipette with a high tip load-
ing force will probably have a high tip ejection force. Remember that multichannel pipettes have
generally higher forces and can be particularly hard on the thumb.

Other Considerations
• Use pipettes with a finger hook that fit comfortably in your hand without gripping tightly.
• Lower the forces associated with volume change by using a pipette with a volume lock, low-
friction volume change mechanism and a large dial with good grip.
• Reduce repetitive pipetting by using electronic pipettes, multichannels and 96-well pipetting
platforms. Take breaks and, if possible, switch hands and stretch arms and hands.

METTLER TOLEDO The Influence of Pipetting on Experimental Results 21

 5. References
Literature References

1 McDonald, G. Hudson, A., Dunn, S., You, H., Baker, G., Whittal, R., Martin, J., Jha, A.,
Edmonson, D., and A. Holt, 2008. Bioactive Contaminants Leach from Disposable Laboratory
Plasticware. Science 332 (5903): 917.

2 Trace organic and inorganic materials. Detection and analysis in pipette tips. 2009. Technical
report. Mettler-Toledo Rainin, LLC. TR-2009-1.

3 BPA Analysis of Polypropylene Beads and Pipette Tips. Impact Analytical technical analysis.
2010.

4 Inhibition of PCR reactions by self-sealing filter tips containing cellulose-gum additives. Rainin
Technical Report 9803. 2008.

5 Pennington, CJ, Edwards, DR. Real-Time PCR Expression Profiling of MMPs and TIMPs. Matrix
Metalloproteinase Protocols, Methods in Molecular Biology 622.

6 Toh, WS, Lee, EH, Richards, M, Cao, T. Invitro Derivation of Chondrogenic Cells from Human
Embryonic Stem Cells. Human Embryonic Stem Cell Protocols, Methods in Molecular Biology
584.

7 Grgicak, CM, Urban, ZM, Cotton, RW. Investigation of Reproducibility and Error Associated
with qPCR Methods using Quantifiler® Duo DNA Quantification Kit. J Forensic Sci, September
2010. Vol. 55, No.5.

8 Morga, B, Arzul, I, Faury, N, Renault, T. Identification of Genes from Flay Oyster Ostrea edulis
as suitable housekeeping genes for quantitative real-time PCR. Institut Français de Recherche
pour l’Exploitation de la Maer (IFREMER).

9 Vallania, FLM, Druley, TE, Ramos, E, et al. High-Throughput Discovery of Rare Insertions and
Deletions in Large Cohorts. Genome Res. 2010 20: 1711-1718.

10 Venegas, V, Wang, J, Dimmock, D, Wong, L-J. Real-Time Quantitative PCR Analysis of

Mitochondrial DNA Content. Current Protocols in Human Genetics, 19.7.1 – 19.7.12, January
2011.

11 Frendewey, D, Chernomorsky, R, Esau, L, Om, J, Xue, Y, Murphy, AJ, Yancolpoulos, GD,

Valenzuela, DM. The Lossof-Allele Assay for ES Cell Screening and Mouse Genotyping.
Methods in Enzymology, Volume 476.

11 Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl
MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guidelines: Minimum Information
for Publication of Quantitative Real-time PCR Experiments. Clin Chem. 2009 Apr; 55(4):611-
22. Epub 2009 Feb 26.

13 Guzman, GD, Shepherd, RP, Walmsley, AM. Immunoassays in Veterinary Plant-Made

Vaccines, Immunoassays in Agricultural Biotechnology. 2011.

22 METTLER TOLEDO The Influence of Pipetting on Experimental Results

14 Alamooti, AA, Ardalan FA, Abdolahi, A, Zeidi, M, Firouzjaie, F. Determination of Lymphocyte
Subsets Reference Values in Healthy Iranian men by a Single Platform Flow Cytometric
method. Cytometry Part A, 77A: 890-894, 2010.

15 ORA Laboratory Procedure, ORA-LAB.5.5, Attachment B, Table 2, Calibration of Equipment.

10/03. Food and Drug Administration.

16 Bertemann, R. Pipet Quality Control: A Microliter of Prevention… American Biotechnology

Laboratory, June 2004.

17 Björksten MG, Almby B, Jansson ES. Hand and shoulder ailments among laboratory
technicians using modern plunger-operated pipettes. Appl Ergon. 1994 Apr;25(2):88-94.

18 McGlothlin JD, Hales TR. Health Hazard Evaluation Report at Scientific Application
International Corporation, Frederick, Maryland. Cincinnati, OH: U.S. Department of Health
and Human Services, Public Health Service, Centers for Disease Control and Prevention,
National Institute for Occupational Safety and Health. HETA Report No. 95-0294-2594.
(August 1996.)

19 Fredriksson K. Laboratory work with automatic pipettes: a study on how pipetting affects the
thumb. Ergonomics. 1995 May;38(5):1067-73.

20 Asundi KR, Bach JM, Rempel DM. Thumb force and muscle loads are influenced
by the design of a mechanical pipette and by pipetting tasks. Hum Factors. 2005
Spring;47(1):67-76.

21 Lichty MG, Janowitz IL, Rempel DM. Ergonomic evaluation of ten single-channel pipettes.
Work. 2011;39(2):177-85.

22 David G, Buckle P. A questionnaire survey of the ergonomic problems associated with

pipettes and their usage with specific reference to work-related upper limb disorders. Appl
Ergon. 1997 Aug;28(4):257-62.

23 Kroemer KH. Cumulative trauma disorders: their recognition and ergonomics measures to
avoid them. Appl Ergon. 1989 Dec;20(4):274-80.

METTLER TOLEDO The Influence of Pipetting on Experimental Results 23

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