BIOTIX INC. THE BIOTIX GUIDE TO PIPETTING · PDF fileBoth types of micropipettes work...

BIOTIX INC.

THE BIOTIX GUIDE TO PIPETTING

Biotix Inc.Technological breakthroughs found only in the most advanced liquid handling tools available. http://biotix.com/ [email protected] +1 858 875 7696

understand and get the most out of your pipettes to get better results in your experiments

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CHAPTER 1

THE HISTORY OF PIPETTE DESIGN

2 Descroizilles’s and Gay-Lussac’s pipettes are most

similar to the modern day serological pipette. Today’s

serological pipettes are either made of disposable plastic or

reusable glass. Like Descroizille’s design, these pipettes

have a gradation scale on the side to accurately measure

and transfer between a fraction of a milliliter and 50 mL of

material. In use, serological pipettes are inserted in to a pipette

plunger apparatus. When depressed, the plunger creates a

partial vacuum that allows the liquid to be drawn up in to the

serological pipette. The user can easily control the amount of

liquid drawn in to the pipette.

3 In the 19th century, Louis Pasteur needed a way to

transfer small amounts of liquid without contamination.

He would take a glass tube, heat the center over a burner,

draw the tube out to make it very thin in the center, and

then snap it in half to yield two fine-tipped pipettes. The

fine-tipped end was placed in liquid and a balloon or rubber

bulb was placed on the larger diameter end. When the bulb

was squeezed, it creates a vacuum that draws liquid into

the pipette. Similar pipettes are still used today in this form

or as an all-in-one plastic version, which is referred to as a

transfer pipette. Typically, a transfer pipette does not have

measurement markings and are not meant for transferring

accurate amounts of liquid.

Like most modern day pipettes, they transfer between a fraction of a milliliter and 50 mL of material.

Francois Descroizilles and Joseph-Louis Gay-Lussac created the first pipettes in the 18th century.

In the 19th century, Louis Pasteur needed a way to transfer small amounts of liquid without contamination.

1 Contrary to popular belief, the earliest pipettes

were created not by Louis Pasteur in the 19th

century but in the 18th century by two French chemists,

Francois Descroizilles and Joseph-Louis Gay-Lussac.

Descroizilles in particular is considered the patriarch of

the volumetric analysis field. He created the precursor

to today’s buret and pipettes called the berthollimetre

and alcalimetre, respectively. In an 1824 paper, Gay-

Lussac made some modifications to the alcalimetre and

coined the resulting instrument a “pipette.”

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http://blogs.discovermagazine.com/bodyhorrors/files/2013/03/13156_loresmouthpipetting.jpg

5 Heinrich Schnitger, a postdoctoral student at the

University of Marburg, Germany was frustrated with

trying to accurately draw small volumes of materials using

the Carlsberg pipette. To overcome this issue, he created

a piston-pump driven glass pipette starting from a syringe.

His design created a more accurate and durable pipette that

didn’t require mouth pipetting. He was awarded a patent for

his design in 1961. Schnitger’s pipette design was adopted

by the scientific device industry and this basic design is still

used today.

Schnitger’s design created a more accurate and durable pipette that didn’t require mouth pipetting.

For his research, Levy created the first reported pipette for small measurements.

4 The Carlsberg pipettes took Pasteur pipettes one

step further. In the 1930s, Kaj Ulrik Linderstrøm–

Lang in the Carlsberg laboratory in Denmark wanted to

study single cell metabolism. He passed the problem

of scaling down the typical laboratory techniques to a

research fellow named Milton Levy. For his research,

Levy created the first reported pipette for small

measurements. Similar to Pasteur, he made these

pipettes by drawing a glass tube over a lit Bunsen

burner and pulling one end to create a thin, capillary

tube. However, each pipette was then calibrated with

mercury or dye to measure a specified volume of

liquid. Also, the liquid was drawn into the pipette by

mouth pipetting—a technique in which the scientist

uses his/her mouth to suck up the liquid into the pipette.

Carlsberg pipettes had a number of limitations—they

were not consistently manufactured (though some were

mass-produced), broke easily, did not ensure accurate

volumes despite calibration, required extensive

experience, and could cause the user to ingest

whatever was being drawn up in to the pipette.

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CHAPTER 2

TYPES OF PIPETTES

While micropipettes have been developed in all sizes to address specific laboratory tasks, mechanistically there

are two main types: Air displacement and positive displacement. In overall basic mechanism, both air and positive

displacement pipettes are similar.

Both types of micropipettes work similarly to Schnitger’s basic pipette design. Both pipettes require depression of

a piston-driven plunger. The user sets the volume to be drawn up prior to pressing the plunger. Depressing and

releasing the plunger creates a vacuum that draws the set amount of liquid up into the pipette. However, because of

slight differences in design and how the sample is drawn up, they are used for very different purposes and materials

in the lab.

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TYPES OF PIPETTES

Air displacement starts with dialining in your volume which moves the internal piston to the correct position

to be able to draw the appropriate volume. Once that volume is set you depress the plunger which moves

the piston down in the pipette. Once you emerge the tip into the liquid is when the vacuum is created and

once you release the button the piston moves up and aspirates the liquid to whatever setting you put on

the pipette. Once you have the sample in the pipette tip and you push the plunger again the piston will

move back towards the position you drew the liquid from. Then the blowout stage which is fully pressing

the top button pushes the piston past its position when you began the draw which creates pressure greater

than the volume drawn making it expel all the remaining liquid.

Figure 1. Comparison of Air Displacement

and Positive Displacement Pipettes.

AIR DISPLACEMENT

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TYPES OF PIPETTES

The downsides of the air displacement mechanism are that the accuracy of the pipettes is affected by

temperature, atmospheric pressure, and density/viscosity of the sample. In addition, the air cushion

can allow highly volatile samples to evaporate into the air space, which can lead to contamination of the

pipette.

Fixed volume Single channel

Manual

Repeat function (repeaters)

Variable/adjustable volume Multi-channel

Electronic

Calibrated to dispense one volume One pipette tip per pipette

Manually press and depress plunger

A larger volume is taken up into a reservoir. The sample volume is taken up and dispensed with a push of a button

Can change and set volume dispensed by pipette

One pipette holds many tips that simultaneously measure and dispense the same volume of liquid (often used with multi-well plates

Pipette operated by battery force

A key feature of air displacement pipettes is that a cushion of air is left between the pipette shaft and the

sample, so the sample is never allowed to come into contact with the shaft. This, in combination with

the use of disposable pipette tips makes air displacement pipettes very convenient for repeated, routine,

accurate pipetting work. In biological labs for example, air displacement pipettes are the instrument of

choice for measuring most aqueous solutions and buffers, including common biological materials like DNA,

RNA and proteins.

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TYPES OF PIPETTES

POSITIVE DISPLACEMENT PIPETTES

Positive displacement pipettes act more like

syringes. A disposable barrel/tip is placed

on to the pipette (see Figure 1). The desired

pipetting volume is selected and the piston

moves down into the disposable barrel. The

piston comes into direct contact with the

sample so no air cushion exists between the

sample and the piston. When the plunger is

released, a partial vacuum draws the liquid

up into the barrel. To release the sample,

the plunger is pressed again and the piston

pushes the liquid out of the barrel. The entire

barrel/tip is disposed of between samples.

A positive displacement pipette applies

constant aspiration force to the sample that

is not affected by the physical characteristics

of the sample. Therefore, they are useful

for accurately pipetting viscous or dense

solutions. They are also used to pipette

corrosive or radioactive samples to prevent

contamination of the pipette as well as cross-

contamination between samples. Positive

displacement pipettes are also used when

working with volatile samples that would be

susceptible to evaporation in the air cushion

of air displacement pipettes.

In comparison to air displacement pipettes,

the types of positive displacement pipettes

are limited. They are generally only available

as repeat or manual, single channel variable

pipettes.

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TYPES OF PIPETTES

Figure 2. Exploded Diagram of an Air

Displacement Pipette.

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TYPES OF PIPETTES

CHAPTER 3

FINDING THE RIGHT PIPETTE TIP

In the days of mouth-drawn Pasteur pipettes (and even earlier), one size of pipette tip had to fit all applications.

But today’s technology has advanced to the point where there many types of tips are available to suit a myriad of

applications. Picking the right tip for your application is important. In fact, the tip is one of the most important factors

in accurate pipetting – it is at least as important as calibration and pipetting technique.

A well-calibrated pipette isn’t very helpful if the wrong type of tip is used. In addition, using the wrong tip can lead to

contamination, and even force you to exert more force while pipetting, leading to repetitive stress injuries.

The sheer variety of available tips can be confusing, but this guide will help you make the best choice for your needs.

Pipetting must be precise and accurate—that is, you

must be able to consistently deliver the exact amount

of material in precisely the right location. This requires

tips that are manufactured to reduce any variation in

volume and sample delivery. That means you should

avoid cheaper tips that may have manufacturing

imperfections such as flashes, protrusions, scratches,

air bubbles, bends or impurities. Also, make sure

there is little tip-to-tip variation in a batch of tips.

Finally, be aware that some lower quality tips may

contain additives in the plastic that can contaminate

samples and affect experiments.

The design of the tip is also critical. While it may

seem like just a piece of plastic, many features of a

pipette tip can improve performance. One example

is BLADE® technology by Biotix in which the distal

end of the pipette tip has an optimal bore size to wall

thickness. This prevents droplet formation reducing

touch-off and save you time with each aspiration.

QUALITY

GENERAL CONSIDERATIONS WHEN CHOOSING A TIP

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Tips should fit snugly on to the end of your pipette.

This ensures precision, avoiding any aspiration of

air into the pipette as you draw samples. Tips should

also release easily from the pipette without falling

off prematurely. Poor fitting tips can lead to sample

leakage and may require additional insertion force

contributing to repetitive stress injury. Biotix tips come

with FlexFit® technology and “flex” at the insertion end

to provide a better fit on a variety pipette types.

Pipette tips represent a small cost compared to

time and reagents. Pipette tip costs can vary greatly

depending on the design of the tip and the materials it

is made from. One way to reduce costs is to purchase

high quality tips delivered in a reload format. Reloads

allow you to rack tips in seconds saving you time, and

come in a multitude of formats to fit your budget.

FIT PRICE

Figures 3 & 4. Packaging

of Bulk Tips.

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UNIVERSAL VERSUS BRAND SPECIFIC

Most pipettes are compatible with universal pipette tips. However many pipette manufacturers recommend using

their brand-specific tips designed for use with their pipettes. In most cases this is unnecessary, but there are a few

pipettes on the market that require a specific pipette tip. When selecting pipettes consider whether or not a specific

tip is required versus universal. The requirement of a specific pipette tip can lead to higher costs and disruptions

in supply. In addition, if brand-specific/pipette-specific tips are used, then each brand of pipette used in the lab will

require purchase of a different tip. Therefore, the most economical choice is to use universal pipettes and tips.

Universal tips are designed to fit most commercially available pipettes containing a standard-diameter barrel. These

tips meet a large number of laboratory requirements, including reagent transfer and high accuracy measurements

and can be used for a wide variety of applications, as long as they are of good quality and fit your pipette snugly.

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Barrier, also called filter, tips contain material near the proximal end of the tip that prevents your sample from

contaminating your pipette or damaging your equipment. The filter works by protecting the pipette barrel from aerosols

and liquids during aspiration of samples. This prevents contamination of the pipette and subsequent samples. Filter

tips are essential when working with toxic or caustic solvents or radioactivity. They are also used when conducting

experiments that require the highest level of purity, such as with PCR or RNA.

BARRIER (OR FILTER) TIPS

Figure 6. Pipette Tip Without Low-Retention Technology

Tip Types

Figure 5. Tip With no Barrier Compared to Tip With

Barrier.

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In many standard pipette tips a tiny amount of residual

liquid will to cling to the sides of the tip after aspiration

and dispensing due to the properties of the resin that the

tip is made. Low retention pipette tips are manufactured

in two ways: from material that is hydrophobic or

batch coated with a hydrophobic material. Typically

manufacturing from a hydrophobic material is superior.

Batch coating can be variable among tips negatively

impacting precision.

LOW RETENTION

Figure 7. Pipette Tip With Low-Retention Technology

Sterile tips are treated with radiation to eliminate

living organisms. They are packaged and shipped in

individual boxes. DNase/RNase-, endotoxin-free tips

undergo additional rounds of sterilization to remove all

contaminants. These tips are typically used for highly

sensitive experiments, such as PCR.

A cheaper alternative to purchasing pre-sterilized tips

is to purchase tips in bulk, and sterilize the racked tips

using an autoclave. This is most useful for tips to be

used in less sensitive, routine applications.

STERILE AND DNASE/RNASE FREE

Figure 8. Sterile, Pre-Racked Pipette Tips.

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Graduated tips contain measurement markings on

the side of the tip. They enable the user to visually

determine that the right volume is aspirated each time.

These graduations are not exact but can help avoid

large mistakes.

Many other tip types are available for specialty

applications. For example, wide orifice/bore tips have

larger openings for pipetting more viscous fluids.

Gel loading tips contain a thinner drawn out tip for

accessing wells.

GRADUATED TIPSOTHER TYPES

Figure 9. Image of Gel-Loading Tip

SELECTING THE BEST TIP FOR YOUR LAB

1. Do you need a specific pipette type for your pipette? Check around there may be multiple companies selling pipette tips compatible with your pipette.

3. Are you performing PCR or a highly sensitive application? Sterile, DNase free/RNase free barrier tips would be the best choice to prevent carry-over and contamination.

2. Are you working with sticky samples such as protein or DNA? You may want to consider low retention tips

4. Are you working with hazardous samples such as human blood, radioactive, or corrosive material? A barrier tip that seals will protect your pipette from contamination.

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CHAPTER 4

CARING FOR AND MAINTAINING YOUR PIPETTE

How you store your pipette when it’s not in use is just as

important as how you use it. For example, using a pipette

stand to store your pipettes prevents the instrument from

being accidentally knocked to the ground and keeps your

pipettes organized. Storing pipettes upright prevents any

liquid that might have accidentally been aspirated into the

barrel from running further into the pipette—possibly even

to the piston.

The pipette is a sophisticated and precise piece of

technology that requires proper care and handling.

Dirt, dust, and residual liquids affect its performance.

Your pipette requires calibration on a regular basis to

maintain its precision and accuracy. Most damage to

pipettes arises from improper handling. For example,

the user drops the pipette on the lab bench and

causes damage to the pipette barrel, or incorrect

pipetting technique/pipette tip choice causing

contamination inside the shaft.

PIPETTING TECHNIQUE ADVICE

STORING OF YOUR PIPETTE

Always make sure your movements are smooth and consistent. This prevents jarring of the internal mechanisms of the pipette.

When not using your pipettes, store them upright and away from liquid.

Proper pipetting technique will prevent damage to the piston and other internal mechanisms of the pipette. It will also keep the pipette barrel free from contamination.

Don’t dial a volume that is outside the limits of your pipette (this can also damage internal mechanisms). Use a pipette with a suitable capacity instead.

Always use a pipette stand that fits your pipettes.

Operate your pipette in a vertical position to prevent liquids running along the shaft of the pipette.

Never store your pipettes on their side, and never store them with liquid in their tips.

Don’t let the piston snap back up into place when aspirating—this can lead to liquid backup and contamination of the shaft’s interior.

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Nearly all pipette manufacturers provide cleaning instructions that won’t harm your pipettes. The manufacturer’s

instructions should contain information about safe products to clean the inside of the pipette, and, for example, how

to clean after using radioactive substances. Because certain cleaning products and procedures can harm different

pipettes, be sure to check their instructions before starting any cleaning procedure. However, there are some general

cleaning steps that are helpful for any pipette:

PROPER CLEANING OF YOUR PIPETTES

Wipe pipettes down with a 70 % ethanol solution every day.

Organic solvents (and proteins) can be removed with a detergent solution, as long as the detergent doesn’t damage the pipette.

Learn how to remove the pipette barrel to clean inside (using the manufacturer’s instructions as a guide).

Autoclaving can be helpful, but for some types of pipettes, only certain parts may be sterilized in this way. Always check manufacturer’s guidelines in advance because autoclaving can destroy pipettes that are not suitable for autoclaving!

Visually inspect your pipettes for damage every time you use them.

You can eliminate DNA by immersing pipette parts in at least 3% (w/v) sodium hypochlorite, for at least 15 minutes. Rinse well with distilled water and allow to dry. Some manufacturers have specialized solutions for removing DNA. RNA degrades quickly, and doesn’t require any special treatment to remove.

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CALIBRATION

Calibrating a pipette involves testing it to determine if the dispensed volume is equal to the measured volume. If

those two values aren’t the same, then your pipette needs calibration. All pipettes are calibrated at the factory, but

repeated use (and maintenance issues) call for periodic recalibration. Manufacturers will disclose precision limits

for their pipettes, but these may be more exact than are necessary for your lab. You must determine the necessary

precision for your experiments.

HOW TO CALIBRATE PIPETTES

1. The calibration testing should take place in a draft-free room at a constant temperature between 20°C to 25°C.

5. Repeat step 4 but dispense the second calibration point’s volume.

2. Relative humidity should be above 45%, especially for volumes under 50 µL. Keep the pipette in this room for at least two hours to acclimate.

6. Verify the weights you’ve collected to be within the published pipette manufacturers systematic error range.

3. Choose two points that are published in the user manual by your pipette manufacturer to calibrate against. Nominal and 10% of nominal are usually published.

7. If the volumes fall outside of the published range, you may do the calibration adjustments yourself or send it out to a repair facility.

4. Using distilled water, dispense the first calibration point’s volume onto an analytical balance. Record the weight. Repeat this process ten times.

Calibrations should be done every 6 to 12 months. If you see worn O-rings, they should also be replaced as needed.

Your lab should have a procedure to regularly do self checks in between scheduled calibrations

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Electronic pipettes should be calibrated by the manufacturer or another specialized and trusted facility, because of the

wide variety of features and construction methods for each type of pipette. For manual pipettes, some adjustments can

be made in the lab using the following tips:

CALIBRATION: DO IT YOURSELF OR SEND IT OUT?

All adjustments are made at the lowest volume.

Place the service tool that came with the pipette into the opening of the calibration nut at the top of the handle.

Place the service tool that came with the pipette into the opening of the calibration nut at the top of the handle.

A service tool should have come with the pipette. If one did not, then you can order this from the manufacturer.

After adjusting, check the calibration again using the process outlined above.

www.Calibrate-It.com is a useful resource when carrying out your own calibrations. If these steps don’t help, then send the

pipette out.

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http://calibrate-it.com/Calibrate-it/#Home

1. Pipette tips drip or leak: This is probably the most commonly encountered pipetting

issue, and can easily occur if tips are loosely fitted, or if inappropriate tips are used. You

can solve this by ensuring that your tips fit your pipettes, and/or by checking the pipette barrel

for damage.

2. The pipette aspirates and dispenses inaccurate volumes: This can occur for a

number of reasons, including pipetting outside the range of your pipette, leaking tips

(see 1. above), or the pipette is in need of calibration.

3. The dispensing button is stiff and doesn’t release smoothly: This may occur if the

seal gets swollen by reagent vapors and can usually be solved by opening the pipette

and allowing it to ventilate. The piston can be lubricated if necessary.

From time to time, your pipette will not behave as it should, and the tips below may help you solve some

of most basic issues you might encounter:

If your pipette stops functioning completely, and the simple tips above don’t help you then don’t

despair, but do send your pipette out for repair!

TROUBLESHOOTING YOUR PIPETTE

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CHAPTER 5

ERGONOMICS AND PIPETTING IN THE LAB

Many scientists do not think about the impact of pipetting on their long-term health. However pipetting has been

identified as a risk for repetitive stress injury (RSI). Ergonomics is the study of how our work and other common

activities affect our physical (in particular our musculoskeletal) health. In the lab, it focuses on how repetitive and

forceful activities, as well as our sitting and standing postures, can cause pain and eventually damage to our bodies.

Much of the work we do in the lab (and many other workplaces) can lead to back pain, RSI and other issues

over time, all depending on the nature of the work. In the lab, we are much more likely to become injured if

we use the wrong equipment, or if we use appropriate equipment in the wrong way.

One common source of RSI and other lab-related work injury stems from pipetting. You may experience RSI

as a numbness around your basal thumb joint, an ache in your wrist, or a general weak feeling in your hand.

These symptoms occur because of the repetitive nature of pipetting and the force you apply when doing

so. The symptoms associated with RSI may also extend into your back and shoulders causing you great

discomfort.

Force in pipetting comes from attaching the pipette tip, dispensing the contents, ejecting pipette tips, and

keeping a tight grip on a poorly designed pipette. Pipetting is usually very repetitive, and any laboratory

worker might go through thousands of pipetting motions every single day. Pipetting for only one hour a day,

over the course of a year, can put you at risk for RSI!

WHY SHOULD WE PRACTICE ERGONOMIC PIPETTING?

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Older pipette models require a lot of manual force on the plunger and the ejector button, which leads to more muscle

strain than modern pipettes.

CHOOSE AN ERGONOMIC PIPETTE AND ERGONOMIC TIPS

Look for more advanced pipettes that have low profile ejector buttons to reduce ejection force.

Use well-fitting tips such as Biotix with a flexible proximal end (“FlexFit”). This flexing end forms a secure seal with your pipette barrel, eliminating banging or rocking. You should not have to twist your wrist or pound the tip into the end of pipette to get a good fit.

Learn how to remove the pipette barrel to clean inside (using the manufacturer’s instructions as a guide).

Ejection forces can vary greatly between pipette types, so it is important to test and select the one with the least force required.

Figure 10. Demonstration of Flexible Fit at

the Proximal End of the Tip.

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BE ERGONOMIC – BE KIND TO YOURSELF!PREPARE YOUR WORKSPACE

Keep an eye on your posture. Sit up straight with your arms straight out.

Make sure your work area is clean, clear and organized.

Using proper pipetting technique and practicing good

posture will go a long way in helping you avoid RSI

and other pipetting-related injuries. You can be kind to

yourself by keeping the following in mind:

As mentioned earlier, RSI is only one-lab related injury, and pipetting is only one cause of RSI. Continuous pain or pain that

doesn’t subside when practicing ergonomic pipetting should be addressed by a doctor. From time to time physiotherapy

may be necessary to treat RSI. Luckily, it’s very easy to reduce the risk of pipetting-associated RSI by following the

ergonomic tips provided here. Don’t be afraid to take that 2-minute break!

Take breaks! If you have hours of pipetting work ahead, don’t do a marathon session. Get up, stretch, walk around a little bit, and then start working again.

Make sure you have a waste disposal bucket nearby on the side that suits you best. Position yourself so that you can pipette without

reaching too far. Don’t hunch over or keep your arms elevated for any extended period of time.

Arrange your most commonly used reagents so that you can reach them easily.

Pour liquids into smaller vessels so they are easier to reach, and keep your pipette tips at bench level (don’t try reaching for them up on a shelf).

Remember that the U.S. Occupational Health and Safety Administration (OSHA) recommends taking a 2-minute break every 20 minutes.

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CHAPTER 6

FURTHERREADING

History of Pipettes:1. Duval C. (1951) Francois Descroizilles, the inventor of volumetric analysis. J. Chem. Educ. 28(10):508–19.

2. The Rockefeller University: Science Outreach Program. Origins of the Pipette: Why Today’s Scientists Don’t Need

to Use Their Mouths.

3. Klingenberg M. (2005) When a common problem meets an ingenious mind. EMBO Reports 6:797-800.

4. JoVE. Introduction to Serological Pipettes and Pipettors.

5. Sella A. (2014) Schnitger’s pipette. Chemistry World.

Pipette Tips:Here are some more references to help with tip selection.

1. Bitesize Bio Biotix article

2. http://biotix.com/technology/

Ergonomic Pipeptting:1. Bitesize Bio Biotix article

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FURTHER READING

http://pubs.acs.org/doi/pdf/10.1021/ed028p508

http://incubator.rockefeller.edu/origins-of-the-pipette-why-todays-scientists-dont-need-to-use-their-mouths/

http://incubator.rockefeller.edu/origins-of-the-pipette-why-todays-scientists-dont-need-to-use-their-mouths/

http://embor.embopress.org/content/6/9/797.long

https://www.jove.com/science-education/5034/introduction-to-serological-pipettes-and-pipettors

https://www.chemistryworld.com/opinion/schnitgers-pipette/7789.article

http://bitesizebio.com/4074/the-pcr-controls-you-must-use/

http://biotix.com/technology/

http://bitesizebio.com/30491/repetitive-strain-injury-pipetting/

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