pipetting techniques
Basic techniques in molecular biology
OBJECTIVE
To practice using a micropipette properly by moving water from a flask to a micro centrifuge tube.
INTRODUCTION
Pipettes
and micropipettes are used to measure and deliver accurate volumes of liquid.
The difference between the two is that micropipettes measure a much smaller
volume, starting at 1 microliter, while pipettes generally start at 1
milliliter.
Both
pipettes and micropipettes are used in various laboratories, including
·
microbiology
·
environmental sciences
·
medical
·
academic, and
·
research labs
Micropipettes are utilized in the laboratory to
transfer small quantities of liquid, usually down to 0.1 uL. They are
most commonly used in chemistry, biology, forensic, pharmaceutical, and drug
discovery labs, among others. Common micropipette sizes used in labs include:
Common Micropipette Sizes
Volume Range
P2
0.2-2 uL
P10
1-10 uL
P20
2-20 uL
P100
20-100 uL
P200
20-200 uL
P1000
100-1000 uL
Not only do micropipettes differ in
size and volume dispensed, but depending on those particular aspects they also
require specific pipette tips. Micropipettes use a disposable pipette tip to
aspirate liquid, note that the tip is the only part of the pipette that makes
contact with the solution. A new tip is utilized for every sample in order
to prevent cross contamination.
The
most essential aspect of a pipette tip is its quality, whether you’re
looking for a filter, low retention, or gel loading tip, make sure that the
pipette tip will perform accordingly and as precise as your micropipette. Make
sure to research the purity of your pipette tips.
Basic parts of a micropipette include plunger button, tip ejector button, volume adjustment dial, volume display, tip ejector, and shaft. They differ in design, weight, plunger force, and overall precision. Depending on your budget and preference, there are plenty of micropipettes in the market that specifically catered to meet our needs.
Figure:1
Glass
pipettes are excellent in chemical resistance. Since dry heat sterilization is
possible, it can be used repeatedly by washing and sterilizing depending on the
type. Plastic pipettes such as polystyrene are basically disposable, so there
is no cleaning time involved and pre-sterilized items are convenient in
preventing contamination. Pipettes are generally used for moving small amounts
of liquid or when measuring and dispensing liquid in mL units. When measuring
less than 1 mL, Micropipettes are more accurate and user-friendly.
Pasteur Pipettes (unit / mL) A pipette made of thin glass with a tapered tip. Some are
plastic. They are often disposed of after use to prevent possible
contamination/cross-contamination and mainly used when you want to handle
solutions individually, such as medium transfer and sampling.  Kaomagma pipettes (unit / mL) It is either made of glass or plastic, has a round bulge
near the top of the pipette body to disperse the bubbles in the liquid, and can
measure formable liquids safely. A rubber bulb is usually attached to the top
of the pipette to act as a pump to aspirate and discharge liquid. 2 mL, 5 mL
capacity types are common.  Measuring Pipettes (Unit / mL) Measuring
Pipettes are made out of glass or plastic with the volume in increments, marked
along the tube, and can fairly accurately measure liquid. In the past, the
method of using this pipette was to put the mouth directly on the open end of
the pipette on top to aspirate the liquid like a straw (a method known as,
"mouth pipetting"), however, this method has the potential danger of
accidental ingestion. Now, these kinds of pipettes are generally used with an
automatic pipette pump (pipette controller), or a rubber suction device (safety
pipette), especially in cases when handling potentially harmful/hazardous reagents and/or bacterial fluids. Depending on the length and scale of the pipette, there are the following types
of pipette designs. ·
Short
Measuring Pipettes ·
Pipettes
that are graduated to the very end of the tip ·
Pipettes
that are graduated for partial delivery  Volumetric Pipettes A pipette made of glass and has a single graduation mark on the glass tube above the bulge in the middle. Although the liquid measurement accuracy is better than other measuring pipettes, accurate measurement requires some skill. In the past, the mouth was used to aspirate the liquid, but now an automatic pipette (pipette controller) or a rubber suction device (safety pipette) is used for safety. If liquid remains in the tip after discharging the liquid, warm up the bulge by hand to expand the air inside to push out the remaining liquid.  Micropipette (Unit / μL) Pipettes
that can measure liquid in microliters (μL) with precision, and are most
commonly used in experiments, research and analysis in the field of life
science. They aspirate and discharge liquid by volumetric displacement of air
by the vertical movement of an internal piston. As for the part that comes into
contact with liquid, disposable plastic tips are mainly attached to the pipette
nozzle and used. There are variable volume types that can be adjusted freely
within the setting range, and fixed types that have the volume set in advance.
Periodic capacity inspections and maintenance are required to maintain accuracy
and repeatability. In addition, pipettes with a volume of 1 mL or 5 mL or more,
may be called a micropipette for convenience. Autoclave (121°C for 20 min.) / UV Resistant / Organic Solvent Resistant  There
are so many types of tips and we must select a suitable one for our experiment. A
standard tip is a multi-purpose tip for many laboratory applications with a
variety of performance requirements that range from high accuracy to reagent
dispensing with greater tolerance. Sterile standard tips are available for
applications demanding the highest level of purity.  Sterile filter
tips A
filter tip is beneficial when the assay is sensitive to cross-contamination, or
if the sample can contaminate the lower part of the pipette. The filter
prevents liquid from accidentally splashing the inside of the pipette, and
reduces aerosols from penetrating the pipette tip cone during pipetting. Filter
tips are recommended for low volume applications in genetic studies, forensics,
PCR, and radioisotope sampling. They are available with either self-sealing
barrier or standard filter tips – both of which are designed to prevent
cross-contamination.  Extended length
pipette tips Extended
length tips allow you to access the bottom of test tubes, reagent bottles,
flasks, and other vessels without touching the shaft of the pipette against the
side of the tube. This adds a layer of security to protect samples, and
virtually eliminates the chance of carryover contamination. The longer tip
length allows you to reach the bottom of long or narrow vessels that standard
tips cannot reach.  Low retention pipette tips Utilizing
polymer technology makes the inner surface of the pipette tip more hydrophobic,
resulting in a significant reduction in sample loss due to adhesion. Benefits
include improved sample delivery and conservation of expensive reagents.  Some pipettes are require different
types of tips and it can be divide as following,
DISCUSSION A pipette is a laboratory instrument used to measure
out or transfer small quantities of liquid, in volumes of milliliters (mL),
microliters (μL). It is used in a wide variety of experiment processes in
chemistry, molecular biology (biotechnology), medical science, experiments in
the field of natural science in general, analysis of food and chemicals, food
safety inspections and inspections in clinical examinations. Using a pipette is an essential part of working in a
science laboratory. Pipettes, specifically micro-pipettes, are used for
accurately measuring and transferring small amounts of solutions from on
container to another. This is useful when creating a dilution series, plating
cell samples. Etc. The first step when using a pipette is setting the
pipette to your desired volume. To do this, locate the small window on our
pipette that has three numbers in it. If our pipette has a lock/unlock switch,
switch it to the unlock position. Then, turning the plunger clockwise or
counter-clockwise until we reach the desired amount. For the picture above, the
pipette being used is a P200 and it is set a 175 uL. It is very important
to stay within the volume limits of your pipette, which depend on its size,
because even though you may be able to get smaller or bigger amounts, the
pipette is not designed to be used with those amounts and it could end up
breaking your pipette. Nearly all pipettes have two 'stops' on the plunger.
Before we start using the pipette, it is a good idea to play with the plunger a
bit to feel these two different stops. The first stop is the stop that changes;
it is the amount that we set the pipette to in the previous step, and it is
shown by the first picture. The second stop, shown in the second picture, is an
extra 'push' that it sometimes needed to remove all of the liquid from the tip.
When to use these two stops with be discussed in later steps. First of all, make sure you have the correct pipette tip size and brand to match our pipette. If the tip doesn't fit on the end of our pipette, it is not the right size. To put a tip on your pipette, open the tip box and place the end of the pipette into one tip. Press down gently, but firmly, to ensure that the tip is firmly on the pipette. Then take the pipette away from the box and the tip should come with it. Close the pipette tip box to help prevent contamination.  Now
that we have a tip on our pipette, we are ready to start pipetting. Press the
plunger down until the first stop - be careful to not go past the first stop.
Depress the plunger BEFORE we put our pipette into the solution - otherwise
bubbles can be created, introducing air that can cause contamination and other
problems.   After
depressing the plunger, carefully place the tip of our pipette into the desired
solution. Try not to touch the sides of the container as we do this. Once the
tip is in the solution, slowly release the plunger and the set amount of
solution will be pulled up into the tip, then remove the tip from the solution
and close the container. If there are any air bubbles in the tip,
start again as this could affect the amount of solution. Place
the tip into the new container, again being careful to not touch the sides of
the container to prevent contamination, and as far into the container we can go
without touching the sides. Slowly push the plunger down all the way to the
first stop, then the second stop to make sure all solution has been pushed out
of the tip. Once done, remove the pipette from the container and close the lid.  Now that you've successfully
measured and transferred our solution, it is time to get rid of the tip. To do
this, press down on the tip ejector button, which is a small button usually
next to the plunger, while holding the pipette over a waste bucket. This will
move a metal or plastic arm down, forcing the tip off of the pipette. It is always
a good idea to change tips when you are changing solutions, or if it touches
any surface; these both reduce risks of contamination.  Two
pipetting techniques are used in the lab: forward mode (also known as standard
mode) and reverse mode. For pipetting most aqueous solutions, forward mode
should be used. It typically yields better accuracy and precision than reverse
mode and is used by manufacturers to calibrate their pipettes. Reverse mode is
recommended for
viscous solutions, because forward mode for this application can result
in under delivery. To
pipette with forward mode, the plunger is pressed to the first stop and the tip
is immersed in the sample. The plunger is then released to aspirate the
aliquot. Finally, the entire contents are dispensed by pressing the plunger to
the second stop, the blow-out step. To
engage reverse mode, the plunger is depressed to the second stop before
immersing in the sample, which effectively over aspirates it. It is then
depressed only to the first stop to deliver sample, leaving some in the tip. Pipetting
should be slow and steady. Operators should pause immediately after aspirating,
with the pipette tip still in the sample liquid. Failure to do so can lead to
inconsistent aspiration volumes because the liquid requires about a second to
settle after aspiration is complete. A pause of about one second is acceptable,
but anything longer than that may result in under delivery. It
is also important to press and release the pipette’s plunger with a consistent,
moderately fast motion. Moving the plunger too fast or too slow will negatively
affect the aspirated volume. Inconsistent speed will decrease precision of the
pipetted aliquots. Holding
the pipette perpendicular to the liquid surface and avoiding touching the
inside walls of the sample container will minimize errors due to surface
tension or partially obstructed flow . Consistency
and care are critical also when dispensing sample. Before dispensing, pipettes
should be checked for droplets on the outside of the tip, with visible droplets
removed very carefully with a lint-free cloth. This should only be done if
absolutely necessary, however. Liquid can be wicked from the tip opening,
causing sample loss and under delivery. Once clean, the tip should be placed
against the receptacle wall and the plunger pressed to the second stop when
using forward mode. The tip should then be slightly dragged up the receptacle
wall to allow all liquid to be drawn from the tip. Repeated actions produce
repeatable results—the plunger should always be pressed and released with
consistent speed and pressure. Tip
immersion depth is important and requires subtlety: if it is too shallow, there
is a risk that air can be aspirated; too deep in the sample, and droplets may
collect on the outside of the tip and more sample might be forced inside. Pipetted volume will determine the
correct immersion depth, generally 2–4 mm below the surface for volumes
from 1 μL to 1000 μL, and 3–6 mm for volumes larger than 1 mL. The pipette
should be held in a vertical position (tilting it may cause a change in sample
volume) and the tip should not be allowed to touch the sides or bottom of the
sample vessel. REFERENCES
·
"Biotechnology Outreach".
Retrieved 3 March 2016. ·
Klingenberg, M (2005). "When a common
problem meets an ingenious mind". EMBO Rep. 6 (9): 797–800.
doi:10.1038/sj.embor.7400520. PMC 1369176. PMID 16138087. ·
Zinnen, Tom (June 2004), The Micropipette
Story, retrieved November 12, 2011 ·
Shohl, Alfred T. (February 1928). "A
Pipet for Micro-Analyses". Journal of the American Chemical Society. 50
(2): 417. doi:10.1021/ja01389a502
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