TA’s notes on Experiment 1


Experiment 1:  Accurate Pipetting of Liquids


There are three main types of tools used in the lab to move liquids from one container to another. These are: pipets, Pasteur pipets and the micropipettes. This exercise aims to get students used to dispensing liquids using these three different instruments.


Pipets: Glass or plastic, calibrated to deliver any amount in the graduated scale from 1-25 mls. Volumes of 5, 10 and 25 ml-pipets are usually common in the lab. Suction of liquid is through bulb or pipetAid.


Pasteur pipet:  A small, tapered glass tube, used with a bulb, and not graduated. It is used to dispense liquid if the volume is not critical. Usually hold about 1 ml.


Micropipette:  Used only with disposable pipette tips. This instrument is often used to dispense small volumes ranging from 1 µl to 1000 µl. To draw liquid up, depress plunger to the first stop point (not all the way down), insert the tip in the liquid to be measured, then slowly release the plunger which pulls the liquid into the tip.  Dispense by pushing the plunger, usually with the tip touching the tip of the receiving container.  Then push the plunger all the way down to expell any residual.



PART I. Practice Experiment


A.  Macropipetting with a PipetAid


First, we will practice dispensing different volumes of liquid using the 10 ml plastic disposable pipet. A PipetAid is provided in each bench to make pipetting easy. This is an electric pipettor that replaces the use of a manual aspirator. It has two buttons that control the flow of liquid in and out of the pipet.  Each is graded so that the farther you push the button, the faster the liquid is drawn up or expelled.



(1)For each group, fill a 250-ml flask halfway with water and obtain one 10-ml pipet.

(2)Using this pipet, practice pipetting with the pipetAid. Get used to the rate at which the fluid is drawn up and expelled. The rate varies depending on how hard you push the buttons.

(3)Learn how to control the rate of flow. Always have one eye on the pipet and at the same time watch the level in the container.

(4)Put less than 10 ml of water in the flask and then draw it all up without taking any air. If you do pull air, measure the volume by the meniscus right below the layer of air bubbles.

(5)Learn how to insert the pipet into a flask without touching the sides and hold it there while withdrawing the liquid. This is a good sterile technique.

(6)Make sure you do not draw the liquid up to the cotton plug and especially not past the cotton plug. This will contaminate your pipettor and requires disassembly and cleaning.


B.  Using a Micropipette


A micropipette is designed to dispense small volumes of liquid. There are different types of micropipettes designed for a particular range of volume. The volume capacity that each micropipette can dispense is indicated at the top near the plunger. For practice, you will use three micropipettes of different volume ranges. 


(1)Use three micropipettes: p20 (1-20µl), p200 (20-200µl) and p1000 (100-1000µl) to cover a range of volumes.  There are two different sizes of tips- yellow or clear for the P20 or P200 and blue for the P1000. 

(2)Put some water in a beaker to use to practice micropipetting.

(3)Set a volume. Make sure that the volume you choose is within the range of the micropipette. Do not push the volume dial beyond the indicated volume range. If you do, this will result to inaccurate measurements and damage to the instrument. 

(4)Load the tip onto the pipettor by firmly pushing the barrel into the tip while it is still in the pipette box.  I usually tap it up and down a few times to make sure it is seated.

(5)Before you put the tip into the liquid, push the plunger down to the first stop. Do this outside of your flask/tube so that you are not pushing potentially contaminated air into your potentially sterile liquid.

(6)Put the tip just below the surface of the liquid, then slowly release the plunger allowing the liquid to be sucked up into the tip.

(7)After filling the tip, take the tip out of the liquid and make sure there are no bubbles or drops on the outside. 

(8)Place the tip against the edge of the tube/dish/liquid into which you are adding it and slowly depress the plunger to expel the liquid.  Push all the way to the first stop and then keep going to the full stop to expell the liquid to its desired container.


IMPORTANT: If you rapidly let go of the plunger, you will pull air along with the fluid up into the barrel. If the barrel gets the media, microorganisms will grow in it causing contamination of most of your experiments. If this happens, the pipetters can be cleaned easily so let us know.


(9)Deposit the tip directly into the sharps container by pressing the ejector button.

(10)Practice using the three micropipettes until you get used to them.



PART II:  Pipette Accuracy and Precision 


Now you will determine whether your micropipettes are functioning correctly and you are using them correctly. You do not want to assume they are correctly measuring volume- you want to be confident that your experiments will be done correctly, so you want to make sure the pipettes are working correctly.  You will measure the accuracy and reproducible each of your pipetter is.  This will be done by weighing the mass of water pipetted at various settings.


(1). Accuracy- Pipette various amounts of water into a disposable weigh boat and determine the volume of water you actually transferred knowing that the density of water is 0.998gram/ml (at room temp); that is, 1 ml of water has a mass of 1 gram so 50µl (0.05ml) volume of water has a mass of 0.05 gram (50mg).

(2) Precision/Reproducibility- pipette the same volume multiple times.  This will allow you to determine the mean volume and see if you get the same reading each time. Try re-using a tip multiple times and see if that affects your measurements. 


You will design your own experiment. Each person should pipette a range of volumes of liquid using each of the 3 micropipettes and measure the corresponding mass on the balance. It is best to design the experiment and set up a grid in your notebook and then take the pipetters to the balance and carry out the measurements.  Make sure you record all your data in your notebook as you do the experiment. This will allow you assess the accuracy of your micropipettor and your technique at the beginning of the semester. It should not change if treated with good care.


Presentation of Data


(1)For this experiment, you each need to make several graphs that display your data; one each from your measurement using p20, p200 and p100 micropipettors. The X axis will be the volume (in µl) and the Y axis will be the Mass (in mg or grams).


(2)You should post your graphs to your website when done so we can check them.  


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Things needed for this experiment:


1.  Make sure all pipetAids in all the benches are working.

2.  There should be a set of Micropipettes [p20, p200 and p1000] per group.

3.  Analytical balance [borrow from Maryke].

4.  plastic sterile 5 and 10 ml pipets.






Accurate Pipetting with a Pipettman


Pipetting techniques

Dispensing with mechanical air-displacement pipettors demands skills and experience to do it right. There are a few things not depending on the technique one should always pay attention to when pipetting:

* The pipettor/tip should be chosen to minimize the air space between the piston and the liquid.

* When filling the tip, the tip should not be placed too deep, but just under the surface of the liquid in the reservoir (2-3 mm).

* Pre-wetting the tip improves both accuracy and precision.

* The pipettor should be held vertically, not at angle.

* The aspiration should be done smoothly, not too quickly.

* Dispense sample by touching the tip end against the sidewall of the receiving vessel to ensure complete sample flow.


Factors affecting pipetting performance

The pipettor tip

The tip is an integral component of the pipetting system and its shape, material properties and fit have a considerable influence on the accuracy of liquid handling. In addition to fitting, most important is to test how the tip wets, and whether there are droplets remaining after the sample is dispensed. To ensure accurate pipetting results, only tips specified by the manufacturer should be used. Cheap, poorly fitting tips not designed for the pipettor can result in serious measurement errors. If using others than tips specified by the manufacturer, one should always test the performance before beginning the analysis. Especially the performance of filter tips used in wide variety of applications vary a lot depending on the pore size and material of the filter in addition to the properties of the tip.  One should also keep in mind that there is no such product as a universal tip.


Environmental conditions

Sources of error from the environment include temperature (differences in temperature between the pipettor, fluid and the ambient temperature), air pressure, and humidity. The single greatest contributor to error is temperature, especially if working with air displacement pipettors (Joyce and Tyler, 1973; Lohner et al., 1996). As an example, increasing the temperature of the liquid from 5°C to 28°C while other elements (pipettor and tip) are kept constant (22°C), pipetting of 1 ml can have up to 6% error in volume. An ideal environment for pipetting maintains ambient temperature within 1°C, including all parts of the liquid handling system.


Inaccuracy and imprecision

Precision is an agreement between replicate measurements. Precision has no numerical value, it is quantified by the imprecision. So high precision i.e. small imprecision, means very little variation between the repeated measurements on the same sample. To achieve it you require a precision instrument, but you must also follow good laboratory practice - cleanliness and consistent correct handling.

On the other hand, it is possible to be very consistent, but consistently wrong. Inaccuracy is the numerical difference between the mean of a set of replicate measurements and the true value - so high accuracy i.e. small inaccuracy means a very little difference between your mean sample and the true value. Accuracy is achieved by careful calibration of a precision instrument. What is needed, of course, is both precision and accuracy.


Formulas:

Expected value: what you set the pipette to measure

Measured value:  what the balance says you measured

Standard deviation: the statistical variation in your measurements- Excel has a function to calculate this





Accuracy (% error) = 100 x (Mean of measured value - Expected value)/Expected value



Precision (% CV) = standard deviation/mean x 100



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