Serological pipette
Serological pipettes come in a variety of sizes, generally from 0.5 ml - 25 ml and are made of glass (nondisposable) or plastic (disposable). In general, we will use disposable pipettes and we will always use them with a mechanical pipette pump. NEVER pipette by mouth!
Fig. 1 Serological pipette pump
Note: sometimes the white rubber seal (into which the pipettes are inserted) detaches from the pipette pump when the pipette is removed and remains stuck on the end of the pipette. Before you discard disposable serological pipettes, check to be sure that you are not throwing away the seal (Fig. 1) - it can easily be re-attached to the pump.
Micropipetter (a.k.a. the "Pipetman")
Fig. 2. Parts of a digital micropipetter
The micropipetter is an adjustable digital microliter (µl) pipette (Fig. 2). There are several different models and sizes available, but you will be using only 3 of these, the P-20 micropipetter, the P-200, and the P-1000. The number on each indicates the maximum volume (in µl) that can be measured with the micropipette. NEVER adjust the micropipette for a setting above this number! Exceeding the maximum volume will break the micropipetter, which costs over $250 to replace.
The volume indicator consists of a three-number dial and is read from top to bottom. The three digits indicate the volume selected and are colored black and/or red. The black numbers on the P-20 pipette show microliters; the red numbers show tenths of microliters. The P-200 pipette has only black numbers and thus shows only the number of microliters. The P-1000 has a red digit to represent one thousand microliliters, followed by black numbers to indicate hundreds and tens of microliters.
Note: the first (red) digit on the P-1000 will always read zero, except in the case when it is set for its maximum volume of 1000 microliters. In that case, it will read 1, 0, 0.
It is possible to measure some volumes using more than one type of micropipetter. For example, 20 µl could be measured by setting the P20 to 2,0,0 or the P200 to 0,2,0 or the P1000 to 0,0,2. However, the most precise measurements will be made using the smallest possible pipetter. In this example, that would be the P20.
1. To set the volume: Hold the Pipetman in one hand and turn the volume adjustment knob with the other hand until the correct volume shows on the digital indicator. DO NOT ADJUST THE DIAL PAST THE MAXIMUM SETTING OR FORCE THE WHEEL WHEN IT DOES NOT TURN EASILY.
Fig. 3 - Pipette tips. Left: the P20 plastic shaft goes far into the small tips. Center: the P200 shaft attaches to the end of the small tips. Right: the P1000 uses large tips.
2. Attach a disposable tip to the pipette shaft. YOU MUST ALWAYS HAVE A TIP IN PLACE BEFORE USING A MICROPIPETTER!!! The tips come in two sizes (Fig. 3). The small size fits either the P20 or the P200. The large size fits only the P1000. Press firmly to ensure an airtight seal. Remove the tip from its storage box slowly to prevent accidentally jerking out the entire rack of tips and scattering them on the floor.
Fig. 4 Left: first stop. Center: up position. Right: second stop.
3. Depress the plunger to the first stop (Fig. 4, left). This part of the stroke produces the calibrated volume displayed on the digital volume indicator. Do NOT press all the way to the second stop (Fig. 4, right)! Doing so is one of the most common pipetting mistakes and will result not only in inaccurate measurements, but may waste an entire bench's worth of a critical reagent.
4. Holding the Pipetman vertically, immerse the disposable tip into the liquid to be measured.
5. Allow the plunger to return slowly to the up position (Fig. 4, center). NEVER LET IT SNAP UP!
Fig. 5. Left: Full pipette tip. Right: Liquid that would have remained in tip if plunger were only depressed to the first stop.
6. Withdraw the tip from the liquid and pull the filled tip along the side of the stock container to remove excess liquid from the outside of the tip. You should be able to see liquid in the pipette tip (Fig. 5, left).
7. Dispense the sample: Touch the tip against the side wall of the receiving vessel and depress the plunger slowly all the way to the second stop (bottom of stroke, Fig. 4, right), expelling any residual liquid in the tip (Fig. 5, right). Frequently, very small volumes must be mixed with larger volumes of water or a solution. In this case, the small volume should be expelled with the tip below the surface of the solution. The residual liquid coating the inside of the tip can then be rinsed out by drawing solution into the tip and expelling it several times. Drawing solutions in and out of the tip is also useful for mixing microliter quantities.
8. With the plunger fully depressed, withdraw the Pipetman from the vessel carefully, THEN allow the plunger to return to the up position. Unless the same solution is to be measured into a clean container, discard the tip. A fresh tip should be used for each sample to prevent carryover or contamination.
