Displacement Pipetters: Their Care and Use
These instruments have been developed in recent years and have been valuable in manipulating tiny volumes, particularly of solutions of DNA and enzymes used in genetic engineering. They function by drawing fluid into the pipet tip using a thumb-driven piston whose movement is calibrated to draw up a specified volume of liquid. The units of volume are in microliters (µL), also termed lambdas ( l). Since there are a million µLs in a liter, 0.1 mL is 100 µL. The plastic disposable tip is discarded after a single use. The tips are color coded according to their capacity: white for 10 µL, yellow for up to 200 µL, and blue for up to 1000 µL.
The advantage of single-use disposable tips in genetic engineering is that there is no possibility of contamination with endonucleases which would destroy DNA samples. Also, the tips are much less expensive than single-use pipettes. However, the instruments are very expensive (about $200 each), and must be handled and cared for properly. NEVER allow fluid up into the body of the pipetter.
Illustrate a displacement pipette and include all of the following features:
blow out stop
volume of aliquot (in window) capacity range indication
thumb knob (to adjust volume)
tip ejector button
- Select the pipetter whose range is appropriate for the volume you wish to measure out.
2. Set the volume as follows:
a. Loosen the lock ring at the base of the thumb plunger.
b Rotate the thumb knob until the desired number of µL appears in the window . [Do not go above the specified range of
the pipetter or you may damage the instrument.]
c. Snug the lock ring. (Do not over-tighten.)
3. Attach a fresh tip to the pipetter:
a: Open the box of sterile tips.
b: Press the pipetter firmly down onto one of the tips, tap once or twice to firm the fitting.
c: Pull out the attached tip, immediately close the box to maintain sterility.
4. Draw up the desired volume of fluid:
a: Before you insert the tip into the sample solution, gently depress the plunger to the first (calibrated) stop and hold in
place. DO NOT GO PAST THE CALIBRATED STOP.
b: Insert the pipetter into the sample solution so that the tip is about 3 mm below the surface.
c: Allow the plunger to slowly and smoothly return to its original position. Note that fluid is drawn up into the tip (no
bubbles). DO NOT LET FLUID ENTER PIPETTER BODY.
d: Withdraw the pipetter from the sample vessel, flame and re-cap vessel to maintain sterility.
- Deliver the fluid into the receiving vessel:
a: Insert the pipet into the receiving vessel so that the tip a: is 3 mm below the surface of the receiving fluid, or b: touches
the bottom of the empty vessel.
b: Depress the plunger slowly and smoothly until the first stop is felt.
c: Press more firmly on the thumb plunger to blow out the remainder of fluid. You should see a bubble expelled.
KEEP THE PLUNGER HELD DOWN.
d: Withdraw the pipetter from the vessel, dragging it along the side to leave any adhered fluid.
e: Allow the plunger to return to its normal position.
6. Discard the used tip (unless you are to deliver additional aliquots of the same fluid):
a: Hold the pipetter over a discard vessel.
b: Depress the tip eject button with the thumb until the tip is ejected
PRACTICE USING THE PIPETTER’S FEATURES
Per desk for practice by two students:
2 20-200 uL pipetters
2+ non-sterile tips in a tray
test tube rack with:
2 13 × 100 mm test tubes, half full of water
4 13 × 100 mm test tubes, empty
- Unlock the lock ring, select the volume by rotating the thumb knob. Do NOT exceed the range of the pipet as indicated on the handle top. Relock (gently).
- Gently press the plunger to the first calibrated stop, press more firmly to feel the blow out stop.
- Pick up a tip from a non-sterile practice box by tapping the pipetter firmly into the tip, then return tip to a different hole in the box, ejecting the tip by pressing the tip ejector button.
- Set the pipetter to 100 µL, and draw up this volume of dH2O, deliver into a clean test tube. Change setting to 10 µL, deliver to a second clean test tube. Note how small a volume this is.
Serial Dilution Pipetting Practice
Serial dilutions are regularly used in microbiology when, for instance,initial concentrations of bacteria are orders of magnitude too high to perform a plate count, or for producing a series of regular dilutions as intitering serum. It has two advantages:
- It allows for rapid achievement of a very high dilution factor.
- It requires a relatively small volume of diluent.
It involves a sequential series of dilutions performed as follows:
1. Equal measured volumes of diluentare placed in each of a labeled series of test tubes.
2. A small aliquot ofthe specimen sample is placed in the first tube and mixed.
3. A small aliquot of that dilution is removed with a fresh pipet and added to the second tube.
4. The second tube is then mixed, and an aliquot from it is transferred to the third tube in like manner.
5. The process is continued until the series of dilutions has been completed (a serial dilution).
Notice that the concentration decreases exponentially as the dilution series progresses In the following example, the relative concentrations are 16, 8, 4, 2, and 1. Dilutions of antibodies orserumfor titering are prepared in much the same fashion.
See handout on Dilutions for in-depth explanation of dilutions and sample problems. The handout on sterile delivery with pippettes describes pipette use.
Illustrate the serial dilution process in your notebook with labeled tubes and volumes involved so that you fully understand what you will be doing before you begin the exercise.
Per table of two students,each performing his or her own experiment:
eight 16 x 150 mm tubes
two test tube racks, larger, fingered
eight 5 mL pipettes in 1000 mL beaker
two 16 x 150 mm tubes, with 7 mL MB solution
one Brinkman Pipetor or pipet bulb
one vortex mixer
one spectrophotometer , warmed up
two cuvettes in plastic test tube rack:
Blank with 3 mL dH2O (marked “B”)
one used pipet receptacle (plastic is best)
7 mL of 0.0005 % methylene blue 1 perstudent
(A609 = about 1.00)
distilled water diluent in a repipet, set for 3 mL
Turn on spectrophotometer to warm up.
Set up your work bench with required equipment.
Label the test tube with theoriginal solutionof methylene blue “16x.” Set up four empty 16 x 150 mL dilutiontubesin a test tube rack. Label the tubes 8x, 4x, 2x and 1x toindicate therelative concentration of dye which they will contain.
Aliquot 3.00 mL of dH2O into each of these four labeled dilution tubes, using are pipet. Here is an alternative style repipet.
Brinkman Pipet Bulb To use the Brinkman Pipet Bulb (click on the image to the left for a labeled version): 1. Squeeze air out of bulb to create a partial vacuum
2. Insert a pipet into the pipet recepticle
3. Place tip of pipet well into the liquid
4. Push lever up to draw fluid above desired calibration line. DO NOT DRAW FLUID INTO BULB!
5. Press level down to deliver fluid to new container
6. Press bulb to blow out last of the liquid from the pipet.
Using the pipet bulb, transfer3.00 mL of the original 16x methylene blue solution from tube #16into tube #8, vortex #8 tube to mix well.
(NOTE: If using a 5 mL pipet, 3 mL are contained when themeniscusis just touching the 2 calibration line. You should be leavingabout4 mL in tube #16.
After vortexing, use a cleanpipet to withdraw3.00 mL from tube #8, add it to #4. Mix as before, using a vortex.
Using a clean pipet, withdraw3.00 mL fromtube #4, add it to #2. Mix as before. Here the class isperforming severalof the stages of the serial dilution.
Using a clean pipet, withdraw3.00 mL fromtube #2, add it to #1. Mix as before. When dilutions are done, 3mLshould remain in tubes #8, #4 and #2. Tube #1 should contain 6 mL.
READ AND PLOT THE ABSORBENCY OF THE DILUTION SERIES:
Read the A609 of each dilution against a blank of distilled water. Begin with tube #1,and work yourway up. In this way, you need not wash the cuvette each time, but touch off the last drop before adding the next dilution.
Plot a graph with the relative concentration of methylene blue(indicated by the tubenumber) as the ordinate (X axis) and absorbency at 609 nm as theabscissa(Y axis). Use the blank tube (zero methylene blue with an A609= 0.000) as your first (zero) point.
[It is puzzling that this curvedoesnot strictly adhere to Beer’s law (it should be linear), as ifconcentrated methylene blue absorbs more proportionately thandilute. Do YOU know why this descrepency?]
1Stock solution of methylene blueis 0.3%: Dilute it 0.166 mL into 100 mL in dH2O to produce ~A609of 1.000.
Sterile Technique: Delivery of Liquids by Pipette
Pasteur observed that once a medium is sterilized, it remains sterile unless contaminated from an external source. He used this observation to reject spontaneous generation but it also lays the foundation for sterile (or aseptic) technique, a technique crucial to microbiology and medicine. Several practices are important to the success of this technique:
- Sterilize the instruments and keep them that way: keep instruments off possibly contaminated surfaces, and flame them to incinerate loose material which might fall off. Never lie pipets down on the surface of a workbench.
- Sterilize the field where work is to be performed with 70% EtOH.
- Exclude airborne contamination: keep containers covered as much as possible
- Minimize drafts: no breathing, coughing, etc over sterile materials.
Mastering sterile technique is one of the most important prerequisites to working in a microbiological lab. While the steps may seem overly detailed in the following narrative, early care in learning proper technique establishes good technique for the rest of your life. Compare these detailed steps with the demonstration given by the professor. Patience pays off. Go slowly at the beginning, and verbally (not physically) assist your fellow students as they work through the steps.
- Write out the procedure which you plan to perform in your notebook. Indicate how the sample was prepared and volumes to be transferred. Construct a table of plates to be spread, with plate number, specimen, plate type, dilution factor, aliquot, and two empty columns for colonies counted and final calculated numbers. Label tubes or plates appropriately.
- Loosen all caps, mentally review the steps you will be performing and arrange materials to ensure they are available and easily accessible.
- Select the appropriate pipet for volume to be delivered, and set the volume to be transferred by rotating the plunger. Pick up a sterile tip, reclose the lid on the box of tips immediately.
- Hold displacement pipetter so that the thumb operates the plunger and you keep your little finger on the pipetting hand free for grasping the cap of the vessel.
- Pick up specimen vessel with other hand, grip cap of vessel with little finger of pipet hand and twist the vessel and pull it down away from the cap. Do not lay the cap down.
- Flame lip of vessel.
USING THE PIPET:
- Pass sterile pipet through flame. (Very briefly so that the plastic tip is not melted…)
- DRAW UP SPECIMEN: Immerse tip of pipet 1/2 cm below surface of liquid, brace in place by gently pressing pipet against the lip of the tube while drawing the fluid up. To avoid bubbles, draw the fluid slowly into the tip by slowly raising your thumb.
- TRANSFER ALIQUOT: Touch off excess fluid from tip of pipet, tilt pipet horizontally to prevent dribbling during transfer. (The fluid will draw up slightly into the tip pipet.)
- Reflame the lip of specimen vessel, screw it back into the cap, set vessel down.
- DELIVER ALIQUOT: Pick up receiving vessel. Remove its cap and flame its lip (as in steps 5 and 6). Insert pipet, return to vertical, deliver desired volume, remove pipet, flame lip, replace cap, discard tip into used pipet container for sterilization.