This demonstration presents aseptic laboratory techniques in volume transfers with serological pipettes and micro pipetters at the laboratory bench. Work within the sterile field created by a bun and burner flame. Select the appropriate volume transfer instrument for the application.
Then use them properly without compromising their sterility, while keeping culture reagents and materials sterile aspirate liquids with precision taken together. These procedures are essential to minimize sources of contamination and offer precision for basic experimentation. Initially, it is challenging to precisely set and accurately read instruments, as well as coordinate movements with the instruments and lab materials without compromising the sterility of the liquids being transferred.
Practicing a septic technique in the laboratory is essential for experimental success when working with live specimens, so invest the time to learn these procedures, allowing them to become second nature. When working at the bench, Dr.Chris Reddy, my laboratory coordinator, will demonstrate the protocols Always wash hands thoroughly with antiseptic soap and warm water before starting any laboratory procedures. First wet hands with warm running water.
Then apply and thoroughly distribute soap vigorously. Rub hands generating friction on all surfaces, including thumbs, backs of fingers, backs of the hands, and beneath the fingernails. Then rinse thoroughly to remove residual soap and dry using paper towels dispensed from a holder.
Finally, with a fresh paper towel, shut off the faucet, clear the work area of the laboratory bench. Then remove a pre-moistened disinfectant wipe from the canister and wipe down the entire area. Allow the disinfectant to evaporate using an igniter light.
The bunsen burner. Adjust the flame to render the blue cone in the middle. Arrange all the supplies needed for the procedure near the sterile field of the laboratory bench.
Also, make sure all materials are properly labeled. There are many selections of serological pipettes, plastic or glass, disposable or reusable, plugged or unplugged. The various sizes are calibrated with plugs that function as a barrier to overfilling the pipette.
Note that serological pipettes are also calibrated in two ways. TD pipettes marked with double rings at the top are calibrated for the tip volume not to be delivered. This is not shown here, but the TC pipettes must be blown out since they're calibrated to deliver all of the volume using aseptic technique with an individually wrapped sterile plastic serological pipette, begin at the plugged end and start to carefully peel away the paper.
Do not remove the entire sleeve. Rather protect the tip of the pipette handle only the top of the pipette above the graduation marks a fix a pipette aid such as a bulb pump or gun to the top end of the serological pipette. Hold the pipette aid in one hand and remove the paper sleeve from the plastic pipette with the other.
For glass serological pipettes stored in metal canisters, loosen the top of the canister and remove the cap flame the open ends of both cap and canister. Then place the cap on its side on the disinfected bench. Hold the canister horizontally and shake so the tops of one or two pipettes.
Stick out about an inch. Lay down the canister on its side and remove one pipette without touching the other pipettes. Keep the bottom tip of the pipette free from contact with hands and non-sterile surfaces.
Proceed to affix the pipette aid. Then pass the bottom third of a glass pipette through the blue cone in the bunsen burner flame for one to three seconds. Now remove the cap of the bottle containing sterile media.
Hold it between the ring finger and palm of one hand while manipulating the pipette aid with the thumb index and middle fingers of the same hand with the other hand, hold the bottle at a 45 degree angle and pass the rim of the bottle through the flame of the bunsen burner. Place the sterile pipette tip into the media. Then aspirate using the pipette aid to control flow with the graduation marks.
Monitor the volume drawn into the pipette to precisely read the volume. Hold the pipette vertically and view the liquid meniscus dead on at eye level. Once again, pass the rim of the bottle through the bunsen burner flame.
Then cap it. Set the media bottle aside. Then take a sterile tube and aseptically.
Remove the cap as described earlier. Create a sterile field by flaming the rim of the tube. Dispense the media from the pipette into the tube controlling sample flow so it does not splash.
Flame the rim. Again, replace the cap and set the tube aside. Now remove the pipette aid, discard plastic pipettes in a designated sharps container, immerse glass pipettes in 10%bleach solution.
A micro pipetter affords precise measuring and dispensing of minute volumes with different sized instruments for specific volume ranges. Pre sterilized tubes and plastic tips. Also use a pre-moistened disinfectant wipe to rub down the micro pipetters.Note.
The Volter shows three numbers to be interpreted relative to the type of micro pipetter with accuracy reflected in the smallest graduation. Mark, for example, the P 20 is used for volumes between 2.0 and 20.0 microliters. The top number in black is for tens of microliters.
The second black number denotes the volume in microliters. The red number marks tenths of a microliter, so each graduation mark equals an increment of 0.02 microliters. First, turn the adjustment knob to set the dispenser volume as indicated on the numeric volter.
When decreasing the volume setting on the micro pipetter, slowly dial down the thumb wheel to the graduation mark. When increasing the volume setting, dial up to pass the desired graduation mark by one third of a turn. Then slowly dial down the thumb wheel to reach the intended volume.
Use sterile plastic disposable tips at all times. Fit a tip tightly onto the end of the barrel of the micro pipetter. Press down and twist slightly to ensure an airtight seal.
Hold the micro pipetter in a vertical position. Note the micro pipetter has three positions. Rest, first stop and second stop.
Depress the push button on the plunger from the rest position to the first stop to displace air equal to the volume of the setting. Now immerse the tip into the liquid while holding down the push button to the first stop. Release the push button slowly to aspirate the liquid into the tip.
When the push button reaches the rest position, allow time for the liquid to be drawn into the tip. Then remove the tip from the liquid. Visually verify the extent of liquid drawn up the tip, ensuring no air bubbles.
Place the tip at a 10 to 45 degree angle against the wall of the tube receiving the liquid. Now, slowly depress the push button on the plunger to the first stop. Expelling the liquid.
After a moment, press the push button to the second stop to expel any residual liquid. In the tip, proceed to remove the tip from the liquid and then release the plunger to the rest position. Finally, discard tips into designated sharp's waste container by pressing the ejection button on the micro pipetter.
First turn off the bun burner. After putting away all supplies and reagents, rub a disinfectant wipe on the outside surfaces of the LabWare before moving them to their storage location. Then place contaminated glassware and hazardous waste materials into the proper disposal.
Receptacle proceed to wipe down the entire work area once again, allowing the disinfectant to evaporate. Finally, wash hands thoroughly with antiseptic soap and warm water. Without a septic technique, cultures can become contaminated and delay experimentation.
Aseptic transfer of liquids using serological pipettes is essential in amplifying pure bacterial cultures like this 25 milliliter e coli preparation. Additionally, serological pipettes facilitate aseptic liquid transfer into sterile test tubes. This example demonstrates a pure versus contaminated culture of e coli in point-to-point delivery of media to the tubes.
Sometimes precision volumes are required. Clearly, there is an imprecise transfer of media in this case. This example shows incorrect volume of buffer transferred.
Instead of dispensing 12.5 microliters, the student dispensed 125 microliters. Although the numbers are set identically on the volter, the wrong micro pipetter was selected for the job. After watching this video, you should have a working knowledge of how to apply a septic technique to routine laboratory procedures such as transferring solutions and cultures using serological pipettes and micro pipetters without compromising their sterility.
The goal is for precautionary procedures such as flaming glass, pipette tips, culture tubes, and flasks, as well as working within the confines of a sterile field at the laboratory bench become habitual with every experiment. It is also essential that precise and accurate volumes be measured and dispensed when performing experiments. Learning how to use these volume transfer instruments properly will improve the reliability of experimental results.
