Reverse Pipetting Technique: What it is and When to Use It
Pipetting is a standard practice in most laboratories, regardless of industry or application. For all its commonality, however, there is a score of variants to consider. These include pipette type, number of channels and pipetting technique.
Proper pipetting technique is a paramount skill to ensure accurate and repeatable liquid handling results. The technique is typically dictated by the properties of the liquid being dispensed and how the user aspirates and dispenses the chosen liquid. One of the most common methods is the reverse pipetting technique. Here, we discuss reverse pipetting to determine how it works, when it should be employed and why it is necessary.
What is Reverse Pipetting?
Forward pipetting is considered a standard technique and is ideal for most aqueous solutions. In forward pipetting, the target volume is aspirated and dispensed, and a separate blowout step is used to completely empty the tip by pressing the plunger to the second stop.
In reverse pipetting technique, the pipette aspirates the selected volume plus an excess volume. After dispensing, the excess volume remains in the tip and is then discarded.
Reverse Pipetting with Mechanical Pipette
- Adjust volume of the pipette
- Fit the tip onto the pipette tip cone
- Press the operating button all the way to the second stop
- Place the tip just under the surface of the liquid and smoothly release the operating button, allowing it to return to the starting position. Wait one second
- Carefully withdraw the tip from the liquid
- Press the operating button smoothly to the first stop to deliver the desired volume. The liquid that remains in the tip should not be included in the delivery
- Discard the remaining liquid by pressing the operating button to the second stop
When Should Reverse Pipetting be Used?
Although air displacement pipettes are calibrated with distilled water, laboratory work is full of liquids that have completely different properties from distilled water. Some liquid properties can affect the accuracy of the pipetted volume. Some of the key properties that can affect the delivered volume include volatile liquids or solvents, highly viscous liquids, warm or cold liquid temperatures, foaming liquids and detergent-containing liquids (liquids with low surface tension). Reverse pipetting is the method of choice when it comes to handling challenging liquids such as:
- Volatile Liquids – Liquids and solvents such as ethanol and acetone are prone to dripping due to high vapor pressure. Reverse pipetting volatile liquids reduces dripping by eliminating the impact of any remaining evaporation on dispensing the total target volume. Any additional evaporating liquid escapes from the excess and won’t impact pipetting performance.
- Viscous Liquids – Glycerol and other viscous liquids can be tricky to pipette due to slow moving properties and adherence to tip surfaces. This can interfere with accuracy and repeatability. Accuracy is improved with reverse pipetting because the adhering amount is reduced from the excess amount present in the tip and the complete target volume is dispensed.
- Oils - Reverse pipetting oils leads to more accurate and reproducible dispensing compared to forward pipetting, as oils form a film on plastic. Adherence of the oil reduces accuracy with forward pipetting. In reverse pipetting, the oil adhering to the plastic escapes from the excess volume and improves accuracy.
- Foaming Agents – Solvents and liquids containing protein or detergents are prone to easy foaming. Forward pipetting is discouraged when handling foaming liquids because it can contribute to the formation of bubbles. With reverse pipetting, no blowout is needed to dispense the desired volume accurately and foam buildup is avoided.
As long as the best pipetting practices are being used in the lab, challenging liquids and solvents do not have to pose a threat to workflow accuracy, repeatability and efficiency. Sartorius offers an abundance of resources – from pipetting guides to the Sartorius Pipetting Academy – to ensure the liquid handling process is simple and error-free.
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