How do I pipette viscous liquids?

Introduction

Viscous is another word for thick, but technically still fluid. Glycerol (100%) might be one of the most viscous liquids lab rats will encounter in their day-to-day pipetting. Viscous liquids are some of the hardest to pipette accurately and consistently. 

The first question you should ask yourself is: can I afford to reduce the concentration?

Reducing the concentration by diluting your viscous liquid with a less viscous liquid (like water) will make the resultant liquid less viscous and thus easier to pipette accurately and consistently. Most researchers will opt to make an 80% solution of glycerol before considering pipetting it. Large volumes of diluted glycerol stocks are usually prepared since this generally leads to more accurate concentrations of stock solutions (e.g. creating 1 L of 80% glycerol stock compared to 10 mL).

If you try to pipette 100% glycerol with a Gilson micropipette, only the P1000 and above will have enough pressure and volume to work at all. However, be prepared to clean your pipette afterwards – it WILL get messy. You can also throw any expectation of accuracy out of the window. 

The next bits of advice apply to any kind of automated liquid handler, as well as pipettes. 

1. Slow and steady wins the race

Thicker liquids move slowly. If you aspirate (suck up) liquid too quickly, bubbles of air will be produced. Also, dispense slowly, or residue will be left inside the tip.

2. Keep the tip near the surface

Thick liquids stick to the outside of pipette tips; minimise the distance the tip is immersed in it. Otherwise the volume dispensed will be greater than expected. This also makes aspirating slowly more paramount as you need to move with the liquid as it drops. If you’ve got an automated liquid handler this usually comes in the form of a setting called something like “liquid level tracking”. 

3. Use a wide bore tip

If you pipette viscous liquids often then buy some dedicated wide bore tips. But these tips are expensive. So a bodge has always been to cut the bottom few mm off of a normal pipette tip. This does compromise sterility but…

4. Use a blowout

On a manual pipette this just means to pushing the plunger to the second position & holding it there – remember viscous liquid takes a second to move. This might cause some stress on your thumb if you need to do it repeatedly but it will result in a slightly more accurate transfer. You should also allow a pause between dispensing and applying the blow out, this will allow time for any remaining residue to gather at the bore, making the blow out more effective.

For an automated liquid handler – program a larger volume of blow out (air gap above the liquid) for more viscous liquids and increase the time the robot pauses at that position. 

5. Bigger is better

Aspirate a larger volume of liquid than you intend to dispense. Viscous liquids have a nasty habit of leaving a small volume stuck as residue within the tip that using a blow out may not affect. This is a good tip if there’s a minimum volume you require. 

6. Throw consistency out the window

If you’re using a manual pipette then you won’t be able to achieve any kind of consistency unless you’ve had years of experience at this, with the same pipette. The more viscous the liquid, the more this will be the case. None of the movements you make will ever be the exact same. If consistency is necessary then use as low a concentration as possible, use the same pipette with the same volume and even try to time your movements. Otherwise, considering automation is the logical next step. This comes at a price but most labs consider it to be a necessary investment.

As always the best way to ensure accurate and reliable liquid transfer is to use regularly calibrated and cleaned pipettes with pipette tips from a reputable supplier. Good equipment is the key and maintaining “high quality is not an act, it’s a habit” – Aristotle.