Pipetting: do human experimenters need liquid class information?

Question

I've been working on a protocol standardization project where, among other things, we want protocols to be able to be run equivalently by both humans and robots.

Something that I've noticed in doing so is that there's a radically different amount of information typically assumed needed by a human vs. a robot when it comes to pipetting.

With a robot, it is generally recommended to understand the nature of the liquid thoroughly, specify the liquid class (which includes information on viscosity, volatility, polarity, etc), and do quite a bit of quality control to make sure than when you want to pipette 100 µL you actually get 100 µL. A typical example is shown on this page by Hamilton.

With a human experimenter, however, the protocol pretty much always just says "pipette 100 µL" and assumes that the human can figure out all of the potential issues that we have to be so careful for with robots.

Is this actually a safe assumption, or just something that we don't typically check during protocol development for humans? Is it really intuitively obvious for nearly all liquids, or should our protocols be providing more guidance for humans as well?

Answer 1

In my experience it is very rare to see a protocol for humans that describes how to pipette a certain liquid, but I don't have as many years pipetting as others do.

In general, it is left to the experimenter to observe the behavior of the liquid and act accordingly. It is quite easy to determine when a liquid is leaking (e.g. ethanol) or when it is very viscous (e.g. glycerol), and determine that a change in technique is required. In this case, I would ask someone else in the lab how to deal with the situation. This is something that robots cannot do.

However, not everyone has expert help available, and although it is fairly obvious when a technique isn't working, often the solution is not intuitive. Additionally, the advice of others in the lab could be unhelpful or even inaccurate. For example, I was told to cut the tip off a pipette in order to pipette Tween-20 more easily, however in reading for this answer have discovered that this is not recommended at all! (See eppendorf FAQ linked below).

Therefore, I would say that, unless the liquid is to be pipetted in the same way as water, it is always useful to provide information for those who are not knowledgeable or do not have easy access to expert practitioners.

For some examples of the sorts of problems that experimenters come up against, and therefore which liquids especially could use instructions in protocols, see this set of FAQs from eppendorf on handling liquids.

Answer 2

I work on software that controls one model of robotic pipetter. There are a few reasons why we use complex liquid class definitions:

1. One of the selling points of using a robot is high speed. You can handle a wide range of viscosities if you set a conservative (slow) speed for aspiration/dispense, but for solutions that don't require this, we'd like to be able to move more quickly. In many cases, the different liquid classes are about optimizing the time required while still maintaining the desired precision and accuracy.

2. Robotic pipetters will often dispense multiple times after a single aspiration of a larger volume. To do this accurately, we compensate for a variety of effects, including the effect of the column pressure, which depends on the reagent density. In the end, this is also largely about improving speed.

3. In some applications, precision is especially important and you can use settings for the robot that improve precision. Let's say you have something that is extremely sensitive to getting the right pH. You could pre-titrate a buffer to the correct pH and load that into the system, but if you're trying to produce an assay grid on a microplate, you'd have to make a bunch of buffers. A well-calibrated robotic system can mix from stock buffers to produce the assay directly, since it has the necessary precision for mixing very small volumes.

Not having much personal lab experience since school, I can't speak to how precisely/accurately pipetting is done in labs by human beings. It probably varies quite a bit from lab to lab and likely depends on the skill and tiredness level of the person doing the work. I did it enough times to know that even basic pipetting of aqueous solutions has a bit of a knack to it and was glad when I was done with the labs that required it. In any case, I thought it might be useful to have an idea of why the robotic control seems so much more complicated; it's mostly because robots are trying to do it faster and without the benefit of being able to observe the reagent's behavior while using it.

Answer 3

Just to be clear, it has been many years since I have done real lab work, so I am certainly not an expert on the specific question. However, I have a fair amount of experience writing clear instructions to cover complex (and often bespoke) processes.

As Dan Bryant has explained, robotic systems absolutely need the additional detail in order to complete their tasks optimized for speed (and a control method without the built in feedback loops that come with a human).

My experience writing procedures for humans is that same level of detail will actually be a significant negative, at least on techniques that are considered standard knowledge. Providing what humans consider 'unnecessary detail' tends to result in a problem where your procedure is 'skimmed' for key information, which may result in lower adherence rather than improved results.

Another consideration is your level of expertise - do you know every situation where a task specific pipette is required? Because if you specify it once, you create an expectation that you will specify it every time, so any situation you miss may cause an error, and the probability of error is higher with operators having less experience - the primary audience for detailed procedures.

There are solutions, but they tend to depend on your documentation standards and the documentation tools in use. The simplest is often to move the robot only detail into an appendix (or something similar, such as a collapsing box). This means the person programming the robot will be able to drag the detail out when required, but the 'visual noise' for the humans will be reduced.

Answer 4

In my experience, I've never seen more than "pipette 100 μl" or at most "caution viscous liquid". Also I've only been taught how to pipette in a general manner and never about how to do it with special liquids except for the "cut the tip off" advise so before Noah's answer I had never heard of reverse pipetting or using a multivette to accurately pipette glycerol for example as described on the eppendorf website. However, when pipetting glycerol for example I could tell that I needed to pipette it slowly and I could check visually that I had (about) the right volume using the marks on the tip. So compared to a robot I can use my eyes to adjust and it's good enough in my particular case and with a single tube it is not optimal and it may not hold true with a multichannel pipette so having good and better pipetting habits would be better for reproducibility.

Now if I can see the "cut the tip" advice and the standalone "pipette 100 μl " instruction work to some extend, "commercial" grade protocol would probably have the instruction written as "using a multivette (or using the reverse pipetting technique) pipette 100 μl".

Another thing to consider is the definition of protocol which if we take wikipedia's definition is not just a succession of steps to perform :

In addition to detailed procedures, equipment, and instruments, protocols will also contain study objectives, reasoning for experimental design, reasoning for chosen sample sizes, safety precautions, and how results were calculated and reported, including statistical analysis and any rules for predefining and documenting excluded data to avoid bias.

What we usually consider as a protocol is the "Quick protocol" with only the steps to perform where you're more likely to find the "pipette 100 μl" type instruction. But if you consider the more detailed protocol you'll find more information. I don't have an example for liquid handling but if you consider the qiagen miniprep protocol depending if you take the quick version or the full one you may not be doing the same thing. Let's take step 2 as an example :

• quick protocol :

Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.

• handbook :

Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, because this will result in shearing of genomic DNA and contamination of plasmid. If continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.

If LyseBlue has been added to Buffer P1, the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions, or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.

If we consider only the mix part here, "gently" is the key word. If you've been handed only the quick protocol and you're an inexperience user even just by inverting too strongly you may end up shearing the genomic DNA and thus contaminate your sample what can impact your downstream application.

So the bottom line is that "pipette 100 μl" may be enough for experience users and/or some applications on a quick protocol but you may also want to have the instruction "pipette 100 μl using a multivette (or using the reverse pipetting technique)" on a more detailed protocol for inexperience user or troubleshooting purposes.