Micropipette 101

Why It Is Cool to Be Precise with Tiny Drops of Liquid?

Since the beginning of the previous century, there was an increasing demands of handling liquid of ultra small volumes. In various scientific, medical, and industrial applications, it became crucial to ensure the accuracy in manipulating liquid of these small volumes. For these small volumes, we are referring to liquid in the unit of micro-liters. You’ve probably heard of a liter. It is the 1-L bottle or soda or water that you might grab from a grocery store. Now if you divide the liquid inside into a million tiny equal parts. Right, a million! Then, one of these incredibly small parts is what we call a micro-liter (µL), the “micro-“ means x10-6. Similarly, just for your reference, let’s get familiar with the following metric prefixes “centi-“ means x10-2, “mili-” means x10-3, “nano-“ means x10-9, “pico-“ means x10-12, and “femto-“ means x10-15; on the other hand, “kilo-“ means x103, “mega” means x106, “giga-“ means x109 and “tera-“ means x1012.

Imagine you're cooking and you add just a smidgen too much salt. Boom! Ruined dish. Similarly, in the world of science and tech, the tiniest drop can make all the difference. Here's why being a master of micro-drops matters:

1.Biochemical and Molecular Biology Research: Ever heard of PCR? Polymerase Chain Reaction, like a magic spell that amplified DNA, requires precise volumes of reaction ingredients. The enzymes, nucleotides, primers, and other reagents, even slight variation in volumes could affect its efficiency and the accuracy of the result.

2. Precision Medicine: Creating a new drug is like inventing a cocktail. When studying the effects of new drugs or pharmaceutical agents, it is essential to ensure consistent dosages and concentrations in all test solutions, as even tiny variances may lead to inconsistent result. 

Before the invention of Micropipette, how did people transfer small volume of liquid accurately (besides of the "mouth pipetting")?

The first modern air-displacement pipette was invented by Dr. Heinrich Schnitger in the early 1960s. Before its widespread use, people had to rely on a few other techniques to handle liquid in a micro volume:

1. Capillary tubes: These are thin glass tubes that can hold liquid by capillary action. By warming one end of a capillary tube and then placing it into a liquid, the liquid would be drawn into the tube. The volume drawn up could be controlled to some extent by the duration the tube was kept submerged and its internal diameter. To dispense, the tube could be touched to the side of a receiving vessel or gently blown into.

2. Volumetric Graduated Pipettes: You may have seen this in some laboratories. These are glass pipettes with a bulb in the middle or at the end. The liquid is drawn into the pipette up to a marked line and then it is dispensed. The reason that they are still used nowadays in certain settings is because they are suitable for many application where precision was not critical.

3. Dilution and titration: People have thought of ways to overcome the prevision issue. In some cases, when the exact measurement of a very small volume was challenging, a larger volume would be measured and then diluted to achieve the desired concentration.

There are other methods that could provide ways to handle small volumes. However, comparing with using a modern micropipette, you can tell that they were generally less accurate, less efficient, lack of consistency and more prone to errors. 

Now you can tell why the advent of the micropipette is so crucial for molecular biology and biochemistry and you could always see one in those settings. 

So how exactly does a micropipette work? (What is the mechanism of micropipette?)

In a nutshell, micropipette operate on the principle of air displacement. It is simple yet effective to ensure the accurate and precise delivery of small liquid volumes, and what’s more, the micropipette does not touch the liquid while handling.

Now, let’s dissect into a step-by-step description so you could understand the air displacement mechanism in a micropipette.

1. Piston Movement: The piston is at the heart of the micropipette and it can move up and down inside the barrel, an airtight shaft.

2. Volume Setting: Usually there is a dial or a digital setting on the pipette. Adjusting this setting determines the distance that the piston would move in the next step.

3. Aspiration:

a) Initial Press: You install a disposable plastic tip on the micropipette. You then press down the plunger button at the top of the micropipette, the piston inside the barrel would move down, expelling an equivalent volume of air.

b)  Insertion: Keeping the button pressed down, the tip of the micropipette is then inserted into the liquid to be transferred.

c) Release: When you release the plunger button, it creates a vacuum, resulting in the up movement of the piston and the aspiration of liquid in the set volume into the disposable tip.

4. Dispensing:

a) Expel: When it is ready to dispense, you press the plunger button again to the first stop. This action pushes the piston downward, expel the air, and therefore, push the liquid out of the disposable tip.

b) Extra-Expel: Due to the retention of the liquid to the inner surface of the tip, you may need to do additional pushes to ensure that all the liquid if dispensed from the tip. To make sure that all the liquid is out, usually we submerge the tip into the liquid to dispense.

5. Tip Ejection: After dispensing, the disposable tip can be ejected by using the ejector mechanism to avoid cross-contamination, by pushing the plunger button all the way down to the second stop.

A key point of the mechanisms associated is that the liquid itself does not have a direct contact with the piston or any internal parts of the micropipette. 

Now let's talk about EVOLUTION!

You must have heard of Charles Darwin and his famous book, On the Origin of Species. It's kind of like the epic adventure novel of biology, opening up an uncharted world that reshaped how we see life itself. Picture Darwin, the curious young naturalist, setting sail on the HMS Beagle, not knowing that his voyage would lead him to one of the most groundbreaking ideas in science: evolution by natural selection. His journey took him to far-off lands, from the jungles of Brazil to the windswept plains of Patagonia, and most notably, the Galápagos Islands. Walking on those islands with Darwin, seeing the giant tortoises, the marine iguanas, and those finches – oh, those finches! Each with a beak shaped just so, perfect for the food they ate. It's like a natural toolkit, where each bird had precisely the tool it needed. It wasn't just a "Eureka!" moment. Darwin puzzled over what he saw for years, like a detective piecing together the clues of a great cosmic mystery. He realized that these creatures weren't created in their current form but had changed over time. It's as if nature were an artist, sculpting life through trial and error, keeping what worked and discarding what didn't. Survival of the fittest, as it's often described. But here's where it gets even more exciting – this idea applies to everything alive, from those finches to the flowers in your garden, and yes, even to you and me. We're all part of this grand, unfolding story of life, shaped by millions of years of successes, failures, twists, and turns.

Darwin's ideas were like a spark that ignited a firestorm of curiosity. People began to look at the world with new eyes, seeing connections, adaptations, and the beautiful complexity of life. And the story is far from over; scientists are still exploring, still discovering, and still being inspired by Darwin's adventurous spirit.

Whether it's unlocking the secrets of our DNA, saving endangered species, or even looking for life on other planets, the study of evolution is a thrilling journey into the unknown. It's about understanding where we came from and where we might be going. So next time you see a bird, a tree, or even look in the mirror, remember – you're witnessing a chapter in the greatest story ever told, and who knows? You might just add a line or two yourself!

I would like to share one of my most favorite readings, 40 Years of Evolution, by Peter R. Grant and B. Rosemary Grant. You could find the book on Amazon (Link). I Strongly suggest you to at least take a look at it.

Link to On the Origin of Species   https://www.vliz.be/docs/Zeecijfers/Origin_of_Species.pdf

Genetic variation provides the raw material for evolution. Under its broad scope, there are a few long-standing questions that intrigue the researchers continue to explore. Bear these questions in mind, you will get a sense of what evolution (or evolutionary biology) is about:

1. Species and Speciation Events: What exactly constitutes a species, and how do new species form? 

2. Origin of Multicellularity: How did multicellular life evolve from unicellular organisms? This transition marked a significant step in the complexity of life and remains an area of active research.

3. Genetic and Phenotypic Complexity: How did the complexity in organisms arise from relatively simple beginnings? The leap from to complex organisms, both in genetic material and physical structure, is poorly understood.

4. Nature of Self-Awareness: How did consciousness evolve, and why? Understanding how subjective experiences and self-awareness arose in organisms remains a profound and unresolved question.

What is happening during the experiments?

When *E. coli* bacteria are grown on plates with increasing amounts of ampicillin, they're being challenged to "level up" and find ways to survive. If they do manage to grow on these plates, it means they've likely made some changes (or mutations) to handle the antibiotic. Here's what those changes might be:

Destroy the Antibiotic: Some *E. coli* might develop the ability to produce an enzyme that breaks the ampicillin down, making it useless. It's like they've evolved their own little "antibiotic neutralizer."

Change the Target: The antibiotic works by sticking to certain parts of the bacteria. But if those parts change shape (because of mutations), the antibiotic can't stick properly. Imagine the antibiotic as a magnet and the bacteria as a metal surface. If the bacteria changes its surface, the magnet won't stick.

Pump the Antibiotic Out: Some bacteria might get better at pumping out stuff they don't like, including the antibiotic. It's like having better bouncers at a club door.

Limit Entry: The bacteria could change how permeable their outer layer is, making it hard for the antibiotic to get inside. It's similar to a castle raising its drawbridge to keep out invaders.

When you increase the antibiotic amount gradually, you're essentially challenging the bacteria to adapt or die. Those that do survive have likely undergone one or more of these changes to resist the effects of ampicillin. This is a basic demonstration of evolution in action: only the fittest (in this case, the most resistant) survive.

Possible Experiment: Demonstrating Evolution Through Bacterial Mutation Rates

Objective: To observe how mutations can cause phenotypic changes in bacteria.

Materials:

- Petri dishes with agar (some containing a specific substrate the bacteria can mutate to utilize, such as lactose)

- Bacterial culture (e.g., E. coli strain that can't initially utilize the substrate)

- Inoculating loops or pipettes

- Incubator

Procedure:

Prepare Plates: Set up Petri dishes with and without the specific substrate (e.g., lactose).

Inoculate Plates: Inoculate all plates with the bacterial culture.

Incubate: Place the plates in an incubator at the appropriate temperature for the bacteria.

Observe and Record: Monitor the plates over several days or weeks, noting any changes in growth, particularly on plates containing the substrate.

Expected Outcome: Over time, students may observe growth on the substrate-containing plates, indicating a mutation that allowed the bacteria to utilize the substrate.

Discussion and Analysis

These experiments offer excellent opportunities for students to discuss and analyze the fundamental principles of evolution, such as natural selection, mutations, adaptation, and survival of the fittest. By comparing observations, students can gain insights into how these processes work in real-time, at a microscopic level.

Always ensure that the experiments are conducted following proper safety protocols, considering the nature of the bacteria and the chemicals used. Supervision by a knowledgeable instructor is essential, especially if working with potential pathogenic organisms or hazardous substances.

Possible Experiment: Bacterial Competition and Coexistence

Objective: To observe how different bacterial species compete or coexist in the same environment.

Materials:

- Petri dishes with agar

- Two different bacterial cultures (e.g., E. coli and S. aureus)

- Inoculating loops or pipettes

- Incubator

Procedure:

Prepare Plates with Both Cultures: Inoculate Petri dishes with both bacterial cultures, either mixed or side by side.

Prepare Control Plates: Inoculate separate plates with only one type of bacteria as controls.

Incubate: Incubate all plates at a suitable temperature for both species.

Observe and Record: Examine the plates daily, noting any changes in growth patterns, dominance of one species, or evidence of coexistence.

Expected Outcome: Students might observe competitive exclusion, dominance of one species, or possible coexistence, depending on the bacterial species' interactions.