Counting Microbes, One Dilution at a Time!

Ever looked at an environmental sample tube and wondered, “How many different bacteria are in here, anyway?” That’s exactly what I thought during lab when we did serial dilutions on our cave samples! Serial Dilution is a pretty simple technique to help answer that exact question.

This is a sample serial dilution performed with green food coloring to simulate a real sample

The main point of doing a serial dilution is is to dilute a dense bacterial sample step by step until it's possible to plate and count individual colonies as well as see the variety of bacteria to potentially create a library plate after. When the colonies grow on agar, each individual colony ideally comes from a single bacteria type, so by counting them, you can estimate how many were in the original sample. This is super useful if you're measuring how many bacteria survive an antibiotic or how fast they grow under different conditions.

This is how its done (at least how I learned)....

1. Set Up the Tubes - labeled a series of sterile tubes: 10⁻¹, 10⁻², 10⁻³, up to whatever dilution you needed (I usually do til 10⁻⁵).

2. Add Sterile Water - Into each tube put 900 µL of sterile water into each dilution tube. this is the “blank canvas” for diluting the bacteria.

3. Transfer the Sample- Using a clean pipette tip, add 100 µL of the original culture to the first tube (10⁻¹). Then I mixed it thoroughly by vortex or pipette up and down so the bacteria distribute evenly.

4. Serial Dilution! - From the 10⁻¹ tube, transfer 100 µL into the next tube (10⁻²), mixed again, and repeated this down the line. Each step dilutes the sample 10-fold (in our setup).

5. Plate the Dilutions - From each dilution tube, take 100 µL and plated it on LB agar plates labeled with the dilution factor. Spread the sample evenly with a cell spreader across the whole plate, so the cells can grow into separate colonies.

6. Incubate - The plates went into the incubator (37°C) overnight, giving the bacteria time to form distinct colonies.

7. Count Colonies & Calculate CFU/mL - using the formula below you can find the CFU/mL by counting the colonies on the plate.

Like everything there are pros and cons to this method. I personally love this method and think the pros outweigh the cons but I can acknowledge that we don’t need to stick to the old way forever. There is always room for innovation and waste reduction.

The pros include the fact that this method is pretty reliable when done right, it requires minimal specialized equipment and you get to actually see the living bacterial colonies. I think in general whether you are counting them or not they are just cool to look at.

The cons include that this method is time-consuming because you have to wait for growth, there can be human error in counting which can mess things up if plates are crowded or colonies overlap, and the biggest one is it being kind of wasteful as it uses lots of single use plastic like plastic tubes, plates, pipette tips.

Improving the CFU Game

In recent studies people are trying to improve the CFU game and I found a very interesting paper from 2023 that connects to why we do serial dilutions and what the future might look like.

A research team at University of Colorado Boulder developed a method called the Geometric Viability Assay (GVA) that makes counting microbes way faster and less wasteful. The Geometric Viability Assay works by measuring how microbial growth changes across a gradient of antibiotic concentrations rather than relying on dozens of dilution tubes and plates. Instead of counting colonies one by one, this method uses mathematical modeling and growth patterns to estimate viable cell numbers much more efficiently. Want to know the coolest part? It drastically reduces the number of plates, pipette tips, and plastic waste needed, while still providing accurate viability data.

Overview of the Geometric Viability Assay (GVA) as a modern alternative to traditional CFU counting.

This approach doesn’t completely replace serial dilutions—especially when you need physical colonies for isolation, identification, or building a culture library—but it shows how microbiology is evolving. Traditional CFU counting gives us something incredibly valuable: a direct, visual confirmation of living microbes. Newer methods like GVA build on that foundation by making the process faster, more sustainable, and more scalable for large experiments.

Why Serial Dilutions Are Still A Top Method

Even with new techniques emerging, serial dilution remains a cornerstone of microbiology education and research. It teaches core lab skills like aseptic technique, pipetting accuracy, experimental planning, and data interpretation. It also connects students directly to the organisms they’re studying. To me personally there is something satisfying about seeing real colonies appear!

At the same time, being aware of newer methods encourages scientists to think critically about efficiency, sustainability, and innovation. The future of microbiology likely isn’t about replacing classic techniques, but about combining them with smarter, cleaner, and more eco friendly approaches.

Counting microbes one dilution at a time might feel old-school, but it’s still incredibly powerful. Serial dilutions give us a reliable way to estimate bacterial abundance, assess survival, and explore microbial diversity, all while building foundational lab skills. As new methods like GVA continue to develop, they challenge us to rethink how we count life at the microscopic level and how we can do better for both science and the environment.

Whether you’re plating colonies or modeling growth curves, one thing is clear its always a good idea to count your microbes!