Making Serial Dilutions: Counting the Uncountable

When I first learned about serial dilutions in our antibiotic discovery lab, I was amazed that microbiologists can measure something as tiny and numerous as bacteria using just pipettes, sterile water, and patience. A serial dilution is a way to take a concentrated sample, like a scoop of soil, and dilute it step by step so that we can actually count or test the microbes growing inside it.

It sounds simple, but this technique is the foundation of so many experiments, from finding new antibiotics to checking bacterial contamination in food or water.

Here’s how to do it:

Step 1. Label and Prepare

• Start by labeling five sterile glass tubes as 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵, plus a stock tube labeled with your soil sample ID and date. Its very important to label carefully as one mix-up can ruin your whole dilution series.

Step 2. Make the Stock Solution

• Measure out about 1 gram of your soil sample into a 15 mL conical vial, then add 10 mL of sterile water. After shaking to mix, this will become your stock suspension, which is basically a microbial soup straight from the soil.

Step 3: Prepare the Dilution Tubes

• Each of the five tubes will get 900 µL of sterile water. This setup lets you create a 1:10 dilution with each step. Precision matters here as tiny volume errors can change the concentration by a lot.

Step 4: Perform the Serial Dilutions

1. Using a micropipette, transfer 100 µL of the stock into the 10⁻¹ tube and mix it by flicking the bottom (the “finger vortex” technique).

2. With a clean tip, take 100 µL from 10⁻¹ into 10⁻², mixed, and repeated down the line until the 10⁻⁵ tube.

3. Each step makes the sample ten times more dilute than the last. By the end, we had a gradient from “crowded” to “countable.”

Step 5: Plate the Dilutions

• Label five LB agar plates with your initials, date, soil ID, and dilution number. Then pipet 100 µL from each tube onto its matching plate and use sterile hockey stick spreader to evenly distribute the sample. The plates are incubated at 26 °C for 24–48 hours.

Step 6: Observe the Plates

• After incubation, the pattern is clear: the 10⁻¹ plate is covered in bacterial growth, while the higher dilutions have fewer and fewer colonies. The 10⁻⁵ plate often has just a few distinct colonies which is perfect for isolating pure cultures later. This is a satisfying process to see the math of dilutions come to life in visible dots of bacteria.