By Palmer Owyoung · Updated Mar 24, 2022
Bacteria are cultivated on agar in Petri dishes, forming visible colonies that represent groups of cells. While a simple colony count can provide a quick estimate of bacterial load, quantitative methods such as viable plate counts yield both absolute numbers and qualitative insights into how different conditions affect growth. Because a single dish can contain billions of cells, the culture must first be serially diluted to a level where individual colonies can be counted reliably.
Begin in a sterile 1.5 mL tube. Pipette 10 µL of the starting culture into 90 µL of sterile dilution medium (e.g., phosphate‑buffered saline). Seal the tube, vortex gently, and you have a 10⁻¹ dilution. Repeat the 10 µL transfer into fresh 90 µL of medium to generate a 10⁻² dilution, and so on. Continue until you reach a dilution between 10⁻⁴ and 10⁻¹⁰. Label each tube (10‑1, 10‑2, …) to keep track of the dilution factor.
Take 10 µL from the most dilute tube that still yields countable colonies (typically 30–300 per plate). Spread the inoculum evenly across the agar surface with a glass spreader, rotate the plate 90°, and repeat. Prepare at least three plates per dilution to ensure statistical confidence. Allow the spreader to dry on a flame or bench for a few minutes, then replace the lids and incubate at the optimal temperature for the organism (usually 35–37 °C) for 12–16 h.
After incubation, inspect each plate for a countable number of colonies. Mark the bottom of the dish (not the lid) with a permanent marker at each colony’s position and tally the dots. Choose plates that contain between 30 and 300 colonies for accurate statistics.
Compute colony‑forming units (CFU) per milliliter of the original culture using the formula:
CFU/mL = (number of colonies) × 10 × 10ⁿ
where n is the negative exponent of the dilution factor (e.g., for a 10⁻⁷ dilution, n = 7). The factor of 10 accounts for the 10 µL inoculum volume.
Sterilize the spreader in 70 % ethanol, ignite it with a Bunsen burner flame, allow the alcohol to burn off, and cool it on the dry agar surface. This kills any residual microbes before plating.
Handle all bacterial cultures as potentially hazardous. Follow institutional biosafety guidelines and use appropriate personal protective equipment.
