Calculate CFU per mL

Serial dilution, the spread and pour plate methods, the 30 to 300 countable range, and a worked example of the formula this calculator uses.

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Chapters

  1. 0:15 Counting bacteria on solid media
  2. 1:08 What a colony-forming unit is
  3. 1:59 Serial dilution
  4. 2:51 Spread plate and pour plate
  5. 3:53 The countable range
  6. 4:31 The formula and a worked example

Transcript

0:15 Counting bacteria on solid media

Around 1881, Robert Koch introduced solid culture media, on which bacteria from a mixed sample grow as separate colonies, each a pure culture. His early solid media used gelatin, which melts at body temperature and which some bacteria digest. Fanny Hesse worked as a laboratory assistant to her husband, Walther Hesse. She suggested agar, which stays solid at incubation temperatures. In 1887, Julius Richard Petri, an assistant to Koch, described the shallow covered dish that carries his name. Agar plates in covered dishes made it possible to count bacteria. On a solid medium, a single living cell divides where it lands and, after many divisions, forms a colony that is visible to the eye. The number of colonies on a plate therefore estimates the number of living cells in the volume placed on that plate.

1:08 What a colony-forming unit is

The estimate needs one qualification, because a colony does not always start from a single cell. A chain or a clump of cells that stays together also produces just one colony. For this reason, microbiologists report the result in colony-forming units, abbreviated CFU, rather than in cells. One colony-forming unit is one living cell, or one group of living cells, that gives rise to one colony. The count also includes only cells that can grow on the chosen medium under the chosen conditions. The count leaves out dead cells and living cells that cannot grow on that medium.

1:59 Serial dilution

The plate count itself has a practical limit. An overnight culture of Escherichia coli can hold about a billion cells per milliliter, far more than one plate can show as separate colonies, so the first step is a serial dilution. For a tenfold dilution, the microbiologist transfers 1 milliliter of sample into 9 milliliters of sterile diluent, such as saline. Smaller volumes keep the same ratio, for example 100 microliters into 900 microliters. After mixing, each tube supplies the sample for the next tube in the series. Each transfer lowers the concentration tenfold. After six transfers, the last tube holds a dilution of 10⁻⁶, or one part of the original culture in one million. A fresh sterile pipette tip at each transfer prevents carry-over of cells from a stronger dilution into the next tube.

2:51 Spread plate and pour plate

The diluted samples then go onto agar by the spread plate method or the pour plate method, and each method places a different volume on the plate. In the spread plate method, 0.1 mL of a dilution goes onto the surface of a set agar plate, and a sterile glass or plastic spreader distributes it evenly. In the pour plate method, 1 mL of a dilution goes into an empty sterile dish. Molten agar, cooled to about 45 to 50 °C so that the heat does not kill the cells, then fills the dish, and gentle swirling mixes the sample through the agar. In a pour plate, colonies grow both on the surface and within the agar, where they stay small and lens-shaped. A laboratory normally plates several consecutive dilutions, often in duplicate, because the analyst cannot know in advance which dilution will give a countable plate. The plates then incubate until colonies appear, for example for 24 to 48 hours at 37 °C for Escherichia coli.

3:53 The countable range

After incubation, the dilution series produces plates with very different numbers of colonies, so a rule decides which plates to count. A widely used rule admits only plates with 30 to 300 colonies, and some standards use 25 to 250 instead. Below 30 colonies, random sampling error is large relative to the count, so the estimate is unreliable. On a plate with more than 300 colonies, neighboring colonies merge and compete for nutrients, which lowers the count. The laboratory records such a plate as TNTC and does not use its count.

4:31 The formula and a worked example

With a countable plate chosen, one formula converts its colony count into the concentration of the original sample. CFU per mL equals the number of colonies divided by the product of the dilution and the volume plated, in milliliters. As a worked example, consider a spread-plate series in which 0.1 mL of each dilution went onto a plate. The 10⁻⁵ plate is TNTC, the 10⁻⁶ plate holds 156 colonies, and the 10⁻⁷ plate holds 14 colonies. Only the 10⁻⁶ plate falls inside the range of 30 to 300, so it alone enters the calculation. The denominator comes first. 10⁻⁶ multiplied by 0.1 mL gives 10⁻⁷ mL, which is the volume of the original culture that the plate received. Dividing 156 colonies by 10⁻⁷ mL gives 1.56 × 10⁹ CFU/mL. A plate count carries about two significant figures of precision, so the laboratory reports the result as 1.6 × 10⁹ CFU/mL. The 10⁻⁷ plate offers a rough check. Its 14 colonies correspond to 1.4 × 10⁹ CFU/mL, close to the main result, although that plate lies below the countable range. When replicate plates at the same dilution both fall in the countable range, their mean count goes into the same formula.

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