How to quantify microbes in a sample

Microbiologists often need to calculate how many microbes are in a sample in order to perform a quantitative analysis of the results. In this article, we will learn how to calculate the number of microbes in our soil sample.

A note about scientific notations

Scientific notation is used to express numbers that are too big or too small to be written in decimal form. It is particularly useful to microbiologists as they often work with huge numbers of microbes in their experiments.

The number 256000 is written as 2.56 x 10⁵, and 0.0000256 is 2.56 x 10^-5. It can take a little while to get your head around if you are not familiar with looking at numbers in this format.

Our experiment

Harriet plated 0.1 mL of a 1 x 10^-1 dilution on a tryptic soya agar (TSA) plate. We first need to count the number of colonies that grew on the plate (Figure 1).

Figure 1: Colonies on a plate © University of Reading

It is quite difficult to count all the colonies on this plate. Some are very small and other colonies have merged together. If we mark off the colonies we can see there are at least 143 colonies on this plate (Figure 2).

Figure 2: Colonies on a plate © University of Reading

In our experiment, we cultured approximately 143 colonies from 0.1 mL of a 1 x 10^-1 dilution.

To calculate the total number of colony-forming units (CFU) per mL in the original soil suspension we can use the following formula:

Our equation

For our experiment the equation looks like this:

<img src=”https://ugc.futurelearn.com/uploads/assets/c5/27/c527bac0-9548-4620-bf1d-f5c2d2665691.PNG” alt=”143 divided by (1 x 10^-1 x 0.1) equals 14300 equals 1.43 x 10 ⁴ CFU per mL”>

Our results

We have calculated that there were 1.43 x 10⁴ CFU per mL in our original soil suspension. To make the soil suspension, Harriet added 20 mL of water to 1 g of soil, so if we divide 1 g by 20 mL we find this is equivalent to 0.05 g of soil per mL. To calculate how many bacteria there were per gram of soil we need to divide the number of CFU per mL by the amount of soil per mL:

<img src=”https://ugc.futurelearn.com/uploads/assets/d9/74/d9741c78-a115-4bf5-bdba-baa7ad647dd2.PNG” alt=”Formula 1.43 x 10⁴ divided by 0.05 equals 2.86 x 10⁵ CFU per g”>

We have found there were approximately 2.86 x 10⁵ CFU per g of soil. It is very likely that this is a huge underestimate of the total number of bacteria in the soil sample for several reasons:

1. It was hard to count all the colonies on the plate. Doing this experiment several times (multiple replicates) to generate an average would help improve the accuracy of our results.
2. We incubated the plate in a standard incubator in the air so we could only culture strict aerobes, facultative aerobes and aerotolerant bacteria. We could repeat the experiment and incubate the plates in an anaerobic chamber and control oxygen levels to isolate obligate anaerobes and microaerophiles.
3. Not all species of bacteria are able to grow on tryptic soya agar at 30°C. We could repeat the experiment using different types of agar at different temperatures. It has been estimated that at least 99% of bacterial species do not grow under any laboratory culture conditions.

More accurate estimates from other research studies have shown there are around 1 x 10⁹ to 1 x 10¹¹ bacteria per gram of soil.

The soil sample also contains a multitude of other microbes, including archaea, fungi, protists and viruses but we need to use different methods to culture these organisms.