# Determining the Population Size of Microbes

### Serial Dilutions Serial dilutions are used to count large population densities of microbes.

Counting individual microbes is nearly impossible because of their small size and large population sizes. To count microbes that are numerous in number, scientists perform a serial dilution. Serial dilution is a stepwise dilution of material into increasing proportions of a solution. A known volume is taken from the original bacterial culture, and a series of 1:10, 1:100, and 1:1,000 (or higher) dilutions is made by making a 1:10 dilution from the original stock and then a 1:10 dilution from that until the desired dilution is achieved. A small quantity, or aliquot, from each sample will be plated on separate plates and allowed to grow. Once colonies form, the number of colonies is measured to determine the starting population density. A colony is a visible mass of microbes originating from the replication of a single microbe. Since each colony begins with a single cell, each colony consists of a genetically identical population of cells. This is a colony-forming unit (CFU), defined as each viable microorganism in a sample that is capable of enough replication to form a visible colony.

The plates that result from serial dilutions have a wide range of colony numbers, though at least one plate in the dilution series will have a quantifiable colony density. Highly concentrated bacteria samples grow across the entire surface, making it impossible to identify single colonies. These plates are considered "too numerous to count." In contrast, if a plate has "too few to count" colonies, the probability is high that the calculated population density will be inaccurate. The general consensus in microbiology is that a plate must contain 30–300 colonies to be used to calculate population density. Population density in the original culture is calculated by multiplying the colony number with the dilution factor. A dilution factor is the ratio of the volume of the final solution to the amount of solution taken from the stock. For example, if 1 mL of stock is added to 99 mL of fresh media, the dilution factor is 1:100. When the number of colonies counted on a plate (i.e., colony forming units, or CFUs) is multiplied by the dilution factor, the result is the number of bacteria present in the original volume of bacteria taken from the stock.

#### Serial Dilution A serial dilution is used to dilute a microbial sample enough to obtain single colonies when plating. One milliliter of liquid containing the bacteria is mixed with 9 mL of liquid in order to give the dilution a 1:10 ratio.
A serial dilution iteratively reduces the number of CFUs in a liquid culture 10-fold until the population is reduced to the point where it can be quantified. In each step, 1 mL of culture is transferred to 9 mL of fresh media, which is a 1:10 dilution. Each successive dilution is another 1:10 dilution. To calculate the total dilution factor, each successive dilution must be multiplied. For example, if four serial dilution steps are required before the CFUs are within the required range of 30–300 colonies for accurate calculations, then four 1:10 dilutions result in a final dilution factor of 1:10,000. The colonies on the resulting plate are counted and multiplied by the final dilution factor. If there are 42 CFUs, the original culture concentration was 420,000 cells. This is achieved by multiplying the number of CFUs by the final dilution factor.

### Counting Microbes A quantity of microbes can be measured with a pour plate, spread plate, direct microscope count, and turbidity.
Measuring microbial population numbers can be achieved with methods other than serial dilution. There are two methods to prepare plates that successively separate out individual microbes for future isolation: the pour plate and the spread plate. In a pour plate the microbe sample is mixed with the liquid agar before pouring the plate. A pour plate results in microbes growing within and on top of the media in the plate. In a spread plate the microbe sample is placed in contact with the solid agar surface. Spreading the culture over the plate results in an even distribution of microbes on the surface of the agar. These methods only account for the number of microbes capable of cell division in the original sample.

#### Pour Plate vs. Spread Plate In the pour plate method, the microbe sample is mixed with the liquid agar before pouring the plate, which results in microbes growing within and on top of the media in the plate. In the spread plate method, the microbe sample is spread over the surface of the solid agar, which results in an even distribution of microbes on the surface of the agar.
Direct microscopic counts and turbidity measurements can be used to measure the number of live and dead microbes. A microscope can be used to directly count the total number of cells present in a given volume of culture. To obtain accurate counts, scientists use a hemocytometer. Hemocytometers were originally invented for counting red blood cells. A hemocytometer is a glass slide on which the number of cells in a known volume can be measured. A hemocytometer is formed from an indented chamber with laser-cut precise grid lines outlining a known area. Cells counted within the grids can be multiplied by the volume of the grid to yield cells per milliliter.

The turbidity, or cloudiness, of a sample can be used to indirectly measure microbial populations. Turbidity affects the way light can move through the solution. Increasing cell numbers increase the turbidity of the sample and lower the amount of light that can pass through the sample. This can be measured with a spectrophotometer. A spectrophotometer is a machine that calculates the amount of light that is able to pass through a sample. By using a spectrophotometer to measure the turbidity of the sample, an estimated count can be determined.