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.
Pour Plate vs. Spread Plate
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.