Hi, I'm Norm Pace. I'm a professor at the University of Colorado. I've long been studying the natural microbial world. And indeed, my laboratory in the early 1980s began the use of nucleic acid sequences to explore the natural microbial world without this requirement for cultivation that I'll discuss. It's been a most rewarding outcome. We humans have long made use of microbial processes. Bread, beer, wine, cheese, sausage and so on. But only recently has the world of microbial life been recognized. So how do we study bacteria? First we'll look at how this was done historically, up until quite recently. The history of the study of bacteria began with the Dutch microbiologist Antonie van Leeuwenhoek. The simple microscopes he invented allowed unprecedented views of the microscopic world. Leeuwenhoek looked at many previously unseen subjects. For example, he looked at scrapings from teeth, of people who never cleaned their teeth. The observations produced the first reports of bacteria and of the human microbiota. Microbes that live in and on people. Other pioneers, such as Robert Hooke, joined van Leeuwenhoek to make early discoveries about the microbial world. Microscopes improved over the next 200 years, and staining methods were developed to improve visualization. But in the last analysis, all the early microbiologists could do was to look at them. And you can't say much just by looking. Robert Koch was the first to grow bacteria on solid media in the 1880s. That made it possible to separate different kinds of bacteria. This was important because it allowed microbiologists to study individual kinds of bacteria in pure cultures. At that time, it was not clear that bacteria can cause disease. So Koch is most famous for his proof that a microbe caused a disease, by isolating it from animals with the disease, growing it in pure culture. Then transmitting the disease to a non-diseased animal, by transferring the microbe, and then re-isolating the microbe from the diseased animal. So what is culture of microbes? This means growth of microbes on media that will support them. Single cells spread onto an appropriate medium develop into a colony of large numbers of cells of the same kind that can be studied. Some media can be formulated to grow only particular organisms. Other kinds of media support many kinds of bacteria, and sometimes contain dyes to diff, differentiate different kinds of microbes. A big problem, however, is that not many bacteria can be cultured. Moreover, microbes that grow well in culture may not be the most important or the most abundant in a particular habitat. For example, the bacteria Escherichia coli, E coli, is the most abundant organism readily cultured from the human gut. But E coli turns out not to be at all abundant in the gut microbial community. Most gut microbes are not cultured. Consequently, when you look at a natural sample in a microscope, what you see is not likely to appear on your culture medium. This discrepancy between the numbers of bacteria directly counted microscopically and the numbers cultured on petri dishes of media had long been noted by microbiologists. It was called the great plate count anomaly by Staley and Konopka in 1984. They estimated in their studies of lakewater that only 0.1 to 1% of microscopically observed bacteria are cultured. You usually can't distinguish different bacteria just by looking at them or their cultures. Carl Woese was the first to determine relationships of different bacteria to one another by comparing their DNA sequences. In particular, sequences of ribosomal RNA genes. These genes occur in all organisms, so all of life can be related in this way. The amount of sequence difference between different organisms is a measure of the amount of evolution that separates the organisms. So Woese could make maps based on the sequences that show the relationships of all life forms. Woese's maps not only outline the course of evolution, but also serve as references for determining the organisms that correspond to particular DNA sequences. This made it possible for the first time to find out what kinds of microbes live in different environments, because culture is not necessary to isolate DNA from environmental samples. Thus, an environmental sample can be processed to extract DNA. Then all the ribosomal RNA genes in the sample are isolated, and their sequences are determined. Sequences are then compared to collections of known sequences to identify microbes in the original sample. This allows us to get a true understanding of the microbes in nature, and to investigate whole microbiological communities, and begin to understand microbial ecology. This culture independence approach has been used to study many different environments, from hot springs in the open ocean to the human microbiome. The results of environmental DNA studies have dramatically expanded knowledge of the extent of bacterial diversity. The diagram shows the expansion of the main groups of bacteria, so called phyla of bacteria. In the mid 1980s, only 12 bacterial phyla were known, and all had culture representation. Two decades of DNA studies, however, have expanded the list to about 100 phyla. Two-thirds of which have no cultured examples, and are only detected by DNA sequences. Many of the organisms not detected by culture dominate their respective environments.
