Following the tremendous advancements of the germ theory of disease, the theory that infectious diseases are caused by microorganisms, biologists focused on isolation and identification of organisms. The field of immunology, the study of how diseases spread, emerged as a major branch of microbiology.In 1884 Danish physician Hans Christian Gram developed a staining technique that helps identify bacteria. Gram stain is a method of staining used to differentiate types of bacteria based on cell-wall structure. This stain targets a particular molecule, peptidoglycan, in the cell walls of bacteria. Bacteria with walls rich in peptidoglycan are gram-positive and stain purple, while those with little peptidoglycan are gram-negative and stain pink. This stain can help microbiologists perform preliminary identification of bacteria as being gram-positive or gram-negative, and it is still in use today. In 1928 Scottish physician Alexander Fleming discovered the bacterial growth-inhibiting properties of the fungus Penicillium notatum (now called Penicillium chrysogenum), leading to the development of the world's first antibiotic: penicillin. Notably, his discovery was accidental—he had been studying Staphylococcus bacteria and left his lab for a time while on vacation. When he returned, he discovered that his culture plates had become contaminated with a fungus. In a radius around the fungal growth, no bacteria grew. Fleming correctly deduced that the fungus contained a compound that killed bacteria. Further work by Fleming and other scientists isolated this compound, now known as penicillin. This ushered in the use of antibiotics for treating bacterial infections, a practice that is still in use today and has saved millions of lives. Penicillin and its derivatives remain one of the main classes of antibiotics used to treat bacterial infections. As the Golden Age of Microbiology progressed, it became clear that some diseases arose from pathogens besides bacteria. Filtration techniques were developed to filter bacteria out of media, yet pathogens remained in the filtrate, the substance that had passed through the filter. Dutch microbiologist Martinus Beijerinck applied the term virus to these pathogens. Previously, the word virus had been used to describe any agent that made people sick. Beijerinck characterized viruses as agents of disease that relied on the metabolic processes of the infected individual—the host. Viruses are now known to be acellular infectious agents consisting of a nucleic acid strand within a protein coat. Because viruses are not living things and cannot carry out all of life's processes on their own, they cannot be cultured in a medium, a nutritious substance for microbes to grow, as bacteria can. The field of virology thus relied initially on tissue culture techniques, growing cells in isolation from the organism to which they belong, to isolate and study viruses. Early techniques included filtration and crystallization of proteins and nucleic acids that made up the viruses. The invention of the electron microscope in 1931 allowed for the first direct visualization of viruses. Advances in the understanding of nucleic acids in the 1950s allowed for more research into the makeup of viruses to be undertaken. Modern virology still relies on filtration and microscopy, but genetic sequencing is enhancing these methods. The catalog of all viruses continues to expand today.
Microbiology continued to advance rapidly throughout the 20th century and advances still to this day. Major discoveries in the later half of the 20th century included:
- DNA - In 1953, British researchers Francis Crick and James Watson announced the discovery that DNA contained the heritable genetic information.
- DNA sequencing - The first widely applied method of DNA sequencing is attributed to British biochemist Frederick Sanger in 1977. The initial method was improved several times and automated in the 1990’s. Much more rapid sequencing chemistries were developed in the late 1990’s and early 2000’s. These new chemistries, combined with subsequent improvements, have decreased the cost and effort of sequencing substantially and are collectively called next-generation sequencing.
- PCR - In 1985, the polymerase chain reaction (PCR) was invented by American biotechnologist Kary Mullis and allows rapid replication of specific DNA sequences. Numerous iterations of PCR have resulted in genotyping, genetic fingerprinting, and DNA sequencing technologies.
- Bioinformatics - Bioinformatics is a new field that combines computer science and biology to analyze and interpret biological data. Many mathematical, technological, and software technologies are incorporated into bioinformatics, making an obvious origin elusive, but the application of bioinformatics has increased steadily as DNA sequencing became more commonplace. The size of sequencing datasets produced by next-generation sequencing are massive and a source of continued development of new bioinformatics tools.
Current practices in microbiology have a strong focus on the genetic characteristics of microbes. Modern techniques such as CRISPR (clustered regularly interspaced short palindromic repeats), a genome-editing technology, are expanding areas of application in microbiology. CRISPR along with the protein Cas9 which cleaves the genome was discovered in bacteria and has been applied to genome editing in mammals and plants. In addition, the desire to create biofuels has contributed to the development of microbes that can produce fuels to replace fossil fuels, such as petroleum and coal, and to genetically engineered algae with high lipid contents that can be refined into biofuel. Furthermore, the increased use of antibiotics has led to bacteria that are resistant to most or all known antibiotics, leading researchers to search for other ways of combating bacterial infections.