Early methods for identifying bacteria

By the late nineteenth century it had become common for scientists to type bacteria by cultivating them on agar media, thanks to the efforts of Agelina Hesse, and look at them under the microscope and examine their physical characteristics such as their shape and size. They could also be identified by adding certain substances to the culture media and judging the degree to which they inhibited or promoted their growth. High concentrations of salt, for example, inhibits the growth of most organisms except the Staphylococcus bacterial species (Tsang). Another method was to establish which dyes they absorbed, a technique first devised by Hans Christian Gram, a Danish biologist, in the early 1880s. His method was called Gram staining.

Serotyping

Identifying bacteria entered a new era with the development of serotyping in the 1920s. The foundations for this technique were laid in 1896 when Georges-Ferdinand Widal, a French-Algerian Jewish physician working in Paris, devised a diagnostic test for typhoid after noticing clumps of cells formed when he added small drops of serum taken from an individual infected with typhoid to cultures of Salmonella enterica, a pathogen that can cause fever, abdominal pain, diarrhoea and sometimes vomiting. Such clumping, called agglutination, was easy to spot with the naked eye. The clumping stemmed from antibodies in the blood binding to antigens, receptors found on the surface of the bacteria. Quick and easy to perform on a glass plate, Widal's test paved the way to the development of other tests using serum taken from immunised laboratory animals, called antisera, for diagnosing syphilis, typhus and dysentery (Kirchelle).

Despite their utility, the serological diagnostic tests did not provide a way to distinguish between different bacterial strains. In 1902, however, Fred Neufield, a German bacteriologist and physician working at the Koch Institute in Berlin discovered that antisera added to pneumococcal bacteria, a common cause of pneumonia, not only resulted in the clumping of cells but also made the capsule part of bacteria swell which was easy to see under a microscope (Neufield). Based on this, he developed an elementary typing system for this bacterial species using serum taken from rabbits immunised with heat-killed pneumococci. In 1910 he and his co-workers reported they had found three immunologically distinct strains of pneumococci (Eichmann, Krause; Methot; Neufield, Handel).

It did not take long before other pneumococcal strains were discovered. By 1931 a group of female scientists, working at the Department of Health in New York, reported they had found 32 serotypes in clinical isolates of pneumococci, and were using the typing system to help determine which serum therapy to give to individual patients (Cooper et al). In addition to being a useful tool for selecting the right therapy, serotyping also provided an important epidemiological method for tracking the prevalence of various pneumococcal serotypes, where they had spread and their invasiveness. It also played a key role in the development of pneumococcal vaccines.

Serotyping soon spread beyond being used to distinguish different pneumococci groups, to also being deployed to identify different strains of Streptococci, bacteria that cause many disorders including pneumonia, sepsis and wound infections (Lancefield) and Salmonella, one of the most common causes of food poisoning (Salmonella Subcommittee).

Microbiologists used serotyping to group similar isolates together into particular type classes based on what kinds of antigens were detected on their surfaces. This was important for classification purposes. One of the first classification schemes was developed for Salmonella by Philip Bruce White . With the taxonomy of Salmonella in complete disarray in the 1920s, whereby the same name was being used for several distinct organisms, White set out to rectify the problem using serotyping. In 1926 he published the first schema for classifying Salmonella using this method. His Salmonella schema was subsequently reorganised and systematised as well as further extended by Fritz Kaufmann (1899-1978), a microbiologist at State Serum Institute in Copenhagen. This formed the basis for what is now known as the Kaufmann-White Schema which classified Salmonella based on lipopolysaccharides (O antigen) or H (flagella antigen). The scheme was adopted by the Committee on Salmonella and published in 1934. By this time the schema listed 44 serotypes and bioserotypes (Salmonella Subcommittee). Similar approaches were developed for E. coli based on O antigen and K (capsule) antigens. K or capsule antigens were classically heat-stable and made of polysaccharides.

Phage typing

Serum was not the only tool used to type bacterial strains. So too were bacteriophages (phages for short), viruses which infect bacteria. One of the advantages with phages is that each type is very selective in which bacteria it will attack. Phage typing goes back to 1921 when Oskar Bail, Chair of Hygiene and Bacteriology at the German University of Prague, suggested that phages could be used to differentiate between bacteria. Four years later, Kurt Marcuse hypothesised that phages attacked specific antigenic points on the surface of bacteria which could help with typing.

By 1926 Curt Sonnenschein, a medically trained bacteriologist at Cologne's Institute of Hygiene, had demonstrated that phages could help in the rapid identification of pathogens associated with enteric diseases that were either difficult or impossible to identify with serotyping. His work opened the way to the adoption of phage typing by many bacteriological centres in Germany and Hungary. Despite their pioneering work, the German scientists were largely forgotten following the rise of the Nazi regime (Kirchelle).

Phage typing soon spread beyond Germany and Hungary and in the 1930s the Central Public Health Laboratory in Colindale, London, began to develop international phage typing sets for Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, and Staphylococcus aureus (Kirchelle). By 1946 the Laboratory had adopted phage typing as routine epidemiological tool for investigations into outbreaks of staphylococcal infections behind food poisoning, wound infection and pemphigus neonatorum, a disease of the newborn. Following this, many laboratories around the world adopted the phage typing techniques developed by the Laboratory and in 1953 it helped establish an International Subcommittee to standardise the procedure (Wentworth). Phage typing played a central role in tracking and controlling the transmission of Staphylococcus aureus, which by the 1950s had become a global source of hospital infections and mortality (Hillier).

Both serotyping and phage typing were a core component of public health monitoring into the 1960s. Yet, both methods had some limitations. Firstly, the preparation of antisera and phages was time consuming and difficult to standardise. Phage typing also required the maintenance of an extensive collection of strains. Another difficulty was both serotyping and phage typing had a low throughput and were relatively slow which limited their utility as a tool for surveillance and epidemiological investigations. On top of these problems the methods did not alway provide results that were easy to interpret or compare.

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