Koch’s Postulates—Then and Now Amid challenges, his principles remain useful for confirming microbial roles in diseases and other processes D. Jay Grimes his story begins in the early 1880s. that diseased cells arose only from other dis- The germ theory of disease was fairly eased cells, and strongly opposed the germ the- well established, although some ex- ory of disease. Other skeptics could be cited, but T perts continued to challenge it. For the important point is that Koch was working in example, Theodor Billroth (1829– a controversial area of medicine and experimen- 1894) of Vienna, often called the founding fa- tal research in the late 1800s. ther of abdominal surgery, did not immediately accept the germ theory, but later embraced the Koch’s Early Focus on Tuberculosis concept and became very successful in many Led Him To Frame Postulates pioneering surgeries. Another critic was Max von Pettenkofer (1818–1901) of Bavaria, a In 1880, Robert Koch at age 37 moved his noted experimental hygienist. In 1892, Petten- family to Berlin, where he joined the staff of the kofer openly challenged Robert Koch (1843– Imperial Health Office. He had conducted his 1910) on his theories about Vibrio cholerae by anthrax research about a decade earlier and in drinking the contents of a flask containing the Berlin turned his attentions to the dreaded dis- cholera bacillus. The flask had been given to ease tuberculosis. He developed many tech- Pettenkofer by Koch himself, but Pettenkofer niques in his own laboratory, including how to did not contract cholera! Perhaps the biggest isolate pure cultures along with many applica- critic was the famous physician Rudolph Vir- tions of microscopy, photomicroscopy, and chow (1821–1902) of Berlin. Virchow believed staining. He also fitted his microscope with the oil-immersion lens that Ernst Abbe (1840– 1905) at the Carl Zeiss Company had re- Summary cently developed. At about this time, Edwin Klebs (1834– • When Robert Koch was framing his postulates 1913), a student of germ theory antagonist about infectious disease, several contemporaries Virchow, published a paper that delineated published similar ideas, while prominent skep- what would soon be established as Koch’s tics argued strongly against the germ theory of postulates (see box, p. 224). Klebs’s three disease. rules are strikingly similar to those of Koch • Koch, who studied cholera, and others, from his era and our own, recognized exceptions to his and his students, but Klebs could not verify postulates, particularly in dealing with species- his procedures mainly because he was un- specific pathogens for which suitable animal able to isolate pure cultures, a central re- models are difficult, and sometimes impossible, quirement for both his procedure and to identify. Koch’s postulates. D. Jay Grimes is • The principles underlying Koch’s postulates Koch published his discovery of the tu- Provost and Vice sometimes are being applied, for example, in bercle bacillus in 1882, describing an exact- President for Aca- environmental settings to address questions of ing approach that laid the framework for demic Affairs at microbial causality that are far afield from infec- his postulates. He observed and isolated the The University of tious diseases. microbe in pure culture from numerous pa- Southern Missis- tients, then introduced the microbe into sippi, Hattiesburg. Volume 1, Number 5, 2006 / Microbe Y 223 discovered the anthrax bacillus and defined its Procedure developed by Edwin role in causing anthrax, explaining that this Klebs to demonstrate causal approach laid the framework for his research on relationship between “germs” and tuberculosis. “[W]hether the organisms have disease constant characteristics. .” is the logic underly- ing his first postulate, and he insisted that this 1. Careful microscopic study of the dis- question should be asked during each investiga- eased organ tion of an infectious agent. 2. Isolation and culture of the germ Further in the same text, Koch discussed the associated with the disease basis for his second postulate—asking whether 3. Production of the same disease by “the parasite” is the cause of the illness or a inoculation of this cultured germ symptom. The answer to this question, he into healthy animals added, can be established only under circum- stances “in which the parasite is completely guinea pigs, successfully induced disease in separated from the diseased organism and from them, and then reisolated the microbe from all symptoms of the disease.” He then developed those animals. This work made Koch an instant the basis for his third postulate by stating that hero and his name a household word. “by introducing the isolated parasite into the In 1883, Friedrich Loeffler (1852–1915), one healthy organism the disease can be produced of Koch’s assistants, published a paper on diph- again with all its usual characteristics.” Over the theria (Fig. 1). Loeffler stated that three postulates next 80 pages, Koch further used his anthrax had to be fulfilled to prove that diphtheria is a discovery to explain the etiology of tuberculosis. disease caused by a specific microorganism (see Thus, Koch explained his postulates. How- box, below). He argued that these steps were ever, he did not publish them in the form that we necessary “to demonstrate strictly the parasitic recognize today until 1890 (see. box, p. 225). nature of a disease.” His three postulates and Since then, the postulates have been used ex- the procedures described by Koch a year earlier tensively to elucidate many diseases and are are nearly identical. Based on the work of Klebs presented in all beginning textbooks of micro- and Loeffler, one can question whether Koch’s biology. Whether Koch merely compiled or gen- postulates were his! uinely conceived them, he now is credited for articulating these simple but powerful tenets of Koch’s Formalized His Postulates infectious disease etiology. between 1884 and 1890 Early and More Recent Exceptions In 1884, Koch published a definitive paper on to Koch’s Postulates tuberculosis and more fully outlined his meth- During the next few decades, others uncovered odology. In the first pages, he described how he many exceptions to Koch’s postulates. Indeed, FIGURE 1 Loeffler’s Postulates (see p. 424 in Loeffler, 1883) 1. The organisms must be shown to be constantly present in characteristic form and arrangement in the dis- eased tissue. 2. The organisms which, from their be- havior appear to be responsible for the disease, must be isolated and cul- tivated in purity. 3. The pure cultures must be shown to Friedrich Loeffler and Robert Koch. induce disease experimentally. 224 Y Microbe / Volume 1, Number 5, 2006 it will no doubt become possible to satisfy Koch’s postulates for polymicrobial diseases. Koch’s Postulates as We Know Lysogenic bacteria provide another example Them Today of exceptions to the postulates. For instance, 1. The same organism must be present Loeffler’s diphtheria findings might have looked in every case of the disease. very different had the Corynebacterium diph- 2. The organism must be isolated from theriae strain that he discovered been cured of the diseased host and grown in pure their prophages before they were reintroduced culture. into susceptible hosts. Although we now realize 3. The isolate must cause the disease, that lysogenic bacteria cause many diseases (or when inoculated into a healthy, sus- intoxications), their etiologies are not always ceptible animal. immediately apparent. 4. The organism must be reisolated Another exception worth noting is contami- from the inoculated, diseased ani- nated cultures, and hog cholera provides a good mal. example. Originally thought to be caused by the FIGURE 2 he described one of those first exceptions himself! In 1884, when Koch published his findings on cholera, he noted that, with no animal model for this disease, he could not satisfy postulate three. Today, cholera mod- els do exist, including the rabbit ligated ileal loop assay. Moreover, modern molecular biological techniques and epidemiology of- ten help to overcome seeming obstacles to Koch’s postulates for those analyzing the etiology of infectious diseases. Other seeming exceptions to Koch’s pos- tulates include many of the diseases attrib- utable to viruses, animal parasites, rickett- sia, and chlamydia. In most of these cases, host specificity appears confined to humans, making it difficult if not impossible to verify the third postulate in an animal model. Al- though cell cultures now are available for many viruses, the disease itself cannot be recreated in cells, even if cytopathogenic effects can be observed. As for animal para- sites, intermediate hosts are often available, but human disease symptoms are not always manifest in these animals. Like viruses, rick- ettsia and chlamydia are obligate intracellu- lar parasites, and lack animal models. Polymicrobial causes of diseases have long been suspected, and today this is an emerging field of study. These disease pro- cesses, such as soft tissue abscesses and peri- odontal disease caused by mixed infections with anaerobes, cannot be recreated in other species. However, as microbiologists be- Membrane chambers (A) containing Escherichia coli H10407 were attached to a come more successful in producing func- sunken barge (B) in Nixon’s Harbor, South Bimini. tional mixed cultures, for whatever purpose, Volume 1, Number 5, 2006 / Microbe Y 225 bacterium Salmonella choleraesuis, hog cholera Two decades ago, Rita Colwell and I relied on is now known to be caused by a virus—the hog a variation of Koch’s postulates to show that cholera virus (HCV). HCV belongs to the family Escherichia coli can form somnicells. We sus- Flaviviridae, genus Pestivirus, and infections pended viable, culturable cells of E. coli H10407 with this virus can give rise to swine fever. In the (an enterotoxigenic strain) within membrane late 1890s, hog cholera was thought to be chambers in Nixon Harbor, South Bimini, Ba- caused by the S.