The convergence of thought engendered by Pasteur’s confirmation of the germ theory of disease, along with technological advances in the laboratory spearheaded by Koch, led to the premise by the latter part of the nineteenth century that many of the most notorious illnesses of the time could be ascribed a specific bacterial origin. Traditional rational empiricism—careful observations filtered through the lens of reason—that had been the guiding force of physicians through the ages could at last be wed to science.

The differentiation of individual diseases on a clinical basis had perhaps been most clearly articulated by Thomas Sydenham, the “father of English medicine,” in the seventeenth century. His writings represent detailed observations on the clinical manifestations of a variety of well-recognized syndromes—many of them infectious in origin—of the time, including smallpox, plague, pertussis—“whooping cough”—and measles as well as descriptions of noninfectious entities such as gout—which the author himself suffered from for most of his adult life—and chorea, a bizarre movement disorder of the nervous system. Although classic and elegant, his observations were made prior to the application of the germ theory; in fact, Sydenham died just a decade after Leeuwenhoek’s initial description of animalcules in rainwater. Thus, they were either silent on the etiology of these infectious maladies or, as in the case of smallpox—which he described in his famous book on the management of fevers as due to a type of “renovation” of the blood—were frankly wrong.¹

The movement towards associating individual diseases with specific causes gained adherents in the eighteenth and early nineteenth centuries, largely through the study of pathological anatomy. It was this discipline that allowed practitioners of the art to correlate symptoms—what the patient complains of—and signs—findings on physical examination—with observations noted at the time of pathological examination, either following surgical extirpation of lesions or necropsy. As viewing techniques improved via better lenses and microscopes, gross findings—those visualized by the naked eye—could be correlated with observations made under the microscope, further enhancing the classification of disease processes. In this fashion, the knowledge gaps in medicine began to close as the tools with which to study the science improved. For infectious diseases, these tools derived from the experimental revelations that came from Paris and Berlin, centers of medicine of the era, and they would galvanize a flurry of microbiologic discovery.

Koch’s approach in Berlin was more technically oriented than Pasteur’s in Paris. After laying spontaneous generation to rest in favor of the germ theory, Pasteur, as we shall see later, turned his attention, following a series of serendipitous laboratory observations, to immunization—vaccination—as the principal means of protection against microbial invaders. He would pursue this path for the remainder of his illustrious career. However, Koch, perhaps due to his training as a physician and his experiences caring for the wounded in the Franco-Prussian War in 1870, took a more utilitarian approach towards the new science of microbiology. He was a practical man who believed that connecting specific bacteria to certain diseases would permit the rational use of hygiene to prevent these infections. Thus, his work became heavily focused on the development of laboratory methods to isolate bacteria in pure culture and on staining techniques with which to better visualize the organisms.

The dividends of Koch’s approach rapidly accrued to the fledgling field of microbiology. One of his disciples, Paul Ehrlich, who would later be recognized as a co-founder of the field of immunology and would figure prominently in our story, devoted much of his early career in Koch’s lab to developing the fundamental tenets of the technical aspects of staining bacteria so that microorganisms could be viewed under the microscope and differentiated from surrounding tissues and inflammation.² Ehrlich’s work with methylene blue dye facilitated the visualization of bacilli in the lesions of tuberculosis, eventually leading Koch to prove, in 1882, a bacterial etiology of this great killer of the time. Christian Gram, a Danish physician also working in Berlin in the 1880s, extended Ehrlich’s methods to demonstrate that adding an iodine solution to the violet dye and then exposing the sample to alcohol could differentially stain types of bacteria. This became the basis for the Gram stain, still widely used today in hospital laboratories to distinguish among categories of bacteria.

Koch, meanwhile, relentlessly pursued the idea of obtaining pure growth of a single bacterial type in artificial culture media.² The importance of this lay in its necessity in proving causality; the definitive proof that a specific microbe was the cause of a specific disease would require the isolation of the germ in pure culture from affected specimens of an individual or animal suffering from said malady. Although he initially recovered pure cultures of anthrax bacteria by injecting the organism into the blood of mice and then successively transferring the infected blood from mouse to mouse—a phenomenon known as “serial passage” and now understood to be related to the genetic selection of favored bacteria, analogous to Darwin’s notion of natural selection—Koch was convinced that a technique was needed for the pure culture of bacteria using artificial substances—in vitro—that was therefore less labor intensive and less expensive.

Building upon a series of trial-and-error experiments, Koch soon devised a method for growing pure cultures of bacteria on a nutrient-fortified, gelatin base. He demonstrated the success of this technique at the International Medical Congress in London in 1881, attended by the most prominent men in the field—Lister and Pasteur among them—and instantly became a celebrity scientist.² Koch’s “plating technique” underwent a few minor modifications early on. First, the gelatin, which tended to melt at the temperature of the human body that bacteria tend to favor, was replaced with a different solidifying agent—agar—an algae-derived substance that was well known at the time in culinary circles for its use in jam-making and was suggested by the wife of one of his co-workers. Agar possessed the dual advantages of possessing a high melting point, thereby allowing it to remain solid at the 98.⁶°F body temperatures and having the ability to retain its liquid form at temperatures that permit it to be comfortably poured into laboratory glassware before solidification.

Richard Petri, one of Koch’s assistants, instituted another minor, albeit critical modification to the technique in 1887. Petri conceived of a transparent, covered dish apparatus into which the liquid agar was poured, thus allowing for the repeated visual examination of the cultures without risking contamination by airborne organisms. Koch’s technique for culturing bacteria using agar in these novel “Petri” dishes represented one of the simplest yet most significant advances in the history of medicine. It provides a classic example of how a seemingly trivial adjustment can propel a field forward by revolutionizing the tools of its trade. The technique remains in daily use today in microbiology labs and hospitals throughout the world, a testament to its lasting legacy.

Now, armed with the laboratory tools needed to determine whether specific germs were associated with specific diseases and heeding the newly minted criteria set down by Koch in 1882 to ascribe a bacterial etiology to an illness, physicians and scientists of the late nineteenth century presided over an unparalleled period of prolific discovery in the arena of medical microbiology. Within a span of twenty-five years, the last quarter-century of the 1800s, the specific bacterial etiologies of a number of the most dreaded diseases of that epoch were elucidated.

The list of infectious diseases with newly assigned bacterial causes reads like a “Who’s-Who” scroll of lethality. Beginning with Koch’s groundbreaking work with anthrax in 1876, that period witnessed the confirmation of bacterial causes of tuberculosis—the “captain of all these men of death”—in 1882; cholera, the elusive pathogen that terrorized the world in recurring pandemics of the nineteenth century, in 1883; typhoid, a disease of significant military import, in 1884; diphtheria, the cruelest of the childhood diseases—killing its young victims by suffocation—also in 1884; lobar pneumonia due to the pneumococcus, the most common and deadly bacterial infection of century’s end, in 1887; tetanus, the cause of lockjaw and battlefield-related deaths, in 1889; and in 1894, plague, the culprit of recurring, explosive pandemics—most notably the Black Death of the fourteenth century—that altered the geopolitical history of Europe.³ Additionally, malaria, the cause of millions of deaths around the world, was proven to be of microbial origin—though not a bacterium but a protozoal parasite—in 1896.

The nineteenth century ended with another discovery that would herald the next great era of medical microbiology. Agricultural scientists working contemporaneously in the Netherlands and in Russia determined that a filterable agent—too small to be trapped by the common devices used to detain bacteria—was responsible for the economically devastating “mosaic disease” of tobacco plants. These previously unidentified filterable agents, soon to be known as viruses, were beyond visualization by the microscopes of the time; hence, they were “submicroscopic.” Viruses would be shown—over the first quarter of the dawning twentieth century—to be causally linked to at least 65 diseases of animals and humans.⁴ Their discovery would eventually usher in a new discipline within microbiology and stimulate the next great period of scientific breakthroughs.