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Bacteriology To The Future

During his time at the University of Kansas in Lawrence, his years at the Medical School’s campus in Rosedale, and for decades after his association with KU ended, Dr. Marshall A. Barber compiled a record of exceptional professional achievement.

He circled the globe to fight malaria, and advised the US military on disease management and prevention techniques in both World Wars. In Kansas, he worked with noted public health advocate (and future KU School of Medicine dean) Dr. Samuel Crumbine to ban the common drinking cup and conduct water-quality tests on the Kaw.

And at the University of Kansas, this KU alum, who also earned three degrees from Harvard, became the first formally trained bacteriologist to teach courses in this then-nascent field. Along the way, he found time to serve as one of the first regular contributors to the Graduate Magazine, the earliest predecessor of today’s Kansas Alumni. He even donated some 18 residential lots he owned in Rosedale to KU, to be sold and used for the Medical School’s benefit.

But ironically, Barber’s most important contribution – his invention of the micropipette and related devices in the early 1900s that enabled the capture and manipulation of single bacteria, and provided conclusive proof of the germ theory of disease – was all but forgotten until the early 1980s when a pair of KU School of Medicine researchers “rediscovered” this unsung Jayhawk scientist.

As the two scholars learned, Barber fashioned his micropipettes from tiny, hollow glass capillary tubes in his laboratory in the original Blake Hall on KU’s Lawrence campus. These remarkably delicate, ultra-fine-pointed instruments allowed him, with aid of a microscope, to isolate, then capture, a single unicellular bacterium of anthrax from specimen culture. Once accomplished, Barber then used his pipette to inject the bacterium into a lab animal. When it contracted anthrax after being exposed to this single bacillus, Barber successfully demonstrated what the renowned French chemist and microbiologist Louis Pasteur never could – namely, that single microorganisms can cause disease.

First publicized in the Journal of the Kansas Medical Society in 1904, Barber presented his “pipette method” to an initially skeptical scientific audience at an international conference in Washington, DC, four years later. The demonstration was effective, and Barber soon won fast and lasting converts.

Yet as Barber knew, and as his peers were quick to discover, micropipettes held value far beyond their wielder’s ability to prove a theorem. Indeed, by allowing scientists to analyze and manipulate, then later dissect and alter, individual cells of all types, Barber’s instruments hastened, and in many ways enabled, our present-day understanding of the very building blocks of life. Accordingly, the fields of embryology (the study of embryos) and cytology (the study of cells) owe him a substantial debt. As does contemporary stem-cell research, in vitro fertilization, and animal and human cloning which, for better or worse, would be impossible were it not for the advances Barber and his “pipette method” made possible.

Born November 22, 1868, in Crown Point, Indiana, Marshall Albert Barber came with his parents, Marshall Luke and Rosannah (Andrews) Barber, to Kansas sometime in the 1870s. Settling near Burlington, just southeast of Emporia, the Barbers were a farming family. Young Marshall, when not helping out in the fields, attended the town’s one-room Prairie View Schoolhouse.

He soon outgrew these environs, both physically and intellectually. In 1887, at the age of 19, Barber advanced to the University of Kansas in Lawrence where he studied natural history and took an AB degree four years later. Following KU, Barber went on to Harvard, where he earned a second bachelor’s degree, plus a master’s. He then returned to the Sunflower State. On September 5, 1894, Barber began his professional career at KU as an instructor in botany and bacteriology – the latter being a relatively new scientific field.

Bacteriology had emerged as a distinct discipline in Europe during the mid-nineteenth century. Its tenets were based on the work of such men as Louis Pasteur, Robert Koch, John Snow and Joseph Lister.

In 1876, Pasteur put forth the germ theory of disease – which maintained that diseases are caused by microorganisms – and demonstrated it in animals.

Koch proved it applied to human beings as well. Koch had established the role of bacteria in both Tbc and cholera, in 1882 and 1883, respectively. His Postulates (now more properly called the Henle-Koch Postulates by medical historians) were the accepted proof connecting a bacterium to a disease.

Snow, who linked polluted water to cholera outbreaks, was a founder of epidemiology (the study of epidemics). And Lister, remembered today by the mouthwash derived from his name, was the era’s leading champion of antiseptic surgery.

By the 1880s, the work of these and other pioneering European bacteriologists was beginning to achieve acceptance among American scientists and academicians as well. At the University of Kansas, for instance, Professor William Chase Stevens, a botanist by training, offered the first bacteriology course in 1890. Four years later, the appointment of Marshall Barber as KU’s first purposefully hired and specially trained bacteriologist, was thus in line with these developments.

Quickly climbing Mount Oread’s academic ladder, Barber was promoted from instructor to assistant professor of botany and bacteriology in 1896, then achieved associate professor status in 1898. But it was in 1899, the year the University founded its two-year School of Medicine, when Barber’s classes in bacteriology began to attain real prominence.

Nineteen years earlier, KU had established a one-year Preparatory Medical Course at the Lawrence campus for aspiring physicians (who would then have to complete their education elsewhere). This rudimentary core curriculum was basically a repackaging of basic science courses – such as biology, chemistry, anatomy, and toxicology – that were already being taught at the University. When KU advanced to a two-year medical school however, it began offering a significantly expanded range of subjects. Among these new required courses were pharmacy, embryology, psychology, medical jurisprudence and, finally, bacteriology – taught by Professor Barber.

It was in this role, as a member of the fledgling Med School’s faculty, that Barber began work sometime in 1902 on the device that would come to be known as the micropipette. (For Barber, 1902 was also marred by personal tragedy when his wife Florence died after only a single year of marriage.)

Like most turn-of-the-century bacteriologists, Barber’s education and understanding of the nature of disease centered on Pasteur and Koch’s germ theory. Using increasingly powerful microscopes, scientists had long been able to observe microorganisms but, as yet, none had been able to prove categorically that they did, in fact, spread disease. What was needed was a means to isolate a single, unicellular bacterium – of anthrax, for example – capture it by some means, then implant or inject it into a test animal to determine whether the subject would then contract the disease. Up to this point, however, instruments of sufficient precision, capable of manipulating cells measured in microns (or millionths of a meter) had not yet been invented.

Enter Marshall Barber. Between 1902 and 1904, he developed a process of creating implements that would permit scientists to do just this – to separate, and then work with, single-celled microorganisms. The key device was the micropipette.

To make a micropipette, Barber used readily accessible materials: a thin-walled glass capillary tube, measuring approximately 10 cm in length and 4 mm in diameter; a Bunsen gas burner; and a pair of small forceps. After lighting the burner and reducing the flame to no higher than 2 mm, Barber’s first step was to take the capillary tube in both hands (one at each end) and hold it over the heat. Then, he slowly pulled his hands apart, as the tube began to soften, until the middle section narrowed to about a half-millimeter in diameter, at which point the tube was removed from the flame and bent so that it separated into two pieces. Each of the tubes now had one relatively wide portion (called the shank) and one small, needle-like point.

Next, Barber took the shank end of one of the tubes in his right hand and, in his left, picked up the forceps. With both hands resting on the table, he then re-positioned the tube over the flame and very slowly and very gently, using the forceps, began stretching the needle-like end to an even finer – and microscopically narrow – tip. Eventually, the glass tube would stretch to the point that it would separate again, this time naturally. What Barber was left with, then, in his right hand, was a micropipette.

What enabled Barber to capture individual bacteria with his micropipettes were the tools’ extraordinarily fine points, some of which were no wider than few microns. (Compare this to a single human hair, which typically measures from 50-100 microns in diameter.) Even more remarkable was how the microorganism’s actual capture was accomplished. Under a compound microscope and, at least initially, with only his hands, Barber placed the glass pipette’s tip into a specimen culture. Attached to the pipette’s shank was a length of rubber tubing, and using mouth suction to create a vacuum, he was able to create the pressure necessary to draw the bacterium into this narrowest of openings. (More remarkable still is the anecdotal evidence that Barber had a “rather pronounced tremor” in one of his hands, which would have made his ability to craft and maneuver such delicate instruments all the more astonishing.)

The immediate and most important early consequence of Barber’s work was that he was able to confirm, at long last, the germ theory of disease. Using his micropipettes, Barber succeeded in capturing a single anthrax bacterium from culture. He then proceeded to inject the lone bacillus into a mouse’s peritoneal cavity (the space within the abdomen that contains the intestines, the stomach and the liver). In due course, the mouse became infected with anthrax, just as the germ theory postulated, thus proving it.

For the time being, though, few outside Kansas medical circles were aware of Barber’s achievement. Aside from a 1904 article he wrote for the Journal of the Kansas Medical Society, titled “A New Method of Isolating Micro-organisms” and containing a brief description of how he forged micropipettes, it seems Barber made no other attempts to publicize his invention or his proof of the germ theory to a wider professional audience. For whatever reason, his research stayed largely confined to his Blake Hall laboratory.

Barber was, however, soon to be on the move in other ways, as was the University of Kansas as a whole. In 1905, KU expanded its Medical School to a full, four-year institution with its Clinical Department campus on “Goat Hill” in Rosedale, an area now part of present-day Kansas City, Kansas. (At this point, and indeed until 1962, the KU School of Medicine would be split between two campuses. Medical students underwent the initial semesters of background scientific instruction on Mount Oread, then moved to Rosedale for the final semesters of more specialized, hands-on clinical training.)

In 1906, when the Medical School’s Eleanor Taylor Bell Memorial Hospital officially opened, Barber himself migrated to Rosedale to become chair of the new Department of Bacteriology and Pathology, as well as director of clinical laboratories. He was also elevated to full professor of bacteriology. The following year, Barber added a third Harvard University degree to his résumé – a PhD in bacteriology.

By 1908, it had been roughly four years since Barber had published anything regarding his micropipettes. Nonetheless, word was beginning to get out. And not surprisingly, many leading scientific lights of the early 1900s were more than a little suspicious of Barber’s claims. These skeptics questioned how he could isolate single microorganisms using implements he had hand-fashioned in a Kansas laboratory, with tips so narrow that they could manipulate micron-sized cells.

Perhaps the most prominent disbeliever was the eminent German physician Dr. Robert Koch himself, a 1905 Nobel Prize winner in medicine. More than just about anyone else at the time, Koch was considered the “Great Man of Science,” mainly for his discovery of the tuberculosis and cholera bacilli, as well as for co-founding, along with Pasteur, the field of bacteriology and developing the germ theory of disease. In fact, upon first hearing of Barber’s claims, Koch was somewhat dubious.

This apparent skepticism notwithstanding, there is also evidence Koch may have harbored the hope that Barber’s pipette technique was indeed as revolutionary as claimed. According to KU School of Medicine professor and institutional historian Dr. Ralph H. Major, “[When] the illustrious Robert Koch visited America [in 1908] there were two things he wished to see – the organisms Howard Ricketts had found in Rocky Mountain spotted fever and Barber’s method of isolating individual bacteria.”

The opportunity to do the latter occurred on September 28, 1908, in Washington, DC at the Sixth International Congress on Tuberculosis. Before these illustrious attendees, Barber decided to formally unveil his “pipette method” to a somewhat dubious scientific community. (Barber chose this setting because of the audience. His pipettes had little bearing on TB research and he did not officially contribute to the conference.)

With Koch in the audience, Barber showed how he fashioned his micropipettes and how, with them, one could isolate a single microorganism from culture, infect a test animal, and thereby prove the germ theory. In so doing, he made instant converts out of his erstwhile doubters. Most prominent among these was Dr. Koch himself, who thereafter publicly praised “the very excellent and efficient technique of Dr. Barber.”

The demonstrations Barber gave in DC actually involved more than just his pipettes. Between 1904 and 1908, he also had designed and built devices he called micromanipulators – essentially elaborate mechanical pipette holders that were attached to the stage of a compound microscope. By securing the micropipette horizontally into the manipulator so that the tip sat right under the microscope’s lens, the pipette could then be moved very precisely in any direction simply by adjusting the manipulator’s calibrated dials. The other element of the technique was to attach a “hanging droplet” of a biological solution from the bottom of a slide’s cover slip in order to observe and gain access to the organisms with his micropipettes under constant humidity.

Upon returning to Kansas following his tuberculosis congress presentation, Barber was soon deluged with requests from scientists and researchers worldwide, all wanting to purchase his micropipettes and micromanipulators. Fortunately, he had recently partnered with one C.W. White, employed as the University’s “instrument maker,” who was building the delicate manipulators in his Blake Hall workshop to be attached to the stage of commercial monocular microscopes. As for the individual pipettes, however, Barber continued to fashion those himself in his research facilities at Rosedale.

According to a December 7, 1908, article in the Kansas City Times, White had come to KU three years earlier to do “instrument work” for Professor Lucien Blake who, at the time, was engaged in underwater signaling experiments on the Kaw River. Now, White was devoted to Barber’s creations, rushing to fill orders emanating “from all parts of the United States and Europe” – one of which, incidentally, came from Dr. Robert Koch in Berlin. “It is no exaggeration to say,” remarked the Times, “that by these instruments the work of the bacteriologist has been practically revolutionized, and the accuracy of his work increased many fold.”

Barber, however, was not quite through revolutionizing his field. Having given his possibly troublesome hands a break with his micromanipulators – necessity is, after all, the mother of invention – over the next three years (from 1908-11), he developed another apparatus to relieve his mouth as well. Known as microinjectors, these devices featured a regular micropipette with a section of rubber tubing attached to the shank. Instead of placing the tube in the mouth to create the negative or positive pressure needed to draw substances into (or drive them out of) the pipette, the tube was attached to a glass column filled with mercury. By alternatively heating or cooling the mercury, Barber could cause an exact and measurable rise or fall of pressure – a vast improvement over his essentially hit-or-miss mouth suction days.

As the name implies, with his microinjectors and the attendant precision they allowed, Barber was able to inject (or conversely extract) “known amounts of fluid into living cells.” Indeed, by the early 1910s, his research had advanced beyond merely isolating and capturing microorganisms. Now he was actually dissecting and altering individual cells.

Beyond his training and professional exploits in bacteriology, Barber possessed considerable expertise in the field of botany as well, having taught KU classes in this subject for many years. Thus, one especially interesting application of his “pipette method” involved experimenting with plant cells. Using all three of his micro-implements – the pipettes, the manipulators and the injectors – Barber successfully implanted foreign microorganisms into various types of plant cells. And when the cells died, Barber was able to show that they possessed no internal defense against microorganisms; that instead, plant cells rely on their comparatively thick outer walls to protect them against bacteria.

Beginning with his micropipettes and culminating in this elaborate “microinjection technique,” Barber had made substantial contributions to the biological sciences that were as in demand as they were useful. That said, he never advertised or sought patents for any of his inventions, nor did he apparently profit to any significant degree from their sale. At every turn, he published detailed illustrations of how they were built, explained in depth how they worked, and even personally instructed visiting researchers in their usage.

In addition to his work with micropipettes while at KU, Barber also collaborated with noted Kansas public health advocate (and future Medical School dean) Dr. Samuel J. Crumbine in the latter’s successful efforts to ban the common drinking cup. The two men again joined forces to conduct water-quality tests on the Kaw. Because of their work – which proved that sewage-ridden rivers did not purify themselves every seven miles, as conventional wisdom then believed – Kansas residents statewide came to have much cleaner and safer drinking water.

In 1911, after almost 17 years on the KU faculty, Barber decided he would leave the University and allow science to take him on what turned out to be a decades-long globetrotting crusade against malaria. Beginning at the Bureau of Science in the Philippines, he would later travel throughout Central and South America, to North Africa, Greece, India, Singapore and Russia – first under the auspices of the International Health Bureau, then for the Rockefeller Foundation – helping foreign governments and peoples combat this near-universal scourge. (Incidentally, in 1946, the first book ever published by the newly-founded University Press of Kansas was Barber’s sweeping autobiographical account of his experiences, titled A Malariologist in Many Lands.)

Between trips, Barber found time to help his own country as well. During World War I, he served as a major in the US Army’s Sanitary Corps, which was responsible for disease management and prevention. And in the Second World War, he advised Secretary of War Henry Stimson on tropical diseases and also gave lectures at the Army Medical School in Washington, DC. For his valued assistance, Barber received a citation from the War Department which, in part, praised him as “one of the most noted malariologists in the world.”

Indeed, by the time the 84-year-old Barber died on January 15, 1953, in El Cajon, California (followed by burial in Lawrence’s Oak Hill Cemetery), he was far better known for his anti-malarial work than for his micropipettes, manipulators and injectors. In the intervening years, other scientists had further perfected his instruments, improving their precision and ease of handling, and had gone on to make long strides of their own. This “next generation” of scientists employed micropipettes for such uses as the microinjection of starfish sperm into unfertilized eggs and, only a year before Barber’s death, the cloning of frogs by nuclear transfer.

Barber’s inventions and seminal contributions to scientific advancement may well have shaken “the grounds of biological sciences and contributed substantially to the emergence of a new experimental biology,” as one scholar has observed. But there was one major problem. By the early 1980s, Barber himself was fading into obscurity.

Although the KU School of Medicine’s Dr. Ralph Major described the micropipette in the second volume of his History of Medicine (published in 1954), and the Med School’s Dr. Robert Hudson told the Barber story in covering the concept of one-bacterium--one-disease in his lectures, Barber was on the verge of becoming a forgotten man.

That this didn’t happen is due in some part to the work of former KU Medical Center graduate student Daniel A. Terreros. While doing research for his PhD dissertation in physiology at the Linda Hall Library in Kansas City, Missouri, Terreros came across an obscure reference to Marshall Barber and his “pipette method.”

Intrigued because of Barber’s KU pedigree, Terreros – who is also an MD and presently head of the pathology research division at Texas Tech – investigated further. In conjunction with KU School of Medicine’s Dr. Jared Grantham, a leading researcher of kidney diseases and a nephrologist in the Departments of Medicine and Biochemistry and Molecular Biology, the two began to “rediscover” this long unsung scientific pioneer. Soon after, they co-authored a comprehensive article titled “Marshall Barber and the Origins of Micropipette Methods,” which was published in the March 1982 edition of the American Journal of Physiology.

According to their research, Barber’s methodology essentially left KU for more than half a century. Physiologists at Cornell and Woods-Hole Marine laboratory appear to have been among the first to adapt it to their purposes. They in turn taught the methodology to visiting scientists from the Cambridge University in Britain and the Max Planck Institute in Germany, who applied it with some modifications to study the mechanisms by which cells can use electrical currents to communicate, absorb substrates, and transport ions. Guided by Dr. Alan Lloyd Hodgkin and others at Cambridge, and Drs. Erwin Neher and Bert Sakmann at Max Planck, these applications ultimately resulted in two Nobel Prizes.

Eventually, these techniques made their way back across the Atlantic, and ultimately to Kansas, albeit unwittingly. By the late 1960s, scientists at the National Institutes of Health in Bethesda, Maryland, were using micropipettes to flow fluid into nerves (known scientifically as micro-perfusion of axons) and to measure electrical currents across the cellular membranes to study how nerves transmit impulses.

NIH researchers in nephrology – the science that examines the functions and diseases of kidneys – were also using this technique. One of these researchers was Dr. Jared Grantham, then a newly minted MD from the KU School of Medicine. Working with Dr. Maurice Berg under Dr. Jack Orloff, they developed the in vitro-perfusion of mechanically isolated renal tubules.

When Grantham returned to the KU Medical Center in 1969, he brought the NIH methodology with him. Since this was essentially an adaptation of the Barber micropipette methodology, the Kansan’s turn-of-the-century contribution to science had finally returned to the Sunflower State – not that anyone realized it at the time. But over the next decade, this renal technique Grantham employed started to revolutionize the understanding of the functions and disease processes residing on each segment of the nephron. (Nephrons are the functional units of the kidney; there are some two million of them in each set of human kidneys.)

As a result, numerous students, fellows, and visiting scientists began to frequent Grantham’s laboratory and lectures to learn more. One of these attendees was a young Colombian student named Daniel Terreros, who had made Grantham’s acquaintance abroad and eventually followed him back to the KU Medical Center for further study. It was during this period that Terreros stumbled across a brief notation by Homer Smith, the founder of American nephrology, about Barber’s invention of pipettes in Kansas.

The rest is history – or perhaps the restoration of history. For more than 20 years, Grantham and Terreros have been working together to research this topic and shed more light on Barber’s contributions. Dr. Grantham himself has taken frequent opportunities, through formal talks and papers, to help modern-day audiences (particularly Kansas ones) appreciate just how significant Barber’s impact on science was – and, indeed, continues to be. In Grantham’s estimation, the advances he and other renal physiologists have made using Barber’s apparatuses and their mechanical descendents would alone warrant lasting acclaim for their originator. Scientists are now able, for example, to “stick pipettes into kidney tubes to tell how [and how well] the kidney converts plasma into urine,” a process that had long been a mystery.

In 2002, there emerged another promising sign that even more contemporary scientists are beginning to credit Barber’s work. That year, two molecular biologists, Vladimir Korzh and Uwe Sträle, wrote an article for the biological journal Differentiation called “Marshall Barber and the Century of Microinjection: From Cloning of Bacteria to Cloning of Everything.”

According to Korzh and Sträle, Barber’s successful removal of the nuclei from individual cells fundamentally changed “the nature of cytology and embryology” and prepared “the foundation [for] all future efforts in animal cloning by nuclear transfer.” Human cloning, too, they hastened to add, can be accomplished using this same nuclear replacement technique – though both Grantham and Terreros believe Barber would have rejected this particular use of his methodology.

Back in 1922, New York University’s Dr. Robert Chambers, a fellow bacteriologist who had collaborated on research with Barber, sought to briefly summarize his colleague’s importance to the ever-broadening and diversifying field of biological science. “Operative work on the living cell,” Chambers noted, “has long been the aim of investigators in cytology and in experimental embryology. It was not, however, till Barber developed his [micropipette] method that any serious attempt could be made to dissect cells under magnification” and thus “observe in detail the various steps of the operation.”


Source Notes

[Source Notes: The documents and materials consulted for this article can be found in the University of Kansas Medical Center Archives. They include the following: Marshall A. Barber, “A New Method of Isolating Micro-organisms,” Journal of the Kansas Medical Society, vol. 4 (1904), pp. 489-494; Marshall A. Barber, “The Pipette Method in the Isolation of Single Micro-organisms and in the Inoculation of Substances into Living Cells,” Philippine Journal of Science, vol. 9 (1914), pp. 308-360; D.A. Terreros and J.J. Grantham, “Marshall Barber and the Origins of Micropipette Methods,” American Journal of Physiology vol. 242 (1982), pp. F293-F296; Vladimir Zorzh and Uwe Sträle, “Marshall Barber and the Century of Microinjection: From Cloning of Bacteria to Cloning of Everything,” Differentiation 70 (2002), pp. 221-226; Jared J. Grantham, M.D., “Research Contributions of Marshall Barber: Development of Micropipette Technology for Renal Research” (undated); Jared J. Grantham, M.D., “Lecture on Marshall Barber,” given April 21, 1982, to the Federation of American Societies for Experimental Biology; Transactions of the Sixth International Congress on Tuberculosis (William F. Fell Company, 1908), pp. 214-215; Kansas City Times (December 7, 1908); University Daily Kansan (January 25, 1938); University Daily Kansan (May 16, 1940); Lawrence Journal-World (February 12, 1945); Kansas City Star (May, 25, 1946); Kansas City Times (January 17, 1953); Kansas City Star (June 18, 1953); Clifford Griffin, The University of Kansas: A History (University Press of Kansas, 1974), p. 318; Thomas N. Bonner, The Kansas Doctor: A Century of Pioneering (University Press of Kansas, 1959), pp. 105, 139-42. The editors wish to thank Dr. Jared J. Grantham and Dr. Daniel A. Terreros for their invaluable assistance in the preparation of this article.]