Experiment Eleven (7 page)

A year earlier, he had been pressed to start such a project by his son,
Byron, who was then finishing his degree in bacteriology at the University of Pennsylvania Medical School. In a letter, Byron had offered to do a summer project at Rutgers, first testing microbes against the nonvirulent strains of
Mycobacteria
in Waksman's lab and then, if an active agent was found, testing it against the virulent human form in animals. As Waksman did not have animal-testing facilities in his department, that risky second test would have to be done elsewhere. But Waksman was still not ready. “
The time has not come yet
,” he replied to his son.

In their competing versions of what happened next, both Waksman and Schatz later claimed that they were the one to first take the TB project seriously. Waksman said he finally decided to go ahead with the project “
early in 1943
.” Schatz said that on his return from the army in June he knew exactly what he wanted to do for his Ph.D., and he chose the most ambitious research project that any Waksman graduate had ever suggested. He would find a new antibiotic that would cure the diseases his military comrades had died from, and that included tuberculosis.

The evidence does not resolve this disagreement, but seems to favor Waksman. He was certainly moving closer to testing his antibiotics against the TB germ. On June 1, according to his expense records, he went to New York City to meet Dr. Leroy Gardner of the Trudeau Sanatorium at Saranac Lake. The topic was “the problem of
bacteriostatic substances
in relation to tuberculosis.” On June 18, Waksman wrote to Dr. Florence Seibert at the Henry Phipps Institute in Philadelphia, where they carried out research on TB. Dr. Seibert had invented the first reliable tuberculosis test for humans. Waksman asked for fifty to one hundred grams of
dried cells of the human TB
H37 germ, and also a culture which could be used for growing the organism. Dr. Seibert
sent one of each
, but warned that the culture was old and of uncertain viability—the dried cells had been alive nine years before being dried in 1943. In other words, she did not know whether these strains would produce meaningful results if used as a test for TB.

Schatz was discharged from the army on June 15 and officially started work in Waksman's department on June 30. Whether Waksman's letter to Dr. Seibert was prompted by a conversation with Schatz after his discharge from the army is not known.

In any case, Schatz did not begin his experiments testing his candidate
antibiotics against
Mycobacterium
; that was to be the second part of his Ph.D. He started by enriching soil in pots with
E. coli
, as Boyd Woodruff had done to find actinomycin.

Schatz's first experiment, as noted in his 1943 lab notebook, was on June 30 and dealt with a “general survey of the occurrence of antagonistic microorganisms.” He isolated bacteria, fungi, and actinomycetes which might be responsible for destroying
E. coli
, then tested them by the streak test to see whether they produced the clear zones. On July 23, he noted in his lab notebook, he gave up the soil-enrichment method and instead switched to the agar plate method used by Dr. Kocholaty when he found streptothricin. This meant random testing of soil samples against known disease-causing bacteria. In experiments started at the beginning of August, Schatz noted that “some molds are apparently antagonistic immediately upon isolation, but they seem to lose this property upon repeated culture on artificial media.” In other words, he still hadn't found anything worthy of isolation and further experiment.

Every researcher knew that no matter how great the effort, luck was always involved in discovery, in biology especially. Alexander Fleming discovered penicillin in 1928 when a fungus spore fell by a happy accident into one of his petri dishes and he noticed the clear zones of antagonism. Too many researchers went on testing for years and never found a microbe capable of killing off a disease. Only the single-minded, obsessive researcher, the kind willing to give up a full life, the “
true devotee
to science,” in Einstein's terms, would have a chance of success. Albert Schatz was such a researcher. Fleming used to describe his microbe experiments as “
playing about
,” and Schatz knew the feeling well. He loved spotting a likely microbe, one that he thought might make a good candidate to produce an antibiotic. He loved fussing over his precious molds as they blossomed into striking, beautiful sculptures of red, blue, yellow, and gray-green. He was so fascinated by the potential power of his friendly bacteria that it seldom felt like work; even the routine, the drudgery, seemed like play. No great skills were needed, he was the first to admit. At this early stage, it was just about a steady hand and a good eye. The basic techniques, which he had learned quickly, were known as “
silly simple
” by one of Dr. Waksman's graduates.

Schatz already understood that his chances of finding a useful antibiotic were remote at best, and the human TB strain posed a special problem. There was a good reason why others had failed. Of the disease-causing microbes, the deceptively simple cell of
Mycobacterium tuberculosis
presented one of the greatest challenges.

Waksman and Schatz working in the laboratory at Rutgers. (Special Collections and University Archives, Rutgers University Libraries
)

Bacteria are divided into two groups on the basis of their reaction to a stain first used in 1884 by a Danish bacteriologist, Hans Christian Gram. Those cells that retain the stain are called Gram-positive; those that don't, Gram-negative. The distinction is important for diagnostic purposes and is due to a basic difference in the cell structure. Gram-negative cells, which cause typhoid and cholera, have an extra outer layer, making them tougher for antibiotics to penetrate. Penicillin, for example, is effective against Gram-positive bacteria such as
Staphylococcus
, the common cause of blood poisoning, but not against Gram-negative
Salmonella
, which can cause typhoid, or
Vibrio cholerae
, which causes cholera. The TB microbe is even more difficult to penetrate, having an extra layer of protection, a waxy wall preventing the entry of unwanted chemicals.

Schatz was determined to find antibiotics which destroyed the Gram-negative bacteria and that also destroyed the TB germ. After ten experiments he had no promising results, so that was when he switched to a third method—random selection. This is the least complicated of the three methods, relying to a greater extent than the other methods on chance. He took samples of soil, compost, or stable manure, added tap water to make a liquid mix, then put drops of the mix on a petri dish of agar containing a food microbes are known to thrive on—egg albumin. Then he incubated the dish and watched the microbes grow. By mid-October, Schatz had selected two strains from the gray-green
Actinomyces griseus
. One strain, 18-16, excreted an antibiotic active against the Gram-negative bacteria
E. coli
. Another strain came from the culture of the chicken's throat given him by Jones. It was also active against
E. coli
. Schatz named it D-1, for Doris. On October 19, Schatz sealed the test tube containing the 18-16 strain and gave it to his mother.

On October 20, Schatz began isolating his new antibiotic in Experiment 25—the “Collection of Active Material of 18-16.” The entry ends with a note: “Material taken over by Dr. W. & E.B.” E.B. was Elizabeth Bugie, who was working under Dr. Waksman in the upstairs lab. She began testing the strain in different mixes of nutrients to find the best one for producing the new antibiotic. The favorite seemed to be a meat extract.

It was “impossible to set down in words the
excitement that prevailed
in the laboratory during those ensuing days,” according to the book
Miracles from Microbes: The Road to Streptomycin
by Samuel Epstein and Beryl Williams, who had already collaborated on several books for young readers. The book was published by Rutgers University Press in 1946. Schatz did not agree with the Epstein and Williams account—at least of his mood, which was not excitable, he said, because of the work yet to be done. Many years after he wrote in the margin of his copy of the book, “Not true, the results were
in vitro
tests. Nobody knew the toxicity.” He was right, of course. No one could know, until tests on animals were done, whether his new antibiotic would be toxic to humans like others produced so far in Waksman's lab.

Waksman gave a sample of the new antibiotic to Doris Jones in Poultry Pathology for the first animal tests—on chick embryos, still in the egg, infected with fowl typhoid that would normally kill them. Jones injected them with five to ten milligrams of the new antibiotic and many of them
hatched. She was so new at this technique that she had never watched them peck their way out of the shells. Then she had to kill them for an autopsy to see if the typhoid bacteria had been completely destroyed. Before the operation, she hosed down the walls of the autopsy room, she recalled later. It was the only method she had of trying to rid the autopsy room of dust-containing bacteria that might have contaminated her experiment. “But
I'm paralyzed
, I can't squeeze the scissors through the necks of those little beings,” she wrote. “Dr. Beaudette mocks me. He won't help. It takes days. Finally, I squeeze and the autopsy proceeds as tears run down.” The new antibiotic had worked; no bacteria were left in the chick's organs or blood.

By this point Waksman's lab must have been buzzing with word of the discovery, and the question was what to call the new antibiotic. The naming of it would be the cause of yet another unresolved disagreement between Waksman and Schultz. Each claimed that they had thought of the name first. Waksman was the first to use the name streptomycin in a document, according to the archives. In a letter, he informed Randolph Major, Merck's research director, of the discovery on October 28, saying it could “tentatively be
designated as streptomycin
.” Schatz claims that he was always going to
call his discovery streptomycin
, but the name does not appear in Schatz's lab notebook until December 14.

Toward the end of October Schatz started to test his new antibiotic from the two strains, 18-16 and D-1, against the harmless strains of mycobacteria in the department's collection. On November 8, he began Experiment 29, which he titled “Bacterial Action of 18-16 Concentration Upon TB.” A week later, on November 18, he started Experiment 30, which was designed to test D-1 on TB. The results were promising, but the tests were again against TB from the department's collection and, therefore, non-pathogenic. The real test was yet to come.

AS SCHATZ BEGAN
his Experiment 30, a distinguished visitor arrived at the Department of Soil Microbiology. He was Dr. William Feldman, a veterinary pathologist at the Mayo Clinic in Rochester, Minnesota, one of the most famous private clinics in America. Its founder, William Worrall Mayo, a British immigrant, had opened the clinic as a frontier practice in 1846. By the 1930s, the Mayo Clinic was well known for its work in trying to find
a cure for tuberculosis. Feldman and a colleague, Corwin Hinshaw, were the principal researchers.

Feldman had immigrated to the United States from Scotland in 1894 at the age of two, and he had grown up in Colorado knowing about TB. His mother had recalled, from her own
childhood in Glasgow
, the suffering caused by the disease. For his doctorate at Colorado State University, Feldman had taken part in a nationwide effort to eradicate TB in cattle, a cause of often-fatal tuberculosis in children. He had also written a book on tuberculosis in birds. His partner, Hinshaw, was also a Westerner, a zoologist and medical doctor with expertise in parasites and bacteria. He had been at the Mayo Clinic since 1933, working on pulmonary diseases, especially pneumonia. Their mix of disciplines was excellent for testing new drugs on animals; they made a perfect team.

For more than a decade they had tried a variety of drugs, including compounds of gold and arsenic and the latest sulfa compounds, on guinea pigs infected with the human TB strain. Results from the sulfa drugs were encouraging. They treated about a hundred patients with drugs that showed unmistakable promise. One of the sulfas, Promin, gave the first hint that the waxy outer wall of
M. tuberculosis
could be breached, but did not destroy the germ. Nothing seemed to halt the onward march of the TB microbe, which nestled in inaccessible places in the lungs of patients suffering from the disease. Feldman felt that he and Hinshaw had a “
foot in the door
,” as Feldman put it, but their peers thought they were “
wasting their time
”; the sulfa drugs might halt the infection, but would never completely destroy TB. This only made the two scientists more determined and stubborn.

They devised more rigorous tests, insisting that virulent and nonvirulent TB germs had different characteristics and that any new drug had to work against the virulent type before they would even consider animal tests. They used guinea pigs because they are highly susceptible to human TB and, once infected, are severely hit by the disease; if a drug worked, it should also work in humans.