History of Microbiology

Milton Wainwright
University of Sheffield
Joshua Lederbergl
The Rockefeller University

I. Observations without Application ness of the nature and etiology of disease, with the
II. The Spontaneous Generation Controversy result that the majority of the traditional killer dis-
III. Tools of the Trade eases have now been conquered. Similar strides
IV. Microorganisms as Causal Agents of have been made in the use of microorganisms in
Disease industry. and more recently attempts are being made
V. Chemotherapy and Antibiosis to apply our knowledge of microbial ecology and
VI. Microbial Metabolism and Applied physiology to help solve environmental problems. A
Microbiology dramatic development and broadening of the subject
VII. Nutrition, Comparative Biochemistry, and of microbiology has taken place since World War II.
Other Aspects of Metabolism Microbial genetics, molecular biology, and bio-
VIII. Microbial Genetics technology in particular have blossomed. It is to be
IX. Viruses and Lysogeny: The Plasmid hoped that these developments are sufficiently op-
Concept portune to enable us to conquer the latest specter of
X. Virology disease facing us, namely AIDS. Any account of the
XI. Mycology and Protozoology, history of a discipline is. by its very nature, a per-
Microbiology’s Cinderellas sonal view: hopefully, what follows includes all the
XII. Modern Period major highlights in the development of our science.
The period approximating 1930-1950 was a ‘vi-
cennium” of extraordinary transformation of micro-
Glossary biology, just prior to the landmark publication on
the structure of DNA by Watson and Crick in 1953.
Antibiotics Antimicrobial agents produced b) We have important milestones for the vicennium:
living organisms Jordan and Falk (1928) and “System of Bacteriol-
Bacterial genetics Study of genetic elements and ogy” (1930) at its start are magisterial reviews of
hereditary in bacteria prior knowledge and thought. Dubos (1945) and
Chemotherapy Systemic use of chemical agents Burner (1945) anticipate the modern era. and Werk-
to treat microbial infections man and Wilson (1951) and Gunsalus and Stanier
Molecular biology Science concerned with DNA (1962) document its early and continued progress in
and protein synthesis of living organisms monographic detail. The AnnunI Review of Micru-
Monoclonal antibodies Specific antibodies pro- biology, starting in 1947 (and several other Annual
duced by in vitro clones of B cells hybridized Reviews), and Bacteriological Reviews. starting in
with cancerous cells 1937, offer invaluable snapshots of the contempo-
rary state of the art. These works can be consulted
for many of the pertinent bibliographic citations,
and they will be explicitly repeated here only when
ALTHOUGH MICROORGANISMS were first ob- important for the argument.
served using primitive microscopes as early as the This account will center on the fundamental biol-
late 16OOs, the science of microbiology is barely 150 ogy of microbes and give scant attention to continu-
years old. In this time, major developments have ing advances in the isolation of etiological agents of
been made in our understanding of microbial physi- disease and of vaccines and immunodiagnostic pro-
olog),. ecology, and systematics. This knowledge cedures. Most of the agents of common bacterial
has been successfuly applied to broaden our aware- infections had been characterized by ” 1930,” but

IFor the period from 1930.

Encyclopedia of Microbiology, Volume 2 419

Copyright C 1992 by Academic Press. Inc. All rights of reproduction in any form reserlred
420 History of Microbiology

the vicennium was distinguished b! important work croscopes (Fig. 2) to examine microorganisms in
on the classification of enteric (diarrhea]) bacteria rainwater. well water. and seawater as well as water
and, above all. by the isolation and nev,’ stud! of infused u ith peppercorns. His observations 1%ere
viruses and rickettsia with methods such as culti- forwarded to the Royal Society in London on Octo-
vation virus in the chick embr),o (Kilbourne. 1987). ber 1676 and were later published in the Society’s
Philosopi~icr~l T~~rr~strc~rious. In 1683. van Leeuwen-
hoek contributed a second letter to the Society de-
scribing his various microscopical investigations. in-
I. Observations without cluding novel obser\,ations on bacteria present in the
Application scurf of teeth. Published in 1683. these observations
include the first dra\+ ings of bacteria ever to appear.
Macroscopic manifestations of microbial gro\\ th These drawings are >til extant and cleal-ly show that
such as bacterial and algal 4limes ha\.e been recag- van Leeuwenhoek observed bacilli. streptococci.
nized since antiquity. However. it \\as the Dutch and man\; other characteristic forms of bacteria.
microscopist van Leeuljenhoek (Fig. I ) \+ ho pro- van Leeuu,enhoek’s meticulous drawings also she\\,
vided the first observations of bacteria at the micro- protozoa such as Vo~ric,c~llr/, VO/UO.\-, and C‘//gl~)/~tr.
scopic level. van Leeuisenhoek. a draper in Delft. At about the hame time, Huygens also reported ob-
Holland. ground his own lense\ lo make micro- servations on a number of free-living protozoa. in-
scopes \+zith short-focal length lenses gi\,ing magni- cluding species of P~i/,rr/llc,c,i,l/rl. Van Leeuwcnhoek
fications of betw.een x.30 and x266. Dr\carte\ had also gains credit for describing the first parasitic
earlier described a similar crude form of micro- protozoan. when in 1681 he observed his ou’n fecal
scope. but the quality of his lenbe\ did not allo\\ stools during a bout of diarrhea and described large
for magnifications sufficient to bee bacteria. \‘an populations of what later became known ;is Gir,rtlitr
Leeuwenhoek. in contrast. used his homemade mi- I~it~lblitr.
 History of Microbiology 421

Although van Leeuwenhoek also observed yeast moved by Pouchet’s work to offer a prize to anyone
cells in beer, the first illustrations of filamentous who could settle the controversy once and for all.
microscopic fungi were provided by Robert Hooke, Despite discouragement from his friends who cau-
again in a letter to the Royal Society. this time in tioned against becoming embroiled in the con-
1667. However. the most important early work on troversy, Louis Pasteur (Fig. 3) realized that if mi-
molds appeared in the following century when the crobiology was to advance as a rational science the
Tuscan botanist. Pietro Antonio hlicheli described idea that microorganisms arose spontaneously
some 900 species. including important genera such would need to be experimentally defeated.
as Asper-gillrrs and Marco,-. It is also worth noting Pasteur’s studies were published in memoir in
that molds have been used from ancient times to 1861 and effortlessly took the prize offered by the
treat infections, an approach termed mold therapy, Academy. He first of all showed that when air is
which was based on folk medicine rather than on any filtered through cotton wool, large numbers of
scientific rationale. microorganisms are held back. Pasteur then suc-
Because the connection between microorganisms cessfully repeated Schwann’s work, but his most
and fermentation or disease was never made during famous and successful experiments involved the
this period. observations made by the first microsco- use of swan-necked flasks, with which he showed
pists had surprisingly little impact on human affairs. that heat-sterilized infusions could be kept sterile
Despite this. Cicero and the Renaissance scholar in an open flask as long as the open part was tor-
Fracastorius had previously suggested that fevers tuous enough to allow any microorganism to set-
might be caused by minute animals. collectively de- tle on the sides of the tubes before reaching the
scribed as conragium vivum, but it was to be many liquid.
centuries before the role of microorganisms in dis- It is often assumed that Pasteur’s experiments
ease became recognized, eventually to replace the
view that disease resulted from odors or other invisi-
ble “miasmas.”

II. The Spontaneous

Generation Controversy
The view that life arises tie t~oco from inanimate
objects was widely held from the hliddle Ages until
remarkably recent times: Van Helmont even pro-
vides us with a recipe for the production of mice.
The tenacity with which spontaneous generation lin-
gered on is highlighted by the fact that H. Charlton
Bastian. one of the concept’s chief proponents, died
in 1915, still totally convinced of its merit. Although
a scientific rationale was apparently provided to ac-
count for spontaneous generation by Needham and
Buffon as early as 1745. these ideas were quickly
dismissed by Spallanzani in the following year. Fur-
ther developments then had to await the work of
Schwann. who in 1837 showed that “air which had
been heated then cooled left unchanged a meat broth
which had been boiled.” Yet by the middle of the
seventeenth century, the concept of spontaneous
generation held on tenaciously. Then, in 1858,Pou-
chet published a paper entitled “Proto-organisms
. . . Borne Spontaneously in Artificial Air and Oxy-
gen Gas.” The French Academy, of Sciences was Figure 3 Louis Pasteur (1822-1895).
422 History of Microbiology

immediately brought about the defeat of the theory alization of microorganisms to occur. The staining of
of spontaneous generation, but this is far from true. histolo$cal specimens was first carried out by the
Pouchet. for one, remained convinced that Pasteur’s German botanist Ferdinand Cohn in 1849, his work
experiments did not defeat the concept. The con- being based on vegetable dyes such as carmine and
troversy continued over the next quarter of a decade hematoxylin. By 1877. Robert Koch (Fig. 4) was
or so. Proponents of Pasteur’s views included Brit- using methylene blue to stain bacteria. a process in
ish scientists such as Huxley, William Roberts. John which he developed the standard techniques of pre-
Tyndall, and the American Jeffries Wyman. The paring dried films. and with the aid of coverslips was
main counter-arguments were provided by the last preparing permanent preparations. By 1882. Koch
and most dedicated of the important hetero- had succeeded in staining the tubercle bacillus with
genesists, H. Charlton Bastian. How this rearguard methy+ene blue, employing heat to encourage the
action by Bastian and others nearly carried the day stain to penetrate the waxy envelope. Two years
is remarkable. Hovvever, experiments by the mathe- later. the Danish pathologist Hans Christian Gram
matician and physicist John Tyndall on the existence introduced his famous stain. which allowed bacteria
of heat-stable forms of certain bacteria (the removal to be characterized as gram-positive if they retained
of which involved the process of repeated heating the violet dye or gram-negative if they did not. This
and rest, referred to as tyndalization) finally con- distinction was later to be correlated with differ-
vinced the scientific establishment of the error of ences in biochemical and morphological characteris-
Bastian’s arguments. Bastian summed up his views tics. allowing bacteria to be classified into the two
on spontaneous generation in his book Tl~e EL’o~u- broad groupings still in use today.
riorr ofLifr (published as late as 1905). and then died Differential staining techniques soon follou,ed. al-
in 1915. still a confirmed believer. lowing Frederick Loeffler in 1890 to demonstrate the
presence of bacterial flagella. During this period,
rapid developments occurred in methods for identi-

III. Tools of the Trade

The science of microbiology needed two major de-
velopments to assure its progress. The first involved
improvements in microscopes and associated means
by which microorganisms could be better visual-
ized. and the second involved developing methods
for culturing microorganisms, thereby ironically
liberating the science from total dependence on
microscope-based observation.
Compound microscopes first began to appear in
Germany at the end of the sixteenth century. and
during the following century Robert Hooke devel-
oped instruments with magnifications of 3-500x.
Although Hooke made major advances in observing
microorganisms, he also recognized cellular struc-
ture in a variety of life forms. His microscopes, like
those of his contemporaries. suffered from chro-
matic aberration (whereby a ring of colored light
prevents accurate focusing on small objects such as
bacteria). It was not until the early nineteenth cen-
tury, when achromatic lenses were introduced by
Professor Amici of the University of Medina. that
this problem was solved, thereby enabling the light
microscope to be developed to its full potential.
The next major development was the introduction
of staining procedures. which allowed the fine visu- Figure4 Robert Koch (l&l&1YlOJ.

._,. ._ .._-“.“.l”-.” -. -,_.-. ..)

^ .._
History of Microbiology

fying bacteria and demonstrating their involvement mycologists, for example, have to spend hours peel-
as causal agents of specific diseases. ing and boiling potatoes when potato dextrose agar
The light microscope was eventually developed was available ready to rehydrate, sterilize, and use.
to its theoretical limits and further progress in mi- None of the preceding developments in media
croscopy had to await the appearance of the ultra- preparation would have been useful without the in-
violet microscope in 1919 (which for the first time troduction of an efficient means of sterilization. Pas-
allowed certain elementary viruses to be seen). teur’s colleague, Chamberland, developed auto-
Then, in 1934, the Belgian physicist Marton built the claves-essentially large pressure cookers-in
first electron microscope, which achieved magnifi- 1884. More recently, gamma rays and ethylene ox-
cations of 2-300,000x, compared to 1200x and ide sterilization have allowed for the introduction
2500x achieved by the light and ultraviolet micro- of factory-sterilized plastics including Petri dishes,
scopes, respectively. A further major development another relatively simple development that has,
in microscope technology came in 1965 with the nevertheless, had a marked stimulatory effect on
introduction of the scanning electron microscope. the recent progress of microbiology. [See SYERIL-
The first semisynthetic medium designed for culti- IZATION.]
vating bacteria was introduced in 1860 by Pasteur
and consisted of ammonium salts, yeast ash. and
candy sugar. Prior to this, meat broths had been
used for bacterial growth medium, an approach that IV. Microorganisms as Causal
persisted well into this century in the laboratories Agents of Disease
devoted to medical bacteriology. Mycologists, too,
tended to rely on undefined media such as potato In 1788, an epidemic of smallpox broke out in the
dextrose agar, although the introduction of Czapek English county of Gloucestershire. Edward Jenner,
Dox medium eventually provided an ideal defined a country doctor and pupil of the famous anatomist
substrate on which molds could be grown. John Hunter, decided to try and prevent his patients
In 1872. Ferdinand Cohn developed the idea of the from contracting the disease by employing the stan-
basal medium, to which various additions could be dard method of inoculation using a mild dose of the
made as required. These early media were always infection. Jenner, who had suffered under the blood
liquid-based and it was not until the introduction purgers and inoculists in his youth, was himself im-
first of gelatine and then agar in 1882 that the use of mune to smallpox. He aimed to make the traditional
solid media became commonplace. The latter intro- inoculation method as rational and reliable as he
duction of silica gel media then allowed for rapid could. While on his regular rounds, he was surprised
advances to be made in the study of chemolithotro- to find that patients who had already suffered from
phic bacteria such as Thiobacillrrs thioosidcttls. cowpox did not react in the normal way to inocula-
By 1887, a simple and prosaic development revo- tion with smallpox. Although Jenner was aware of
lutionized microbiology when Petri. one of Koch’s the old wives’ tale suggesting that cowpox gave
assistants, introduced the Petri dish. This simple protection against the disease, it was not until 1796
invention provided a far more versatile means of nearly a quarter of a century after he had first heard
culturing microorganisms than did use of the bulky these suggestions. that he decided to act. His first
bell jars employed previously. experimental inoculation involved a local boy
From 1898 onward, the Dutch school of microbio- named James Phipps, who, after receiving cowpox,
logists led by Beijerinck developed the art of enrich- became immune to smallpox. In June 1798, Jenner
ment culture. which led to the isolation of both nitri- presented a paper on his work to the Royal Society,
fying and cellulolytic bacteria. Studies on gas and the effect was remarkable- within a few years,
gangrene during the first war encouraged McIntosh vaccination was commonplace.
and Fildes to develop the anaerobic jar. A vast array Despite Jenner’s breakthrough, there was still no
of selective media were then developed that in- convincing explanation to account for the appear-
volved amendments such as tetrathionate broth, ance and spread of infections, and by the mid-1800s
tellurite. and crude penicillin. Finally. the introduc- there was still little that could be done to counter
tion of central media supplies after the war liberated infectious disease. Childbed or puerperal fever was
the microbiologist and their technicians from the a particularly terrible blight that affected every one
tedium of preparing media in-house. No longer did of the lying-in hospitals in Europe. During a single
424 History of Microbiology

month in 1856 in a Paris hospital. 3 1 recent mothers Lister published his findings in 71re Lctrzcet in 1867.
died of the infection. Vienna of the 1830s had a In contrast to Semmelweiss’s efforts. Lister’s work
particularly bad reputation for this disease. despite attracted immediate attention-The age of antisep-
having one of the most enlightened hospitals in Eu- tic surgery was soon underway.
rope. It was here that the Hungarian doctor, Ignaz Once it became realized that microscopic organ-
Semmelweiss joined the staff of the lying-in clinic of isms present in the air were responsible for transmit-
the Vienna General Hospital in 1844. In his first few ting disease. the next important development was to
months of practice, he heard yet another wives’ tale. isolate these organisms and then conclusively dem-
this one associating the high death rate from onstrate their role as causal agents of any given
childbed fever found in the teaching division of the disease. Yet. some authorities continued to argue
hospital with the high frequency of examination by that microorganisms u’ere not the cause of disease,
doctors and their students. Semmelweiss began to but merely grew on the weakened infection site. In
collect statistics and soon became aware that the May 1882. Robert Koch dismissed this view when
highest rates of infection and mortality occurred in he announced the discovery of the tubercle bacillus;
the teaching clinic. This information led him to sur- the search for other disease-causing microorganisms
mise that the contagion was being transmitted by the then gathered momentum. The introduction by
doctors.and medical students. many of whom exam- Koch of his famous postulates finally established a
ined the wombs of patients without washing their means of conclusively demonstrating the involve-
hands. even after coming directly from mortuary ment of a microorganism as a causal agent of a given
duty. Semmelvveiss suggested that anyone examin- disease. and the way lay open to disease prevention
ing patients should first wash their hands in chlorine and cure.
water. The results of this simple remedy w’ere phe- Major developments were next made in our un-
nomenally successful. with mortality rates being re- derstanding of immunity. The first rational attempts
duced from around 11 to 3% within I yr. Semmel- to produce artificial active immunity was made by
Weiss was slow to write an account of his work, but Pasteur in 1880 during his work on fowl cholera. By
eventually in 1857 he provided a rambling and highly 1882. the Russian biologist Metchnikoff had made
egotistical survey of his vvork. which completely the first observations of cellular immunity and
failed to make any impression. coined the term phagocyte. By 1891. Ehrlich had
Eventually. however. the view that infection was distinguished between active and passive immunity.
spread by some organic particle did at last become and 6 years later Kraus published the first account of
widely accepted. although the exact nature of such precipitation reactions when immune sera were
particles was unknown. The effect of this ignorance added to cell-free filtrates of homologous bacterial
was devastating: during the Crimean War of 1853- cultures.
1826. for example, a single regiment of the British Nearly 250 million people have been vaccinated
Armv lost 2162 men. with 1713 dying not from against tuberculosis with the bacille Calmette-
wounds or the effects of trauma but from disease. Guerin (BCG) vaccine. yet its originator, Charles
The infamous hospital diseases of erysipelas. pye- Calmette. remains a largely unknown figure. Cal-
mia. septicemia. and gangrene made surgical wards mette. a disciple of Pasteur, was the first Director of
nightmares of suffering and death. The causes and the Pasteur Institute in Lille. France, and later be-
mechanisms of disease transmission remained es- came Assistant Director of the Pasteur Institute in
sentially unknown. By 1865. however. Pasteur had Paris. With Guerin. he set about to prepare a protec-
concluded that disease must be airborne. a view that tive vaccine against tuberculosis. He spent 13 years
galvanized the English surgeon Joseph Lister into developing an attenuated virus. which by not recov-
action. Lister reasoned that he could reduce mortal- ering its lost virulence remained both stable and
ity due to sepsis by covering wounds with dressings safe. This vaccine. BCG. was first used in 1921. but
containing chemicals that killed these airborne because of considerable resistance to its use was
germs without preventing the entry of air. He knew not widely accepted until after Calmette’s death in
that carbolic acid had recently been used to sterilize 1933.
sewage. and with the help of the chemist Anderson Modern developments in immunology include the
he obtained a supply of the sweet-smelling dark liq- work of F. Ma&t-lane Burnet. who in 1957 pub-
uid that was commonly called German creosote. lished his clonal selection hypothesis.
History of Microbiology

V. Chemotherapy and Antibiosis prontosil. This compound had a dramatic effect on

lobar pneumonia in humans, reducing death rates by
The origin and early development of the concept of by two-thirds. In the same year as its discovery,
chemotherapy is somewhat unusual in that it can be the French scientist Trefouel showed that the active
credited to the work of one man, the German chem- ingredient of prontosil was not the chromophore,
ist Paul Ehrlich. Ehrlich had the vision to apply his but the sulphonamide moeity tsulfanilamide). Sul-
knowledge of specific staining of bacteria to the phonamides were widely used with success to treat
search for chemical compounds that would inhibit bacterial infections from the mid-1930s until the
the growth of pathogenic bacteria in uiuo. In 1891. he middle of the following decade.
showed that methylene blue was useful for the treat- The concept of chemotherapy reached its zenith
ment of malaria, but because this dye showed no with the sulphonamides. but such compounds were
advantage over quinine it was not widely used. By soon eclipsed by the arrival of first penicillin and
1902. Ehrlich was concentrating his attention on the then a range of other antibiotics.
organic arsenic compounds. which he hoped would Antibiotics (cf. Waksman. MacFarlane. Wilson)
defeat experimental trypanosomiasis in mice. At had a spectacular beginning with the famous discov-
this point, he and his Japanese bacteriologist assis- ery of penicillin by Fleming in 1928, a mold spore
tant Shiga found that atoxyl (sodium arsanilate) was having accidentally lodged on agar plates seeded
ineffective against mouse trypanosomiasis. This with staphylococci. The story of the discovery of
turned out to be a somewhat inexplicable error when penicillin by Alexander Fleming is probably the best
the British bacteriologist Thomas was soon to show known in the history of medicine, although much
that atoxyl was in fact extremely effective against that has been written on the subject borders on fairy
trypanosomiasis in mice. tale. The important point about Fleming’s initial ob-
A second equally inexplicable error followed servation. made during the late summer of 1928, was
when Schaudinn and Hoffman concluded that that it represented an extremely rare phenomenon,
Treponemn pallidurn was a protozoan. Ironically, not merely an example of microbial antagonism. but
this error proved productive because it pointed to one of bacterial lysis brought about by mold contam-
the likelihood that the antiprotozoal agent atoxyl. or inant. Fleming probably initially thought that he had
a similar compound. might cure syphilis. In 1906. discovered a fungal variant of lysozyme. a lytic sub-
Robert Koch used atoxyl to treat trypanosomiasis in stance that he had previously found in various body
humans. This was the year in which Ehrlich became fluids. It was this lytic phenomenon that distin-
director of the newly opened George Speyer Insti- guished Fleming’s observations from the numerous
tute. which was devoted to chemotherapy research. observations of microbial antagonism that had been
It was here that the first major chemotherapeutic reported since Pastuer’s time. It is likely that had he
agent salvarsan was developed. Salvarsan was first observed microbial antagonism. rather than lysis,
discovered in 1907 and was initially found to be Fleming would have ignored his observations. re-
inactive against the experimental mouse trypanoso- garding them as an example of common phenome-
miasis sy’stem. Then in 1909. a young Japanese sci- non that w’as of little interest.
entist. Hata. joined Ehrlich’s laboratory. bringing Fleming. however. understood the significance of
with him a sy’stem that he had developed for the what he observed. He soon showed that the contam-
artificial transmission of 7. prrl/ic/irrnr in rabbits. To inant produced the antibacterial substance in culture
his evident surprise. Hata found that salvarsan was broth. which he called penicillin. Then. with help
in fact effective against syphilis in mice: by 1909. the from various surgeon colleagues. Fleming used
drug was proving spectacularly successful in treat- crude penicillin-rich filtrates to treat superficial bac-
ing the disease in humans. terial infections. unfortunately without much suc-
Following Ehrlich’s death in 1915. research con- cess. The first documented cures with penicillin
tinued into chemotherapy. but little progress was were in fact achieved (using the crude broths) by a
made, with the exception that in 1932 Atebrin be- former student of Fleming’s, Cecil George Paine,
came available as the first synthetic drug for pro- who worked at Sheffield University.
phylactic use against malaria. The ne.\t major ad- In his first famous paper on penicillin, Fleming
vance in chemotherapy came in 1935. when Domagk detailed its properties and antibacterial spectrum
discovered the antibacterial effect of the red dye and suggested that it. or a similar substance. might
426 History of Microbiology

find a use in medicine. Unfortunately, neither he nor substances. Tyorhricin (Dubos, 1939; cf. Crease,
his colleagues could purify penicillin, an obvious 1989) was the first antibiotic to be clinically applica-
necessary first step for its successful introduction ble, but its systemic toxicity limited its application
into medicine. It is worth pointing out, however, to topical treatment. In Waksman’s hands, the same
that Fleming was not alone in being unable to paradigm led to the discovery of streptomycin (1944),
achieve this essential purification step: other at- which when used in conjunction with periodic acid-
tempts such as those made by famous fungal product Schiff and isoniazid helped to defeat tuberculosis.
biochemist Harold Raistrick also proved unsuc- Thereafter, a continued stream of new antibiotics
cessful. with untold human benefit. It would be some time
Fleming’s notebooks show that despite being before the mode of action of antibiotics would even
unable to purify penicillin. he continued working on begin to be understood (cf. Gottlieb and Shaw, 1967)
crude penicillin throughout the 1930s. during which and to allow rational principles to assist in their
time he also attempted to isolate other microor- improvement.
ganisms capable of producing antibacterial prod- Although Waksman received the Nobel Prize for
ucts. Unfortunately, this work was not published streptomycin, his triumph was marred by his tardy
and the medical potential of antibioisis remained un- treatment of the codiscoverer of the antibiotic, Al-
developed until the discovery of gramicidin in 1939. bert Schatz. Schatz, one of Waksman’s graduate
This substance, which was discovered by Rene Du- students. successfully sued Waksman and Rutgers
bos, was unfortunately too toxic for intravenous for a share of the royalties for streptomycin. His
use: therefore. it was limited to use on a number of later attempts to gain a share of the Nobel Prize for
superficial infections. his work (he was senior author on the first strep-
At about the time when gramicidin was being first tomycin papers and coassignee with Waksman of
developed, Florey, Chain, and Heatley managed to the streptomycin patents) were, however. unsuc-
purify penicillin and demonstrate its remarkable an- cessful.
tibacterial effects when used systematically. The Streptomycin was soon followed by antibiotics
isolation of the antibiotic from the crude culture such as chloramphenicol, neomycin, tetracycline,
filtrates was a formidable chemical task, but it was and the first effective antifungal antibiotic, nystatin
undertaken successfully in the late 1930s by Florey (discovered by Elizabeth Hazen and Rachel
and Chain in England. Industrial production of peni- Brown). Penicillinase-resistant penicillins such as
cillin soon followed as a joint U.S.-British war methicillin then appeared, followed by semisyn-
project. For this to be feasible required a substantial thetic penicillins, and finally broad-spectrum com-
effort in strain improvement. which was conducted. pounds like ampicillin.
however. along empirical rather than rational ge-
netic lines (Wilson 1976). This was nevertheless the
forerunner of the modern fermentation industry and
biotechnology: its antecedents had been the produc- VI. Microbial Metabolism and
tion of butanol and acetone as munitions solvents Applied Microbiology
during World War I and the peacetime production of
citric acid by a mold fermentation. Penicillin’s intro- Developments in the study of microbial metabolism
duction into medicine as the first successful antibi- were, from the outset. closely associated with at-
otic stimulated the search for similar compounds. A tempts to use microorganisms for industrial pur-
particularly successful antibiotic screening pro- poses, a trend that continues in modern biotech-
gram, devoted to soil actinomycetes, was carried nology. It is not surprising then to find that the first
out by Selman Waksman and his students at Rutgers. scientific paper devoted to microbial metabolism
The first major product of this research. actinomy- (appearingin 18.57)can also be reiarded as the first
tin. was. like gramicidin. too toxic to be of medical citation in applied microbiology or biotechnology.
use as an antibiotic. although it was later used as an Again Pasteur was responsible for this development,
anticancer agent. the paper being devoted to an explanation of the
S. Waksman and R. J. Dubos had been studying causes of the repeated failures of industrial alcohol
the biochemical and ecological interrelations of soil fermentations. This was an important paper for two
microbes. The role of secreted antibiotics in ecologi- reasons: first, because it laid the foundation of the
cal competition provided a rational for seeking these view, later to be amply validated. that microbial

.“., . . ^__..“.,,. _. _d
History of Microbiology 427
activity was responsible for many industrially im- olism. By 1930, Karstrom had established the con-
portant fermentations, and. second. because it intro- cept of constitutive and adaptive enzymes. By now,
duced quantitative treatment of data on microbial microbiology had begun to be a cornerstone of bio-
growth and metabolism. chemistry and the boundaries between the subjects
Pasteur also addressed problems associated with were soon blurred. In 1941, Lipmann advanced the
the microbiology of wine-making. Among the sug- concept of the high-energy bond, and major devel-
gestions that he made was a method for improving opments in theories on the working of enzymes
the keeping qualities of wine by heating it to 68°C for came from Monod’s lab.
IO min. followed by rapid cooling, a process subse- While most of the early developments in microbial
quently referred to as pasteurization. By 1872. Pas- metabolism were centered on bacteria, fungal me-
teur’s work had been developed to the point where tabolism, because of its importance to many indus-
Ferdinand Cohn could suggest that microorganisms trial fermentation (e.g., citric acid production), was
play a major role in the biological cy,cling of the by no means neglected. The seminal work in this
elements responsible for soil fertility and the proper area came in 1940, when Jackson Foster published
functioning of natural ecosystems. The first fruits of his “Chemical Activities of the Fungi.” Studies on
Cohn’s theory came in 1888 when the Dutch micro- fungal metabolism obviously gained impetus follow-
biologist Beijerinck isolated the symbiotic N-fixing ing the introduction, while the isolation of antibio-
bacterium Rlli:obi/,rn from the root nodules of le- tics such as streptomycin also gave a boost to the
gumes. During these studies. Beijerinck also devel- study of a neglected group of organisms-the acti-
oped the enrichment technique to the isolation of nomycetes. It was Selman Waksman who initiated
microorganisms, an approach later to be refined and work in these organisms during the early part of this
developed by the Dutch school of microbiologists. century. a period when the actinomycetes were re-
Many of the following breakthroughs in microbial garded as fungi rather than bacteria.
metabolism were associated with studies on soil mi-
crobiology and its association with soil fertility. In
1889, Winogradsky described the autotrophic iron VII. Nutrition, Comparative
and sulfur bacteria. and in the following year the
free-living N-fixing Azorobrrcter- and the nitrite- Biochemistry, and Other
oxidizing bacterium Nitrobrlcro-. Although many of Aspects of Metabolism
Winogradsky’s so-called pure cultures appear to
have been contaminated, his work was nevertheless Microbes. first yeast and then bacteria, played an
important because he was the first to appreciate the important part in the discovery of vitamins and other
concept of chemoautotrophy and to relate this growth factors. Growth could be measured in test
growth strategy to the major natural cycles. It was a tubes far more expeditiously and economically than
lack of appreciation of this concept that had hin- in mice. rats. or humans. Conversely, the realization
dered the work of others interested in processes that microbes shared virtually all of the complex
such as nitrification. Despite this. the soil chemist growth factor requirements of animals was an im-
Warrington nevertheless did important work on the portant impetus to “comparative biochemistry,”
factors that influence this process in agricultural the view that they had a common evolution and a
soils. similar underlying architecture. One of the essential
Waksman and Joffe isolated and described T. amino acids, methionine, was first discovered by
tllioosidrltz.5 in 1922. and over the next quarter of a Mueller (1922) as a growth factor required by diph-
century, major contributions to the science of mi- theria bacilli. Mueller joined a school founded by
crobial physiology came from, among others, Wie- Twort (1911), including Lwoff, Fildes, Knight, and
land, who in 1900 demonstrated the importance of Tatum, that made nutrition a branch of general bio-
biological oxidations using microorganisms. Other chemistry. They perceived that the requirement for
work of note came from Marjorie Stephenson and a growth factor belied a loss or deficiency of syn-
J. H. Quastel on enzymes. In 1914, A. J. Kluyver thetic power: lacking internal synthesis. the organ-
published an important article entitled “Unity and ism had to look to the nutrient environment for sup-
Diversity in the Metabolism of Micro-organisms,” a ply of substance. This also implied that organisms
paper that demonstrated the fundamental unity un- with simple nutrition had to be empowered with
derlaying the apparent diversity of microbial metab- complex biosynthetic capability-leaving us humili-

. _.. .- _...
428 History of Microbiology

ated by our species’ inferiority to Escller.ichirr co/i, This in turn led to concepts and experiments on the
but that in turn is less capable than the green plant! genetic underpinnings of metabolism.
Besides the practical utility of these fiildings. they The birth of molecular biology followed the work
led to a well-founded respect for the complexity of of Watson and Crick in 1853. when microbiology
microbial cells. entered into a new phase. allowing it to overlap with
By “1930,” a number of growth factors had been many other sciences, leading to the appearance of
shown to be important in bacterial nutrition. includ- numerous exciting developments.
ing factors V and X, later sholvn to be diphospho- The major conceptual theme of change in microbi-
pyridine-nucleotide and heme. respectively. for he- ology during the vicennium was the convergence of
mophilic bacteria: MycoDacre/.il//,~ pIl/c>i factor. later the discipline with general biology. As noted by Du-
shown to be vitamin K. for M. pserrdotrthe,-c,rrlosis; bos (1945),
and tryptophane for Sal/?~or~ella rxphi (Fildes. 1936).
Starting with the work of W. H. Peterson. H. Wood. To the biologist of the nineteenth century,
E. Snell. and E. L. Tatum at the University of Wis- bacteria appeared as the most primitive ex-
consin and B.C.J.G. Knight and P. Fildes in Eng- pression of cellular organization, the very limit
land. a number of bacteria1 growth factors were of life. Speaking of what he considered “the
identified with B vitamins (extensively cataloged by smallest, and at the same time the simplest and
Johnson and Johnson, 1945). By “1950.” most of lowest of all living forms,” Ferdinand Cohn
the known trace growth factors had been identified asserted: “They form the boundary line of life:
and associated with nutritional requirements of par- beyond them. life does not exist, so far at least
ticular bacteria, as also had been most of the amino as our microscopic expedients reach: and
acids and a host of other metabolites (Snell. these are not small.” The minute dimensions
1951). During the vicennium. most of the vita- of bacteria were considered by many to be
mins were also identified as co-enzymes. playing a incompatible with any significant morphologi-
role in the function of specific metabolic enzymes cal differentiation; it encouraged the physical
[e.g., thiamin for keto-acid decarboxylases, niacin chemist to treat the bacterial cell as a simple
for dehydrogenases, pyridoxal for transaminases. colloidal system and the biochemist to regard it
pantothenate in the citric acid cycle (Schlenk. as a “bag of enzymes.”
1951)]. The 20 canonical amino acids were listed
and could be shown to be incorporated into bacterial Still dominated by the medical importance of mi-
protein. crobes, the views of microbiologists in “1930” had
A host of other biochemical pathways were also not evolved much further, although “System”
detailed with the help of new methodologies of ra- (1930) does have a brief chapter on bacterial cy-
dioisotopic tracers and chromatography. Of special tology and allusion to ongoing controversy over the
significance in bacterial metabolism was the demon- existence of nuclear structures. Far more attention
stration of heterotrophic assimilation of CO,. This is given to the Gram stain!
view of CO- as an anabolite was contrary to its usual While a few differences in the detail of interme-
image as a waste product. The specific requirements diary metabolism and biosynthetic options have
for CO2 as a nutrient helped to clear up difficulties in been discovered (e.g., for lysine), it remains true
the cultivation of fastidious bacteria and eventually that pathways conveniently noted in bacteria have
of tissue cells. usually been reliable predictors of the same steps in
By 1941, microbiology and genetics overlapped higher plants and animals. It is possible today to
when G. W. Beadle. Tatum. and coworkers at Stan- relate this functional conservatism to evolutionary
ford University began to use the red bread mold affinity with currently available tools of DNA se-
Ne~rrosporrr ~YNSSCI,an approach in which mutants quencing.
were employed to help elucidate genetic mecha-
nisms, thereby allowing a number of microbial path-
A. Induced Enzyme Formation, or
ways to be worked out for the first time.
In due course, especially after Beadle and Tatum
“Enzymatic Adaptation”
(1941). the power of synthesis came to be under- One of the most intriguing phenomena of bacterial
stood as the capability of individual specific genes. physiology is the plasticity of enzyme expression
History of Microbiology 429

dependent on the chemical environment. For exam- was the neutralization of an endogenous repressor
ple. E. &i grown on a glucose medium exhibits very that inhibited the expression of the lactase gene in
low levels of p-galactosidase (lactase). When glu- the absence of an inducer (Jacob, 1965).
cose is replaced by lactose. there is a growth delay The simultaneous induction of several steps in a
followed by the abundant production of lactase. metabolic pathway. usually by an early substrate,
Thousands of comparable examples are now known. was exploited to delineate the later steps. notably in
and the pursuit of the mechanism of this phenome- the oxidation of aromatic compounds by pseudo-
non has been of outstanding importance in the devel- monads.
opment of molecular genetics. Anecdotal reports of Among technical innovations, one of the most in-
enzyme adaptation can be traced back to Wortmann genious was the chemostat (Novick and Szilard,
(1882. cited in Karstrom. 1930): they were collected. 1950). This allowed microbial populations to be
together with new experimental observations. by maintained for the first time in a well-defined steady
Henning Karstrom for his doctoral dissertation in state; albeit under limitation for one specific nu-
Virtanen’s laboratory in Helsinki. In this turning- trient.
point review [Karstrom. 1930. followed by the more
accessible Karstrom. 1937; Dubos 1940 (1945)],
bacterial enzymes are classified as constitutive or
adaptive according to their independence. or other-
wise, of the cultural environment. Except for glu- VIII. Microbial Genetics
cose metabolism. most sugar-splitting enzymes are
adaptive-resulting in substantial biosynthetic During the last two decades of the nineteenth cen-
economy for a bacterium or yeast that may only tury, it was realized that bacterial species were not
rarely encounter, say. maltose now, or lactose next as stable as had first been thought. Pure line cultures
week. During the vicennium. the work of Stephen- that had been maintained for many generations sud-
son and Yudkin (1963) and Gale (1943) furnished denly underwent dramatic changes in morphology,
additional clearcut examples of the adaptive re- metabolic properties. and pathogenicity. As more
sponse, and Dubos (1945) offers a critical ap- pure cultures were obtained, this variability. or dis-
praisal of the fundamental biological issues. Several sociation as it was called, became even more appar-
theories allowed for the stabilization of preformed ent. Then in 1925, R. M. Mellon published a paper
enzyme by a substrate, or a Le Chatelier-like prin- describing a primitive from of sexuality in coli-
ciple of mass action. to encourage enzyme synthe- typhoid bacteria. This work had little contemporary
sis. They shared the presumption that the enzyme impact on the contemporary view that bacteria were
molecule itself was the receptor of the inducing sub- anucleate organisms that reproduced without sexu-
strate. Other hypotheses lent the substrate an in- ality by binary fission.
structive role in shaping the specificity of the en- Bacterial genetics was substantially nonexistent
zyme. Further progress would depend on the in 1930. As late as 1942, the eminent British biologist
postulation of an enzyme-forming system distinct Julian Huxley would suggest of bacteria that “the
from the enzyme-and this would emerge under the entire organism appears to function both as soma
impetus of genetic studies to be described later. At and germ plasm and evolution must be a matter of
the very end of the vicennium. Lederberg et r/l. alteration in the reaction system as a whole” (Hux-
( I95 I) described a noninducing substrate of lactqse, ley. 1942. Such ideas gave little encouragement to
the analog altrose-P-D-galactoside. which pointed to efforts to dissect out individual genes along the Men-
a separation of those specificities. This substrate delian lines that had been so successful with DI-o-
also allowed the selection of constitutive-lactase soyhil~ and other animals and plants. Some work
formers. showing that lactose w’as not required for with fungi had gotten off to a promising start early in
the conformation of the enzyme. but that the latter the century (Blakeselee, 1902). Authentic but spo-
could be derived directly from the genetic consti- radic observations of bacterial mutation (Beijerinck,
tution. The debate continued until the mid-1950s 1901) were outnumbered by wooly-minded specula-
(see Lederberg. 1956.p. 51: Monod. 1956):it was tions that embraced variations of colony form as
mooted by the spectacular progress of the Pasteur manifestations of cellular life cycles among the bac-
lnstitute group in showing that enzyme induction teria (see Dubos, 1935; Lederberg, 1992). These
 430 History of Microbiology

clouds of speculation probably discouraged more se- clones, and mutant cells, which are counted when
rious-minded experimentation. you plate a population with the selecting phage.
Mention has already been made of the impact of Luria (1984) in his charming book “A Slot Ma-
the work of Beadle and Tatum on mutants in Nelr~~ chine. A Broken Test Tube,” recounts how his ob-
sporc~ on our understanding of microbial physiology. servation of a jackpot in a gambling den inspired his
However. by initiating the field of biochemical ge- premonition of the skewed statistics that would gov-
netics, these studies had even greater impact on the ern the numbers of mutants. The fit of experimental
science of genetics. Prior to 1941, genetic research numbers to those statistics is subject to great theo-
was dominated by work on the fruit fly. Drosopl~il~~ retical uncertainty, but they were a corroboration of
l,lelanogrrsre,-. Much was learned from studying the clonal model. One of the first articles on bacteria
morphological mutations in this organism, but ef- to be published in Generics, the paper promptly at-
forts to disentangle the biochemical basis of these tracted broad attention and was widely regarded as
characteristics resulted only in frustration. Beadle having proved “that bacteria have genes.” The gist
and his microbial biochemist colleague Tatum of the demonstration was that mutations to phage
turned their attention to studying the red bread mold resistance agree with a clonal distribution and, thus,
N. o-rrssn and soon obtained mutants with nutri- render more likely their “preadaptive” occurrence,
tional defects such as blocks in the biosynthesis of that is, within the growth of the population rather
vitamins like pyridoxine and thiamine. This allows than at the time of the challenge with the selective
for rapid improvements to be made in genetic analy- agent. It therefore harkens more to Darwin than to
sis. an approach that was subsequently extended by Mendel: nevertheless, it was a turning point in ge-
other workers using bacteria. The first fruits of such neticists’ appreciation of bacteria. The statistical
application came in 1943 when Luria and Delbriick methods, which are helpful in the quantitative esti-
showed by means of their “fluctuation test” that mation of mutation rate, have been improved
spontaneous mutations occurred in bacteria, to both (Sarkan, 1991).
phage resistance and streptomycin resistance at sim- The themes of nutrition and mutation among mi-
ilar frequencies, as had been observed in other or- crobes had occasional false starts, with observations
ganisms. of strain variability and the “training” of exacting
The study of bacterial genetics was dramatically bacteria to dispense with growth factors (Knight,
advanced during the 1940s following the recognition 1936). However, lacking a conceptual framework of
of antibiotic resistance in pathogenic bacteria. Here “genes in bacteria,” these had little fruit prior to the
was a practical problem. the solution to which pro- work of Beadle and Tatum (1941) on Neurospora.
vided an obvious impetus to studies aimed at deter- Beadle had begun his research program with Eph-
mining its cause. [See ANTIBIOTIC RESISTANCE.] russi on the genes for eye color in Drosophilrt (Bur-
Bacteria did of course suffer from the serious ian, 1989). Tatum was engaged to do the biochemical
methodological constraint of the apparent lack of work but found the material almost intractable-
any recombinational (sexual or crossing1 mecha- When he approached success, he was scooped by
nism by which to analyze and reconstitute gene Butenandt on the identification of kynurenine as a
combinations. They would prove, however. to be pigment precursor. Nor was it clear how much
marvelous material for mutation studies (cf.. e.g.. closer to the primary gene product this chemistry
Ames. 1975) once the concepts were clarified. for would bring them. The following account is taken
which a major turning point was the work of Luria from J. Lederberg’s memoir on E. L. Tatum, who
and Delbriick (1943). In a fashion that reminds one was his teacher from 1946 to 1947 (Lederberg,
of Gregor Mendel, they studied bacterial mutation 1990).
by quantitative counts. They used resistance to
(bacteriojphage as the marker. Like resistance to This jarring experience, to have such pains-
antibiotics. or growth on a nutritionally deprived taking work overtaken in so facile a fashion,
medium, the phage is an environmental agent that impelled Beadle and Tatum to seek another
makes it easy to count exceptional cells against a organism more tractable than D~osop/?i/~ for
preponderant background that can be selectively biochemical studies of gene action.
wiped out. Most importantly, they distinguished be- In Winter Quarter 1941, Tatum offered a
tween mutational events, which engender resistant new graduate course in comparative biochem-
History of Microbiology 431

istry. In it, he called upon his postdoctorate a mistake to focus too sharply on the numerical 1: 1
experience with Kogl in Utrecht, in 1937, and assertion; more important was the general assump-
recounting the nutrition of yeasts and fungi, tion of simplicity, and that the details of gene ex-
some of which exhibited well-defined blocks in pression could be learned as an outcome of such
vitamin biosynthesis. Beadle, attending some studies-as indeed they were (see also Horowitz,
of these lectures, recalled the elegant work on 1990).
the segregation of morphological mutant fac- The recruitment of Nerrrospora for what have be-
tors in Neur-ospouc that he had heard from come classical genetic studies offered further en-
B. 0. Dodge in 1932. The conjunction was that couragement that bacteria, albeit somewhat more
Nerlrosporcr had an ideal life-cycle for genetic primitive, might be handled in similar fashion. By
analysis with the immediate manifestation of 1944, Gray and Tatum had produced nutritional mu-
segregating genes in the string of ascospores. tants in bacteria. including some in a strain that has
Nerrrospor-n also proved to be readily cultured dominated bacterial genetics ever since, namely E.
on a well defined medium, requiring only biotin co/i strain K-12. These mutants were soon to be put
as a supplement. By February 1941, the team to a most striking use.
was X-raying Nellrospora and seeking mutants In 1944,O. T. Avery and his colleagues concluded
with specific biosynthetic defects, namely nu- that the transforming principle involved in transfor-
tritional requirements for exogenous growth mation in pneumococci was DNA. This was a major
factors. breakthrough, because until then it was thought that
the significant part of the nucleoprotein of the chro-
Harvesting nutritional mutants in microor- mosome molecule was the protein, the nucleic acid
ganisms in those days was painstaking hand merely acting as a sort of binding agent. The role of
labor: it meant examining single-spore cultures DNA was initially puzzling, because it was difficult
isolated from irradiated parents, one by one. to see how a polymer that contained only four bases
for their nutritional properties. No one could could possibly code for the complex phenotype of
have predicted how many thousands of cul- even the simplest of organisms. Meanwhile, classic
tures would have to be tested to discover the genetic approaches were yielding a wealth of new
first mutant: isolate #299 in fact required py- discoveries. In 1945. Tatum showed that the mutant
ridoxine. Furthermore, the trait segregated in rate of bacteria could be increased using X-rays,
crosses according to simple Mendelian prin- whereas 2 yr later, Tatum and Lederberg demon-
ciples, which foretold that it could in due strated genetic recombination between two nutri-
course be mapped onto a specific chromosome tionally defective strains of E. cdi KIZ.
of the fungus. Therewith Nellrospo,~r moved The first gene map of E. coli K12 appeared, and
to center stage as an object of genetic experi- over the next few years progress was made in ex-
mentation. plaining the phenomena of conjugation, transduc-
tion. and transformation. William Hayes, working at
In their first paper, they remarked “that there the postgraduate medical school in Hammersmith
must exist orders of directness of gene control rang- announced in 1952 his discovery that in conjugation
ing from one-to-one relations to relations of great recombination occurred due to the one-way transfer
complexity.” The characteristics of mutations af- ofgenetic material, and during the same year Leder-
fecting metabolic steps spoke to a direct and simple berg and Cavalli coined the terms fertility plus (F’)
role of genes in the control of enzymes. These were for donor cells and fertility minus (F-1 for recipient
therefore hypothesized to be the primary products cells. The recognition of these mating types made it
of genes. Indeed, insome cases, genes might them- clear that conjugation was a primitive form of sexu-
selves be enzymes. This was an assertion of what ality, with the recipient F- cell being the zygote.
came to be labeled the one-gene : one-enzyme the- More advances came when Lederberg, Cavalli, and
ory, which has become the canonical foundation of Lederberg discovered high-frequency recombi-
modern molecular genetics. albeit with substantial nant mutants from the Ff type of E. co/i K12. a
correction and elaboration of detail. especially with finding that was subsequently confirmed by Hayes.
regard to the intermediating role of messenger RNA. These mutant strains (Hfr) differed from the wild-
which could hardly be thought of in 1941. It would be type F’ strains. first in transferring various genetic
 432 History of Microbiology

markers at a rate hundreds of times greater than the cell to another, but this interpretation was inevitably
original strains and. second. in not producing an dimmed by the poor general understanding of bacte-
alteration in the mating type of the recipient cell. rial genetics at that time.
However. although the frequency of transfer of the This vagueness was compounded by two out-
various markers differed. it was the same for any standing misinterpretations: (1) that the transmissi-
given strain of Hfr. ble agent was the polysaccharide itself and (2) that
Between 1955 and 1958. Jacob and Wollman used the agent was a “specific mutagen.” Concerning the
their famous “interrupted mating experiment” to first, it is sometimes overlooked that Griffith under-
determine the mechanism of gene transfer in E. coli stood the distinction well enough. Better than many
K12. Jacob and Wollman coined the term episome, of his followers, he had at least the germ of a genetic
and in 1963 Cairns confirmed the circular nature of theory: “By S substance I mean that specific protein
the bacteria1 chromosome using autoradiography. structure of the virulent pneumococcus which en-
Bacterial genetics further progressed following the ables it to manufacture a specific soluble carbohy-
report published in 1961 by Watanabe explaining drate.” In regard to the second misinterpretation,
infectious drug resistance. Dobzhansky wrote that “. . . we are dealing with
authentic cases of induction of specific mutations by
specific treatments-a feat which geneticists have
A. The Pneumococcus Transformation vainly tried to accomplish in higher organisms.”
What might be regarded as the first major break- This formally correct attribution, from a most in-
through in microbial genetics came in 1928 when fluential source, obfuscates the idea that the agent
Griffith published on detailing “transformation” is the genetic information. Muller had much
in pneumococci, a study that laid the foundation greater clarity: In his 1946 Pilgrim Trust Lecture
for later work by Avery and his colleagues. A fur- to the Royal Society, he remarked,
ther development in our understanding of trans-
formation came in 1933, when Alloway showed . . . in the PII(~IIII~OCO(.CIIS case the ex-
that rough type 1 cells could be changed into ge- tracted “transforming agent” may really have
netically stable smooth type II cells, by growing had its genetic proteins still tightly bound to
them in the presence of a cell-free extract of a the polymerized nucleic acid; that is. there
heat-killed broth culture of smooth type II cells. were. in effect. still viable bacterial “chromo-
This work demonstrated the existence of a soluble somes” or parts of chromosomes floating free
“transforming agent.” [See GENETIC TRANSFOR- in the medium used. These might. in my opin-
MATION, MECHANISMS.] ion, have penetrated the capsuleless bacteria
Apart from cataclysmic happenings in global war. and in part at least taken root there. perhaps
1944 will also be remembered for the publication of after having undergone a kind of crossing over
“Studies on the Chemical Nature of the Substance with the chromosomes of the host. In view of
Inducing Transformation of Pneumococcal Types.” the transfer of only a part of the genetic mate-
by Avery. MacLeod. and McCarty.’ The pneumo- rial at a time, at least in the viruses. a method
coccus transformation was stumbled upon by Fred appears to be provided whereby the gene con-
Griffith in London. in 1918. in the course of his stitution of these forms can be analyzed. much
studies on the serosystematics of pneumonia. Ex- as in the cross-breeding test on higher organ-
tracts of one serotype evidently could transform isms. However, unlike what has so far been
cells of another into the type of the frst. In retro- possible in higher organisms, viable chromo-
spect, it is hard to imagine any interpretation other some threads could also be obtained from
than the transmission of a gene from one bacterial these lower forms for in vitro observation.
chemical analysis. and determination of the
genetic effects of treatment.

’ It is a\vkuard ta have such ;i nondescript tern1 BSX*transfer.

mation” .~pplied to such an impel-tant. \pecilic phenomenon. Hut
Other “classical” geneticists had virtually noth-
uhen it \\as first discovered and named. there \\a\, no \rarrant to ing to say about Griffith’s work and would have
five it an!: narrouer connotation. .4\,er-y had the po~,er of nc\r judged themselves incompetent to assess its experi-
coinage but \has hardly the likely perwnality. mental validity. They began to pay closer attention
History of Microbiology 433

after 1944. but again had little training in bacterial date settled so important a question as the chemical
chemistry to enable them to form critical judgments identity of the gene as pure DNA (versus a complex
about the claims presented them. nucleoprotein). Avery himself had cause to worry-
In Avery’s world, however, Griffith w’as a central There had been much resistance to his earlier proofs
figure and his observations could not be ignored. His that pneumococcal polysaccharides, free of protein,
basic observations were confirmed in Avery’s labo- were immunogenic. Wendell Stanley’s first claims
ratory (see Dubos, 1986), and in due course Avery that crystalline tobacco mosaic virus was pure
felt compelled to pursue the chemical extraction and protein had to be subject to humiliating correction
identification of the substance responsible for the when ribonucleic acid was also found therein. We
transformation. Sixteen years after Griffith, this was should recall that when most biologists of that era
achieved, and DNA was thrust into the scientific used terms such as protein, nucleic acid, or nucleo-
consciousness as the substance of the gene. protein, it can hardly be assumed that they had to-
In retrospect, it is difficult to give proper credit to day’s crisp connotations of defined chemical struc-
the logical validity of a large range of alternative ture. These issues could only be settled by the few
interpretations and to reconstruct the confusions experts who had worked with these materials exper-
about what was meant by “gene” and “genetic.” imentally-and it was a daunting task to prove that
Recall that until 1951 the only marker observed in there were too few molecules of any contaminating
transformation was the capsular polysaccharide, the protein in the “DNA” to account for its genetic
biosynthesis of which was itself subject to many specificity. Maclyn McCarty’s meticulous work
conjectures [e.g., about the role of starter fragments continued to provide ever more persuasive evidence
in self-assembly (discussed by Lederberg. 1956)]. that it was DNA, and the contemporaneous studies
Avery undoubtedly somewhat intimidated by of Chat-gaff showed that DNA was far more complex
Dobzhansky’s authority. was reluctant to put his than Levene had figured it to be and, therefore,
speculations about the genetic significance of trans- capable of the subtlety demanded of a “gene.” Rig-
formation in print; his famous letter to his brother orous proof about “DNA alone” was really not fur-
Roy surfaced only years later. There. but not in the nished prior to the production of genetically active
paper, he remarks that the . . . [transforming sub- synthetic DNA three decades later. By 1952,
stance is] thereafter reduplicated in the daughter Hershey and Chase gave evidence from an indepen-
cells and after innumerable transfers [it] can be re- dent quarter that DNA alone penetrated the phage-
covered far in excess of the amount originally infected cell. In the following year. the structural
used. . . . Sounds like a virus-may be a gene. But models of DNA as a double helix (Watson and
with mechanisms 1 am not now concerned-One Crick. 1953) lent final plausibility to “DNA alone.”
step at a time-and the first is. what is the chemical This episode is sometimes painted as unreason-
nature of the transforming principle? Someone else able resistance to a new idea (Stent, 1972). This is
can work out the rest (quoted in Dubos, 1976). AS hardly a fair assessment of a controversy that was
late as 194S, so distinguished a geneticist as G. W. settled within 9 years and that required the emer-
Beadle still referred to the phenomenon as a gence of a new class of workers, and conversion of
“first success in transmuting genes in predetermined some of the old ones. to deal with new techniques
ways” (note transmuting. not transmitting!). This and experimental materials. That controversy con-
obscuration of the pneumococcus transformation tinued is appropriate to the spirit of scientific skep-
became less troublesome with the overall develop- ticism-more to worry about when challenging new
ment of bacterial genetics. ideas are merely ignored.
Indeed. the controversy raged on the chemical All these discoveries. taken together, gave sub-
claim that the substance was DNA (and nothing stance to Luria’s vision of the virus as a genetic
else!). [This story is detailed by Judson (1979) and in element that is coordinated with the genome of the
McCarty’s personal memoir (1987).] Alfred hlirsky. host. but with pathogenetic consequence that has
Avery’s colleague at the Rockefeller Institute. was a evolved to suit the needs of the parasite. The host
vocal critic of the chemical identification of the may also co-evolve to reach an equilibrium compati-
transforming agent. Some believe he was quite per- ble with the survival of both partners-a general
suaded that this was an instance of gene transfer. but principle in the evolution of pathogenicity (Th.
the more reluctant to concede that the evidence to Smith. 1934).
434 History of Microbiology

Prospects of cytoplasmic heredity fascinated Hershey (1946): Different phage genomes can
many workers, even during the working out of the undergo genetic recombination, enabling the
nuclear (Mendelian) basis of microbial biology, per- construction of linkage maps. These would
haps as a carryover of Huxley’s idea of the persis- eventually be constructed in ultimate detail,
tent soma. In the course of the discussion. there matching the DNA sequence of the nucleotides.
were angry ripostes as to whether a given entity was
really a plasmagene. or perhaps a virus, or perhaps a Viruses were defined by Luria (1953, p.) as “sub-
symbiont. The term and concept “plasmid” was microscopic entities. capable of being introduced
introduced (in 1952) to stress the operational vacuity into specific living cells and of reproducing inside
of those distinctions. A particle could be at the same such cells only.” He pointed out that this is a meth-
time a virus (if one focuses on pathology), or sym- odological rather than taxonomic criterion; such a
boint. or plasmagene (if one focuses on the genetic definition might well embrace a wide range of di-
role). As a prophage. it may even be integrated into verse entities. By 1950, he insisted that the phages
the chromosome. w,ith a potential reappearance exhibited “parasitism at the genetic level,” taking
later. And it would be impossible to say whether a over the metabolic direction of the host cell and
virus had evolved its pathogenicity, having once exploiting a wide repertoire of its genetic capabili-
been a benign organelle. or vice versa, or both at ties. Whether or not other viruses, in plant and
different evolutionary epochs. One might even re- animal cells, would share these attributes remained
vive Altmann’s old picture of the mitochondria as to be seen (Luria, 1953; Adams, 1959; Hayes, 1964;
originally symbiotic bacteria. an allusion founded Galpern, 1988; Burner, 1945).
merely on the limitations of cytological analysis.
The vicennium worked a transformation-the
B. Lysogeny
“biologization” of the microbe. It was an extraordi-
narily exciting and fertile time. with new phenomena Not long after the Twort-d’Herelle discovery of the
to be found in every culture dish. One could even bacteriophages (1915-1917), bacterial cultures were
learn to treasure one’s contaminations. found that appeared to have established a durable
symbiosis with a resident phage. The Delbruck
school tended to dismiss these as contaminants, de-
spite persuasive arguments of Burnet and Lush
IX. Viruses and Lysogeny: The (1936). Lwoff and Gutmann (1950) reentered the
controversy and showed that lysogenic Bclcilli car-
Plasmid Concept ried a “prophage,” a genetic capability of producing
the phage. At the same time, Lederberg and Leder-
A. Biology of the Virus
berg (1951, 1953) had discovered that E. co/i K-12
The cardinal discovery for virology was the isola- was lysogenic, for a phage they named “lambda,” as
tion and crystallization of the tobacco mosaic virus a parallel (or so they thought) for the kappa particles
(Stanley, 1935). which sharpened many questions in Pcrru~zeci~~rr. Crosses of lysogenic with sensitive
about this boundary of living existence (Pirie, strains, however, showed that the capacity to pro-
1937). A more convenient system for virus biology duce lambda segregated in close linkage with a chro-
proved, however, to be the viruses attacking bac- mosomal marker (gal): therefore, they invoked
terial hosts, the (bacterio)phages, especially in the Lwoffs concept and terminology of prophage.
hands of the Delbrtick school (Adams, 19.59). However, the working out of that story, and of the
Their life cycle was worked out in some detail, phenomena of phage-mediate transduction, belongs
eventually culminating in two cardinal experi- to the next era.
ments:

Hershey and Chase (1951): The DNA of the

attacking phage particle is sufficient to initiate X. Virology
infection. The DNA (not the entire phage)
replicates in the host bacterium and then The term virus was originally an unspecific term
generates the capsid and assembles itself into coined by Pasteur to mean any living organism that
mature, infectious phage particles. caused disease. This terminology was used well into

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^ .__... - I...

,.._^.
History of Microbiology 435

the 1930s; thus, the word antivirus was used by sis. The application of Enders’ tissue culture tech-
Besredka to refer to bacterial filtrates that could niques led to the isolation of many other viruses: in
apparently cure infections. 1954, the year when he received the Nobel Prize,
The realization that disease could be transmitted Enders himself, for example, succeeded in isolating
by inoculation of cell-free lesions from plant and the measles virus.
animal infections led to the introduction of the con- The introduction of the electron microscope in
cept of “filterable virus.” Iwanowski’s discovery in 1934 proved a great asset to research on viruses. In
1892 of tobacco mosaic disease in plants is usually 1956, Watson and Crick proposed on theoretical
credited as the first demonstration that a filterable grounds that virus particles must be made up of a
virus could cause disease. Then in 1898. Loeffler nucleic acid core and a surrounding shell comprised
and Frosch showed that a filterable virus was appar- of protein subunits, a structure later seen in 1959
ently the cause of foot and mouth disease. In the under the electron microscope by Horne and Nag-
same year, S. M. Chapman introduced the use of ington.
fertile hens eggs as a means of cultivating viruses. Antibiotics aided virus research, allowing for con-
This approach was later to be used by Pyton Rous tamination-free studies, so that by 1949, poliomyeli-
in his work on the fowl sarcoma that bears his name. tis virus could be grown on nonneural tissues such as
By 1915, a new class of virus affecting bacteria but minced monkey kidney.
neither plants nor animals. was discovered by F. W. In 1952, the name of the patient Helen Lane be-
Twort. His observations were extended in 1917 by came cryptically immortalized when Gay and his
D. Herrelle, who over the next 13 years published a colleagues established the famous continuous cell
series of papers on what was initially called the line of HeLa cells, derived from a carcinoma of the
Twort-Herelle phenomenon, but which later be- patient’s cervix uterus. Then. in the following year,
came known as bacteriophage. Scherer succeeded in growing poliomyelitis virus in
The development first of the ultraviolet micro- these cells.
scope and then of tissue culture techniques in the In 1954, Younger published his technique for
1920s added impetus to research on virus structure growing trypsinized cells in monolayers on glass.
and cultivation. Maitland’s work in 1928 was a major This allowed viral infection of cells to be recognized
advance in tissue culture techniques, but because of by detecting the cytopathic effect, which allowed for
the tedious nature and lack of antibiotics to control the routine screening for the presence of viruses.
bacterial contaminants they were not widely
adopted.
By 193 1, the potential of the fertile egg for cultur-
ing viruses was finally appreciated in the work first XI. Mycology and Protozoology,
of Goodpasture and then of the Australian Macfar- Microbiology’s Cinderellas
lane Burnet. Burnet used this approach to culture
the influenza virus. which previously had to be Filamentous fungi and protozoa (i.e.. molds and
grown in ferrets. animacules) were observed soon after the earliest
John Enders did much to develop the art of cultur- microscopes were developed. Studies of these or-
ing viruses. which finally enabled the development ganisms continued largely unnoticed as bacteriology
of a range of vaccines. Enders’ outstanding contri- developed. The fact that neither of these groups of
bution to the study of viruses began with his work on microorganisms cause major diseases in the devel-
mumps when he showed that a virus could be grown oped world tended to hinder the rapid development
in chick embryos and. after successive generations. of both mycology and protozoology. The principle
would loose its ability to cause the disease while motivation for studying fungi came from their ability
retaining the capacity to immunize against it. In this to infect important crop plants. This resulted in a
way, the modified virus could be used to prepare close association between mycology and botany,
vaccines to control the disease. Prior to 1949. for with the unfortunate result that many microbio-
example. the poliomyelitis virus could only be prop- logists in the past, as today, regard fungi as lying
agated in monkeys. Enders showed that the virus outside the orbit of their subject.
could be grown in culture of nonnervous tissues, and As early as 1767. Torgioni-Tozetti advanced the
by using this technique Salk developed his famous view that rust diseases of cereals are caused by mi-
vaccine. which essentially defeated infantile paraly- croscopic fungi, but experimental proof of the role
436 History of Microbiology

of fungi as phytopathogens had to await the mono- man Goldfuss in 1817. By 1836, Alfred Donne work-
graph by Prevost, who in 1807 described experimen- ing in Paris had shown that a flagellate was respon-
tal smut infections. Prevost also showed that fungal sible for vaginal discharge in women. It was,
infections could be prevented by soaking seeds in a however, the colonial expansion of the European
solution of copper sulfate and thus. he inadvertently powers that provided the stimulus to studies in med-
became the originator of the pesticide industry. It ical protozoology. The first observations of para-
was Anton de Bary. however. who did the most to sites in the blood of malaria sufferers was made in
develop the science of plant pathology. 1880 by Alphonse Laveran. A long list of diseases
During the early part of this century, attention were then shown to be caused by protozoa including
was also focused on the role that fungi play in soil Texas cattle fever in 1893. Malta fever in 189.5, ma-
fertility. It soon became evident that while fungi are laria in 1898, sleeping sickness in 1902.
not as metabolically diverse as soil bacteria. they Protozoa have yet to be widely used in industrial
nevertheless play an important role, principally as microbiology and biotechnology. and their role in
agents of decay of organic forms of carbon and nitro- the environment has been subject to only limited
gen, in the degradation of leaf litter and humus. study; therefore, the history of the development of
Waksman and his colleagues were particularly ac- protozoology in these areas will have to await future
tive in demonstrating the role played by fungi in developments.
soils.
Waksman was also one of the first microbiologists
to appreciate the industrial importance of molds, he
and his group investigating the production of butyric XII. The Modern Period
acid and butyl alcohol from starch-rich materials,
and then, in 1930. examined lactic acid production What then of the landmarks of the recent history of
by species of Rhixprrs. The foundation for the de- science? Without a doubt. the most obvious devel-
velopment of studies on mold metabolism was laid opment in our science that has taken place since the
by Raistrick and his numerous collaborators work- last war has been the rise in the status of a single
ing at the London School of Hygiene and Tropical organism, the colon bacterium E. coli. Using this
Medicine during the 1920s. single organism, scientists such as Niremberg.
Ringworm was the first human disease to be Holley, Jacob, and Monod have revolutionized our
shown to be caused by fungi: described in 1839 by thinking on biology. One practical outcome of this
Schoenlein. it was soon followed by the recognition work was the development of an E. co/i strain by W.
by the Swede F. T. Berg that Ctrlldidrrrrlbictrus was Gilbert and others in 1978 that produces human in-
the causal agent of thrush. Medical mycology was sulin.
slow to develop, however, and it was not until 1910 A perusal of the list of awards for the Nobel Prize
tha Sabouraud introduced a medium suitable for the for research in microbiology in the widest sense
isolation and growth of pathogenic fungi. Systemic shows that since 1958 particular recognition has
mycoses were discovered at the turn of this century, been given to work on genetics, virology, and immu-
while it was as late as 1934 before Monbreun conclu- nology. Knowledge derived from such studies have
sively demonstrated that histoplasmosis is caused had a profound effect on our understanding of the
by Histoplrrsnrcr crrpsrrl~~~rrn~. Medical mycology has life process, and recent developments in biotech-
tended to lag behind other aspects of medical micro- nology have provided real benefits in our lives. [See
biology. although the importance of fungal infec- BIOTECHNOLOGY INDUSTRY: A PERSPECTIVE.]
tions such as pneumocystis pneumonia and candidi- The key technique that has made genetic engi-
asis in the AIDS syndrome is likely to accelerate neering possible was devised by Herbert Boyer and
developments in this area of the subject. Stanley Cohen. Boyer working at the University of
The development of protozoology as a science is California collaborated with Cohen of Stanford Uni-
almost exclusively devoted to the role of protozoa as versity to develop a method of splicing genes from a
agents of disease. Although initially referred to as donor into a recipient bacterium. In 1973. they took
animacules, by 1764 Wrisberg had introduced the a gene from the plasmid of one organism and spliced
term infusoria, while the first generic name for a it into a plasmid from another to produce recombi-
protozoan, Prrromecium, was introduced by Hill in nant DNA. When inserted into a recipient bacte-
1752. The term protozoa was first used by the Ger- rium, the foreign genes not only survived but also
History of Microbiology 437
affected the host in the way it had affected the do- Collard. P. (1976). “The Development of Microbiology.”
nor, and was also copied as the cell divides. Boyer Cambridge University Press, Cambridge.
Dubos, R. J. (1945). “The Bacterial Cell in Its Relation to
later used this approach to insert genes from human
Problems of Virulence. Immunity and Chemotherapy.”
proteins into bacteria, and, thus, heralded a bio- Harvard University Press. Cambridge, Massachusetts.
technological revolution. A similar revolution was Dubos, R. J. (1976). “The Professor, The Institute and
initiated by the production of monoclonal antibodies DNA.” Rockefeller University Press, New York.
by Kohler and Milstein (Interestingly, what might be Gunsalus, I. C., and Stanier, R. Y. (eds). (1962). “The Bac-
teria: A Treatise on Structure and Function. Biosynthesis.”
termed “natural monoclonal antibodies” had been
Academic Press, New York.
observed a few years earlier by Joseph Sinkovicks.) Jordan, E. O., and Falk, 1. S. (eds). (1928). “The Newer
A microbiologist who left science even as late as Knowledge of Bacteriology and Immunology.” University
the mid-1970s to follow other pursuits would now of Chicago Press. Chicago, Illinois.
hardly recognize his or her former subject. Studies Judson, H. F. (1979). “The Eighth Day of Creation-makers of
the revolution in biology.” Simon and Schuster, New York.
on the genetics and molecular biology of microor-
Lechevali, H. A., and Solotorovsky, M. (1965). “Three Cen-
ganisms have made particularly rapid progress in the turies of Microbiology.” McGraw-Hill. New York.
intervening years. We have also seen major im- Lederberg, J. (1987). “Genetic Recombination in Bacteria: A
provements in the way we apply microorganisms in Discovery Account.” Ann. Rev. Genet. 21, 23-46.
biotechnology and. more recently. to address envi- Lederberg, J. (1990). “Edward Lawrie Tatum. Biographical
Memoirs.” Nat. Acad. Sci. 59,357-386.
ronmental problems. The appearance of AIDS has
Lederberg. J. (1991). “The Gene (H. J. Muller 1917).” Ge-
once and for all shattered our cozy belief that we had netics l29,313-316.
all but conquered infectious disease. HIV will un- Lederberg. J. (1992). “Bacterial Variation since Pasteur;
doubtedly not be the last new infectious agent to Rummaging in the Attic: Antiquarian Ideas of Transmis-
confront us in the future: if for no other reason than sible Heredity, 1880-1940.” A.544 News 58(5), 261-265.
Medical Research Council (Great Britain). (1930). ‘*A System
to combat such infections. our science will need to
of Bacteriology in Relation to Medicine.” His hlajesty’s
continue to develop at the rapid rate seen in the past Stationery Office. London.
few decades. Summers, W. C. (1991). “From Culture as Organism to Or-
ganism as Cell: Historical Origins of Bacterial Genetics.”
J. Hisf. Biol. 24, 171-190.
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