History of Microbiology

Milton Wainwright
University of Sheffield
Joshua Lederbergl
The Rockefeller University

I. Observations without Application
II. The Spontaneous Generation Controversy
III. Tools of the Trade
IV. Microorganisms as Causal Agents of
  Disease

V. Chemotherapy and Antibiosis
VI. Microbial Metabolism and Applied
  Microbiology

VII. Nutrition, Comparative Biochemistry, and
  Other Aspects of Metabolism
VIII. Microbial Genetics
IX. Viruses and Lysogeny: The Plasmid
   Concept
X. Virology
XI. Mycology and Protozoology,
  Microbiology's Cinderellas
XII. Modern Period

ness of the nature and etiology of disease, with the
result that the majority of the traditional killer dis-
eases have now been conquered. Similar strides
have been made in the use of microorganisms in
industry. and more recently attempts are being made
to apply our knowledge of microbial ecology and
physiology to help solve environmental problems. A
dramatic development and broadening of the subject
of microbiology has taken place since World War II.
Microbial genetics, molecular biology, and bio-
technology in particular have blossomed. It is to be
hoped that these developments are sufficiently op-
portune to enable us to conquer the latest specter of
disease facing us, namely AIDS. Any account of the
history of a discipline is. by its very nature, a per-
sonal view: hopefully, what follows includes all the
major highlights in the development of our science.

Glossary

Antibiotics Antimicrobial agents produced b)
living organisms

Bacterial genetics Study of genetic elements and
hereditary in bacteria

Chemotherapy Systemic use of chemical agents
to treat microbial infections
Molecular biology Science concerned with DNA
and protein synthesis of living organisms
Monoclonal antibodies Specific antibodies pro-
duced by in vitro clones of B cells hybridized
with cancerous cells

ALTHOUGH MICROORGANISMS were first ob-
served using primitive microscopes as early as the
late 16OOs, the science of microbiology is barely 150
years old. In this time, major developments have
been made in our understanding of microbial physi-
olog),. ecology, and systematics. This knowledge
has been successfuly applied to broaden our aware-

The period approximating 1930-1950 was a `vi-
cennium" of extraordinary transformation of micro-
biology, just prior to the landmark publication on
the structure of DNA by Watson and Crick in 1953.
We have important milestones for the vicennium:
Jordan and Falk (1928) and "System of Bacteriol-
ogy" (1930) at its start are magisterial reviews of
prior knowledge and thought. Dubos (1945) and
Burner (1945) anticipate the modern era. and Werk-
man and Wilson (1951) and Gunsalus and Stanier
(1962) document its early and continued progress in
monographic detail. The AnnunI Review of Micru-
biology, starting in 1947 (and several other Annual
Reviews), and Bacteriological Reviews. starting in
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
important for the argument.

This account will center on the fundamental biol-
ogy of microbes and give scant attention to continu-
ing advances in the isolation of etiological agents of
disease and of vaccines and immunodiagnostic pro-
cedures. Most of the agents of common bacterial
infections had been characterized by " 1930," but

IFor the period from 1930.
Encyclopedia of Microbiology, Volume 2

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

419


420                                   History of Microbiology

the vicennium was distinguished b! important work
on the classification of enteric (diarrhea]) bacteria
and, above all. by the isolation and nev,' stud! of
viruses and rickettsia with methods such as culti-
vation virus in the chick embr),o (Kilbourne. 1987).

I. Observations without
Application

Macroscopic manifestations of microbial gro\\ th
such as bacterial and algal 4limes ha\.e been recag-
nized since antiquity. However. it \\as the Dutch
microscopist van Leeuljenhoek (Fig. I ) \+ ho pro-
vided the first observations of bacteria at the micro-
scopic level. van Leeuisenhoek. a draper in Delft.
Holland. ground his own lense\ lo make micro-
scopes \+zith short-focal length lenses gi\,ing magni-
fications of betw.een x.30 and x266. Dr\carte\ had
earlier described a similar crude form of micro-
scope. but the quality of his lenbe\ did not allo\\
for magnifications sufficient to bee bacteria. \`an
Leeuwenhoek. in contrast. used his homemade mi-

croscopes (Fig. 2) to examine microorganisms in
rainwater. well water. and seawater as well as water
infused u ith peppercorns. His observations 1% ere
forwarded to the Royal Society in London on Octo-
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-
cluding novel obser\,ations on bacteria present in the
scurf of teeth. Published in 1683. these observations
include the first dra\+ ings of bacteria ever to appear.
These drawings are >til extant and cleal-ly show that
van Leeuwenhoek observed bacilli. streptococci.
and man\; other characteristic forms of bacteria.
van Leeuu,enhoek's meticulous drawings also she\\,
protozoa such as Vo~ric,c~llr/, VO/UO.\-, and C`//gl~)/~tr.
At about the hame time, Huygens also reported ob-
servations on a number of free-living protozoa. in-
cluding species of P~i/,rr/llc,c,i,l/rl. Van Leeuwcnhoek
also gains credit for describing the first parasitic
protozoan. when in 1681 he observed his ou'n fecal
stools during a bout of diarrhea and described large
populations of what later became known ;is Gir,rtlitr
I~it~lblitr.


History of Microbiology

421

Although van Leeuwenhoek also observed yeast
cells in beer, the first illustrations of filamentous
microscopic fungi were provided by Robert Hooke,
again in a letter to the Royal Society. this time in
1667. However. the most important early work on
molds appeared in the following century when the
Tuscan botanist. Pietro Antonio hlicheli described
some 900 species. including important genera such
as Asper-gillrrs and Marco,-. It is also worth noting
that molds have been used from ancient times to
treat infections, an approach termed mold therapy,
which was based on folk medicine rather than on any
scientific rationale.

Because the connection between microorganisms
and fermentation or disease was never made during
this period. observations made by the first microsco-
pists had surprisingly little impact on human affairs.
Despite this. Cicero and the Renaissance scholar
Fracastorius had previously suggested that fevers
might be caused by minute animals. collectively de-
scribed as conragium vivum, but it was to be many
centuries before the role of microorganisms in dis-
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

moved by Pouchet's work to offer a prize to anyone
who could settle the controversy once and for all.
Despite discouragement from his friends who cau-
tioned against becoming embroiled in the con-
troversy, Louis Pasteur (Fig. 3) realized that if mi-
crobiology was to advance as a rational science the
idea that microorganisms arose spontaneously
would need to be experimentally defeated.

Pasteur's studies were published in memoir in
1861 and effortlessly took the prize offered by the
Academy. He first of all showed that when air is
filtered through cotton wool, large numbers of
microorganisms are held back. Pasteur then suc-
cessfully repeated Schwann's work, but his most
famous and successful experiments involved the
use of swan-necked flasks, with which he showed
that heat-sterilized infusions could be kept sterile
in an open flask as long as the open part was tor-
tuous enough to allow any microorganism to set-
tle on the sides of the tubes before reaching the
liquid.

It is often assumed that Pasteur's experiments

Figure 3 Louis Pasteur (1822-1895).


422                                       History of Microbiology

immediately brought about the defeat of the theory
of spontaneous generation, but this is far from true.
Pouchet. for one, remained convinced that Pasteur's
experiments did not defeat the concept. The con-
troversy continued over the next quarter of a decade
or so. Proponents of Pasteur's views included Brit-
ish scientists such as Huxley, William Roberts. John
Tyndall, and the American Jeffries Wyman. The
main counter-arguments were provided by the last
and most dedicated of the important hetero-
genesists, H. Charlton Bastian. How this rearguard
action by Bastian and others nearly carried the day
is remarkable. Hovvever, experiments by the mathe-
matician and physicist John Tyndall on the existence
of heat-stable forms of certain bacteria (the removal
of which involved the process of repeated heating
and rest, referred to as tyndalization) finally con-
vinced the scientific establishment of the error of
Bastian's arguments. Bastian summed up his views
on spontaneous generation in his book Tl~e EL'o~u-
riorr ofLifr (published as late as 1905). and then died
in 1915. still a confirmed believer.

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-

alization of microorganisms to occur. The staining of
histolo$cal specimens was first carried out by the
German botanist Ferdinand Cohn in 1849, his work
being based on vegetable dyes such as carmine and
hematoxylin. By 1877. Robert Koch (Fig. 4) was
using methylene blue to stain bacteria. a process in
which he developed the standard techniques of pre-
paring dried films. and with the aid of coverslips was
preparing permanent preparations. By 1882. Koch
had succeeded in staining the tubercle bacillus with
methy+ene blue, employing heat to encourage the
stain to penetrate the waxy envelope. Two years
later. the Danish pathologist Hans Christian Gram
introduced his famous stain. which allowed bacteria
to be characterized as gram-positive if they retained
the violet dye or gram-negative if they did not. This
distinction was later to be correlated with differ-
ences in biochemical and morphological characteris-
tics. allowing bacteria to be classified into the two
broad groupings still in use today.

Differential staining techniques soon follou,ed. al-
lowing Frederick Loeffler in 1890 to demonstrate the
presence of bacterial flagella. During this period,
rapid developments occurred in methods for identi-

Figure 4 Robert Koch (l&l&1YlOJ.

^ .._                                  ._,. ._    .._-".".l"-." -.    -,_.-. ..)


History of Microbiology

fying bacteria and demonstrating their involvement
as causal agents of specific diseases.

The light microscope was eventually developed
to its theoretical limits and further progress in mi-
croscopy had to await the appearance of the ultra-
violet microscope in 1919 (which for the first time
allowed certain elementary viruses to be seen).
Then, in 1934, the Belgian physicist Marton built the
first electron microscope, which achieved magnifi-
cations of 2-300,000x, compared to 1200x and
2500x achieved by the light and ultraviolet micro-
scopes, respectively. A further major development
in microscope technology came in 1965 with the
introduction of the scanning electron microscope.

The first semisynthetic medium designed for culti-
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
persisted well into this century in the laboratories
devoted to medical bacteriology. Mycologists, too,
tended to rely on undefined media such as potato
dextrose agar, although the introduction of Czapek
Dox medium eventually provided an ideal defined
substrate on which molds could be grown.

In 1872. Ferdinand Cohn developed the idea of the
basal medium, to which various additions could be
made as required. These early media were always
liquid-based and it was not until the introduction
first of gelatine and then agar in 1882 that the use of
solid media became commonplace. The latter intro-
duction of silica gel media then allowed for rapid
advances to be made in the study of chemolithotro-
phic bacteria such as Thiobacillrrs thioosidcttls.

By 1887, a simple and prosaic development revo-
lutionized microbiology when Petri. one of Koch's
assistants, introduced the Petri dish. This simple
invention provided a far more versatile means of
culturing microorganisms than did use of the bulky
bell jars employed previously.

From 1898 onward, the Dutch school of microbio-
logists led by Beijerinck developed the art of enrich-
ment culture. which led to the isolation of both nitri-
fying and cellulolytic bacteria. Studies on gas
gangrene during the first war encouraged McIntosh
and Fildes to develop the anaerobic jar. A vast array
of selective media were then developed that in-
volved amendments such as tetrathionate broth,
tellurite. and crude penicillin. Finally. the introduc-
tion of central media supplies after the war liberated
the microbiologist and their technicians from the
tedium of preparing media in-house. No longer did

mycologists, for example, have to spend hours peel-
ing and boiling potatoes when potato dextrose agar
was available ready to rehydrate, sterilize, and use.

None of the preceding developments in media
preparation would have been useful without the in-
troduction of an efficient means of sterilization. Pas-
teur's colleague, Chamberland, developed auto-
claves-essentially large pressure cookers-in
1884. More recently, gamma rays and ethylene ox-
ide sterilization have allowed for the introduction
of factory-sterilized plastics including Petri dishes,
another relatively simple development that has,
nevertheless, had a marked stimulatory effect on
the recent progress of microbiology. [See SYERIL-
IZATION.]

IV. Microorganisms as Causal
  Agents of Disease

In 1788, an epidemic of smallpox broke out in the
English county of Gloucestershire. Edward Jenner,
a country doctor and pupil of the famous anatomist
John Hunter, decided to try and prevent his patients
from contracting the disease by employing the stan-
dard method of inoculation using a mild dose of the
infection. Jenner, who had suffered under the blood
purgers and inoculists in his youth, was himself im-
mune to smallpox. He aimed to make the traditional
inoculation method as rational and reliable as he
could. While on his regular rounds, he was surprised
to find that patients who had already suffered from
cowpox did not react in the normal way to inocula-
tion with smallpox. Although Jenner was aware of
the old wives' tale suggesting that cowpox gave
protection against the disease, it was not until 1796
nearly a quarter of a century after he had first heard
these suggestions. that he decided to act. His first
experimental inoculation involved a local boy
named James Phipps, who, after receiving cowpox,
became immune to smallpox. In June 1798, Jenner
presented a paper on his work to the Royal Society,
and the effect was remarkable- within a few years,
vaccination was commonplace.

Despite Jenner's breakthrough, there was still no
convincing explanation to account for the appear-
ance and spread of infections, and by the mid-1800s
there was still little that could be done to counter
infectious disease. Childbed or puerperal fever was
a particularly terrible blight that affected every one
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
died of the infection. Vienna of the 1830s had a
particularly bad reputation for this disease. despite
having one of the most enlightened hospitals in Eu-
rope. It was here that the Hungarian doctor, Ignaz
Semmelweiss joined the staff of the lying-in clinic of
the Vienna General Hospital in 1844. In his first few
months of practice, he heard yet another wives' tale.
this one associating the high death rate from
childbed fever found in the teaching division of the
hospital with the high frequency of examination by
doctors and their students. Semmelweiss began to
collect statistics and soon became aware that the
highest rates of infection and mortality occurred in
the teaching clinic. This information led him to sur-
mise that the contagion was being transmitted by the
doctors.and medical students. many of whom exam-
ined the wombs of patients without washing their
hands. even after coming directly from mortuary
duty. Semmelvveiss suggested that anyone examin-
ing patients should first wash their hands in chlorine
water. The results of this simple remedy w'ere phe-
nomenally successful. with mortality rates being re-
duced from around 11 to 3% within I yr. Semmel-
Weiss was slow to write an account of his work, but
eventually in 1857 he provided a rambling and highly
egotistical survey of his vvork. which completely
failed to make any impression.

Eventually. however. the view that infection was
spread by some organic particle did at last become
widely accepted. although the exact nature of such
particles was unknown. The effect of this ignorance
was devastating: during the Crimean War of 1853-
1826. for example, a single regiment of the British
Armv lost 2162 men. with 1713 dying not from
wounds or the effects of trauma but from disease.
The infamous hospital diseases of erysipelas. pye-
mia. septicemia. and gangrene made surgical wards
nightmares of suffering and death. The causes and
mechanisms of disease transmission remained es-
sentially unknown. By 1865. however. Pasteur had
concluded that disease must be airborne. a view that
galvanized the English surgeon Joseph Lister into
action. Lister reasoned that he could reduce mortal-
ity due to sepsis by covering wounds with dressings
containing chemicals that killed these airborne
germs without preventing the entry of air. He knew
that carbolic acid had recently been used to sterilize
sewage. and with the help of the chemist Anderson
he obtained a supply of the sweet-smelling dark liq-
uid that was commonly called German creosote.

Lister published his findings in 71re Lctrzcet in 1867.
In contrast to Semmelweiss's efforts. Lister's work
attracted immediate attention-The age of antisep-
tic surgery was soon underway.

Once it became realized that microscopic organ-
isms present in the air were responsible for transmit-
ting disease. the next important development was to
isolate these organisms and then conclusively dem-
onstrate their role as causal agents of any given
disease. Yet. some authorities continued to argue
that microorganisms u'ere not the cause of disease,
but merely grew on the weakened infection site. In
May 1882. Robert Koch dismissed this view when
he announced the discovery of the tubercle bacillus;
the search for other disease-causing microorganisms
then gathered momentum. The introduction by
Koch of his famous postulates finally established a
means of conclusively demonstrating the involve-
ment of a microorganism as a causal agent of a given
disease. and the way lay open to disease prevention
and cure.

Major developments were next made in our un-
derstanding of immunity. The first rational attempts
to produce artificial active immunity was made by
Pasteur in 1880 during his work on fowl cholera. By
1882. the Russian biologist Metchnikoff had made
the first observations of cellular immunity and
coined the term phagocyte. By 1891. Ehrlich had
distinguished between active and passive immunity.
and 6 years later Kraus published the first account of
precipitation reactions when immune sera were
added to cell-free filtrates of homologous bacterial
cultures.

Nearly 250 million people have been vaccinated
against tuberculosis with the bacille Calmette-
Guerin (BCG) vaccine. yet its originator, Charles
Calmette. remains a largely unknown figure. Cal-
mette. a disciple of Pasteur, was the first Director of
the Pasteur Institute in Lille. France, and later be-
came Assistant Director of the Pasteur Institute in
Paris. With Guerin. he set about to prepare a protec-
tive vaccine against tuberculosis. He spent 13 years
developing an attenuated virus. which by not recov-
ering its lost virulence remained both stable and
safe. This vaccine. BCG. was first used in 1921. but
because of considerable resistance to its use was
not widely accepted until after Calmette's death in
1933.

Modern developments in immunology include the
work of F. Ma&t-lane Burnet. who in 1957 pub-
lished his clonal selection hypothesis.


History of Microbiology

V. Chemotherapy and Antibiosis

The origin and early development of the concept of
chemotherapy is somewhat unusual in that it can be
credited to the work of one man, the German chem-
ist Paul Ehrlich. Ehrlich had the vision to apply his
knowledge of specific staining of bacteria to the
search for chemical compounds that would inhibit
the growth of pathogenic bacteria in uiuo. In 1891. he
showed that methylene blue was useful for the treat-
ment of malaria, but because this dye showed no
advantage over quinine it was not widely used. By
1902. Ehrlich was concentrating his attention on the
organic arsenic compounds. which he hoped would
defeat experimental trypanosomiasis in mice. At
this point, he and his Japanese bacteriologist assis-
tant Shiga found that atoxyl (sodium arsanilate) was
ineffective against mouse trypanosomiasis. This
turned out to be a somewhat inexplicable error when
the British bacteriologist Thomas was soon to show
that atoxyl was in fact extremely effective against
trypanosomiasis in mice.

A second equally inexplicable error followed
when Schaudinn and Hoffman concluded that
Treponemn pallidurn was a protozoan. Ironically,
this error proved productive because it pointed to
the likelihood that the antiprotozoal agent atoxyl. or
a similar compound. might cure syphilis. In 1906.
Robert Koch used atoxyl to treat trypanosomiasis in
humans. This was the year in which Ehrlich became
director of the newly opened George Speyer Insti-
tute. which was devoted to chemotherapy research.
It was here that the first major chemotherapeutic
agent salvarsan was developed. Salvarsan was first
discovered in 1907 and was initially found to be
inactive against the experimental mouse trypanoso-
miasis sy'stem. Then in 1909. a young Japanese sci-
entist. Hata. joined Ehrlich's laboratory. bringing
with him a sy'stem that he had developed for the
artificial transmission of 7. prrl/ic/irrnr in rabbits. To
his evident surprise. Hata found that salvarsan was
in fact effective against syphilis in mice: by 1909. the
drug was proving spectacularly successful in treat-
ing the disease in humans.

Following Ehrlich's death in 1915. research con-
tinued into chemotherapy. but little progress was
made, with the exception that in 1932 Atebrin be-
came available as the first synthetic drug for pro-
phylactic use against malaria. The ne.\t major ad-
vance in chemotherapy came in 1935. when Domagk
discovered the antibacterial effect of the red dye

prontosil. This compound had a dramatic effect on
lobar pneumonia in humans, reducing death rates by
by two-thirds. In the same year as its discovery,
the French scientist Trefouel showed that the active
ingredient of prontosil was not the chromophore,
but the sulphonamide moeity tsulfanilamide). Sul-
phonamides were widely used with success to treat
bacterial infections from the mid-1930s until the
middle of the following decade.

The concept of chemotherapy reached its zenith
with the sulphonamides. but such compounds were
soon eclipsed by the arrival of first penicillin and
then a range of other antibiotics.

Antibiotics (cf. Waksman. MacFarlane. Wilson)
had a spectacular beginning with the famous discov-
ery of penicillin by Fleming in 1928, a mold spore
having accidentally lodged on agar plates seeded
with staphylococci. The story of the discovery of
penicillin by Alexander Fleming is probably the best
known in the history of medicine, although much
that has been written on the subject borders on fairy
tale. The important point about Fleming's initial ob-
servation. made during the late summer of 1928, was
that it represented an extremely rare phenomenon,
not merely an example of microbial antagonism. but
one of bacterial lysis brought about by mold contam-
inant. Fleming probably initially thought that he had
discovered a fungal variant of lysozyme. a lytic sub-
stance that he had previously found in various body
fluids. It was this lytic phenomenon that distin-
guished Fleming's observations from the numerous
observations of microbial antagonism that had been
reported since Pastuer's time. It is likely that had he
observed microbial antagonism. rather than lysis,
Fleming would have ignored his observations. re-
garding them as an example of common phenome-
non that w'as of little interest.

Fleming. however. understood the significance of
what he observed. He soon showed that the contam-
inant produced the antibacterial substance in culture
broth. which he called penicillin. Then. with help
from various surgeon colleagues. Fleming used
crude penicillin-rich filtrates to treat superficial bac-
terial infections. unfortunately without much suc-
cess. The first documented cures with penicillin
were in fact achieved (using the crude broths) by a
former student of Fleming's, Cecil George Paine,
who worked at Sheffield University.

In his first famous paper on penicillin, Fleming
detailed its properties and antibacterial spectrum
and suggested that it. or a similar substance. might


426                                       History of Microbiology

find a use in medicine. Unfortunately, neither he nor
his colleagues could purify penicillin, an obvious
necessary first step for its successful introduction
into medicine. It is worth pointing out, however,
that Fleming was not alone in being unable to
achieve this essential purification step: other at-
tempts such as those made by famous fungal product
biochemist Harold Raistrick also proved unsuc-
cessful.

Fleming's notebooks show that despite being
unable to purify penicillin. he continued working on
crude penicillin throughout the 1930s. during which
time he also attempted to isolate other microor-
ganisms capable of producing antibacterial prod-
ucts. Unfortunately, this work was not published
and the medical potential of antibioisis remained un-
developed until the discovery of gramicidin in 1939.
This substance, which was discovered by Rene Du-
bos, was unfortunately too toxic for intravenous
use: therefore. it was limited to use on a number of
superficial infections.

At about the time when gramicidin was being first
developed, Florey, Chain, and Heatley managed to
purify penicillin and demonstrate its remarkable an-
tibacterial effects when used systematically. The
isolation of the antibiotic from the crude culture
filtrates was a formidable chemical task, but it was
undertaken successfully in the late 1930s by Florey
and Chain in England. Industrial production of peni-
cillin soon followed as a joint U.S.-British war
project. For this to be feasible required a substantial
effort in strain improvement. which was conducted.
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-
tion of butanol and acetone as munitions solvents
during World War I and the peacetime production of
citric acid by a mold fermentation. Penicillin's intro-
duction into medicine as the first successful antibi-
otic stimulated the search for similar compounds. A
particularly successful antibiotic screening pro-
gram, devoted to soil actinomycetes, was carried
out by Selman Waksman and his students at Rutgers.
The first major product of this research. actinomy-
tin. was. like gramicidin. too toxic to be of medical
use as an antibiotic. although it was later used as an
anticancer agent.

S. Waksman and R. J. Dubos had been studying
the biochemical and ecological interrelations of soil
microbes. The role of secreted antibiotics in ecologi-
cal competition provided a rational for seeking these

substances. Tyorhricin (Dubos, 1939; cf. Crease,
1989) was the first antibiotic to be clinically applica-
ble, but its systemic toxicity limited its application
to topical treatment. In Waksman's hands, the same
paradigm led to the discovery of streptomycin (1944),
which when used in conjunction with periodic acid-
Schiff and isoniazid helped to defeat tuberculosis.
Thereafter, a continued stream of new antibiotics
with untold human benefit. It would be some time
before the mode of action of antibiotics would even
begin to be understood (cf. Gottlieb and Shaw, 1967)
and to allow rational principles to assist in their
improvement.

Although Waksman received the Nobel Prize for
streptomycin, his triumph was marred by his tardy
treatment of the codiscoverer of the antibiotic, Al-
bert Schatz. Schatz, one of Waksman's graduate
students. successfully sued Waksman and Rutgers
for a share of the royalties for streptomycin. His
later attempts to gain a share of the Nobel Prize for
his work (he was senior author on the first strep-
tomycin papers and coassignee with Waksman of
the streptomycin patents) were, however. unsuc-
cessful.

Streptomycin was soon followed by antibiotics
such as chloramphenicol, neomycin, tetracycline,
and the first effective antifungal antibiotic, nystatin
(discovered by Elizabeth Hazen and Rachel
Brown). Penicillinase-resistant penicillins such as
methicillin then appeared, followed by semisyn-
thetic penicillins, and finally broad-spectrum com-
pounds like ampicillin.

VI. Microbial Metabolism and
Applied Microbiology

Developments in the study of microbial metabolism
were, from the outset. closely associated with at-
tempts to use microorganisms for industrial pur-
poses, a trend that continues in modern biotech-
nology. It is not surprising then to find that the first
scientific paper devoted to microbial metabolism
(appearing in 18.57) can also be reiarded as the first
citation in applied microbiology or biotechnology.
Again Pasteur was responsible for this development,
the paper being devoted to an explanation of the
causes of the repeated failures of industrial alcohol
fermentations. This was an important paper for two
reasons: first, because it laid the foundation of the
view, later to be amply validated. that microbial

                                                                           ^__..". ,,. _.  _d
.".,        . .


History of Microbiology                                              427

activity was responsible for many industrially im-
portant fermentations, and. second. because it intro-
duced quantitative treatment of data on microbial
growth and metabolism.

Pasteur also addressed problems associated with
the microbiology of wine-making. Among the sug-
gestions that he made was a method for improving
the keeping qualities of wine by heating it to 68oC for
IO min. followed by rapid cooling, a process subse-
quently referred to as pasteurization. By 1872. Pas-
teur's work had been developed to the point where
Ferdinand Cohn could suggest that microorganisms
play a major role in the biological cy,cling of the
elements responsible for soil fertility and the proper
functioning of natural ecosystems. The first fruits of
Cohn's theory came in 1888 when the Dutch micro-
biologist Beijerinck isolated the symbiotic N-fixing
bacterium Rlli:obi/,rn from the root nodules of le-
gumes. During these studies. Beijerinck also devel-
oped the enrichment technique to the isolation of
microorganisms, an approach later to be refined and
developed by the Dutch school of microbiologists.

Many of the following breakthroughs in microbial
metabolism were associated with studies on soil mi-
crobiology and its association with soil fertility. In
1889, Winogradsky described the autotrophic iron
and sulfur bacteria. and in the following year the
free-living N-fixing Azorobrrcter- and the nitrite-
oxidizing bacterium Nitrobrlcro-. Although many of
Winogradsky's so-called pure cultures appear to
have been contaminated, his work was nevertheless
important because he was the first to appreciate the
concept of chemoautotrophy and to relate this
growth strategy to the major natural cycles. It was a
lack of appreciation of this concept that had hin-
dered the work of others interested in processes
such as nitrification. Despite this. the soil chemist
Warrington nevertheless did important work on the
factors that influence this process in agricultural
soils.

Waksman and Joffe isolated and described T.
tllioosidrltz.5 in 1922. and over the next quarter of a
century, major contributions to the science of mi-
crobial physiology came from, among others, Wie-
land, who in 1900 demonstrated the importance of
biological oxidations using microorganisms. Other
work of note came from Marjorie Stephenson and
J. H. Quastel on enzymes. In 1914, A. J. Kluyver
published an important article entitled "Unity and
Diversity in the Metabolism of Micro-organisms," a
paper that demonstrated the fundamental unity un-
derlaying the apparent diversity of microbial metab-

olism. By 1930, Karstrom had established the con-
cept of constitutive and adaptive enzymes. By now,
microbiology had begun to be a cornerstone of bio-
chemistry and the boundaries between the subjects
were soon blurred. In 1941, Lipmann advanced the
concept of the high-energy bond, and major devel-
opments in theories on the working of enzymes
came from Monod's lab.

While most of the early developments in microbial
metabolism were centered on bacteria, fungal me-
tabolism, because of its importance to many indus-
trial fermentation (e.g., citric acid production), was
by no means neglected. The seminal work in this
area came in 1940, when Jackson Foster published
his "Chemical Activities of the Fungi." Studies on
fungal metabolism obviously gained impetus follow-
ing the introduction, while the isolation of antibio-
tics such as streptomycin also gave a boost to the
study of a neglected group of organisms-the acti-
nomycetes. It was Selman Waksman who initiated
work in these organisms during the early part of this
century. a period when the actinomycetes were re-
garded as fungi rather than bacteria.

VII. Nutrition, Comparative
  Biochemistry, and Other
  Aspects of Metabolism

Microbes. first yeast and then bacteria, played an
important part in the discovery of vitamins and other
growth factors. Growth could be measured in test
tubes far more expeditiously and economically than
in mice. rats. or humans. Conversely, the realization
that microbes shared virtually all of the complex
growth factor requirements of animals was an im-
portant impetus to "comparative biochemistry,"
the view that they had a common evolution and a
similar underlying architecture. One of the essential
amino acids, methionine, was first discovered by
Mueller (1922) as a growth factor required by diph-
theria bacilli. Mueller joined a school founded by
Twort (1911), including Lwoff, Fildes, Knight, and
Tatum, that made nutrition a branch of general bio-
chemistry. They perceived that the requirement for
a growth factor belied a loss or deficiency of syn-
thetic power: lacking internal synthesis. the organ-
ism had to look to the nutrient environment for sup-
ply of substance. This also implied that organisms
with simple nutrition had to be empowered with
complex biosynthetic capability-leaving us humili-

.       _.. .- _...


428                                    History of Microbiology

ated by our species' inferiority to Escller.ichirr co/i,
but that in turn is less capable than the green plant!
Besides the practical utility of these fiildings. they
led to a well-founded respect for the complexity of
microbial cells.

By "1930," a number of growth factors had been
shown to be important in bacterial nutrition. includ-
ing factors V and X, later sholvn to be diphospho-
pyridine-nucleotide and heme. respectively. for he-
mophilic bacteria: MycoDacre/.il//,~ pIl/c>i factor. later
shown to be vitamin K. for M. pserrdotrthe,-c,rrlosis;
and tryptophane for Sal/?~or~ella rxphi (Fildes. 1936).
Starting with the work of W. H. Peterson. H. Wood.
E. Snell. and E. L. Tatum at the University of Wis-
consin and B.C.J.G. Knight and P. Fildes in Eng-
land. a number of bacteria1 growth factors were
identified with B vitamins (extensively cataloged by
Johnson and Johnson, 1945). By "1950." most of
the known trace growth factors had been identified
and associated with nutritional requirements of par-
ticular bacteria, as also had been most of the amino
acids and a host of other metabolites (Snell.
1951). During the vicennium. most of the vita-
mins were also identified as co-enzymes. playing a
role in the function of specific metabolic enzymes
[e.g., thiamin for keto-acid decarboxylases, niacin
for dehydrogenases, pyridoxal for transaminases.
pantothenate in the citric acid cycle (Schlenk.
1951)]. The 20 canonical amino acids were listed
and could be shown to be incorporated into bacterial
protein.

A host of other biochemical pathways were also
detailed with the help of new methodologies of ra-
dioisotopic tracers and chromatography. Of special
significance in bacterial metabolism was the demon-
stration of heterotrophic assimilation of CO,. This
view of CO- as an anabolite was contrary to its usual
image as a waste product. The specific requirements
for CO2 as a nutrient helped to clear up difficulties in
the cultivation of fastidious bacteria and eventually
of tissue cells.

By 1941, microbiology and genetics overlapped
when G. W. Beadle. Tatum. and coworkers at Stan-
ford University began to use the red bread mold
Ne~rrosporrr ~YNSSCI, an approach in which mutants
were employed to help elucidate genetic mecha-
nisms, thereby allowing a number of microbial path-
ways to be worked out for the first time.

In due course, especially after Beadle and Tatum
(1941). the power of synthesis came to be under-
stood as the capability of individual specific genes.

This in turn led to concepts and experiments on the
genetic underpinnings of metabolism.
The birth of molecular biology followed the work
of Watson and Crick in 1853. when microbiology
entered into a new phase. allowing it to overlap with
many other sciences, leading to the appearance of
numerous exciting developments.
The major conceptual theme of change in microbi-
ology during the vicennium was the convergence of
the discipline with general biology. As noted by Du-
bos (1945),

To the biologist of the nineteenth century,
bacteria appeared as the most primitive ex-
pression of cellular organization, the very limit
of life. Speaking of what he considered "the
smallest, and at the same time the simplest and
lowest of all living forms," Ferdinand Cohn
asserted: "They form the boundary line of life:
beyond them. life does not exist, so far at least
as our microscopic expedients reach: and
these are not small." The minute dimensions
of bacteria were considered by many to be
incompatible with any significant morphologi-
cal differentiation; it encouraged the physical
chemist to treat the bacterial cell as a simple
colloidal system and the biochemist to regard it
as a "bag of enzymes."

Still dominated by the medical importance of mi-
crobes, the views of microbiologists in "1930" had
not evolved much further, although "System"
(1930) does have a brief chapter on bacterial cy-
tology and allusion to ongoing controversy over the
existence of nuclear structures. Far more attention
is given to the Gram stain!

While a few differences in the detail of interme-
diary metabolism and biosynthetic options have
been discovered (e.g., for lysine), it remains true
that pathways conveniently noted in bacteria have
usually been reliable predictors of the same steps in
higher plants and animals. It is possible today to
relate this functional conservatism to evolutionary
affinity with currently available tools of DNA se-
quencing.

A. Induced Enzyme Formation, or
"Enzymatic Adaptation"
One of the most intriguing phenomena of bacterial
physiology is the plasticity of enzyme expression


History of Microbiology                                              429

dependent on the chemical environment. For exam-
ple. E. &i grown on a glucose medium exhibits very
low levels of p-galactosidase (lactase). When glu-
cose is replaced by lactose. there is a growth delay
followed by the abundant production of lactase.
Thousands of comparable examples are now known.
and the pursuit of the mechanism of this phenome-
non has been of outstanding importance in the devel-
opment of molecular genetics. Anecdotal reports of
enzyme adaptation can be traced back to Wortmann
(1882. cited in Karstrom. 1930): they were collected.
together with new experimental observations. by
Henning Karstrom for his doctoral dissertation in
Virtanen's laboratory in Helsinki. In this turning-
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-
cose metabolism. most sugar-splitting enzymes are
adaptive-resulting  in substantial biosynthetic
economy for a bacterium or yeast that may only
rarely encounter, say. maltose now, or lactose next
week. During the vicennium. the work of Stephen-
son and Yudkin (1963) and Gale (1943) furnished
additional clearcut examples of the adaptive re-
sponse, and Dubos (1945) offers a critical ap-
praisal of the fundamental biological issues. Several
theories allowed for the stabilization of preformed
enzyme by a substrate, or a Le Chatelier-like prin-
ciple of mass action. to encourage enzyme synthe-
sis. They shared the presumption that the enzyme
molecule itself was the receptor of the inducing sub-
strate. Other hypotheses lent the substrate an in-
structive role in shaping the specificity of the en-
zyme. Further progress would depend on the
postulation of an enzyme-forming system distinct
from the enzyme-and this would emerge under the
impetus of genetic studies to be described later. At
the very end of the vicennium. Lederberg et r/l.
( I95 I) described a noninducing substrate of lactqse,
the analog altrose-P-D-galactoside. which pointed to
a separation of those specificities. This substrate
also allowed the selection of constitutive-lactase
formers. showing that lactose w'as not required for
the conformation of the enzyme. but that the latter
could be derived directly from the genetic consti-
tution. The debate continued until the mid-1950s
(see Lederberg. 1956. p. 51: Monod. 1956): it was
mooted by the spectacular progress of the Pasteur
lnstitute group in showing that enzyme induction

was the neutralization of an endogenous repressor
that inhibited the expression of the lactase gene in
the absence of an inducer (Jacob, 1965).

The simultaneous induction of several steps in a
metabolic pathway. usually by an early substrate,
was exploited to delineate the later steps. notably in
the oxidation of aromatic compounds by pseudo-
monads.

Among technical innovations, one of the most in-
genious was the chemostat (Novick and Szilard,
1950). This allowed microbial populations to be
maintained for the first time in a well-defined steady
state; albeit under limitation for one specific nu-
trient.

VIII. Microbial Genetics

During the last two decades of the nineteenth cen-
tury, it was realized that bacterial species were not
as stable as had first been thought. Pure line cultures
that had been maintained for many generations sud-
denly underwent dramatic changes in morphology,
metabolic properties. and pathogenicity. As more
pure cultures were obtained, this variability. or dis-
sociation as it was called, became even more appar-
ent. Then in 1925, R. M. Mellon published a paper
describing a primitive from of sexuality in coli-
typhoid bacteria. This work had little contemporary
impact on the contemporary view that bacteria were
anucleate organisms that reproduced without sexu-
ality by binary fission.

Bacterial genetics was substantially nonexistent
in 1930. As late as 1942, the eminent British biologist
Julian Huxley would suggest of bacteria that "the
entire organism appears to function both as soma
and germ plasm and evolution must be a matter of
alteration in the reaction system as a whole" (Hux-
ley. 1942. Such ideas gave little encouragement to
efforts to dissect out individual genes along the Men-
delian lines that had been so successful with DI-o-
soyhil~ and other animals and plants. Some work
with fungi had gotten off to a promising start early in
the century (Blakeselee, 1902). Authentic but spo-
radic observations of bacterial mutation (Beijerinck,
1901) were outnumbered by wooly-minded specula-
tions that embraced variations of colony form as
manifestations of cellular life cycles among the bac-
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
the work of Beadle and Tatum on mutants in Nelr~~
sporc~ on our understanding of microbial physiology.
However. by initiating the field of biochemical ge-
netics, these studies had even greater impact on the
science of genetics. Prior to 1941, genetic research
was dominated by work on the fruit fly. Drosopl~il~~
l,lelanogrrsre,-. Much was learned from studying
morphological mutations in this organism, but ef-
forts to disentangle the biochemical basis of these
characteristics resulted only in frustration. Beadle
and his microbial biochemist colleague Tatum
turned their attention to studying the red bread mold
N. o-rrssn and soon obtained mutants with nutri-
tional defects such as blocks in the biosynthesis of
vitamins like pyridoxine and thiamine. This allows
for rapid improvements to be made in genetic analy-
sis. an approach that was subsequently extended by
other workers using bacteria. The first fruits of such
application came in 1943 when Luria and Delbriick
showed by means of their "fluctuation test" that
spontaneous mutations occurred in bacteria, to both
phage resistance and streptomycin resistance at sim-
ilar frequencies, as had been observed in other or-
ganisms.

Luria (1984) in his charming book "A Slot Ma-
chine. A Broken Test Tube," recounts how his ob-
servation of a jackpot in a gambling den inspired his
premonition of the skewed statistics that would gov-
ern the numbers of mutants. The fit of experimental
numbers to those statistics is subject to great theo-
retical uncertainty, but they were a corroboration of
the clonal model. One of the first articles on bacteria
to be published in Generics, the paper promptly at-
tracted broad attention and was widely regarded as
having proved "that bacteria have genes." The gist
of the demonstration was that mutations to phage
resistance agree with a clonal distribution and, thus,
render more likely their "preadaptive" occurrence,
that is, within the growth of the population rather
than at the time of the challenge with the selective
agent. It therefore harkens more to Darwin than to
Mendel: nevertheless, it was a turning point in ge-
neticists' appreciation of bacteria. The statistical
methods, which are helpful in the quantitative esti-
mation of mutation rate, have been improved
(Sarkan, 1991).

The study of bacterial genetics was dramatically
advanced during the 1940s following the recognition
of antibiotic resistance in pathogenic bacteria. Here
was a practical problem. the solution to which pro-
vided an obvious impetus to studies aimed at deter-
mining its cause. [See ANTIBIOTIC RESISTANCE.]

Bacteria did of course suffer from the serious
methodological constraint of the apparent lack of
any recombinational (sexual or crossing1 mecha-
nism by which to analyze and reconstitute gene
combinations. They would prove, however. to be
marvelous material for mutation studies (cf.. e.g..
Ames. 1975) once the concepts were clarified. for
which a major turning point was the work of Luria
and Delbriick (1943). In a fashion that reminds one
of Gregor Mendel, they studied bacterial mutation
by quantitative counts. They used resistance to
(bacteriojphage as the marker. Like resistance to
antibiotics. or growth on a nutritionally deprived
medium, the phage is an environmental agent that
makes it easy to count exceptional cells against a
preponderant background that can be selectively
wiped out. Most importantly, they distinguished be-
tween mutational events, which engender resistant

The themes of nutrition and mutation among mi-
crobes had occasional false starts, with observations
of strain variability and the "training" of exacting
bacteria to dispense with growth factors (Knight,
1936). However, lacking a conceptual framework of
"genes in bacteria," these had little fruit prior to the
work of Beadle and Tatum (1941) on Neurospora.
Beadle had begun his research program with Eph-
russi on the genes for eye color in Drosophilrt (Bur-
ian, 1989). Tatum was engaged to do the biochemical
work but found the material almost intractable-
When he approached success, he was scooped by
Butenandt on the identification of kynurenine as a
pigment precursor. Nor was it clear how much
closer to the primary gene product this chemistry
would bring them. The following account is taken
from J. Lederberg's memoir on E. L. Tatum, who
was his teacher from 1946 to 1947 (Lederberg,
1990).

This jarring experience, to have such pains-
taking work overtaken in so facile a fashion,
impelled Beadle and Tatum to seek another
organism more tractable than D~osop/?i/~ for
biochemical studies of gene action.
In Winter Quarter 1941, Tatum offered a
new graduate course in comparative biochem-


History of Microbiology

431

istry. In it, he called upon his postdoctorate
experience with Kogl in Utrecht, in 1937, and
recounting the nutrition of yeasts and fungi,
some of which exhibited well-defined blocks in
vitamin biosynthesis. Beadle, attending some
of these lectures, recalled the elegant work on
the segregation of morphological mutant fac-
tors in Neur-ospouc that he had heard from
B. 0. Dodge in 1932. The conjunction was that
Nerlrosporcr had an ideal life-cycle for genetic
analysis with the immediate manifestation of
segregating genes in the string of ascospores.
Nerrrospor-n also proved to be readily cultured
on a well defined medium, requiring only biotin
as a supplement. By February 1941, the team
was X-raying Nellrospora and seeking mutants
with specific biosynthetic defects, namely nu-
tritional requirements for exogenous growth
factors.

Harvesting nutritional mutants in microor-
ganisms in those days was painstaking hand
labor: it meant examining single-spore cultures
isolated from irradiated parents, one by one.
for their nutritional properties. No one could
have predicted how many thousands of cul-
tures would have to be tested to discover the
first mutant: isolate #299 in fact required py-
ridoxine. Furthermore, the trait segregated in
crosses according to simple Mendelian prin-
ciples, which foretold that it could in due
course be mapped onto a specific chromosome
of the fungus. Therewith Nellrospo,~r moved
to center stage as an object of genetic experi-
mentation.

In their first paper, they remarked "that there
must exist orders of directness of gene control rang-
ing from one-to-one relations to relations of great
complexity." The characteristics of mutations af-
fecting metabolic steps spoke to a direct and simple
role of genes in the control of enzymes. These were
therefore hypothesized to be the primary products
of genes. Indeed, insome cases, genes might them-
selves be enzymes. This was an assertion of what
came to be labeled the one-gene : one-enzyme the-
ory, which has become the canonical foundation of
modern molecular genetics. albeit with substantial
correction and elaboration of detail. especially with
regard to the intermediating role of messenger RNA.
which could hardly be thought of in 1941. It would be

a mistake to focus too sharply on the numerical 1 : 1
assertion; more important was the general assump-
tion of simplicity, and that the details of gene ex-
pression could be learned as an outcome of such
studies-as indeed they were (see also Horowitz,
1990).

The recruitment of Nerrrospora for what have be-
come classical genetic studies offered further en-
couragement that bacteria, albeit somewhat more
primitive, might be handled in similar fashion. By
1944, Gray and Tatum had produced nutritional mu-
tants in bacteria. including some in a strain that has
dominated bacterial genetics ever since, namely E.
co/i strain K-12. These mutants were soon to be put
to a most striking use.

In 1944,O. T. Avery and his colleagues concluded
that the transforming principle involved in transfor-
mation in pneumococci was DNA. This was a major
breakthrough, because until then it was thought that
the significant part of the nucleoprotein of the chro-
mosome molecule was the protein, the nucleic acid
merely acting as a sort of binding agent. The role of
DNA was initially puzzling, because it was difficult
to see how a polymer that contained only four bases
could possibly code for the complex phenotype of
even the simplest of organisms. Meanwhile, classic
genetic approaches were yielding a wealth of new
discoveries. In 1945. Tatum showed that the mutant
rate of bacteria could be increased using X-rays,
whereas 2 yr later, Tatum and Lederberg demon-
strated genetic recombination between two nutri-
tionally defective strains of E. cdi KIZ.

The first gene map of E. coli K12 appeared, and
over the next few years progress was made in ex-
plaining the phenomena of conjugation, transduc-
tion. and transformation. William Hayes, working at
the postgraduate medical school in Hammersmith
announced in 1952 his discovery that in conjugation
recombination occurred due to the one-way transfer
ofgenetic material, and during the same year Leder-
berg and Cavalli coined the terms fertility plus (F')
for donor cells and fertility minus (F-1 for recipient
cells. The recognition of these mating types made it
clear that conjugation was a primitive form of sexu-
ality, with the recipient F- cell being the zygote.
More advances came when Lederberg, Cavalli, and
Lederberg discovered high-frequency recombi-
nant mutants from the Ff type of E. co/i K12. a
finding that was subsequently confirmed by Hayes.
These mutant strains (Hfr) differed from the wild-
type F' strains. first in transferring various genetic


432                                     History of Microbiology

markers at a rate hundreds of times greater than the
original strains and. second. in not producing an
alteration in the mating type of the recipient cell.
However. although the frequency of transfer of the
various markers differed. it was the same for any
given strain of Hfr.

Between 1955 and 1958. Jacob and Wollman used
their famous "interrupted mating experiment" to
determine the mechanism of gene transfer in E. coli
K12. Jacob and Wollman coined the term episome,
and in 1963 Cairns confirmed the circular nature of
the bacteria1 chromosome using autoradiography.
Bacterial genetics further progressed following the
report published in 1961 by Watanabe explaining
infectious drug resistance.

A. The Pneumococcus Transformation

What might be regarded as the first major break-
through in microbial genetics came in 1928 when
Griffith published on detailing "transformation"
in pneumococci, a study that laid the foundation
for later work by Avery and his colleagues. A fur-
ther development in our understanding of trans-
formation came in 1933, when Alloway showed
that rough type 1 cells could be changed into ge-
netically stable smooth type II cells, by growing
them in the presence of a cell-free extract of a
heat-killed broth culture of smooth type II cells.
This work demonstrated the existence of a soluble
"transforming agent." [See GENETIC TRANSFOR-
MATION, MECHANISMS.]

Apart from cataclysmic happenings in global war.
1944 will also be remembered for the publication of
"Studies on the Chemical Nature of the Substance
Inducing Transformation of Pneumococcal Types."
by Avery. MacLeod. and McCarty.' The pneumo-
coccus transformation was stumbled upon by Fred
Griffith in London. in 1918. in the course of his
studies on the serosystematics of pneumonia. Ex-
tracts of one serotype evidently could transform
cells of another into the type of the frst. In retro-
spect, it is hard to imagine any interpretation other
than the transmission of a gene from one bacterial

' It is a\vkuard ta have such ;i nondescript tern1 BS X*transfer.
mation" .~pplied to such an impel-tant. \pecilic phenomenon. Hut
uhen it \\as first discovered and named. there \\a\, no \rarrant to
five it an!: narrouer connotation. .4\,er-y had the po~,er of nc\r
coinage but \has hardly the likely perwnality.

cell to another, but this interpretation was inevitably
dimmed by the poor general understanding of bacte-
rial genetics at that time.

This vagueness was compounded by two out-
standing misinterpretations: (1) that the transmissi-
ble agent was the polysaccharide itself and (2) that
the agent was a "specific mutagen." Concerning the
first, it is sometimes overlooked that Griffith under-
stood the distinction well enough. Better than many
of his followers, he had at least the germ of a genetic
theory: "By S substance I mean that specific protein
structure of the virulent pneumococcus which en-
ables it to manufacture a specific soluble carbohy-
drate." In regard to the second misinterpretation,
Dobzhansky wrote that ". . . we are dealing with
authentic cases of induction of specific mutations by
specific treatments-a feat which geneticists have
vainly tried to accomplish in higher organisms."
This formally correct attribution, from a most in-
fluential source, obfuscates the idea that the agent
is the genetic information. Muller had much
greater clarity: In his 1946 Pilgrim Trust Lecture
to the Royal Society, he remarked,

   . . . in the PII(~IIII~OCO(.CIIS case the ex-
tracted "transforming agent" may really have
had its genetic proteins still tightly bound to
the polymerized nucleic acid; that is. there
were. in effect. still viable bacterial "chromo-
somes" or parts of chromosomes floating free
in the medium used. These might. in my opin-
ion, have penetrated the capsuleless bacteria
and in part at least taken root there. perhaps
after having undergone a kind of crossing over
with the chromosomes of the host. In view of
the transfer of only a part of the genetic mate-
rial at a time, at least in the viruses. a method
appears to be provided whereby the gene con-
stitution of these forms can be analyzed. much
as in the cross-breeding test on higher organ-
isms. However, unlike what has so far been
possible in higher organisms, viable chromo-
some threads could also be obtained from
these lower forms for in vitro observation.
chemical analysis. and determination of the
genetic effects of treatment.

Other "classical" geneticists had virtually noth-
ing to say about Griffith's work and would have
judged themselves incompetent to assess its experi-
mental validity. They began to pay closer attention


History of Microbiology

433

after 1944. but again had little training in bacterial
chemistry to enable them to form critical judgments
about the claims presented them.

In Avery's world, however, Griffith w'as a central
figure and his observations could not be ignored. His
basic observations were confirmed in Avery's labo-
ratory (see Dubos, 1986), and in due course Avery
felt compelled to pursue the chemical extraction and
identification of the substance responsible for the
transformation. Sixteen years after Griffith, this was
achieved, and DNA was thrust into the scientific
consciousness as the substance of the gene.

In retrospect, it is difficult to give proper credit to
the logical validity of a large range of alternative
interpretations and to reconstruct the confusions
about what was meant by "gene" and "genetic."
Recall that until 1951 the only marker observed in
transformation was the capsular polysaccharide, the
biosynthesis of which was itself subject to many
conjectures [e.g., about the role of starter fragments
in self-assembly (discussed by Lederberg. 1956)].
Avery undoubtedly somewhat intimidated by
Dobzhansky's authority. was reluctant to put his
speculations about the genetic significance of trans-
formation in print; his famous letter to his brother
Roy surfaced only years later. There. but not in the
paper, he remarks that the . . . [transforming sub-
stance is] thereafter reduplicated in the daughter
cells and after innumerable transfers [it] can be re-
covered far in excess of the amount originally
used. . . . Sounds like a virus-may be a gene. But
with mechanisms 1 am not now concerned-One
step at a time-and the first is. what is the chemical
nature of the transforming principle? Someone else
can work out the rest (quoted in Dubos, 1976). AS
late as 194S, so distinguished a geneticist as G. W.
Beadle still referred to the phenomenon as a
"first success in transmuting genes in predetermined
ways" (note transmuting. not transmitting!). This
obscuration of the pneumococcus transformation
became less troublesome with the overall develop-
ment of bacterial genetics.

date settled so important a question as the chemical
identity of the gene as pure DNA (versus a complex
nucleoprotein). Avery himself had cause to worry-
There had been much resistance to his earlier proofs
that pneumococcal polysaccharides, free of protein,
were immunogenic. Wendell Stanley's first claims
that crystalline tobacco mosaic virus was pure
protein had to be subject to humiliating correction
when ribonucleic acid was also found therein. We
should recall that when most biologists of that era
used terms such as protein, nucleic acid, or nucleo-
protein, it can hardly be assumed that they had to-
day's crisp connotations of defined chemical struc-
ture. These issues could only be settled by the few
experts who had worked with these materials exper-
imentally-and it was a daunting task to prove that
there were too few molecules of any contaminating
protein in the "DNA" to account for its genetic
specificity. Maclyn McCarty's meticulous work
continued to provide ever more persuasive evidence
that it was DNA, and the contemporaneous studies
of Chat-gaff showed that DNA was far more complex
than Levene had figured it to be and, therefore,
capable of the subtlety demanded of a "gene." Rig-
orous proof about "DNA alone" was really not fur-
nished prior to the production of genetically active
synthetic DNA three decades later. By 1952,
Hershey and Chase gave evidence from an indepen-
dent quarter that DNA alone penetrated the phage-
infected cell. In the following year. the structural
models of DNA as a double helix (Watson and
Crick. 1953) lent final plausibility to "DNA alone."

This episode is sometimes painted as unreason-
able resistance to a new idea (Stent, 1972). This is
hardly a fair assessment of a controversy that was
settled within 9 years and that required the emer-
gence of a new class of workers, and conversion of
some of the old ones. to deal with new techniques
and experimental materials. That controversy con-
tinued is appropriate to the spirit of scientific skep-
ticism-more to worry about when challenging new
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
many workers, even during the working out of the
nuclear (Mendelian) basis of microbial biology, per-
haps as a carryover of Huxley's idea of the persis-
tent soma. In the course of the discussion. there
were angry ripostes as to whether a given entity was
really a plasmagene. or perhaps a virus, or perhaps a
symbiont. The term and concept "plasmid" was
introduced (in 1952) to stress the operational vacuity
of those distinctions. A particle could be at the same
time a virus (if one focuses on pathology), or sym-
boint. or plasmagene (if one focuses on the genetic
role). As a prophage. it may even be integrated into
the chromosome. w,ith a potential reappearance
later. And it would be impossible to say whether a
virus had evolved its pathogenicity, having once
been a benign organelle. or vice versa, or both at
different evolutionary epochs. One might even re-
vive Altmann's old picture of the mitochondria as
originally symbiotic bacteria. an allusion founded
merely on the limitations of cytological analysis.

The vicennium worked a transformation-the
"biologization" of the microbe. It was an extraordi-
narily exciting and fertile time. with new phenomena
to be found in every culture dish. One could even
learn to treasure one's contaminations.

IX. Viruses and Lysogeny: The
Plasmid Concept
A. Biology of the Virus

The cardinal discovery for virology was the isola-
tion and crystallization of the tobacco mosaic virus
(Stanley, 1935). which sharpened many questions
about this boundary of living existence (Pirie,
1937). A more convenient system for virus biology
proved, however, to be the viruses attacking bac-
terial hosts, the (bacterio)phages, especially in the
hands of the Delbrtick school (Adams, 19.59).
Their life cycle was worked out in some detail,
eventually culminating in two cardinal experi-
ments:

Hershey and Chase (1951): The DNA of the
attacking phage particle is sufficient to initiate
infection. The DNA (not the entire phage)
replicates in the host bacterium and then
generates the capsid and assembles itself into
mature, infectious phage particles.

Hershey (1946): Different phage genomes can
undergo genetic recombination, enabling the
construction of linkage maps. These would
eventually be constructed in ultimate detail,
matching the DNA sequence of the nucleotides.

Viruses were defined by Luria (1953, p.) as "sub-
microscopic entities. capable of being introduced
into specific living cells and of reproducing inside
such cells only." He pointed out that this is a meth-
odological rather than taxonomic criterion; such a
definition might well embrace a wide range of di-
verse entities. By 1950, he insisted that the phages
exhibited "parasitism at the genetic level," taking
over the metabolic direction of the host cell and
exploiting a wide repertoire of its genetic capabili-
ties. Whether or not other viruses, in plant and
animal cells, would share these attributes remained
to be seen (Luria, 1953; Adams, 1959; Hayes, 1964;
Galpern, 1988; Burner, 1945).

B. Lysogeny

Not long after the Twort-d'Herelle discovery of the
bacteriophages (1915-1917), bacterial cultures were
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
(1936). Lwoff and Gutmann (1950) reentered the
controversy and showed that lysogenic Bclcilli car-
ried a "prophage," a genetic capability of producing
the phage. At the same time, Lederberg and Leder-
berg (1951, 1953) had discovered that E. co/i K-12
was lysogenic, for a phage they named "lambda," as
a parallel (or so they thought) for the kappa particles
in Pcrru~zeci~~rr. Crosses of lysogenic with sensitive
strains, however, showed that the capacity to pro-
duce lambda segregated in close linkage with a chro-
mosomal marker (gal): therefore, they invoked
Lwoffs concept and terminology of prophage.
However, the working out of that story, and of the
phenomena of phage-mediate transduction, belongs
to the next era.

X. Virology

The term virus was originally an unspecific term
coined by Pasteur to mean any living organism that
caused disease. This terminology was used well into

                                                                   ^ .__... - I... _y_, _ --., ^__""__".- I"_ .-.-
                
,.._^.


History of Microbiology                                             435

the 1930s; thus, the word antivirus was used by
Besredka to refer to bacterial filtrates that could
apparently cure infections.

The realization that disease could be transmitted
by inoculation of cell-free lesions from plant and
animal infections led to the introduction of the con-
cept of "filterable virus." Iwanowski's discovery in
1892 of tobacco mosaic disease in plants is usually
credited as the first demonstration that a filterable
virus could cause disease. Then in 1898. Loeffler
and Frosch showed that a filterable virus was appar-
ently the cause of foot and mouth disease. In the
same year, S. M. Chapman introduced the use of
fertile hens eggs as a means of cultivating viruses.
This approach was later to be used by Pyton Rous
in his work on the fowl sarcoma that bears his name.
By 1915, a new class of virus affecting bacteria but
neither plants nor animals. was discovered by F. W.
Twort. His observations were extended in 1917 by
D. Herrelle, who over the next 13 years published a
series of papers on what was initially called the
Twort-Herelle phenomenon, but which later be-
came known as bacteriophage.

The development first of the ultraviolet micro-
scope and then of tissue culture techniques in the
1920s added impetus to research on virus structure
and cultivation. Maitland's work in 1928 was a major
advance in tissue culture techniques, but because of
the tedious nature and lack of antibiotics to control
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
of Goodpasture and then of the Australian Macfar-
lane Burnet. Burnet used this approach to culture
the influenza virus. which previously had to be
grown in ferrets.

John Enders did much to develop the art of cultur-
ing viruses. which finally enabled the development
of a range of vaccines. Enders' outstanding contri-
bution to the study of viruses began with his work on
mumps when he showed that a virus could be grown
in chick embryos and. after successive generations.
would loose its ability to cause the disease while
retaining the capacity to immunize against it. In this
way, the modified virus could be used to prepare
vaccines to control the disease. Prior to 1949. for
example. the poliomyelitis virus could only be prop-
agated in monkeys. Enders showed that the virus
could be grown in culture of nonnervous tissues, and
by using this technique Salk developed his famous
vaccine. which essentially defeated infantile paraly-

sis. The application of Enders' tissue culture tech-
niques led to the isolation of many other viruses: in
1954, the year when he received the Nobel Prize,
Enders himself, for example, succeeded in isolating
the measles virus.

The introduction of the electron microscope in
1934 proved a great asset to research on viruses. In
1956, Watson and Crick proposed on theoretical
grounds that virus particles must be made up of a
nucleic acid core and a surrounding shell comprised
of protein subunits, a structure later seen in 1959
under the electron microscope by Horne and Nag-
ington.

Antibiotics aided virus research, allowing for con-
tamination-free studies, so that by 1949, poliomyeli-
tis virus could be grown on nonneural tissues such as
minced monkey kidney.

In 1952, the name of the patient Helen Lane be-
came cryptically immortalized when Gay and his
colleagues established the famous continuous cell
line of HeLa cells, derived from a carcinoma of the
patient's cervix uterus. Then. in the following year,
Scherer succeeded in growing poliomyelitis virus in
these cells.

In 1954, Younger published his technique for
growing trypsinized cells in monolayers on glass.
This allowed viral infection of cells to be recognized
by detecting the cytopathic effect, which allowed for
the routine screening for the presence of viruses.

XI. Mycology and Protozoology,
Microbiology's Cinderellas

Filamentous fungi and protozoa (i.e.. molds and
animacules) were observed soon after the earliest
microscopes were developed. Studies of these or-
ganisms continued largely unnoticed as bacteriology
developed. The fact that neither of these groups of
microorganisms cause major diseases in the devel-
oped world tended to hinder the rapid development
of both mycology and protozoology. The principle
motivation for studying fungi came from their ability
to infect important crop plants. This resulted in a
close association between mycology and botany,
with the unfortunate result that many microbio-
logists in the past, as today, regard fungi as lying
outside the orbit of their subject.

As early as 1767. Torgioni-Tozetti advanced the
view that rust diseases of cereals are caused by mi-
croscopic fungi, but experimental proof of the role


436                                    History of Microbiology

of fungi as phytopathogens had to await the mono-
graph by Prevost, who in 1807 described experimen-
tal smut infections. Prevost also showed that fungal
infections could be prevented by soaking seeds in a
solution of copper sulfate and thus. he inadvertently
became the originator of the pesticide industry. It
was Anton de Bary. however. who did the most to
develop the science of plant pathology.

During the early part of this century, attention
was also focused on the role that fungi play in soil
fertility. It soon became evident that while fungi are
not as metabolically diverse as soil bacteria. they
nevertheless play an important role, principally as
agents of decay of organic forms of carbon and nitro-
gen, in the degradation of leaf litter and humus.
Waksman and his colleagues were particularly ac-
tive in demonstrating the role played by fungi in
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
acid and butyl alcohol from starch-rich materials,
and then, in 1930. examined lactic acid production
by species of Rhixprrs. The foundation for the de-
velopment of studies on mold metabolism was laid
by Raistrick and his numerous collaborators work-
ing at the London School of Hygiene and Tropical
Medicine during the 1920s.

Ringworm was the first human disease to be
shown to be caused by fungi: described in 1839 by
Schoenlein. it was soon followed by the recognition
by the Swede F. T. Berg that Ctrlldidrr rrlbictrus was
the causal agent of thrush. Medical mycology was
slow to develop, however, and it was not until 1910
tha Sabouraud introduced a medium suitable for the
isolation and growth of pathogenic fungi. Systemic
mycoses were discovered at the turn of this century,
while it was as late as 1934 before Monbreun conclu-
sively demonstrated that histoplasmosis is caused
by Histoplrrsnrcr crrpsrrl~~~rrn~. Medical mycology has
tended to lag behind other aspects of medical micro-
biology. although the importance of fungal infec-
tions such as pneumocystis pneumonia and candidi-
asis in the AIDS syndrome is likely to accelerate
developments in this area of the subject.

The development of protozoology as a science is
almost exclusively devoted to the role of protozoa as
agents of disease. Although initially referred to as
animacules, by 1764 Wrisberg had introduced the
term infusoria, while the first generic name for a
protozoan, Prrromecium, was introduced by Hill in
1752. The term protozoa was first used by the Ger-

man Goldfuss in 1817. By 1836, Alfred Donne work-
ing in Paris had shown that a flagellate was respon-
sible for vaginal discharge in women. It was,
however, the colonial expansion of the European
powers that provided the stimulus to studies in med-
ical protozoology. The first observations of para-
sites in the blood of malaria sufferers was made in
1880 by Alphonse Laveran. A long list of diseases
were then shown to be caused by protozoa including
Texas cattle fever in 1893. Malta fever in 189.5, ma-
laria in 1898, sleeping sickness in 1902.
Protozoa have yet to be widely used in industrial
microbiology and biotechnology. and their role in
the environment has been subject to only limited
study; therefore, the history of the development of
protozoology in these areas will have to await future
developments.

XII. The Modern Period

What then of the landmarks of the recent history of
science? Without a doubt. the most obvious devel-
opment in our science that has taken place since the
last war has been the rise in the status of a single
organism, the colon bacterium E. coli. Using this
single organism, scientists such as Niremberg.
Holley, Jacob, and Monod have revolutionized our
thinking on biology. One practical outcome of this
work was the development of an E. co/i strain by W.
Gilbert and others in 1978 that produces human in-
sulin.

A perusal of the list of awards for the Nobel Prize
for research in microbiology in the widest sense
shows that since 1958 particular recognition has
been given to work on genetics, virology, and immu-
nology. Knowledge derived from such studies have
had a profound effect on our understanding of the
life process, and recent developments in biotech-
nology have provided real benefits in our lives. [See
BIOTECHNOLOGY INDUSTRY: A PERSPECTIVE.]

The key technique that has made genetic engi-
neering possible was devised by Herbert Boyer and
Stanley Cohen. Boyer working at the University of
California collaborated with Cohen of Stanford Uni-
versity to develop a method of splicing genes from a
donor into a recipient bacterium. In 1973. they took
a gene from the plasmid of one organism and spliced
it into a plasmid from another to produce recombi-
nant DNA. When inserted into a recipient bacte-
rium, the foreign genes not only survived but also


History of Microbiology

affected the host in the way it had affected the do-
nor, and was also copied as the cell divides. Boyer
later used this approach to insert genes from human
proteins into bacteria, and, thus, heralded a bio-
technological revolution. A similar revolution was
initiated by the production of monoclonal antibodies
by Kohler and Milstein (Interestingly, what might be
termed "natural monoclonal antibodies" had been
observed a few years earlier by Joseph Sinkovicks.)

A microbiologist who left science even as late as
the mid-1970s to follow other pursuits would now
hardly recognize his or her former subject. Studies
on the genetics and molecular biology of microor-
ganisms have made particularly rapid progress in the
intervening years. We have also seen major im-
provements in the way we apply microorganisms in
biotechnology and. more recently. to address envi-
ronmental problems. The appearance of AIDS has
once and for all shattered our cozy belief that we had
all but conquered infectious disease. HIV will un-
doubtedly not be the last new infectious agent to
confront us in the future: if for no other reason than
to combat such infections. our science will need to
continue to develop at the rapid rate seen in the past
few decades.

Bibliography

Amsterdamska, 0. (1991).  Stabilizing instability: the contro-
versy over cyclo_eenic theories of bacterial variation during
the interwar period. J. Hist. Biol. 24, 191-222.
Brock. T. (1990). "The Emergence of Bacterial Genetics."
Cold Spring Harbor Laboratory Press.
Burnet. F. M. (1935). "Virus as Organism. Evolutionary and
Ecological Aspects of Some Human Virus Diseases." Har-
vard University Press, Cambridge. Massachusetts.

437

Collard. P. (1976). "The Development of Microbiology."
Cambridge University Press, Cambridge.
Dubos, R. J. (1945). "The Bacterial Cell in Its Relation to
Problems of Virulence. Immunity and Chemotherapy."
Harvard University Press. Cambridge, Massachusetts.
Dubos, R. J. (1976). "The Professor, The Institute and
DNA." Rockefeller University Press, New York.
Gunsalus, I. C., and Stanier, R. Y. (eds). (1962). "The Bac-
teria: A Treatise on Structure and Function. Biosynthesis."
Academic Press, New York.

Jordan, E. O., and Falk, 1. S. (eds). (1928). "The Newer
Knowledge of Bacteriology and Immunology." University
of Chicago Press. Chicago, Illinois.
Judson, H. F. (1979). "The Eighth Day of Creation-makers of
the revolution in biology." Simon and Schuster, New York.
Lechevali, H. A., and Solotorovsky, M. (1965). "Three Cen-
turies of Microbiology." McGraw-Hill. New York.
Lederberg, J. (1987). "Genetic Recombination in Bacteria: A
Discovery Account." Ann. Rev. Genet. 21, 23-46.
Lederberg, J. (1990). "Edward Lawrie Tatum. Biographical
Memoirs." Nat. Acad. Sci. 59,357-386.
Lederberg. J. (1991). "The Gene (H. J. Muller 1917)." Ge-
netics l29,313-316.

Lederberg. J. (1992). "Bacterial Variation since Pasteur;
Rummaging in the Attic: Antiquarian Ideas of Transmis-
sible Heredity, 1880-1940." A.544 News 58(5), 261-265.
Medical Research Council (Great Britain). (1930). `*A System
of Bacteriology in Relation to Medicine." His hlajesty's
Stationery Office. London.

Summers, W. C. (1991). "From Culture as Organism to Or-
ganism as Cell: Historical Origins of Bacterial Genetics."
J. Hisf. Biol. 24, 171-190.

%`ainwright, M., and Swan. H. T. (1986). Medical History 30,
32-56.

Wainwright. M. (1990). "Miracle Cure-The Story of Anti-
biotics." Blackwell, Oxford.
Weatherall. M. (1990). "In Search of a Cure." Oxford Univer-
sity Press, Oxford.

Werkman, C. H., and Wilson P. W. (eds). (1951). "Bacterial
Physiology." Academic Press, New York.