DISCOVERY OF MICROORGANISMS

Even before microorganisms were seen, some investigators suspected their existence
and responsibility for disease.

The earliest microscopic observations appear to have been made between 1625 and
1630 on bees and weevils by the Italian Francesco Stelluti, using a microscope probably
supplied by Galileo.

In 1665 the first drawing of a microorganism was published in Robert Hooke’s

Micrographia.

However, the first person to publish extensive, accurate observations of microorganisms

was the amateur microscopist Antony van Leeuwenhoek (1632–1723) of Delft, the
Netherlands (figure 1.3a).

Leeuwenhoek earned his living as a draper and haberdasher (a dealer in men’s clothing
and accessories) but spent much of his spare time constructing simple microscopes
composed of double convex glass lenses held between two silver plates (figure 1.3b).
As important as Leeuwenhoek’s observations were, the development of microbiology
essentially languished for the next 200 years.

Little progress was made primarily because microscopic observations of

microorganisms do not provide sufficient information to understand their biology.

For the discipline to develop, techniques for isolating and culturing microbes in the
laboratory were needed.

Many of these techniques began to be developed as scientists grappled with the

conflict over the theory of spontaneous generation.

This conflict and the subsequent studies on the role played by microorganisms in
causing disease ultimately led to what is now called the golden age of microbiology.
From earliest times, people had believed in spontaneous generation—that living
organisms could develop from nonliving matter.

Even Aristotle (384–322 bce) thought some of the simpler invertebrates could arise by
spontaneous generation.

This view finally was challenged by the Italian physician Francesco Redi (1626–1697),
who carried out a series of experiments on decaying meat and its ability to produce
maggots spontaneously.

Redi placed meat in three containers. One was uncovered, a second was covered with
paper, and the third was covered with fine gauze that would exclude flies.

Flies laid their eggs on the uncovered meat and maggots developed. The other two
pieces of meat did not produce maggots spontaneously. However, flies were attracted to
the gauze-covered container and laid their eggs on the gauze; these eggs produced
maggots.

Thus the generation of maggots by decaying meat resulted from the presence of fly
eggs, and meat did not spontaneously generate maggots as previously believed. Similar
experiments by others helped discredit the theory for larger organisms.
Leeuwenhoek’s discovery of microorganisms renewed the controversy.

Some proposed that microorganisms arose by spontaneous generation even though

larger organisms did not.

They pointed out that boiled extracts of hay or meat gave rise to microorganisms after
sitting for a while.

In 1748 the English priest John Needham (1713–1781) reported the results of his
experiments on spontaneous generation.

Needham boiled mutton broth in flasks that he then tightly stoppered. Eventually many
of the flasks became cloudy and contained microorganisms. He thought organic
matter contained a vital force that could confer the properties of life on nonliving
matter.

A few years later, the Italian priest and naturalist Lazzaro Spallanzani (1729–1799)
improved on Needham’s experimental design by first sealing glass flasks that contained
water and seeds.

If the sealed flasks were placed in boiling water for three-quarters of an hour, no growth
took place as long as the flasks remained sealed.
 He proposed that air carried germs to the culture medium but also commented that
the external air might be required for growth of animals already in the medium.

The supporters of spontaneous generation maintained that heating the

air in sealed flasks destroyed its ability to support life.

Several investigators attempted to counter such arguments. Theodore Schwann

(1810–1882) allowed air to enter a flask containing a sterile nutrient solution after
the air had passed through a red-hot tube.

The flask remained sterile. Subsequently Georg Friedrich Schroder (1810–1885) and
Theodor von Dusch (1824–1890) allowed air to enter a flask of heat-sterilized
medium after it had passed through sterile cotton wool.

No growth occurred in the medium even though the air had not been heated. Despite
these experiments, the French naturalist Felix Pouchet (1800–1872) claimed in 1859
to have carried out experiments conclusively proving that microbial growth could
occur without air contamination. This claim provoked Louis Pasteur (1822–1895)
to settle the matter.
Pasteur first filtered air through cotton and
found that objects resembling plant spores had
been trapped.

If a piece of the cotton was placed in sterile

medium after air had been filtered through it,
microbial growth occurred.

Next he placed nutrient solutions in flasks,

heated their necks in a flame, and drew them out
into a variety of curves.

The swan neck flasks that he produced in this

way had necks open to the atmosphere (figure
1.5).
Pasteur then boiled the solutions for a few
minutes and allowed them to cool. No growth
took place even though the contents of the
flasks were exposed to the air.

Pasteur pointed out that no growth occurred

because dust and germs had been trapped on the
walls of the curved necks.

If the necks were broken, growth commenced

immediately. Pasteur had not only resolved the
controversy by 1861 but also had shown how to
keep solutions sterile.
Support for the idea that microorganisms cause disease—that is, the germ
theory of disease—began to accumulate in the early nineteenth century from
diverse fields.
Indirect evidence for the germ theory of disease came from the
work of the English surgeon Joseph Lister (1827–1912) on the
prevention of wound infections.

Lister, impressed with Pasteur’s studies on the involvement of

microorganisms in fermentation and putrefaction, developed a
system of antiseptic surgery designed to prevent
microorganisms from entering wounds.

Instruments were heat sterilized, and phenol was used on

surgical dressings and at times sprayed over the surgical area.
The approach was remarkably successful and transformed
surgery.

It also provided strong indirect evidence for the role of

microorganisms in disease because phenol, which kills bacteria,
also prevented wound infections.
Koch’s Postulates
The first direct demonstration of the role of bacteria in causing disease came from the
study of anthrax by the German physician Robert Koch (1843–1910).

Koch (figure 1.6) used the criteria proposed by his former teacher Jacob Henle (1809–
1885) to establish the relationship between Bacillus anthracis and anthrax; he
published his findings in 1876.

Koch injected healthy mice with material from diseased animals, and the mice became
ill.

After transferring anthrax by inoculation through a series of 20 mice, he incubated a

piece of spleen containing the anthrax bacillus in beef serum.

The bacilli grew, reproduced, and produced endospores.

When the isolated bacilli or their spores were injected into healthy mice, anthrax developed.

His criteria for proving the causal relationship between a microorganism and a specific
disease are known as Koch’s postulates.

Koch’s proof that B. anthracis caused anthrax was independently confirmed by Pasteur and
his coworkers.

They discovered that after burial of dead animals,

anthrax spores survived and were brought to the
surface by earthworms. Healthy animals then ingested
the spores and became ill.

After completing his anthrax studies, Koch fully

outlined his postulates in his work on the cause of
tuberculosis (table 1.1).
In 1884 he reported that this disease was caused by the rod-shaped bacterium
Mycobacterium tuberculosis, and he was awarded the Nobel Prize in Physiology or
Medicine in 1905 for his work.

Koch’s postulates were quickly adopted by others and used to connect many diseases to
their causative agent.
Development of Techniques for Studying Microbial
Pathogens
During Koch’s studies on bacterial diseases, it became necessary to isolate suspected
bacterial pathogens in pure culture—a culture containing only one type of
microorganism.

At first, Koch cultured bacteria on the sterile surfaces of cut, boiled potatoes, but the
bacteria would not always grow well.

Eventually he developed culture media using meat extracts and protein digests,
reasoning these were similar to body fluids.

Initially he tried to solidify the media by adding gelatin.

Separate bacterial colonies developed after the surface of the solidified medium had
been streaked with a bacterial sample. The sample could also be mixed with liquefied
gelatin medium. When the gelatin medium hardened, individual bacteria produced
separate colonies.

Despite its advantages, gelatin was not an ideal solidifying agent because it can be
digested by many microbes and melts at temperatures above 28°C.
A better alternative was provided by Fanny Eilshemius Hesse (1850–1934), the
wife of Walther Hesse (1846–1911), one of Koch’s assistants.

She suggested the use of agar as a solidifying agent, which she used to make jellies.
Agar was not attacked by most bacteria.

Furthermore, it did not melt until reaching a temperature of 100°C and, once melted,
did not solidify until reaching a temperature of 50°C; this eliminated the need to
handle boiling liquid.

Some of the media developed by Koch and his associates, such as nutrient broth and
nutrient agar, are still widely used.

Another important tool developed in Koch’s laboratory was a container for

holding solidified media—the petri dish (plate), named after Richard Petri
(1852–1921), who devised it.

These developments directly stimulated progress in all areas of microbiology.

Immunological Studies
In this period, progress also was made in determining how animals resisted disease
and in developing techniques for protecting humans and livestock against pathogens.

During studies on chicken cholera, Pasteur and Pierre Roux (1853–1933) discovered
that incubating the cultures for long intervals between transfers would attenuate the
bacteria, which meant they had lost their ability to cause the disease.

If the chickens were injected with these attenuated cultures, they remained healthy
and developed the ability to resist the disease when exposed to virulent cultures.

Pasteur called the attenuated culture a vaccine (Latin vacca, cow) in honor of
Edward Jenner (1749–1823) because, many years earlier, Jenner had used material
from cowpox lesions to protect people against smallpox.

Shortly after this, Pasteur and Chamberland developed an attenuated anthrax

vaccine.

Pasteur also prepared a rabies vaccine using an attenuated strain of Rabies virus.
During the course of these studies, Joseph Meister, a nine-year-old boy who had
been bitten by a rabid dog, was brought to Pasteur.
Since the boy’s death was certain in the absence of treatment, Pasteur agreed to try
vaccination. Joseph was injected 13 times over the next 10 days with increasingly
virulent preparations of the attenuated virus.

He survived. In gratitude for Pasteur’s development of vaccines, people from around

the world contributed to the construction of the Pasteur Institute in Paris, France.

One of the initial tasks of the institute was vaccine production.

DEVELOPMENT OF INDUSTRIAL
MICROBIOLOGY AND MICROBIAL ECOLOGY
Although humans had unknowingly exploited microbes for thousands of years,
industrial microbiology developed in large part from the work of Louis Pasteur and
others on the alcoholic fermentations that yielded wine and other alcoholic beverages.

In 1837, when Theodore Schwann and others proposed that yeast cells were
responsible for the conversion of sugars to alcohol, the leading chemists of the time
believed microorganisms were not involved.

They were convinced that fermentation was due to a chemical instability that
degraded the sugars to alcohol.

Pasteur did not agree; he believed that fermentations were carried out by living
organisms.

In 1856 M. Bigo, an industrialist in Lille, France, where Pasteur worked, requested

Pasteur’s assistance. His business produced ethanol from the fermentation of beet
sugars, and the alcohol yields had recently declined and the product had become sour.
Pasteur discovered that the fermentation was failing because the yeast normally
responsible for alcohol formation had been replaced by bacteria that produced acid
rather than ethanol.

In solving this practical problem, Pasteur demonstrated that all fermentations were due
to the activities of specific yeasts and bacteria, and he published several papers on
fermentation between 1857 and 1860.

His success led to a study of wine diseases and the development of pasteurization to
preserve wine during storage.

Pasteur’s studies on fermentation continued for almost 20 years.

One of his most important discoveries was that some fermentative microorganisms
were anaerobic and could live only in the absence of oxygen, whereas others were able
to live either aerobically or anaerobically.