Lecture 1 - Milestones in Microbiology

A/Prof Lily Pereg

1.1 Introduction
Nowadays Microbiology is an advanced field of research and microbiologists use
sophisticated, state of the art, tools to study microorganisms, their origin, roles,
applications and more. Nevertheless, most of the current techniques have developed
based on past inventions and use the same basic principles. It is important to
understand these basic ideas and discoveries in Microbiology in order to grasp current
practices and theory. The best way to do it is to follow the pathways of discovery and
developments in Microbiology and the background to different discoveries. As we
progress through this unit, you will realize that some of the original methods are still
in use today, with little or no modifications.

1.2 Milestones in Microbiology

The following list shows selected events & individuals, who contributed to the
development of the science of microbiology. Note that there was almost a 200-year
gap between the first morphological descriptions of microorganisms and the advances
contributed by Pasteur and Koch in the mid to late 19th century.

Hooke (1665) Invention of compound light microscope

Van Leeuwenhoek First to observe & describe bacteria, using hand-held

(1632-1723) lenses

Jenner (1798) Cowpox vaccine for smallpox

Cohn (1828-1898) Bacterial differentiation into spores

Spore resistance to heating
Methods for preventing culture contaminations
Lister (1867) Sterilization of surgical instruments

Pasteur (1822-1895) Disproved theory of spontaneous generation

Koch (1843-1910)
Significance of microbes in wine production
Significance of yeast for beer fermentation
Development of rabies vaccine
Germ theory of disease
Identified causative agents of tuberculosis & anthrax
Use of agar (solid, inert) to solidify medium for
microbial growth
Ross & Grassi (1898)
Beijerinck (1851-1931)
Winogradsky (1856-1953)
Gram (1884)
Petri (1887)
Fleming (1928)
Avery, MacLeod & McCarty DNA as the genetic material
(1944)
Watson & Crick (1953)
Nirenberg & Khorana (1966)
Arber & Smith (1970)
Woese (1977)
Smallpox (1979)
Prusiner (1982)
Montagnier & Gallo (1983)
Vaccine for hepatitis
(1986)
Mullins (1988)
Venter et al (1995)
Yeast genome (1996)
Escherichia coli (1997)
Homo sapiens (2001)
Staphylococcus aureus
(2001)
2004
2006
Malaria caused by protozoa (carried by mosquitoes)
Importance of bacteria in the soil
Nitrogen fixing bacteria
Development of the Gram stain method [gram-
positive (purple) & gram–negative bacteria (pink)]
Use of Petri dish for growth of microbes
Discovery of penicillin, developed by Florey & Chain
(1940)
Structure of DNA, double helix
Deciphering of the genetic code
Discovery of restriction enzymes, that cut DNA at
very specific points
Discovery of new microbial group, the Archaea
Officially eradicated, the first microbial disease ever
to be deliberately eradicated (vaccine), last case in
1977 in Somalia
Characterization of prions (causative agent of mad
cow disease)
Isolation of HIV, causative agent of AIDS
B Produced in yeast cells
Polymerase chain reaction, PCR
fluenzae
First eukaryotic DNA completely sequenced, yeast
Complete sequence of E. coli DNA
Complete sequence of human DNA
Complete sequence of S. aureus MRSA DNA
Complete sequences of 152 microorganisms
Complete sequences of 353 bacterial genomes
http://cmr.tigr.org
In 1665, Robert Hooke reported that living organisms consisted of small “boxes” or
“cells” as he called them. Using a crude microscope he made drawings of molds and
fungal spores. However, the first person to observe living microorganisms and make
relatively accurate drawings of common bacteria was the Dutch merchant, Anton van
Leeuwenhoek (1632-1723). Leeuwenhoek, who was the official wine taster of Delft
and a cloth maker and tailor, constructed simple magnifying glasses and glass
microscopes with which to examine fabrics. His microscopes were capable of
showing detail down to about 1000 nm (1 nm = 10-9 m) on a carefully sited and
illuminated object. His microscopes were handheld, single lens microscopes with
either natural or candle light as the light source. Under these relatively primitive
conditions, he was still able to obtain magnifications of around 200 to 300 times.

We will cover the modern light microscope towards the end of the lecture, however,
just for a comparison note down what magnifications can be achieved by modern
light microscopes. What about the electron microscope?

In a series of letters and drawings submitted to the Royal Society of London, van
Leeuwenhoek presented detailed drawings of microorganisms including rod and cocci
shaped bacteria. He called these microscopic organisms “animacules”, because he
believed they were tiny living animals. These observations marked the birth of the
science of Microbiology. However, it was not until Louis Pasteur and Robert Koch,
some 200 years later, that the concepts of microorganisms contributing to wine and
beer fermentation and as causative agents of disease were accepted.

Drawings of bacteria by van Leeuwenhoek

From Madigan et al., 14th ed. Figure 1.14 (13th ed., Figure 1.13)

1.3 Contributions by Pasteur

Humans have known the technology for baking, brewing and wine making for many
thousands of years. However, it was not until the work of Louis Pasteur (1822-1895)
in the latter half of the 19th century that we have known that these processes, based on
fermentation, are in fact due to the fermentation of sugars to carbon dioxide, water
and ethanol by the microscopic yeast Saccharomyces (Latin for sugar fungus)
cerevisiae (Latin for brewer). It was Pasteur who first unequivocally demonstrated
that wine and beer fermentation was the result of the presence of particular
microorganisms and not that fermentation produced microorganisms, as some
believed at the time.

Read Madigan et al., 14th or 13th ed, Chapter 1, Section: 1.7 Pasteur
and the defeat of Spontaneous Generation.
Answer the following questions:
1. What is the Spontaneous Generation theory and how did Pasteur
disprove it?
2. What major accomplishments (major milestones in microbiology)
he achieved in this experiment?

Pasteur demonstrated, for example, the presence of budding yeast cells in vats of good
wine and rod-shaped bacteria (Lactobacillus) in vats with sour wine. He showed that
yeasts were responsible for the fermentation of sugars to alcohol and the rod-shaped
bacteria produced lactic acid that caused the production of sour wine. Pasteur further
went on to demonstrate that the souring of milk was also due to microorganisms and
showed that heating for short periods could be used to kill such harmful microbes.
This process of pasteurization is widely used today in the food industry.

Following his success with fermentation, Pasteur was approached by the French silk
industry to solve the problem of a silkworm disease. He demonstrated that the disease
was due to a protozoan infection and proposed that the selection of protozoan-free
silkworms would eliminate the problem.

Pasteur made numerous contributions to the development of the Science of

Microbiology. Two of the most famous were his work on rabies and his definitive
experiments to disprove the concept of spontaneous generation.

1.4 Contributions by Koch

At about the same time as Pasteur was making fundamental discoveries on the role of
microorganisms in fermentation and related areas, the German physician, Robert
Koch (1842-1920) was developing the germ theory of disease. Koch and his
colleagues used specific solid media for the cultivation of microorganisms and
developed the concept of pure cultures of microbes. These pure cultures (as opposed
to mixed cultures containing many different types of microbes) allowed the study of a
particular microorganism and established the relationship between a particular
microbe and an infectious disease (germ theory of disease).

Gelatin (a protein) was initially employed as an inert base solid medium for microbial
growth but disadvantages included susceptibility to bacterial degradation and
liquefaction at relatively low temperatures (>37°C). On the other hand, the adoption
of agar, a complex polysaccharide from red algae, as a solidifying agent and the use
of the Petri dish for the cultivation of microorganisms was a quantum leap in the
development of microbiological techniques for the study of microorganisms. The
chemical properties of agar greatly extended the temperature range to well above
40°C at which microbes could be cultivated. These techniques together with light
microscopy and specific stains, allowed Koch and his colleagues to determine the
causative organism for several important diseases, including cholera (Vibrio sp,
Gram-negative rod), anthrax (Bacillus anthracis, Grampositive rod) and tuberculosis
(Mycobacterium tuberculosis, pink rod on acid-fast staining). Koch first showed that
anthrax, a deadly disease, which afflicts cattle and sheep and which, can be
transmitted to humans, is caused by a rod-shaped bacterium, Bacillus anthracis. He
demonstrated that this bacterium was the causative agent of anthrax by infecting mice
with the anthrax bacteria. The mice subsequently developed the disease and he was
able to isolate the Bacillus bacterium from the blood of the infected mice.

Koch and his colleagues were the first to prove unequivocally that one kind of disease
was caused by one particular kind of microbe. Until today Koch’s postulates need to
be satisfied in order to differentiate cause and effect in infectious disease (to prove
that a certain microbial species is the causative agent, or pathogen, of a specific
disease). The following figure shows the procedure to be taken to prove a suspected
pathogen.

Koch’s Postulates
From Madigan et al., 14th ed., Figure 1.20 (13th ed., Figure 1.19)
 Read Madigan et al., 14th or 13th ed, Chapter 1, Section: 1.8 Koch,
Infectious Disease, and Pure Culture Microbiology. Read also the
Microbial Sidebar if you use the 13th ed (pg. 45).
Answer the following questions:
1. In the practical sessions of MICR220 you will examine microbial
colonies on Petri dishes containing solid media. This is an old
technique. Who was the first to use it?
2. What is the origin of each microbial colony you will see growing on
the Petri dish?
3. What is a pure culture?
4. How do the developments of solid media and pure cultures relate to
the birth of microbial systematics?
5. You observed lesions on the skin of fish in your aquarium. You
suspect the lesions are the result of a bacterial infection. How would
you try to isolate possible microbial pathogens from the skin
lesions?
6. You found an exceptionally large number of a particular bacterium
in the lesions. How would you prove that this particular bacterium is
the actual pathogen causing the disease (in contrast to a secondary
infection)?

1.5 Beijerinck, Winogradsky and Gram

With the advance of Microbiology, focus of research broadened to include microbial

diversity, in particular in soil and water, and the microbial metabolic processes in
these habitats. Beijerinck (1851-1931) and Wingradsky (1856-1953) had great
contributions to these fields by the formulation of enrichment culture techniques and
the development of the important concept of chemolithotrophy.

Gram, in 1884, developed a staining technique (Gram staining technique) that can
differentiate between bacteria having specific differences in their cell walls. Some
bacteria will stain red (Gram negative) and other purple (Gram positive). The
technique is still being used in the initial step of bacterial characterization and
identification. We will use the technique in our practical sessions.

Read Madigan et al., 14th or 13th ed, Chapter 1, Section: 1.9 The Rise of
Microbial Diversity
Answer the following questions:
1. Explain the term “enrichment culture”. What is being enriched and
how is it being achieved?
2. Explain how the development of enrichment culture techniques
advanced the study of microbial diversity.
3. Explain the term “chemolithotrophy”. How did Winogradsky find
out that chemolithotrophic bacteria were autotrophs?
1.6 The development of microscopes

The need to see microorganisms makes the microscope the most basic and essential
tool for any microbiologist, thus making the invention of the microscope probably the
most important milestone in the history of Microbiology. Today microbiologists are
equipped with a large variety of microscopes designed for different purposes.

All microscopes employ lenses that magnify the image. Resolution rather than
magnification is the limiting factor in our ability to see small objects. Resolution is
the ability to distinguish two adjacent objects as distinct and separate. Resolution is a
function of the physical properties of the light and thus cannot be increased without a
limit.

The light microscope

The resolution limits of the light microscope, which uses visible light to illuminate
cell structures, are about 0.2 µm. Magnifications of about x2000 are the upper limits
of light microscopes.

Read Madigan et al., 14th or 13th ed, Chapter 2, Sections 2.1 and 2.2.

Answer the following questions:

1. Explain the terms magnification and resolution.
2. What determines the useful upper limit of magnification for a bright
field microscope?
3. Based on section 2.2, suggest ways to improving contrast in light
microscopy.

Electron Microscopes

Electron microscopes use electrons to image cells and cells structures. The
wavelength of electrons is much shorter than that of visible light. As wavelength
affects resolution, the resolving power (and thus useful magnification) of the electron
microscopes are much higher than that of the light microscope, enabling viewing
structures at the molecular level.

Read Madigan et al., 14th or 13th ed, Chapter 2, Section: 2.4 Electron
microscopy.
Answer the following questions:
1. What two types of electron microscopes exist? Which one
would you use to observe a cluster of cells and which one would
you use to observe internal cellular structures?
2. What is a light micrograph? What is an electron micrograph?
Which one has greater resolution? Briefly explain.
1.7 The development of the Autoclave

Another very important development, used daily by microbiologists is the autoclave!

We have seen before how highly important is the ability to sterilize media and
instruments to get pure cultures, as well as in many human and veterinary medical
professions to prevent cross contamination of pathogens among patients.

Pasteur used heat to sterilize his media. The autoclave is a variation of this technique,
it is a sealed heating chamber that uses steam under pressure, producing temperatures
above 121 deg C, killing microorganism and their survival forms (bacterial and fungal
spores).
Summary of Lecture 1
On completion of this lecture, you should have an understanding and give examples
of:

• Contribution of the microscope to Microbiology

• Pure cultures
• The disproving of the spontaneous generation theory
• Koch’s Postulates
• Culture enrichment techniques
• Contribution of Hooke, Van Leeuwenhoek Pasteur, Koch, Beijerinck,
Wingradsky and Gram to the advancement of Microbiology

Reference and recommended information source

Madigan et al., 14th or 13th ed., Chapters 1 & 2

Revision Questions – Lecture 1

1. If microscopes had never been invented, explain how the existence of
microorganisms might still have been suspected.
2. Why was knowledge of how to obtain a pure culture important for
development of the science of Microbiology?
3. You have obtained a soil sample from the roots of plants. Briefly list what
would you do to isolate pure cultures of as many different microorganisms as
possible.
4. This time you are studying the microbial flora from the gut of humans. In
general, would you use the same media (and growth conditions) you used for
isolating microbes from the soil? Briefly explain your answer.