MICROBIOLOGY

 Microbiology is the study of living organisms of microscopic size.

 The term microbiology was given by French chemist Louis Pasteur (1822-95).
 Microbiology is said to have its roots in the great expansion and development of the
biological sciences that took place after 1850.
 The term microbe was first used by Sedillot (1878).

The Discovery Era

 Robert Hooke, a 17th-century English scientist, was the first to use a lens to observe the
smallest unit of tissues he called “cells.” Soon after, the Dutch amateur biologist Anton
van Leeuwenhoek observed what he called “animalcules” with the use of his homemade
microscopes.
 Antonie van Leeuwenhoek (1632-1723) of Delft, Holland (Netherland) was the first
person to observe and accurately describe microorganisms (bacteria and protozoa) called
‘animalcules’ (little animals) in 1676.
 Actually he was a Dutch linen merchant but spent much of his spare time constructing
simple microscopes composed of double convex lenses held between two silver plates.
He constructed over 250 small powerful microscopes that could magnify around 50-300
times.
 Leeuwenhoek was the first person to produce precise and correct descriptions of bacteria
and protozoa using a microscope he made himself. Because of this extraordinary
contribution to microbiology, Antonie van Leeuwenhoek is considered as the “Father of
microbiology”.
 Antonie van Leeuwenhoek is also considered to be the father of bacteriology and
protozoology (protistology).
 He wrote over 200 letters which were transmitted as a series of letters from 1674-1723 to
Royal Society in London during a 50 years period.

Transition Period

 When microorganisms were known to exist, most scientists believed that such simple life
forms could surely arise through spontaneous generation. That is to say life was thought
 to spring spontaneously from mud and lakes or anywhere with sufficient nutrients. This
concept was so compelling that it persisted until late into the 19th century.
 The main aspects were to solve the controversy over a spontaneous generation which
includes experimentations mainly of Francesco Redi, John Needham, Lazzaro
Spallanzani, and Nicolas Appert, etc, and to know the disease transmission which
mainly includes the work of Ignaz Semmelweis and John Snow.
 Francesco Redi (1626-1697): The ancient belief in spontaneous generation was first of
all challenged by Redi, an Italian physician, who carried out a series of experiments on
decaying meat and its ability to produce maggots spontaneously.
 John Needham (1713-1781): He was probably the greatest supporter of the theory of
spontaneous generation. He proposed that tiny organisms the animalcules arose
spontaneously on his mutton gravy. He covered the flasks with cork as done by Redi and
even heated some flasks. Still the microbes appeared on mutton broth.
 Lazzaro Spallanzani (1729-1799): He was an Italian Naturalist who attempted to refute
Needham’s experiment. He boiled beef broth for longer period, removed the air from the
flask and then sealed the container. Followed incubation no growth was observed by him
in these flasks. He showed that the heated nutrients could still grow animalcules when
exposed to air by simply making a small crack in the neck. Thus Spallanzani disproved
the doctrine of spontaneous generation.
 Nicolas Appert followed the idea of Spallanzani’s work. He was a French wine maker
who showed that soups and liquids can be preserved by heating them extensively in thick
champagine bottles.
 Ignaz Semmelweis and John Snow were the two persons who showed a growing
awareness of the mode of disease transmission.
 Two German scholars Schulze (1815-1873) and Theodor Schwan (1810-1882) viewed
that air was the source of microbes and sought to prove this by passing air through hot
glass tubes or strong chemicals into boiled infusions in flasks. The infusion in both the
cases remained free from the microbes.
 George Schroeder and Theodor Von Dusch (1854) were the first to introduce the idea
of using cotton plugs for plugging microbial culture tubes.
  Darwin (1859) in his book, ‘Origin of the Species’ showed that the human body could be
conceived as a creature susceptible to the laws of nature. He was of the opinion that
disease may be a biological phenomenon, rather than any magic.

The Golden Age

 The Golden age of microbiology began with the work of Louis Pasteur and Robert Koch
who had their own research institute. More important there was an acceptance of their
work by the scientific community throughout the world and a willingness to continue and
expand the work. During this period, we see the real beginning of microbiology as a
discipline of biology.
 The concept of spontaneous generation was finally put to rest by the French chemist
Louis Pasteur in an inspired set of experiments involving a goosenecked flask. When he
boiled broth in a flask with a straight neck and left it exposed to air, organisms grew.
When he did this with his goose-necked flask, nothing grew. The S-shape of this second
flask trapped dust particles from the air, preventing them from reaching the broth. By
showing that he could allow air to get into the flask but not the particles in the air, Pasteur
proved that it was the organisms in the dust that were growing in the broth.
 Pasteur, thus in 1858 finally resolved the controversy of spontaneous generation versus
biogenesis and proved that microorganisms are not spontaneously generated from
inanimate matter but arise from other microorganisms.
 He also found that fermentation of fruits and grains, resulting in alcohol, was brought
about by microbes and also determined that bacteria were responsible for the spoilage of
wine during fermentation. Pasteur in 1862 suggested that mild heating at 62.8°C (145°F)
for 30 minutes rather than boiling was enough to destroy the undesirable organisms
without ruining the taste of the product, the process was called Pasteurization.
Pasteurization was introduced into the United States on a commercial basis in 1892. His
work led to the development of the germ theory of disease.
 Louis Pasteur is known as the “Father of Modern Microbiology / Father of
Bacteriology.
 John Tyndall (1820 – 1893): An English physicist, deal a final blow to spontaneous
generation in 1877. He conducted experiments in an aseptically designed box to prove
 that dust indeed carried the germs. He demonstrated that if no dust was present, sterile
broth remained free of microbial growth for indefinite period even if it was directly
exposed to air. He discovered highly resistant bacterial structure, later known as
endospore, in the infusion of hay. Prolonged boiling or intermittent heating was necessary
to kill these spores, to make the infusion completely sterilized, a process known as
Tyndallisation.
 Around the same time that Pasteur was doing his experiments, a doctor named Robert
Koch was working on finding the causes of some very nasty animal diseases (first
anthrax, and then tuberculosis). He gave the first direct demonstration of the role of
bacteria in causing disease. He was a german physician who first of all isolated anthrax
bacillus (Bacillus anthracis, the cause of anthrax) in 1876. He perfected the technique of
isolating bacteria in pure culture. He also introduced the use of solid culture media in
1881 by using gelatin as a solidifying agent. In 1882 he discovered Mycobacterium
tuberculosis. He proposed Koch postulate which were published in 1884 and are the
corner stone of the germ theory of diseases and are still in use today to prove the etiology
(specific cause) of an infectious disease.

Koch’s four postulates are:

 The organism causing the disease can be found in sick individuals but not in healthy
ones.
 The organism can be isolated and grown in pure culture.
 The organism must cause the disease when it is introduced into a healthy animal.
 The organism must be recovered from the infected animal and shown to be the same as
the organism that was introduced.
 The combined efforts of many scientists and most importantly Louis Pasteur and Robert
Koch established the Germ theory of disease. The idea that invisible microorganisms are
the cause of disease is called germ theory. This was another of the important
contributions of Pasteur to microbiology. It emerged not only from his experiments
disproving spontaneous generation but also from his search for the infectious organism
(typhoid) that caused the deaths of three of his daughters.
  Fanny Angelina Hesse (1850-1934) one of Koch’s assistant first proposed the use of
agar in culture media. Agar was superior to gelatin because of its higher melting (i.e.
96°C) and solidifying (i.e. 40-45°C) points than gelatin and was not attacked by most
bacteria. Koch’s another assistant Richard Petri in 1887 developed the Petri dish (plate), a
container used for solid culture media. Thus contribution of Robert Koch, Fanny
Angelina Hesse and Richard Petri made possible the isolation of pure cultures of
microorganisms and directly stimulated progress in all areas of microbiology.

Development in Medicine and Surgery

 Lord Joseph Lister (1827-1912): A famous English surgeon is known for his notable
contribution to the antiseptic treatment for the prevention and cure of wound infections.
Lister concluded that wound infections too were due to microorganisms. In 1867, he
developed a system of antiseptic surgery designed to prevent microorganisms from
entering wounds by the application of phenol on surgical dressings and at times it was
sprayed over the surgical areas.
 Because of this notable contribution, Joseph Lister is known as the Father of Antiseptic
surgery.

Development of Vaccines

 Edward Jenner (1749-1823) an English physician was the first to prevent small pox. He
was impressed by the observation that countryside milk maid who contacted cowpox
(Cowpox is a milder disease caused by a virus closely related to small pox) while milking
were subsequently immune to small pox. On May 14th , 1796 he proved that inoculating
people with pus from cowpox lesions provided protection against small pox. Jenner in 1798,
published his results on 23 successful vaccinators. Eventually this process was known as
vaccination, based on the latin word ‘Vacca’ meaning cow. Thus the use of cow pox virus to
protect small pox disease in humans became popular replacing the risky technique of
immunizing with actual small pox material.
 Jenner’s experimental significance was realized by Pasteur who next applied this principle to
the prevention of anthrax and it worked. He called the attenuated cultures vaccines (Vacca =
cow) and the process as vaccination. Encouraged by the successful prevention of anthrax by
vaccination, Pasteur marched ahead towards the service of humanity by making a vaccine for
 hydrophobia or rabies (a disease transmitted to people by bites of dogs and other animals).
As with Jenner’s vaccination for small pox, principle of the preventive treatment of rabies
also worked fully which laid the foundation of modern immunization programme against
many dreaded diseases like diphtheria, tetanus, pertussis, polio and measles etc.
 Elie Metchnikoff (1845-1916) proposed the phagocytic theory of immunity in 1883. He
discovered that some blood leukocytes, white blood cells (WBC) protect against disease by
engulfing disease causing bacteria. These cells were called phagocytes and the process
phagocytosis. Thus human blood cells also confer immunity, referred to as cellular
immunity.
 The credit for the discovery of this first ‘wonder drug’ penicillin in 1929 goes to Sir
Alexander Fleming of England, a Scottish physician and bacteriologist. Fleming had been
actually interested in searching something that would kill pathogens ever since working on
wound infections during the first world war (1914-1918).
 Antibiotics were discovered completely by accident in the 1920s, when a solid culture in a
Petri dish (called a plate) of bacteria was left to sit around longer than usual. As will happen
with any food source left sitting around, it became moldy, growing a patch of fuzzy fungus.
The colonies in the area around the fungal colony were smaller in size and seemed to be
growing poorly compared to the bacteria on the rest of the plate. The compound found to be
responsible for this antibacterial action was named penicillin. The first antibiotic, penicillin
was later used to treat people suffering from a variety of bacterial infections and to prevent
bacterial infection in burn victims, among many other applications. In this way, Sir
Alexander Fleming in 1929 discovered the first antibiotic penicillin.

Important Contributors in Microbiology

Louis Pasteur

 Louis Pasteur is known as the “Father of Modern Microbiology / Father of Bacteriology. He

has many contributions to microbiology:
 He has proposed the principles of fermentation for the preservation of food.
 He introduced sterilization techniques and developed steam sterilizers, hot air oven, and
autoclave.
 He described the method of pasteurization of milk.
  He had also contributed for designing the vaccines against several diseases such as anthrax,
fowl cholera, and rabies
 He disproved the theory of spontaneous generation of disease and postulated the ‘germ
theory of disease’. He stated that disease cannot be caused by bad air or vapor, but it is
produced by the microorganisms present in the air.
 Liquid media concept- He used nutrient broth to grow microorganisms.
 He was the founder of the Pasteur Institute, Paris.

Robert Koch

 Robert Koch provided remarkable contributions to the field of microbiology:

 He used solid media for the culture of bacteria-Eilshemius Hesse, the wife of Walther Hesse,
one of Koch’s assistants had suggested the use of agar as a solidifying agent.
 He also introduced methods for isolation of bacteria in pure culture.
 Described the hanging drop method for testing motility.
 Discovered bacteria such as the anthrax bacilli, tubercle bacilli, and cholera bacilli.
 Introduced staining techniques by using aniline dye.
 Koch’s phenomenon: Robert Koch observed that guinea pigs already infected with tubercle
bacillus developed a hypersensitivity reaction when injected with tubercle bacilli or its
protein. This reaction is called Koch’s phenomenon.
 According to Koch’s postulates, a microorganism can be accepted as the causative agent of
an infectious disease only if the following conditions are fulfilled:
 i. The microorganism should be constantly associated with the lesions of the disease.
 Ii. It should be possible to isolate the organism in pure culture from the lesions of the disease.
 Iii. The same disease must result when the isolated microorganism is inoculated into a
suitable laboratory animal.
 Iv. It should be possible to re-isolate the organism in pure culture from the lesions produced
in the experimental animals.

Other Important Contributors in Microbiology

1. Antonie Philips van Leeuwenhoek: Discovered single-lens microscope and named organisms as
‘Little animalcules’.
2. Edward Jenner: Developed the first vaccine of the world, the smallpox vaccine by using the
cowpox virus.

3. Joseph Lister: Joseph Lister is considered to be the father of antiseptic surgery. He used carbolic
acid during surgery.

4. Hans Christian Gram: He developed a ‘Gram stain’.

5. Ernst Ruska: He was the founder of the electron microscope.

6. Alexander Fleming: He discovered the antibiotic penicillin.

7. Elie Metchnikoff: He described phagocytosis and termed phagocytes.

8. Kleinberger: He described the existence of L forms of bacteria.

MICROBIAL WORLD

Methods for microbial diversity studies

Methods for studying microbial diversity have been developed over the years and can be divided
into traditional research methods and modern molecular biology methods. The traditional
microbial community analysis method is based on microbial isolation and pure culture, and the
community structure is understood through microscopic observation and physiological and
biochemical characteristics study of pure microorganisms, and several non-culture methods are
gradually developed in response to the limitations of culture methods to study the types and
quantities of microorganisms. These methods are broadly classified as biochemistry, physiology,
etc.
Modern molecular biology techniques mainly include denaturant gradient gel electrophoresis
(DGGE)/temperature gradient gel electrophoresis (TGGE), restriction fragment length
polymorphism (RFLP)/terminal restriction fragment length polymorphism (T-RFLP), Single
Strand Conformation Polymorphism (SSCP), DNA microarrays, fluorescent in situ hybridization
(FISH), metagenomic analysis, high-throughput technology etc.

1) Denaturant gradient gel electrophoresis/temperature gradient gel

electrophoresis(DGGE/TGGE)

DGGE is used to analyze the diversity and relative richness of microorganisms by typing PCR
products by electrophoresis. The principle of electrophoresis is used to separate DNA fragments
by either a temperature or chemical gradient to denature the sample as it moves across an
acrylamide gel. DNA segments which are same in length but have different base-pair sequences
can be separated using these techniques. The separation is based on the difference in mobility of
partially melted DNA molecules in acrylamide gels containing a linear gradient of DNA
denaturants (urea and formamide). Hence, DNA sequences having a difference in only one base-
pair can be separated by DGGE. TGGE employs the same principle as DGGE, but in this
method, the gradient is temperature rather than chemical denaturants. DGGE was originally
developed as a technique to study mutational sites in DNA sequences. In 1993, Muyzer et al.
(1993) first applied DGGE to the study of microbial genetic diversity. PCR-DGGE a culture-
independent approach that has been used to analyze microbial community structure across
different fields, such as food microbiology, oral microbiology, soil microorganisms,
environmental microbiology, and other areas (Sidira et al., 2014; de Paula et al., 2014; Jonathan
et al., 2013; Zhong et al., 2014).

2) Restriction fragment length polymorphism/ terminal restriction fragment length

polymorphism (RFLP/T-RFLP)

Another approach to study microbial diversity is restriction fragment length polymorphism

(RFLP). PCR-RFLP is a combination of PCR technology, RFLP analysis and electrophoresis.
First, the target DNA fragment to be detected is replicated and amplified, and then the amplified
product is digested by DNA restriction endonuclease. Finally, it is analyzed by electrophoresis
whether the target DNA fragment is cut or not. Another variant of this technique is terminal
RFLP that addresses some of the limitations of RFLP by providing an alternate method for rapid
analysis of microbial community diversity in different environments. Kanokratana P et al. (2004)
used RFLP to study prokaryotic diversity in the Bor Khlueng hot springs, Thailand; Everroad R
C et al. (2012) used T-RFLP analysis to study the effect of temperature on microbial diversity in
Nakabusa hot springs, Japan, in order to better understand the biogeography and relationship
between temperature and community structure within microbial mats. Based on the principal of
RFLP, there are other techniques that were used sporadically. They are RISA and the automated
version called ARISA. Here, the intergenic spacer region between the 16S and 23S ribosomal
subunits is amplified by PCR and separated on a polyacrylamide gel under denaturing
conditions. This is found to have differentiating potential for bacterial strains and closely related
species (Fisher and Triplett, 1999).

3) Single strand conformation polymorphism (SSCP)

Single strand conformation polymorphism (SSCP) also relies on electrophoretic separation based
on differences in DNA sequences and allows differentiation of DNA molecules having the same
length but different nucleotide sequences. This technique was originally developed to detect
known or novel polymorphisms or point mutations in DNA (Peters et al., 2000). In this method,
single-stranded DNA separation on polyacrylamide gel was based on differences in mobility
resulted from their folded secondary structure (Heteroduplex) (Lee et al., 1996). As formation of
folded secondary structure or heteroduplex and hence mobility is dependent on the DNA. SSCP
also suffers from the limitations as like DGGE, and hence the same DNA sequence can produce
multiple bands on the gel. However, it does not require gradient gel and has been used for
diversity studies (Stach et al., 2001).

4) Microarrays

Gene microarrays have been used to analyze the composition and diversity of microorganisms in
samples for nearly 20 years, and have undergone several generations of improvement. The
commonly used microarrays include PhyloChip (used to identify microorganisms and their
phylogenetic relationships and analyze the diversity of microorganisms) and Functional GeoChip
(used to study the diversity of functional genes and the activity of functional microorganisms).
Microarray is a chip filled with probes (known short sequences for hybridization with sample
DNA), which provide phylogenetic information or functional property information of organisms,
or both. When the sequence in the sample (the fluorescence-labeled PCR product of the target
fragment of DNA or RNA of pure bacteria and environmental samples or the fluorescence-
labeled PCR product of random primers) hybridizes with the probe, the relative fluorescence
ratio of the sequence matched with the probe can be calculated, so as to obtain the diversity and
relative richness information of microorganisms. Gene microarray method is especially suitable
for identifying the differences of representative microorganisms or microbial communities
between different times, places and treatments. In addition, PhyloChip can also quantify the
changes of related functional genes such as C, N, S and P cycles, degradation of organic
pollutants and stress response. Aronson et al. (2013) used the third generation PhyloChip(16S
rRNA gene microarray, which can provide about 60,000 different OTU information) to study the
microorganisms related to methane cycle in pine forest soil under different experimental
conditions.

5) Fluorescent in situ hybridization (FISH)

FISH is one of the most popular methods for DNA hybridization. Nucleic acid hybridization
using specific probes is an important qualitative and quantitative tool in molecular bacterial
ecology (Clegg et al., 2000). These hybridization techniques can be done on extracted DNA or
RNA, or in situ. The sample is lysed to release all nucleic acids. Dot-blot hybridization with
specific and universal oligonucleotide primers is used to quantify rRNA sequences of interest
relative to total rRNA. The relative abundance may represent changes in the abundance in the
population or changes in the activity and hence the amount of rRNA content (Theron and Cloete,
2000). Spatial distribution of bacterial communities in different environments such as biofilms
can be determined using FISH (Schramm et al., 1996).

6) Metagenomic analysis

Metagenomics is defined as the functional and sequence-based analysis of the collective

microbial genomes that are contained in an environmental sample (Zeyaullah et al., 2009). This
contains all the genomes from coexisting microbes-called microbial communities. This is further
subjected to the sequencing to know about the species composition in the sample. This provides
a comprehensive view of the genetic diversity, species composition, evolution, and interactions
with the environment of natural microbial communities (Simon and Daniel, 2011).
7) High-throughput technology

Sanger sequencing has been used for decades, and the sequencing quality is high, with a
sequence length of 750—1000 bp. This method can’t measure the mixed sequence, so it is
necessary to establish a clone library first, that is, transfer the target sequence to the host cell for
culture to form a single strain, and then sequence the target sequence in the single strain. It is
time-consuming and expensive to analyze environmental samples with high microbial diversity.

On the basis of Sanger sequencing, high-throughput sequencing, also called next-generation

sequencing (NGS), was developed. This method used PCR products of microbial target genes as
samples to sequence, and tens of thousands to millions of sequences were obtained in one
reaction, which greatly improved the breadth and depth of sequencing. At present, amplicon
sequencing is commonly used methods for microbial diversity analysis in environmental
samples, namely, 16S/18S/ITS sequencing. These high-throughput sequencing methods can
identify the sequences of different samples by labeling the primers of each sample, and can
simultaneously analyze multiple environmental samples and obtain a large number of sequences.