in the form of prebiotics
Module 2: Introduction to Microbiology
Microbiology
- the scientific study of
microorganisms (living things that
are too small to be seen with the
naked eye).
- concerned primarily with the
agents of infectious disease, the
immune response, the search for
chemotherapeutic agents and
bacterial metabolism” in the early
part of the twentieth century
Why study microbes?
- Microbes benefit humans and
animals
- Microbes aid in nutrient recycling
- Microbes as food for humans and
other organisms
- Microbes as sources of medicine
- Microbes cause diseases
How do trillions of microbes affect every
stage of our life - from birth to old age as
the impact of these microorganisms on
our well-being becomes clearer, scientist
say new remedies for disease are likely to
emerge
- “ innate as a child’s temperament
might be related to whether the
bacteria in an infant’s gut are
predominantly from one genus: the
… Bifidobacterium … with the
highest proportion of
Bifidobacterium organisms at two
months were more likely at six
months to exhibit a trait the
researchers called “positive
emotionality.”
- “In the not-too-distant future,
according to the most enthusiastic
researchers, it might be routine to
deliver a dose of healthy microbes
(compounds that act as a
substrate on which beneficial
microbes can grow), probiotics (the
beneficial microbes themselves),
or fecal transplants (microbe-rich
feces from healthy donors)—
helping us realize the promise of
operating at top form, from the
inside out.
Progressive increase of physical activity
level generates changes in intestinal
microbiota
- exercise affiliated with greater
microbial diversity in the intestine
…generally regarded as a marker
of health.
- diverse microbiomes seem to do a
better job fighting off illness and
staving off chronic disease,
- the microbiota is an important
component of whether or not our
bodies even respond to exercise –
exercise resistance
a. associated with a diabetic disease
course
b. SCFA caused improvement in their
insulin sensitivity after a regimen of
physical activity.
c. Diabetis 2 – resistance to insulin
Aids in the digestion of cellulose in
Ruminants - Nutrient Recycling
Nitrogen Cycle
 Bioremediation Brief History

- Definition: Use of living organisms A. First Observations

to transform, destroy, or
immobilize contaminants
- Goal: Detoxification of the parent
compounds and conversion to
products that are no longer
hazardous to human health and
the environment

Fermentation of Products
Robert Hooke (1635-1703)
- an English mathematician and
natural historian coined the term
“cells” to describe the “little boxes”
he observed in examining cork
slices with a compound
microscope.
- the first to make a known
description of microorganisms and
recorded in his book
Micrographia.

Antony van Leeuwenhoek (1973)

Pathogens - a draper and an amateur
microscope builder.
- he learned lens grinding as a
hobby and made over 100 simple
microscopes each capable of
magnifying an image about 300
times.
- the first person to publish
extensive and accurate
observations of microorganisms.
- known as the father of bacteriology
- “Animalcules”

Spontaneous Generation
Vaccine Efficacy
- The Greek philosopher Aristotle
- efficacy is a measurement of how
(384–322 BC) was one of the
much a vaccine lowers the risk of
earliest recorded scholars to
an outcome.
articulate the theory of
- Vaccinated group has only 18
spontaneous generation, the
covid-19 cases vs. 63 covid-19
notion that life can arise from
cases under the placebo ---- a
nonliving matter.
drop of 45 cases or 71.4 % =
efficacy
 - Aristotle proposed that life arose
from nonliving material if the
material contained pneuma (“vital
heat”).
- As evidence, he noted several
instances of the appearance of
animals from environments
previously devoid of such animals,
such as the seemingly sudden
appearance of fish in a new puddle
of water
- Sparked by the discovery of
”animalcules’ by Leeuwenhoek.
- A long-held theory that life could
arise spontaneously from non-
living or decaying organic matter.
Francesco Redi (1626-1678)
- Strong opponent of Abiogenesis
- Italian physician Francesco Redi
(1626–1697), performed an
experiment in 1668 that was one of
the first to refute the idea that
maggots (the larvae of flies)
spontaneously generate on meat
left out in the open air
John Needham (1713 - 1781)
- published a report of his own
experiments, in which he briefly
boiled broth infused with plant or
animal matter, hoping to kill all
preexisting microbes. He then
sealed the flasks.
- Abiogenesis occurred due to the
random “clumping of organic
molecules”
Abiogenesis
Lazaro Spallanzani (1729-1799)
- Microorganisms in the air entered
in Needham’s experiment
- did not agree with Needham’s
conclusions, however, and
performed hundreds of carefully
executed experiments using
heated broth.
- As in Needham’s experiment,
broth in sealed jars and unsealed
jars was infused with plant and
animal matter.
- Spallanzani’s results contradicted
the findings of Needham: Heated
but sealed flasks remained clear,
without any signs of spontaneous
growth, unless the flasks were
subsequently opened to the air.
- This suggested that microbes were
introduced into these flasks from
the air.
- In response to Spallanzani’s
findings, Needham argued that life
originates from a “life force” that
was destroyed during
Spallanzani’s extended boiling.
- Any subsequent sealing of the
flasks then prevented new life
force from entering and causing
spontaneous generation
Rudolf Virchow (1821-1902)
- Cells arise from pre-existing living
things
Louis Pasteur (1822-1895)
 - Swan-neck flasks show the
spontaneous generation does not
occur
1876
Golden Age of Microbiology (1857-1914)
1858
- Pasteur 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.
1862
- Pasteur 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. This led to
the development of the germ
theory of disease. Became known
as the “Father of Modern
Microbiology / Father of
Bacteriology.
1867
- Lord Joseph Lister (1827-1912)
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
- Robert Koch worked on finding the
causes of some very nasty animal
diseases (first anthrax (1876), and
then tuberculosis (1882)). He gave
the first direct demonstration of the
role of bacteria in causing disease.
- 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:
1. The organism causing the disease
can be found in sick individuals but
not in healthy ones.
2. The organism can be isolated and
grown in pure culture.
3. The organism must cause the
disease when it is introduced into a
healthy animal.
4. The organism must be recovered
from the infected animal and
shown to be the same as the
organism that was introduced.
PPT NOTES

Fermentation and pasteurization
- microorganisms called yeasts
convert the sugars to alcohol in the
absence of air, - fermentation used
to make wine and beer – by
Pasteur
- Wine souring is spoilage (alcohol
turn to vinegar).
- heat the beer and wine to kill most
of the bacteria that caused the
spoilage - pasteurization.
- Importance of discovery: Aerobes
and anaerobes & Links disease
and microbes
Germ Theory of disease
- A theory that proposes that
microorganisms are the cause of
many diseases
- 1835; Bassi proved silkworm
disease caused by a fungus.
- 1865; Pasteur found protozoan as
recent cause.
- 1840; Sammelweis - physicians
with undisinfected hands transmits
infection causing childbirth fever
- 1860; used carbolic acid (phenol)
to treat surgical wounds (aseptic
surgery)
- 1870; Koch’s Postulates linking
microbes and disease.
Koch’s Postulates
- Koch's Postulates are used to
prove the cause of an infectious
disease
- An organism can be isolated from
a host suffering from the disease
- The organism can be cultured in
the laboratory
- The organism causes the same
disease when introduced into
another host
- The organism can be re-isolated
from that host
Koch and Pure Cultures (Other
Discoveries)
- Used potato slice before – able to
grow pure culture.
- Then gelatinized solutions
- Latest - agar was suggested by
Walter Hesse (originally used by
Fannie Hesse) – able to
- Richard Petri (1887) - development
of the transparent double-sided
dishes that bear his name.
- Petri dishes can be stacked and
sterilized separately from the
medium.
- Colonies retained access to air
without direct exposure to air and
could easily be manipulated for
further study.
Limits of Application of Koch’s
Postulates
- Agents do not cause disease to
other non-human hosts.
- Not all diseases have microbial
origins: Genetic, Degenerative,
Congenital, nutritional def.
- Some microbes are not culturable
in the lab.
- Treponema / Rickettsia /
Chlamydia / viruses
- Some pathogens cause many
different diseases in one or many
hosts.
- Ethical considerations in
introducing pathogen to healthy
host
1877
- John Tyndall made the final blow
to spontaneous generation.
 - 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 Tyndallization.
1881
- Fanne Eilshemius 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.
1883
- Elie Metchnikoff (1845-1916)
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.
1887
- 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,
Fannie Hesse and Richard Petri
made possible the isolation of pure
cultures of microorganisms and
directly stimulated progress in all
areas of microbiology.
- Winogradsky discovered bacterial
sulfide oxidation for which he first
became renowned, including the
first known form of lithotrophy. His
work on nitrogen cycling includes
chemosynthesis and the
Winogradsky column.
1890
- Emile Roux (1853-1933) and
Alexandre Yersin discovered
tetanus (lock jaw) antitoxin. Only
about a week after the
announcement of the discovery of
tetanus antitoxin, Von Behring in
1890 reorted on immunization
against diphtheria by diphtheria
antitoxin.
- The discovery of toxin-antitoxin
relationship was very important to
the development of science of
immunology.
1892
- Dmitri Ivanowski made the first
evidence of the filterability of a
pathogenic agent, the virus of
tobacco mosaic disease.
- His work had launched the
emergence of virology.
1898
- Beijerinck demonstrated that
tobacco mosaic virus is caused by
an infectious agent smaller than a
bacterium.
1904
- Paul Ehrlich (1854-1915) found
that the dye Trypan Red was
active against the trypanosome
that causes African sleeping
sickness and could be used
therapeutically.
 - This dye with antimicrobial activity
was referred to as a ‘magic bullet’.
1910
- Ehrlich in collaboration with
Sakahiro Hata, a japanese
physician, introduced the drug
Salvarsan (arsenobenzol) as a
treatment for syphilis caused by
Treponema pallidum.
1935
- Gerhard Domagk experimented
with numerous synthetic dyes and
reported that Prontosil, a red dye
used for staining leather, was
active against pathogenic,
Streptococci and Staphylococci in
mice even though it had no effect
against that same infectious agent
in a test tube.
1928
- Sir Alexander Fleming discovered
a ‘wonder drug’ called penicillin
Modern Developments in Microbiology
(Brooks 2013)
1931
- Knoll and Ruska developed the
first prototype electron microscope
capable of fourhundred-power
magnification.
1941
- Beadle and Tatum proposed the
one gene-one enzyme hypothesis.
This is the idea that genes act
through the production of
enzymes, with each gene
responsible for producing a single
enzyme that in turn affects a single
step in a metabolic pathway.
1943
- Luria and Delbruck demonstrate
that in bacteria, genetic mutation
arises in the absence of selection,
rather than being a response to
selection.
1944
- Waksman discovered another
antibiotic, streptomycin produced
by two strains of actinomycete,
Streptomyces griseus
- Avery, MacLeod and McCarty
demonstrated that DNA is the
substance that causes bacterial
transformation, in an era when it
had been widely believed that it
was proteins that served the
function of carrying genetic
information. It was first described
in Griffith’s experiment of 1928.
1953
- Watson and Crick completed their
DNA model, which is now
accepted as the first correct model
of the double-helix
1960
- Joseph Gall and Mary Lou Pardue
developed radioactively labelled
hybridisation probes. Hybridisation
probes are DNA or RNA fragments
which can bind to complementary
sequences in the microbial
chromosome.
- More user-friendly fluorophores
replaced the radioactive labels
leading to the development of
fluorescence in situ hybridisation
(FISH).
1977
- Carl Woese studied ribosomal
genes that led to the first
scientifically based tree of life.
- His work paved the way for a new
method of identifying microbes
based on the nucleotide sequence
of the genes encoding the small
 16S ribosomal RNA subunit for
bacteria and the 18S rRNA subunit
for eukaryotic organisms such as
fungi.
- World Health Organization
eradicates smallpox
1983
- Luc Montaigner and Robert Gallo -
HIV as causative agent of AIDS.
- Kary Mullis developed polymerase
chain reaction (PCR) that enables
a target stretch of DNA to be
copied thousands or millions of
times.
vaccines for cholera, anthrax,
rabies
Act of Vaccination by Jenner
- Edward Jenner used cowpox-
infected material obtained from the
hand of Sarah Nemes, a milkmaid
from his home village of Berkley in
Gloucestershire to successfully
vaccinate 8 years old James
Phipps
- Jenner challenged the boy by
deliberately inoculating him with
material from a real case of small
pox. He did not become infected!

Gram Staining
- Gram was searching for a method
that would allow visualization of
cocci in tissue sections of lungs of
those who had died of pneumonia
Chemotherapy
- the use of substances (natural or
1984
synthetic) to treat disease.
- Marshall discovered Helicobacter
- In its non-oncological use, the term
pylori
may also refer to antibiotics
(antibacterial chemotherapy).
1995
- Principle: some chemicals are
- First complete genetic sequence of
more toxic to microbes than their
a bacterium is published
hosts
- Contributors: Paul Ehrlich (”magic
bullet” Salvarsan against syphilis)
PPT NOTES
(1910) Domagk (Sulfonamides)
Vaccination
(1930s) & Alexander Fleming
- process of administering
(Penicillin – first antibiotic, 1928)
pathogens that cannot reproduce
(due to being weakened or dead)
in a healthy person or animal, the
intent is to confer immunity against
a targeted disease agent (or
related).
- Contributor: Edward Jenner (1796)
(use of cowpox virus to immunize
against smallpox virus) – first one
- 1880: – Pasteur discovered how
vaccines work; developed
 virus in test tube cultures of human
Second Golden Age (1943-1970) tissues. 1954

- Microbial Genetics Era - a race to Alfred Hershey and Martha Chase

the discovery of DNA as the suggest that the only DNA is needed for
genetic material. viral replication (1950’s) - DNA as the
- Important discoveries: genetic material rather than protein
Transformation in bacteria,
Nucleotides make up DNA, DNA Joshua Lederberg and Esther Lederberg
as the genetic material, DNA publish their replica plating method and
structure provide firm evidence that mutations in
bacteria yielding resistance to antibiotics
Griffth’s Experiment - Stepping Stone and viruses are not induced by the
for the discovery of genetic material presence of selective agents.
(1928)

- Oswald Avery, Colin MacLeod,

and Maclyn McCarty show that
DNA is the transforming material in
cells. (1944)
- Albert Schatz, E. Bugie, and - James Watson and Francis Crick
Selman Waksman discover publish a description of the
streptomycin, soon to be used double-helix structure of DNA
against tuberculosis (1952) (1953)

Joshua Lederberg and Edward L.

Tatum publish on conjugation in
bacteria.

- Microbiologist John Franklin

Enders, virologist Thomas H.
Weller and physician Frederick
Chapman Robbins together
develop a technique to grow polio
- Peter Mitchell proposes the
chemiosmotic theory in which a
 molecular process is coupled to
the transport of protons across a
biological membrane 1978
- Francois Jacob, David Perrin,
Carmen Sanchez and Jacques
Monod propose the operon
concept for control of bacteria
gene action.
- Marshall Nirenberg and J.H.
Matthaei observe that a synthetic
polynucleotide, poly U, directs the
synthesis of a polypeptide
composed only of phenylalanine
Modern Developments
Microbial Genetics
- Microorganisms have served as
important biochemical and genetic
model systems
- Understanding the molecular role
of DNA in the hereditary process
occurred as a consequence of
studies employing
microorganisms.
- Recombinant DNA and
engineering, DNA technology
Bacteria for insulin , Yeast for hepa
vaccine
- Howard Temin and David
Baltimore independently discover
reverse transcriptase in RNA
viruses 1975
- Stanley Cohen, Annie Chang,
Robert Helling, and Herbert Boyer
show that if DNA is broken into
fragments and combined with
plasmid DNA, such recombinant
DNA molecules will reproduce if
inserted into bacterial cells
Fields of Study / Scope
Agricultural Microbiology
- study to combat plant diseases
that attack important food crops,
work on methods to increase soil
fertility and crop yields etc.
- Currently there is a great interest
in using bacterial or viral insect
pathogens as substitute for
chemical pesticides
Microbial Ecology nutrient cycling, geomicrobiology,
- study of biogeochemical cycles microbial diversity and
and bioremediation to reduce bioremediation.
pollution effects - Characterization of key bacterial
habitats such as the rhizosphere
Microbial Physiology and phyllosphere
- The study of how the microbial cell
functions biochemically. Evolutionary Microbiology
- Includes the study of microbial - The study of the evolution of
growth, microbial metabolism and microbes.
microbial cell structure - Includes the study of bacterial
Microbial Genetics systematics and taxonomy.
- The study of how genes are
organized and regulated in Industrial Microbiology
microbes in relation to their cellular - industrial fermentation and
functions. wastewater treatment. Closely
- Closely related to the field of linked to the biotechnology
molecular biology industry.
- brewing, an important application
Genetic Engineering of microbiology.
- arisen from work of microbial - Antibiotics, vitamins,
genetics and molecular biology. pharmaceuticals
Engineered microorganisms are
used to make hormones, Aeromicrobiology
antibiotics, vaccines and other - The study of airborne
products. New genes can be microorganisms.
inserted into plants and animals
Food Microbiology
Medical Microbiology - The study of microorganisms
- The study of the role of microbes causing food spoilage.
in human illness.
- Includes the study of microbial
pathogenesis and epidemiology
and is related to the study of
disease pathology and Pharmaceutical microbiology
immunology - the study of microorganisms
causing pharmaceutical
Veterinary Microbiology contamination and spoilage.
- The study of the role of microbes
in veterinary medicine or animal Parasitology
taxonomy - The study of parasites, their hosts,
and the relationship between them.
Environmental Microbiology - The focus of study is on
- The study of the function and relationship and NOT the
diversity of microbes in their organisms.
natural environments. - Protozoology - Focuses on
- Includes the study of microbial protozoans
ecology, microbe-mediated
- Helminthology Focuses of - Beijerinck and Winogradsky
helminths studied bacteria that inhabit soil
and water.
- In the middle to latter part of the
Key takeaways twentieth century, basic and
applied subdisciplines of
- Microorganisms, which include all microbiology emerged; these have
single-celled microscopic led to the current era of molecular
organisms and the viruses, are microbiology.
essential for the well-being of the - Some of the notable National
planet and its plants and animals. Scientists and Academicians
- Metabolism, growth, and evolution worked on microbiological
are necessary properties of living problems and were given credits to
systems. Cells must coordinate improve health, agriculture and
energy production and ecology
consumption with the flow of
genetic information during cellular
events leading up to cell division.
- Bacteria, Archaea, and Eukarya
are the major phylogenetic
lineages of cells.
- Microorganisms can be both
beneficial and harmful to humans,
although many more
microorganisms are beneficial or
even essential than are harmful.
- Robert Hooke was the first to
describe microorganisms, and
Antoni van Leeuwenhoek was the
first to describe bacteria.
- Louis Pasteur is best remembered
for his ingenious experiments
showing that living organisms do
not arise spontaneously from
nonliving matter. He developed
many concepts and techniques
central to the science of
microbiology, including
sterilization.
- Robert Koch developed a set of Module 3: Taxonomy and Systematics
criteria anchored in
experimentation—Koch’s
postulates—for the study of Introduction
infectious diseases and developed
the first methods for growth of pure - Taxonomy and systematics are
cultures of microorganisms. two concepts related to the study
of diversification of living forms and
 the relationships of living things
through time.
- Taxonomy involves not just
naming organisms but grouping
them with other organisms that
share common properties.
- The main difference between
taxonomy and systematics is that
taxonomy is involved in the
classification and naming of
organisms whereas systematics is
involved in the determination of
evolutionary relationships of
organisms.
- This means systematics ascertain
the sharing of the common
ancestry by din taxonomy, different
organisms are scientifically named
and grouped in different taxonomic
levels. In systematics, organisms
are grouped based on their
evolutionary relationships.
- Taxonomy can be considered as a
branch of systematics. Both
taxonomy and systematics use
morphological, behavioral,
genetics, and the biochemical
observations.
- For more reading on the
difference, refer to this fferent
organisms
History of Classification System
- In the early days, classification
appeared relatively
straightforward, with all living
things apparently fitting into one of
two kingdoms of plants and
animals. Most of us are
accustomed to the Linnaean
system of classification that
assigns every organism a prokaryotic cells and one type of
kingdom, phylum, class, order,
family, genus, and species, which,
among other possibilities, has the
handy mnemonic King Philip Came
Over For Good Soup.
- It was in 1735 when the two-
kingdom system, Plantae and
Animalia was formally introduced
by Swedish botanist Carolus
Linnaeus. This system was
created long before scientists
understood that organisms
evolved.
- The inclusion of microorganism in
the Linnean system became
apparent in 1857 when Carl von
Nägeli, proposed to include
bacteria and fungi in the plant
kingdom. In 1866, Ernst Haeckel
proposed the Kingdom Protista, to
include bacteria, protozoa, algae,
and fungi. The term prokaryote
was then introduced in 1937 by
Edouard Chatton to distinguish
cells having no nucleus from the
nucleated cells of plants and
animals, with the current definition
provided by Roger Stanier in 1961
- “cells in which the nuclear
material (nucleoplasm) is not
surrounded by a nuclear
membrane”.
- In 1968, Robert G.E. Murray
proposed the Kingdom
Prokaryotae. In 1969, Robert H.
Whittaker founded the five-
kingdom system in which
prokaryotes were placed in the
Kingdom Prokaryotae, or Monera,
and eukaryotes comprised the
other four kingdoms. The Kingdom
Prokaryotae had been based on
microscopic observations.
Subsequently, new techniques in
molecular biology revealed that
there are actually two types of
eukaryotic cell. Increased focus on
the cellular and molecular
similarities and dissimilarities
between organisms led to
 proposals for further refinements to
the five-kingdom system proposed
by Robert Whittaker in 1969.
- Because the Linnaean system is
not based on evolution, most
biologists are switching to a
classification system that reflects
the organisms' evolutionary
history. Phylogenetic classification
system names only clades —
groups of organisms that
descended from a common
ancestor
Phylogenetic Tree of Life
- DNA sequence-based
phylogenetic analysis has revealed
that the five kingdoms do not
represent five primary evolutionary
lines. Instead, cellular life on Earth
has evolved along three primary
lineages, called domains (Figure
1). Two of these domains, the
Bacteria and the Archaea, are
exclusively composed of
prokaryotic cells. The Eukarya
contains the eukaryotes including
the plants, animals, fungi, and
protists.
- Molecular studies in the 1970s
revealed that the Archaea differed
from all other bacteria in their 16S
rRNA sequences, as well as in
their cell wall structure, membrane
lipids and aspects of protein
synthesis. These differences were
seen as sufficiently important for
the recognition of a third basic cell
type to add to the prokaryotes and
eukaryotes. This led to the
proposal of a three-domain
scheme of classification, in which
prokaryotes are divided into the
Archaea and the Bacteria.
- Ribosomal RNAs (rRNA), are
excellent tools for discerning
evolutionary relationships because
all cells contain ribosomes (and
thus rRNA), including
microorganisms. Carl Woese, an
American microbiologist,
pioneered the use of comparative
rRNA sequence analysis as a
measure of microbial phylogeny
and, in so doing, revolutionized our
understanding of cellular evolution.
In brief, genes encoding rRNA
from two or more organisms are
sequenced and the sequences
aligned and scored, base-by-base,
for sequence differences and
identities using a computer; the
greater the sequence variation
between any two organisms, the
greater their evolutionary
divergence. Then, using a treeing
algorithm, this divergence is
depicted in the form of a
phylogenetic tree.
- The gene for 16S rRNA which are
highly conserved and highly
variable regions was used to
determine the three lineages.
Based on the analysis, three cell
lineages clearly emerged as cells
were forming 3.5 billion years ago.
This led to the Archaea, the
Bacteria, and what eventually
became the nucleoplasm of the
eukaryotes. The phylogenetic tree
of life reveals two very important
evolutionary facts: (1) all
prokaryotes are not
 phylogenetically closely related, Thermotoga, and the Chloroflexus
and (2) Archaea are actually more group (green non-sulfur bacteria)
closely related to Eukarya than to which do also contain thermophilic
Bacteria. Thus, from the last species. Furthermore after the
universal common ancestor green non-sulfur bacteria, the
(LUCA) of all life forms on Earth, Deinococci, the unique
evolutionary diversification Spirochetes, the phototrophic
diverged to yield the ancestors of green sulfur bacteria, the
the Bacteria and of a second main chemoorganotrophic
lineage. The latter once again Flavobacterium and Cytophaga
diverged to yield the ancestors of groups, the budding Planctomyces
the Archaea, a lineage that and the Verrucomicrobium groups,
retained a prokaryotic cell the Chlamydia, and the genera
structure, and the Eukarya, which Nitrospira and Deferribacter. Other
did not. The universal tree of life major groups include the gram-
shows that LUCA resides very positive bacteria and the
early within the Bacteria domain. Cyanobacteria. The Firmicutes and
the Actinobacteria, which are the
Gram-positive bacteria, come
Domain Bacteria after.

- are shown in such a way that their

phenotypic traits are well known
and studied since they are already
cultured in the laboratory. Although
some lineages are characterized
by unique phenotypic traits, such
as the morphology (the cell shape
of spirochetes, physiology of
cyanobacteria) most species of
bacteria were classified by
molecular analysis since these
bacteria lack phenotypic
cohesiveness, or they share
almost the same traits as the other
species. The largest group, the
Proteobacteria, is a good example
of this, as collectively this group
shows all known forms of microbial
physiology.
- As Figure 2 shows, the most
phylogenetically ancient phylum Domain Archaea
contains the genus Aquifex, which
are hyperthermophilic H2 - Carl Woese, along with his
(Hydrogen) – oxidizing colleagues, constructed the
chemolitotrophs. These were phylogenetic trees for the
followed by the prokaryotes, which showed
Thermodesulfobacterium, evolutionary relatedness. Woese’s
 work also revealed that one group
of prokaryotes are very different
from all the others. This group of
prokaryotes are known as
Archaea, which differ from bacteria
by its cell wall and plasma
membrane chemistry, as well as
the sequences of its 16S rRNA.
- Archaean members show diversity
of both morphological and
physiological characteristics.
However, it was not noticed before
since Archaea remained
unidentified as a separate group
because they did not display any
very obvious morphological
differences from true bacteria like
the main cell shapes for example.
This is a problem before since one
of the first ways of classifying
bacteria are by morphological
characteristics.
- However, as more advances in
microbial research push through,
more unusual shapes of Archaean
members are encountered,
members of the genus Haloarcula
for example, have flattened square
or triangular cells (Figure 3).
Gram-positive and Gram-negative
forms of these members are also
found. However, neither group
possesses a true peptidoglycan.
Some members of the said domain
possess pseudomurein, which is
composed of different substituted
L-amino acids and
polysaccharides. Some archaea,
have cell walls composed of a
layer of proteinaceous subunits
known as an S-layer, which is
directly associated with the cell
membrane. This difference actually
makes the Archaean members not
susceptible to antibiotics such as
penicillin and lysozyme, whose
antibacterial action is specifically
directed to peptidoglycan.
- One of the distinctions of Archaea
are their membranes, in which the
lipid component of the part
contains branched isoprenes
instead of fatty acids. Furthermore,
these isoprenes were joined to
glycerol by ether-linkages rather
than the ester linkages found in
true bacteria. And the last and the
most distinctive characteristic is
that Archaea are found in extreme
environments. Some extreme
thermophiles can live well at over
100 C, while psychrophilic forms of
Archaea can thrive in very cold
environments, like in the Antarctic.
Furthermore, other species of
Archaea are able to live in extreme
alkalinity, salinity, or acidity.
- Initially, scientists thought that
archaea species were only limited
to such extreme environments.
However, recent studies actually
show that species of archaea can
also be observed in the world’s
oceans, and also in terrestrial and
semi terrestrial areas, making up a
part of the bacterial biomass. The
reason that this lay undetected for
so long is that these organisms
cannot as yet be cultured in the
laboratory, and their presence can
only be inferred by the use of
modern DNA-based analysis.
Eukarya
- The oldest known fossils are the
remains of prokaryotes that lived
more than 3.5 billion years ago.
Eukaryotic cells evolved more
recently, about 2.5 billion years
ago. According to the
endosymbiotic theory, eukaryotic
cells evolved from prokaryotic cells
living inside one another, as
endosymbionts (Figure 4.) In fact,
 the similarities between prokaryotic
cells and eukaryotic organelles
provide striking evidence for this
endosymbiotic relationship.
- The original nucleoplasmic cell
was prokaryotic. However,
infoldings in its plasma membrane
may have surrounded the nuclear
region to produce a true nucleus.
Over time, the chromosome of the
nucleoplasm may have acquired
pieces such as transposons. In
some cells, this large chromosome
may have fragmented into smaller
linear chromosomes. Perhaps cells
with linear chromosomes had an
advantage in cell division over
those with a large, unwieldy
circular chromosome. That
nucleoplasmic cell provided the
original host in which
endosymbiotic bacteria developed
into organelles such as
mitochondrion and chloroplast.
However, a major problem with
this hypothesis is that it does not
easily account for the fact that
Bacteria and Eukarya have similar
membrane lipids, in contrast to
those of Archaea. An example of a
modern prokaryote living in a
eukaryotic cell is shown in Figure
5. The cyanobacterium-like cell
and the eukaryotic host require
each other for survival.
Classification and Nomenclature
- Classification is the organization of
- Phylogenetic trees of species in
the domain Eukarya have been
constructed from comparative
sequence analysis of the 18S
rRNA gene, the functional
equivalent of the 16S rRNA gene.
The 18S tree shows some “early-
branching” microbial eukaryotes,
such as the microsporidia and the
diplomonads. By contrast, the
position of these organisms on
multigene phylogenetic trees is
quite different, and shows them to
have arisen during a burst of
evolutionary radiation that led to
most lineages of microbial
eukaryotes. It is likely that this
burst in eukaryotic evolution was
triggered by the onset of oxic
conditions on Earth and
subsequent development of the
ozone shield. The latter would
have greatly expanded the number
of surface habitats available for
colonization. Nevertheless,
although 18S rRNA sequencing
appears to give a skewed view of
eukaryotic microbial evolution, it
still clearly sorts the eukaryotes out
as a distinct domain of life with
evolutionary roots more closely
tied to the Archaea than to the
Bacteria.
organisms into progressively more
inclusive groups on the basis of
either phenotypic similarity or
evolutionary relationship. The
hierarchical nature of classification
is shown in Table 2. A species is
made up of one to several strains,
and similar species are grouped
into genera (singular, genus).
Similar genera are grouped into
 families, families into orders,
orders into classes, up to the
domain, the highest level taxon.
- Nomenclature is the actual naming
of organisms and follows the
binomial system of nomenclature
devised by the Swedish medical
doctor and botanist, Carl Linnaeus,
and used throughout biology;
organisms are given genus names
and species epithets. The names
are Latin or Latinized Greek
derivations, often descriptive of
some key property of the
organism, and are printed in italics.
By classifying organisms into
groups and naming them, we order
the natural microbial world and
make it possible to communicate
effectively about all aspects of
particular organisms, including
their behavior, ecology,
physiology, pathogenesis, and
evolutionary relationships. The
creation of new names must follow
the rules described in The
International Code of
Nomenclature of Bacteria. This
source presents the formal
framework by which Bacteria and
Archaea are to be officially named
and the procedures by which
existing names can be changed,
for example, when new data
warrants taxonomic
rearrangements.
Bergey’s Manual and the Prokaryotes
- Because taxonomy is largely a
matter of scientific judgment, there
is no “official” classification of
Bacteria and Archaea. Presently,
the classification system most
widely accepted by microbiologists
is that of Bergey’s Manual of
Systematic Bacteriology, a major
taxonomic treatment of Bacteria
and Archaea. Widely used,
Bergey’s Manual has served the
community of microbiologists since
1923 and is a compendium of
information on all recognized
prokaryotes. Each chapter, written
by experts, contains tables,
figures, and other systematic
information useful for identification
purposes. A second major source
in bacterial diversity is The
Prokaryotes, a reference that
provides detailed information on
the enrichment, isolation, and
culture of Bacteria and Archaea.
This work is available online by
subscription through university
libraries. Collectively, Bergey’s
Manual and The Prokaryotes offer
microbiologists both the concepts
as well as the details of the biology
of Bacteria and Archaea as we
know it today; they are the primary
resources for microbiologists
characterizing newly isolated
organisms.
- When a new prokaryote is isolated
from nature and thought to be
unique, a decision must be made
as to whether it is sufficiently
different from other prokaryotes to
be described as a new taxon. To
achieve formal validation of
taxonomic standing as a new
genus or species, a detailed
description of the organism’s
characteristics and distinguishing
traits, along with its proposed
 name, must be published, and, as of methods for the identification of
just mentioned, viable cultures of bacteria and description of new
the organism must be deposited in species. This polyphasic approach
at least two international culture to taxonomy uses three kinds of
collections. The manuscript methods—phenotypic, genotypic,
describing and naming a new and phylogenetic— for the
taxon undergoes peer review identification and description of
before publication. A major vehicle bacteria. Phenotypic analysis
for the description of new taxa is examines the morphological,
the International Journal of metabolic, physiological, and
Systematic and Evolutionary chemical characteristics of the cell
Microbiology (IJSEM), the official (Table 3). Genotypic analysis
publication of record for the considers characteristics of the
taxonomy and classification of genome (Table 4). These two
Bacteria and Archaea. In each kinds of analysis group organisms
issue, the IJSEM publishes an based on similarities. They are
approved list of newly validated complemented by phylogenetic
names. By providing validation of analysis, which seeks to place
newly proposed names, organisms within an evolutionary
publication in IJSEM paves the framework.
way for their inclusion in Bergey’s
Manual of Systematic
Bacteriology.

Microbial Systematics

- Systematics is the study of the

diversity of organisms and their
relationships. It links together
phylogenyGE with taxonomy, in
which organisms are
characterized, named, and placed
into groups according to several
defined criteria. Bacterial
taxonomy traditionally has focused
on practical aspects of
identification and description,
activities that have relied heavily
on phenotypic comparisons. At
present, the growing use of genetic
information, especially DNA
sequence data, is increasingly Evolutionary Analysis (Theoretical
allowing taxonomy to reflect Aspects)
phylogenetic relationships as well.
Bacterial taxonomy has changed - The evolutionary history of a group
substantially in the past few of organisms is called its
decades, embracing a combination phylogeny, and a major goal of
 evolutionary analysis is to
understand phylogenetic
relationships. Because we do not
have direct knowledge of the path
of microbial evolution, phylogeny is
inferred indirectly from nucleotide
sequence data. Our premises are
that (1) all organisms are related
by descent, and (2) that the
sequence of DNA in a cell’s
genome is a record of the
organism’s ancestry. Because
evolution is a process of inherited
nucleotide sequence change,
comparative analyses of DNA
sequences allow us to reconstruct
phylogenetic histories. Here, we
examine some of the ways in
which this is carried out.
Genes Employed in Phylogenetic
Analysis
- The most widely used and useful
for defining relationships in
prokaryotes is the gene encoding
16S ribosomal RNA (rRNA) and its
counterpart in eukaryotes, 18S
rRNA, parts of the small subunit of
the ribosome.
Molecular Clocks
- An unresolved question in
phylogenetics is whether DNA
(and protein) sequences change at
a constant rate. The approach to
answering the question focuses on
pairs of homologous sequences—
that is, sequences of shared
evolutionary ancestry that encode
functionally equivalent molecules.
If sequences do change at a
constant rate, such pairs would
serve as an approximate molecular
clock, allowing the time in the past
when the two sequences diverged
from a common ancestral
sequence to be estimated.
Evolutionary Analysis (Analytical Method)
Obtaining DNA Sequences
- Phylogenetic analysis using DNA
sequences relies heavily on the
polymerase chain reaction (PCR)
to obtain sufficient copies of a
gene for reliable sequencing.
Specific oligonucleotide primers
have been designed that bind to
the ends of the gene of interest, or
to DNA flanking the gene, allowing
DNA polymerase to bind to and
copy the gene. The source of DNA
bearing a gene of interest typically
is genomic DNA purified from
particular bacterial strains, but
could be DNA extracted from an
environmental sample. The PCR
product is visualized by agarose
gel electrophoresis, excised from
the gel, extracted and purified from
the agarose, and then sequenced,
often using the same
oligonucleotides as primers for the
sequencing reactions.
Sequence Alignment
- Phylogenetic analysis is based on
homology, that is, analysis of DNA
sequences that are related by
common ancestry. Once the DNA
sequence of a gene is obtained,
the next step in phylogenetic
analysis is to align that sequence
with homologous sequences from
other organisms. By doing this,
nucleotide mismatches and
insertions and deletions, some of
which may be phylogenetically
informative, can be pinpointed.
Phylogenetic Trees
- construction of a phylogenetic tree,
which is a graphic depiction of the
relationships among sequences of
the organisms under study, much
 like a family tree. A phylogenetic
tree is composed of nodes and
branches (Figure 4). The tips of
the branches represent species
that exist now and from which the
sequence data were obtained. The
nodes are points in evolution
where an ancestor diverged into
two new organisms, each of which
then began to evolve along its
separate pathway. The branches
define both the order of descent
and the ancestry of the nodes,
whereas the branch length
represents the number of changes
that have occurred along that
branch
Species Concept in Microbiology
- At present, there is no universally
accepted concept of species for
prokaryotes. Microbial systematics
combines phenotypic, genotypic,
and sequence-based phylogenetic
data within a framework of
standards and guidelines for
describing and identifying
prokaryotes, but the issue of what
actually constitutes a prokaryotic
species remains controversial.
- A prokaryotic species is defined
operationally as a group of strains
sharing a high degree of similarity
in several independent traits. Traits
currently considered most
important for grouping strains
together as a species include 70%
or greater genomic DNA–DNA
hybridization and 97% or greater
identity (3% difference) in 16S
rRNA gene sequence.
Experimental data suggest that
these two criteria are valid,
reliable, and consistent in
identifying new species of
prokaryotes. Based on genotypic
criteria such as these, over 7000
species of Bacteria and Archaea
have been formally recognized.
- What criteria should be used to
define a genus, the next highest
taxon, is more a matter of
judgment, but 16S rRNA gene
sequence differences of more than
5% from all other organisms is
considered good evidence that an
organism constitutes its own
genus. Above the level of genus to
the family, order, and other ranks
of higher taxa, no consensus
ribosomal RNA sequence-based
criteria exist for delineating these
ranks. Table 2 gives an example of
species definition in practice for
the classification of the
phototrophic purple bacterium
Allochromatium warmingii from the
domain down to the species level.
- How do new prokaryotic species
arise? A likely possibility is by the
process of periodic purges and
selection within cell populations.
Imagine a population of bacteria
that originated from a single cell
and that occupies a particular
niche in a habitat. In theory, these
cells are genetically identical. If
cells in this population share a
particular resource (for example, a
key nutrient), the population is
considered an ecotype (Figure 6).
Different ecotypes can coexist in a
habitat, but each is only most
successful within its prime niche in
the habitat. However, within each
ecotype, genes mutate at random
over time as the cells grow. Most
of these mutations are neutral and
have no effect. However, if there is
a beneficial mutation (one that
increases fitness) in a cell in one of
the ecotypes, that cell will produce
more progeny over time, and this
will purge the population of the
original, less well-adapted cells.
Repeated rounds of mutation and
selection in this ecotype lead it to
become more and more distinct
genetically from the other
ecotypes. Then, given enough
time, cells in this lineage will carry
a sufficiently large set of unique
traits that they emerge as their
own species. Selection of strains
bearing beneficial mutations can
proceed gradually, or it can occur
quite suddenly due to rapid
environmental change. Note that
this series of events within an
ecotype has no effect on other
ecotypes, because different
ecotypes do not compete for the
same resources.

Module 4: Functional Anatomy of

Prokaryotic Cell

Morphology of Cells
- Cells are the fundamental
structures as well as functional
units of every organism. Based on
general anatomy, they can be
categorized into two – the
eukaryotic and prokaryotic cells.
The word eukaryotic means “true
nucleus”, from the Greek word eu
meaning true, and karyon, kernel
which refers to the nucleus. On the
other hand, prokaryotic means
“before nucleus”, from the Greek
word pro, meaning before. This
reflects the fact that prokaryotic
cells actually evolved before
eukaryotic cells. Organisms which
are categorized in the domains of
Bacteria and Archaea belong to
the prokaryotic cells. Fungi,
animals, plants and organisms
under the kingdom Protista are
under the category of eukaryotic
cells.
 - In general, cells share similar
characteristics and features. All
cells are bounded by the plasma
membrane, which is the selective
barrier between its cytosol, a
semifluid jelly-like substance
inside, and the external
environment. They also do contain
chromosomes, which carry the
genes made of DNA
(deoxyribonucleic acid) segments;
and complexes or units that
convert instructions from the genes
to proteins, called ribosomes. A
review on the differences between
eukaryotes and prokaryotes is
presented in Table 1 below.
Differences between key structures
and accessory organs of eukaryotic
and prokaryotic cells
- A basic difference between
eukaryotic and prokaryotic cell is
the location of their genetic
material. In a prokaryotic cell, the
genetic material is usually located
in a region that is not membrane
closed, which is called a nucleoid.
On the other hand, the genetic
material possessed by a
eukaryotic cell is contained or
found in a double membrane-
bound organelle called the
nucleus.
Endosymbiotic Hypothesis - A review
- The first simplest life forms on
Earth were the prokaryotes, which
only have simple structures on
their cells. After some million
years, after the rise of atmospheric
oxygen, eukaryotes emerged in
the earth, having complex
structures of organelles and can
support aerobic metabolism as
well as photosynthesis.
- One good explanation on how the
first simple eukaryotic cells have
emerged to earth is the
Endosymbiotic Hypothesis. The
hypothesis states that the
mitochondria and chloroplasts, the
organelles which is responsible for
cellular respiration, came from a
respiring prokaryote and a
cyanobacterium-like prokaryote.
The theory explains that a
prokaryote which is large in size is
able to engulf a bacterium which
can use oxygen, able to perform
cellular respiration. But instead of
digesting the organism, the
engulfed bacteria and the
engulfing prokaryote lived together
as symbionts, therefore living
together. Same concept as to
chloroplasts in which a bacterium
which uses sunlight as energy is
being engulfed by a larger
prokaryote and lived as symbionts.
- This further results into an
evolutionary event wherein the
engulfed bacteria able to perform
cellular respiration and
photosynthesis evolved into
mitochondria and chloroplasts in
the cell, respectively.
- The overall physiology and
characteristics of the mitochondria
and the chloroplasts are able to
support the said hypothesis. One
evidence is that both mitochondria
and chloroplasts possess short
amounts of DNA which are in a
circular form, which is common to
bacteria. Such organelles also
contain ribosomes of 70S type,
which is the same size as bacteria.
Thus, some antibiotics, which
inhibits ribosomal function in
bacteria found in the environment
are also able to inhibit the same in
these organelles - supporting the
hypothesis that the ancestral
eukaryotes may have come from
the symbiosis of two prokaryotic
cells with unique abilities.
The Eukaryotic Cell
- In a rule of thumb, eukaryotic cells
are undeniably more complex and
larger than the prokaryotic cells.
These cells contain a range of
sophisticated subcellular
membrane-bound organelles,
which support the whole unit for
survival. In the world of
microbiology, the common and
major groups of eukaryotes are the
protists (protozoa and algae) and
the fungi. These groups both have
representatives of single-celled
and multicellular eukaryotic cells. A
quick review of the parts and their
function follows.
Nucleus
- Eukaryotic cells have a true
nucleus bounded by a double-layer
membrane. This membrane
contains pores, which function as
“passageways” for messenger
RNA leaving the nucleus out to the
cytoplasm during the event of
protein synthesis.
- As mentioned in Table 1, the
organization of genetic material in
eukaryotes is different from
prokaryotes. The DNA of
eukaryotes is formed into
chromosomes, which occurs in
pairs. This, in fact, is one of the
reasons why eukaryotes are
genetically diploid in at least some
parts of the cell’s life cycle,
whereas prokaryotes are haploid.
Moreover, the genetic material of
eukaryotic chromosomes is linear
instead of a singular closed loop in
prokaryotes. This explains the
presence of free ends in the
eukaryote chromosomes, and the
levels of organization found
therein. The DNA is highly
condensed and wrapped around
proteins called histones. Histones
possess a strong positive charge
responsible in associating with the
negatively charged phosphate
groups on the DNA, giving it a
more compact shape.
Endoplasmic Reticulum
- The endoplasmic reticulum (ER) is
a complex system comprised of
 tubes. It is an extensive network of
membrane that makes up for more
than half the total membrane in
most eukaryotic cells. It comes
from the words endoplasmic,
which means “within the
cytoplasm”, and reticulum, a Latin
word meaning “little net”.
- The endoplasmic reticulum is
differentiated into two types: the
rough endoplasmic reticulum
(RER) and the smooth
endoplasmic reticulum (SER). The
rough endoplasmic reticulum
mainly possesses numerous
ribosomes on its surface, resulting
in a rough, granular appearance
when seen under an electron
microscope, hence, the name. The
areas which do not contain
numerous amounts of ribosomes
are known as the smooth
endoplasmic reticulum. Since the
rough endoplasmic reticulum
possesses numerous amounts of
ribosomes, its function is to mainly
synthesize proteins. Proteins
synthesize in the RER already
have specified final destinations,
which is to the Golgi apparatus, or
will stay in the ER. SER, in
contrast, is involved in a large
array of metabolic processes.
These processes include lipid
synthesis, carbohydrate
metabolism, calcium ion storage,
and detoxification of drugs and
poisons.
Golgi Apparatus
- The Golgi apparatus is a transport
organelle, which consists of a set
of flattened vesicles, called
dictyosomes. The Golgi apparatus
is responsible for the modification,
storage and sending of the
products formed in the
endoplasmic reticulum. Moreover,
the membrane itself is extensive in
cells which are specialized in
secretion.
- A Golgi stack has a distinct
structural directionality. It
comprises of two sides, the cis
face and the trans face.
Respectively, these act as the
receiving and shipping
departments of the Golgi
apparatus. The cis face is the part
which is usually located near the
ER. Transport vesicles from the
ER move to the Golgi apparatus
through the cis face. The trans
face gives rise to vesicles that
“pinch off” and travel to other sites.
Lysosomes
- Lysosomes are membrane bound
organelles which contain digestive
enzymes. They contain vesicles
which possess hydrolytic enzymes
that are responsible for digesting
waste products of the cell. These
hydrolytic enzymes have the
potential to digest the whole cell, in
a process called autolysis.
However, this process is being
regulated by enclosing the
enzymes with the lysosomal
membrane.
- The hydrolytic enzymes and the
lysosomal membrane are
manufactured in the RER and are
distributed by the Golgi apparatus.
These enzymes work at acidic
environments, hence, not very
active when released into the cell
 since the pH of the cytosol is
neutral in nature. However, if large
numbers of these enzymes are
released, this can already destroy
the self by means of self-digestion.
Vacuoles
- Vacuoles are organelles derived
from the Golgi apparatus. These
organelles are responsible for
storing various nutrients and waste
products.

Cell Wall
- The cell wall is the rigid protective
layer that surrounds the cell. In a
general view, not all eukaryotes
possess a cell wall. Some
organisms that do have cell walls
Mitochondria and Chloroplasts
are members of fungi, algae, and
- Mitochondria are generally rod-
plants groups. Its general function
shaped organelles enclosed by a
is to provide form and strength to
double membrane. The inner
the cell. Morphologically, the
surface of the organelle is folded
structures that make up the cell
into finger-like projections called
wall is not the same in all
cristae. These organelles vary in
organisms, and varies depending
numbers depending on which kind
on the eukaryote groupings. The
of cell they are present in,
cell walls of algae, plants and
sometimes in singles or may be
some lower members of fungi are
found in large numbers.
composed of cellulose - a chain of
Mitochondria are sites where
glucose molecules. In fungi such
cellular respiration occurs, which is
as yeasts and mushrooms, chitin,
the metabolic process that uses
a polymer of N-acetylglucosamine
oxygen to generate ATP by
is the primary component of the
extracting energy from fats,
cell wall. Chitin is also observed as
sugars, and other fuels.
a major component of crustacean
- Chloroplasts, which can be
and insect exoskeletons, whose
observed in plants and algae, are
functions are for strength and
specialized cells which are also
rigidity.
sites of photosynthesis. These
organelles are also surrounded by
Plasma Membrane
a double membrane. Inside the
- In eukaryotes without cell walls,
organelle are thylakoids, which are
the plasma membrane becomes
flatted membranous sacs arranged
the outermost layer of the cells. It
into stacks called grana. These
also acts as selective barrier which
thylakoids contain the
allows the passageway of
photosynthetic pigment of the plant
important molecules into the cell.
called the chlorophyll. They are
The plasma membrane is
responsible for converting solar
comprised of lipid structures
energy to chemical energy when it
containing two chains of fatty acids
is used to synthesize organic
and a phosphate group formed into
compounds particularly sugars
a phospholipid bilayer. Each
from carbon dioxide and water.
phospholipid molecule is
composed of two regions: a
 hydrophilic head, which is
composed of the phosphate group
and glycerol; and a hydrophobic
tail, which is composed of the two
fatty acid chains.
- Because of the nature of cell
cytoplasm and its aqueous
surrounding, the plasma
membrane form a bilayer of
phospholipids, with one hydrophilic
layer facing the interior aqueous
cytoplasm, and the other, the
exterior of the cell. It can be
noticed that when a phospholipid is
dropped into a body of water, a
micelle would form, which is an
aggregate with the hydrophilic
head in contact with the solvent,
while the hydrophobic tail is being
protected inside away from the
solvent.
Cytoskeleton
- The cytoskeleton is an extensive
network of fibers that can be
observed in the cytoplasm. Its
function is to support the cell’s
shape and for mechanical support.
It is a very important part of the cell
since some eukaryotes lack cell
wall. The cytoskeleton is
comprised of three types of
molecular structures: microtubules,
microfilaments, and intermediate
filaments.
Flagella and Cilia
- Flagella and cilia are the main
components of cells in regard to
motility. A flagellum is a lash-like
appendage which provides
movement or locomotion in certain
eukaryotic cells called flagellates,
which possess either one or
several flagella. It functions also as
sensory organelles to
temperatures and chemicals in the
surroundings of the cell. Cilia on
the other hand, can be described
as short flagella. Both appendages
are attached to the plasma
membrane and the bases are
anchored to the cell by a basal
body.
The Prokaryotic Cell
- Prokaryote is the classification of
organisms that possess simple
characteristics in their cells. Unlike
a eukaryotic cell, a prokaryotic cell
does not have complex structures,
let alone a complete set found in
eukaryotes. Generally, they are
also much smaller than eukaryotic
cells, typically into a size range of
1-5 micrometers. A representative
picture of a prokaryotes is shown
in Figure 7.
- Due to the small size of these
organisms, it wasn’t until the
invention of the microscope that
scientists were able to observe
such fascinating organisms.
Bacteria, a type of prokaryotes,
were also observed with their
differences in shape and
arrangement. Three basic shapes
are observed: rod (bacillus),
spherical (coccus), and curved,
into spiral shapes (spirillum). See
figure 5.
- Bacterial cells also remain
attached in an arrangement
 depending on the plane of fission,
which is a characteristic of their
genera. Related to this, some can
be in a form of pairs (diplo), groups
of four (tetrad), three-dimensional
cubes (Sarcina), chains
(streptococcus), or in an irregular
“bunch of grape” (staphylococcus)
arrangements.
Flagella and Motility
- The most observed extracellular
- In bacterial cells, there are many
Extracellular Structures
Capsules and Slime Layers
- Most bacteria secretes a
glycocalyx, a slimy or sticky
material on their cell surface made
of polysaccharide or proteins.
Related to its form, it is called a
capsule for a more organized
matrix, otherwise, an easily
- As the flagellum’s function is for
deformed, loosely attached type is
a slime layer. It functions as a
protective layer that enables
bacteria to resist phagocytosis by
the host immune system as what
happens to bacteria with capsules.
Furthermore, it enables some
bacteria to adhere to surfaces,
thus helping in formation of
bacterial biofilms. Its
polysaccharide nature allows it to
bind water and likely resist
desiccation. Figure 9 provides
information on capsular materials
of some bacteria.
structures are the flagella. These
are thin hair-like structures which
are used for motility of a cell,
regardless whether eukaryotic or
prokaryotic.
flagellar arrangements, depending
on the genera of the bacteria.
There may be a single flagellum at
one end (monotrichous), flagellum
on both end (amphitrichous), many
flagella on one end (lophotrichous)
and numerous flagella surrounding
the cell (peritrichous).
locomotion, it greatly relies in
energy driven by the basal body. A
clockwise rotation of a single
flagellum will result into “tumbling”
of the cell, which is directionless.
 On the other hand, if the cell’s
flagella rotates counterclockwise, it
results in “running” to a straight
line.
- Other extracellular structure which
can be noticed in a bacterial cell is
the pili. These are the structures
which resemble short flagella.
However they are not used with
motility, which makes them
different from the flagella. Instead,
they are used for anchorage or
“sticking” of the bacterium in a
specific surface. Attachment pili
are sometimes called fimbriae,
which is a term mostly used in
order to differentiate attachment
pili to a sex pili, which functions in
transfer of genetic information by
means of conjugation.
Plasma Membrane
- Similar to eukaryotes, prokaryotes
also possess a plasma membrane.
The cytoplasm as well as its
contents are enveloped by its
bilayer of phospholipids arranged
like a sandwich, and associated
with a lot of proteins. Its function is
to confine the contents of the cell,
while allowing selective passage of
some substances and molecules in
and out, a function of a
semipermeable membrane.
Analogous to sterols of the cell
membrane are hopanoids, which
strengthen the membranes of
bacteria.
- Note, however, that Archaea, while
also considered prokaryotes, do
have variations in their cell
membranes not found in the other
two domains. Their hydrophobic
sides are attached to the
hydrophilic heads by ether linkage
instead of ester linkages, and are
composed of repeating units of
hydrophobic five-carbon (5-C)
hydrocarbon isoprene (a 20-C unit
is called a phytanyl group; a 40-C
unit is biphytanyl. Hence, the
cytoplasmic membrane of Archaea
can be constructed with glycerol
diether or diglycerol tetraethers,
which is twice the former but the
ends pointing inward become
covalently linked (Figure 11.c).
Lipid monolayers, indeed, serve its
purpose to Archaeans living in
extreme conditions such as the
hyperthermophiles.
Among the critical functions of the
prokaryote membranes are:
Permeability Barrier
- Prevents leakage and functions as
gateway for transport of nutrients
into and out of the cells
Protein Anchor
- Site of many ptroteins involved in
transport, bioenergetics, and
chemotaxis
Energy Conversation
- Site of generation and use of
proton motive force
Additional Notes:
- Small uncharged molecules can
pass through membranes by
simple diffusion except charged
ions. Water, while polar, does
freely pass the membrane in both
directions. The presence of
dedicated transport proteins for
water called aquaporins
accelerates further its movement.
AqpZ of E. coli adjusts movement
of water across its membrane in
varying osmotic conditions.
- Transport proteins help
accumulate solutes against
concentration gradient, which
never would occur had diffusion
been the only mechanism to so. To
fulfill this intention, different
mechanism for transport exist in
prokaryotes. Simple transport
consists only of a membrane-
spanning transport protein; group
translocation involves a series of
proteins in the transport event, and
the ABC system consists of three
components: a substrate-binding
protein, a membrane-integrated
transporter, and an ATP-
hydrolyzing protein (Figure 12).
- All transport systems require
energy in some form, either from
the proton motive force, or ATP, or
some other energy-rich organic
compound. Simple transport
involves transport proteins such as
uniporters, symporters and
antiporters, whose functions is
owed to their names. Lac
permease in E. coli is well-studies
symporter for lactose using the
energy of the protons as the
driving force, which puts is in the
indirect active transport category.
Group translocation modifies the
substance during the uptake by
phosphotransferase system.
Phosphoenolpyruvate (PEP)
provide the energy (phosphate)
which is cascaded from the five-
protein system to glucose (sample
molecule) during transport.
- The ATP-Binding Cassette (ABS
transport system) are found in
gram-negative and employs the
periplasmic-binding proteins,
membrane transporter and ATP-
hydrolyzing proteins. About 200
ABC transport systems have been
identified in prokaryotes for the
uptake of organic compounds
such as sugars and amino acids,
and inorganic nutrients. Gram
positives have ABC transport
systems involving peripheral
proteins of their cell membranes
and do the same just as in the
gram negatives.
Genetic Material
- While there is a specific area
where the genetic materials of
prokaryotes can be found, the
absence of a nuclear membrane
make it indistinguishable. It is,
thus, found in an area called the
nucleoid region.
- Generally, the nucleoid or the
bacterial chromosome of the
prokaryotes is constituted in a
closed circular shape of double
stranded DNA, which is highly
folded and compacted. However,
not all bacteria conform to a
circular structure of DNA. Some
bacteria also do possess linear
chromosomes (Streptomyces
coelicolor for example).
- Unlike eukaryotes, the bacterial
DNA is not packed with histones.
Instead, other bacterial proteins
are associated with their genetic
 material. Some bacteria also
contain additional DNA in a form of
smaller, extrachromosomal
plasmids. These are accessory
genetic materials since they can
survive in its absence. However,
they can still be beneficial as they
may sometimes include survival or
defense genes that encode toxins
or resistance to some antibiotics
that they can pass to another.
Ribosomes
- The ribosomes, along with the
nucleoid, are the fundamental
internal structures of prokaryotic
cells. These structures are
comprised of complex proteins and
RNA. Ribosomes are the site of
protein synthesis, where an mRNA
is being translated into a protein.
- While both prokaryotic and
eukaryotic ribosomes do have
similar function, the size of
prokaryotic ribosomes are much
smaller compared to eukaryotic
ones. Foremost, ribosomes are
measure with Svedberg units (S),
with which an eukaryotic ribosome
has 80S, while a prokaryotic
ribosome has only 70S. All
ribosomes are composed of two
unequal subunits. Prokaryotes are
comprised of a 50S and a 30S
subunit, while eukaryotes are
composed of 60S and 40S
subunits.
Prokaryotic Cell Wall
- Bacteria, like other prokaryotic and
some eukaryotic cells such as
plants, have a protective layer in
their cells which provides structural
rigidity and strength which are
called cell walls. It protects
bacterial cells against lysis, which
can be caused by pressures in the
activities of transport systems of
the cell.
- Generally, species of bacteria are
divided into two major
classifications in regards with their
cell wall structures. These are
called Gram-positive and Gram-
negative bacteria. The
fundamental distinction between
Gram-positive and Gram-negative
bacteria is based on the Gram
stain reaction, which can be
observed in a technique or method
called Gram staining. However, the
results of the Gram stain reaction
is characterized by the
morphological differences between
the cell walls of the two major
bacterial classes. Basically, the
cell wall structure of a Gram-
negative bacteria is very much
more chemically complex,
consisting of two layers. On the
other hand, the cell wall of a Gram-
positive bacteria is comprised of
only a single type of molecule but
very much thicker than the Gram-
negative ones.
Peptidoglycan
- Peptidoglycan is a polysaccharide
which is made up of N-
acetylglucosamine and N-
acetylmuramic acid – which are
two sugar molecules, and amino
acids, particularly L-alanine, D-
alanine, D-glutamic acid, and
either lysine or DAP
 (diaminopimelic acid). Such
molecules are responsible for the
formation of a repeating structure
called the glycan tetrapeptide.
- Peptidoglycan are synthesized in a
manner resulting in a sheet-like
formation surrounding the cell. The
chains of sugars connected by the
glycosidic bonds are linked by the
cross-links of amino acids. This
therefore, provides rigidity of the
layer in both directions since the
glycosidic bonds of the sugar
molecules only provide strength
and rigidity in only a single
direction. Logically, more cross-
linked molecules results in greater
strength and rigidity.
- Peptidoglycan cross linkages differ
between Gram-negative and
Gram-positive bacteria. Gram -
negative bacteria typically obtain
cross linkages by a peptide
formation from the carboxyl group
of D-alanine to the amino group of
DAP. On the other hand, cross
linkages of Gram-positive bacteria
occurs through a peptide
interbridge, in which the numbers
and types of amino acids in that
interbridge vary between species.
Cell Wall of a Gram Positive Bacteria
- In the cell wall of a Gram-positive
bacteria, peptidoglycan comprises
about 90% in the structure itself,
mostly having several sheets
stacked one after another.
Generally, peptidoglycans are
produced by the cell in “cables”,
each having cross-linked glycan
strands. As the peptidoglycan
develops, these “cables” mature
into a stronger cell wall structure
by cross-linking to the cables
themselves. In addition, they
contain acidic polysaccharides
known as teichoic acids, which
contain phosphate groups that are
responsible for an overall negative
charge of the cell surface.
- Gram-negative bacteria typically
has a thinner layer of
peptidoglycan compare to the
Gram-positive bacteria. This
actually makes the cell wall of
Gram-negative bacteria to be less
sturdy. However, the structure is
more complex because there is a
second phospholipid bilayer
observed outside of the
peptidoglycan layer, which is more
permeable than the inner
cytoplasmic membrane. This
actually makes the single layer of
peptidoglycan being sandwiched
by two membrane phospholipid
bilayers. In addition, the second
phospholipid bilayer contains
special types of molecules called
lipopolysaccharides which helps
support the bacterial cell by
protecting it from certain drugs and
antibodies in the immune system.
This outer membrane is connected
from the peptidoglycan layer by
molecules called lipoproteins,
which functions as anchorage and
hold the outer membrane via
 covalent bonds of the bacteria to
itself.
Periplasm and Porins
- While the outer membrane is more
permeable to small molecules, it is
still not permeable to molecules
which are large in size. Moreover,
one function of the outer
membrane is to keep the proteins
whose activities are being done
outside the cytoplasmic membrane
from going away. Therefore it is
contained in a region between the
outer surface of the cytoplasmic
membrane and the inner surface of
the outer membrane called the
periplasm.
- The reason why the outer
membrane is relatively permeable
to some molecules is because of
channels embedded in the
membrane which are known as
porins. These porins are divided
into two types: nonspecific porins
which form water-filled channels
making any small substance to
pass, and specific porins which
contain binding sites for specific
types of molecules.
Cell Wall Structure to Gram Stain
- Gram stains produce different
results in certain bacteria
depending on what structural
differences they have in regards
with their cell walls. In Gram
staining procedure, the crystal
violet-iodine complex forms inside
the bacterial cell. However, by
applying alcohol, the crystal violet-
iodine complex becomes extracted
from the Gram-negative bacteria
but not from the Gram-positive
bacteria. It is because of the
alcohol’s effect, which dehydrates
the thick peptidoglycan layer of the
Gram-positive cell wall, resulting in
the closure of pores in the layer
itself and preventing the escape of
the crystal violet-iodine complex,
hence, the violet coloration.
- On the other hand, the alcohol
readily penetrates the outer
membrane and extracts the crystal
violet complex from the cell itself.
This makes the Gram-negative
bacteria readily accept the
counterstain (usually Safranin)
making Gram negative cells red or
pink when observed under the
microscope.
Endospores
- Certain species of bacteria, such
as Bacillus and Clostridium,
produce structures called
endospores. Endospores are
highly differentiated, dormant
forms of the cells which function as
survival structures that enables the
organism equipped with it highly
resistant to extreme conditions
such as very high temperature,
harsh chemicals, pH, as well as
radiation, and germinate into new
vegetative bacterial cells when
conditions in the surrounding
become favorable to bacteria.
 Module 5: Microscopy and Staining

Microscopy
- Using microscopes to view objects
and areas of objects that cannot
be seen with the naked eye

Important Features of Objectives

Resolving Power/Resolution (R)

- You have two microscopes with

different LR. You have a specimen
consisting of two lines, and the
distance between these two lines
is given as 0.50 μm. If Microscope
A has an LR of 0.45 μm and
Microscope B has an LR of 0.75
μm, under which microscope(s)
can you see the two lines as
separate and distinct?

Answer: Microscope A

Units of Measurement
- 1 micrometer (µm) = 10-6m
- 1 nanometer (nm) = 10-9m
 Dark Field
Types of Microscopes - Microscope field is dark
Light Microscopes - Objects under study are luminous
a. Simple - For specimens that are:
- Only 1 lens ● invisible in the ordinary LM
- Magnification (300x) ● cannot be stained by
b. Compound or complex standard methods
- 2 sets of lenses ● distorted by staining
- Magnification (1000x)

Electron Microscopes
- Electron beams and magnetic
fields
- For objects smaller than 0.2 mm in
diameter
- In vacuum
UV
- Shorter wavelength of light (180
nm- 400nm)
- Image made visible by
photography or TV screen
- Detecting substances (e.g. DNA)

What makes a good microscope?

1. Adequate magnifying power
2. Provide good contrast Fluorescence
3. High resolving power - Modification of UV microscope
4. Serves your purpose - Makes use of flurochromes
- Detection of immunological
Different types of Light Microscopes reactions

Bright Field
- microscopic field is brightly lit
- objects under study are darker
- gross morphology

Phase-Contrast
 - Detailed examination of internal
structure
- Not necessary to fix or stain cells
- Principle is based on variations in
the refractive indices
- Surface features of viruses and
cells
- Reveals a 3-dimensional image

Example of EM stains
- Osmic Acid
Differential Interference Contrast
- Permanganate
- Principle is based on variations in
- Lead
the refractive indices
- Uranium
- Advantage: no diffraction halo
- Lanthanum
associated with phase contrast
- Disadvantage: the three-
Examination of Microorganisms
dimensional appearance may not
(1) Living or Natural State
represent reality
Disadvantage
- Refractive index of cells almost
similar to that of water

What techniques are used?

Wet Mount Technique (WMT)

Transmission Electron Microscope

(TEM)
- Examine viruses
- Ultrastucture in thin sections of the
cells Hanging Drop Technique (HDT)

(2) Stained preparations

Scanning Electron Microscope (SEM) Advantages
 - Provides contrast - Capillary Action
- Slides can be preserved - Ion-exchange
- Specimens are killed
Disadvantages Different staining procedures on simple
- More complicated and tedious to staining
prepare
- More expensive Positive/Direct
- Cells same color as dye
3 Basic Steps in Staining Microorganisms - Methylene Blue
Smear Preparation - Crystal Violet

Smear Preparation
- Smear: a thin dry fil of
microorganisms

Fixation
- Heat Fixation: Direct flame, Steam
Fixation
- Chemical Fixation: Alcohols
Purpose of fixation
- Kills the cells
- Makes the cell sticky
- Increase apparent diameter of Negative/Indirect
cells - Cells colorless or luminous
Staining - Nigrosin
- Application of biological dyes - India Ink
- Dye (stains)
- Organic compound carrying
chromophoric ions

Different Types of Stains Differential Staining

● Basic or Positively Charged Dye - 2 or more dyes/reagents
● Acidic or Negatively Charged - Gram Staining: Gram positive /
Dye Gram negative
● Neutral - Acid fast staining: Diagnosis of
tuberculosis
Mechanisms of Staining
- Absorption
- Adsorption
- Osmosis
 Module 6: Growth and Reproduction in
Prokaryotes

Growth

Increase in cellular constituents that may

result in:
- Increase in cell number
- Increase in cell size

Growth refers to the population growth

rather than growth of individual cells

Consequence of Bacterial Growth

- If Streptococcus pyogenes at the
back of your throat, a sore throat.
- Growth of microorganisms in
refrigerator shortens the shelf life
of the food
- Produce beer, wine, cheese,
Structural Staining
yogurt and other products.
- 2 or more dyes/regeants
- Endospore, Capsule, Flagella,
Bacterial Division Chromosome
Storage Granules
Replication and Partitioning and
Cytokinesis

- Most bacterial chromosomes are

circular
- DNA replication proceeds in both
directions from the origin
- Origins move to opposite ends of
the cell
- Cell elongates
- Septation – formation of cross
walls between daughter cells and
cells separate

Types of bacterial division:

- Binary Fission
- Budding
- Certain actinomycetes by
coniodiospores
- Fragmentation
Peptidoglycan Synthesis and Cell
Division
- New cell wall is synthesized during
bacterial growth by inserting new
glycan units into preexisting wall
material
- A hydrophobic alcohol called
bactoprenol facilitates transport of
new glycan units through the
cytoplasmic membrane to become
part of the growing cell wall (Figure
6.4).
- Transpeptidation bonds the
precursors into the peptidoglycan
fabric
The Mathematics of Growth
- Generation (doubling) time
- Time required for the population to
double in size
- varies depending on species of
microorganism and environmental
conditions
- range is from 10 minutes for some
bacteria to several days for some
eukaryotic microorganisms
- This is calculated during log
growth phase
- Microbial populations show a
characteristic type of growth
pattern called exponential growth,
which is best seen by plotting the
number of cells over time on a
semilogarithmic graph
Converting Arithmetic to Logarithmic
Calculating no. of generation and
generation time
Number of generation
Generation Time
The Growth Cycle
 - Microorganisms show a -
characteristic growth pattern
(Figure 6.8) when inoculated into a
fresh culture medium.
There is usually a lag phase, then
exponential growth commences. As
essential nutrients are depleted or toxic
products build up, growth ceases, and the
population enters the stationary phase. If
incubation continues, cells may begin to
die (the death phase).
(1)Lag Phase
Cell synthesizing new components
- e.g., to replenish spent materials §
e.g., to adapt to new medium or
other conditions
Varies in length
- in some cases can be very short or
even absent
Depends on harshness of medium
- is it selective or enrichment
medium?
- what is the temperature of
medium?
The general rule of thumb is that microbes
adapt to a shift to improved conditions
much more rapidly than they do to a shift
to poorer conditions.
(2)Exponential
- Also called log phase or log growth
phase
- Rate of growth and division is
constant and maximal
- Population is most uniform in
terms of chemical and physical
properties during this phase
- Bacteria from this stage would be
used for studies
(3)Stationery Phase
Closed system population growth ceases
due to
- Nutrient limitation
- accumulation of a waste product.
Limited oxygen availability
- Critical population density reached
- Bacteria die off and liberate some
nutrients
No change in the number of viable cells,
active cells stop reproducing or
reproductive rate is balanced by death
rate
Can last for long period
- microbes in nutrient-poor
environments (like soils and many
aqueous environments) probably
spend most of their time in
stationary phase
(4)Death Phase
Cell numbers begin to decline due
- DNA or protein damage or perhaps
exhaustion of energy reserves
- Accumulation of toxic waste
Bacteria are dying off opposite to log
growth phase
- do not die all at once
Two alternative hypotheses
- cells are Viable But Not Culturable
(VBNC)
- cells alive, but dormant, capable of
new growth when conditions are
right
Continuous Culture
- The culture is kept in exponential
growth phase for as long as
desired by the continuous addition
of new medium and the
simultaneous removal of the same
volume of old medium and cells.
- Under these conditions, all cells
should be growing exponentially
and samples at various times
should be identical
Environmental effects of Microbial Growth temperatures. Organisms that
grow at 0ºC but have optima of
Temperature 20ºC to 40ºC
- Temperature is a major
environmental factor controlling Hyperthermophiles
microbial growth. - Those with optima greater than
- The cardinal temperatures are the 80°C
minimum, optimum, and maximum
temperatures at which each These organisms inhabit hot environments
organism grows up to and including boiling hot springs, as
well as undersea hydrothermal vents that
Microbes based on Optimum can have temperatures in excess of
Temperature 100ºC.
- Psychrophiles - optimum is 15C
and max is below 20 C; Thermophiles and hyperthermophiles
Psychrotolerant, opt. 20C to 40C produce heat-stable macromolecules,
- Psychrotrophs – 0 oC to 35 oC such as Taq polymerase, which is used to
- Mesophiles - optima in the 20 oC automate the repetitive steps in the
to 45 oC range polymerase chain reaction (PCR)
- Thermophiles have optima from 45 technique.
oC to 80 oC
- Extreme thermophiles - optima, Hydrogen Ion Conc (PH)
above 80 C - In water, the hydrogen ion
concentration will range from 1 x
Mesophiles 10- 14 M (a pH of 14) to 1 M (a pH
- which have midrange temperature of 0).
optima, are found in warmblooded - Microbes are found at almost any
animals and in terrestrial and conceivable pH, with most
aquatic environments in temperate common bacteria growing at or
and tropical latitudes. near neutral pH (7).
- The acidity or alkalinity of an
Extremophiles environment can greatly affect
- have evolved to grow optimally microbial growth. Figure 6.22
under very hot or very cold shows the pH scale.
conditions.
Some organisms have evolved to grow
Psychrophiles best at low or high pH, but most
- Organisms with cold temperature organisms grow best between pH 6 and 8.
optima are called psychrophiles,
and the most extreme The internal pH of a cell must stay
representatives inhabit relatively close to neutral even though the
permanently cold environments. external pH is highly acidic or basic.

Psychrotolerant Organisms that grow best at low pH are

- Psychrophiles have evolved called acidophiles; those that grow best at
biomolecules that function best at high pH are called alkaliphiles.
cold temperatures but that can be
unusually sensitive to warm Bacteria Based on PH Req
 - Acidophiles -pH optima 1-5.4
- Neutrophiles - 5.5-7.9
- Alkalophiles - 8.0-11
Osmotic Effect on Bacterial Growth
Osmotic Pressure
- Water availability (water activity) is
a measurement of how much free
water is available.
- Pure water has a water activity of
100% and the activity decreases
as solutes are added to the
solution.
Changes in osmotic concentrations in the
environment may affect microbial cells
Hypotonic solution (lower osmotic
concentration)
- water enters the cell
- cell swells may burst
(plasmoptysis)
Hypertonic (higher osmotic
concentration)
- water leaves the cell
- membrane shrinks from the cell
wall (plasmolysis) may occur
- endures NaCl; organisms can
tolerate some reduction in the
water activity of their environment
but generally grow best in the
absence of the added solute.
Osmophiles
- lives in high sugar
Xerophiles
- microbes in dry environments
Some microorganisms (halophiles) have
evolved to grow best at reduced water
potential, and some (extreme halophiles)
even require high levels of salts for
growth.
Water activity becomes limiting to an
organism when the dissolved solute
concentration in its environment
increases.
To counteract this situation, organisms
produce or accumulate intracellular
compatible solutes (Figure 6.24; Table
6.3) that maintain the cell in positive water
balance.
Amino acid-type and related solutes

Osmotic
Water can be made unavailable by:
- Evaporation
- Freezing
- Bound to solutes
- Cause dehydration
- Water unavailable to enzymes
Carbohydrate-type solutes

Microbes based on Osmotic

Water can be made unavailable by:
- Evaporation
- Freezing
- Bound to solutes
- Cause dehydration
- Water unavailable to enzymes
Alcohol-type solutes
Halotolerant
 - are able to use oxygen in
metabolic processes and generate
more energy per mole of energy
source consumed.

Aerotolerant anaerobes and strict

anaerobes
- do not use oxygen in their
metabolism and typically have a
Oxygen and Microbial Growth lower energy yield and slower
growth rates.
Oxygen
- Many microbes and most
eukaryotes need oxygen for
growth as it is the terminal electron
acceptor

Others microbes can live without it and

some cannot even tolerate its presence.
- Obligate aerobes
- facultative anaerobes A reducing agent such as thioglycolate
- Microaerophiles can be added to a medium to test an
- aerotolerant anaerobes organism's requirement for oxygen
- strict anaerobes
Thioglycollate Test
Aerobes - TA restrict oxygen to the top one-
- require oxygen to live third of the tube. Aerobes will grow
only at the top of the tube (1)
Anaerobes Facultative anaerobes will growth
- do not and may even be killed by throughout (2 and 3) and strict
oxygen anaerobes will only grow at the
bottom of the tube (4).
Facultative
- organisms can live with or without Oxygen Requirements
oxygen

Microaerophiles
- are aerobes that can use oxygen
only when it is present at levels
reduced from that in air.

Aerotolerant anaerobes
- can tolerate oxygen and grow in its
presence even though they cannot
use it.

Strict aerobes and facultative Toxic Forms of Oxygen

anaerobes
 - Several toxic forms of oxygen can
be formed in the cell, but enzymes
are present that can neutralize
most of them Superoxide in
particular seems to be a common
toxic oxygen species

Enzymes that destroy toxic forms of

oxygens

The requirements for growth: Chemical

Requirements

Nitrogen
- In amino acids, proteins
- Most bacteria decompose proteins
- Some bacteria use NH4 + or NO3 -
Special techniques are needed to grow
- A few bacteria use N2 in nitrogen
aerobic and anaerobic microorganisms
fixation

Sulfur
Nutrition and Culture of Microorganisms
- In amino acids, thiamine, biotin
- Most bacteria decompose proteins
Requirements:
- Some bacteria use SO4 2- or H2S
- Macronutrients
- Micronutrients
Phosphorus
- Trace elements
- In DNA, RNA, ATP, and
- Temperature
membranes
- pH
- PO4 3- is a source of phosphorus
- Osmotic Pressure (Aw)
- Oxygen
The roles trace elements play in
metabolism

Cobalt
- Part of vitamin B12, which is used
to carry methyl groups
Macro and Micronutients

Zinc
 - Structural role in many enzymes - Heterotrophs: rely on pre-made
including DNA polymerase organic compounds for carbon
Mo - Lithotrophs: obtain electrons from
- Certain reactions involving inorganic compounds
nitrogen assimilation. Found in
nitrate reductase and nitrogenase Major Nutritional Types of Prokaryotes
Cu
- Catalytic role in some enzymes
that react with oxygen for example
cytochrome oxidase
Mn
- Required by a number of enzymes
in catalytic sites. Certain
photosynthetic enzymes use
manganese to split water into
oxygen and protons.
Ni
Measuring Microbial Growth
- Several different enzymes
including some involved in carbon
Parameters used as a measure of growth
monoxide metabolism, urea
of a population of bacteria. They include:
metabolism and methanogenesis
- Change in cell number.
- Change in the turbidity or light
Some examples for growth factors include
scattering of the culture.
- Vitamins which are non-protein
- Change in the amount of a cell
components of many enzymes
component.
- Amino acids for protein synthesis
- Nucleic acids for DNA and RNA
Microscopic Counts
synthesis
- counting chambers
- electronic counters – flow
The Requirements for Growth: Chemical
cytometry
Requirements
- on membrane filters

Trace Elements
Viable Counting Methods
- Inorganic elements required in
- Spread and pour plate techniques
small amounts
- Membrane filter technique
- Usually as enzyme cofactors
- Turbidity for Most Probable
Number (MPN)
Nutritional Classification of Microbes
- Phototrophic: utilize light as a
Measurement of Cell Mass
source of energy
- Dry Weight Analysis
- Autotrophs: obtain their carbon
- Measurement of cell components
from carbon dioxide
- Turbidity
- Organotrophs: obtain their
electrons from organic compounds
Direct Count: Counting Chambers
- Chemotrophs: obtain energy by
- Easy, inexpensive, and quick
the oxidation of either inorganic or
- Useful for counting both
organic compounds.
eukaryotes and prokaryotes
 - Cannot distinguish living from dead Viable counting: Alive or Dead
cells Whether or not a cell is alive or dead isn’t
always clear cut in microbiology
Direct Counts on Membrane Fillers - Cells can exist in a variety of
- Cells filtered through special states between ‘fully viable’ and
membrane that provides dark ‘actually dead’
background for observing cells - VBNC (Viable but not culturable)
- Cells are stained with fluorescent
dyes
- Useful for counting bacteria
- With certain dyes, can distinguish
living from dead cells

Direct Count: Flow Cytometry

- Microbial suspension forced
through small orifice with a laser
light beam
- Movement of microbe through
orifice impacts electric current that
flows through orifice
- Instances of disruption of current
are counted = result in count of
individual cells.

Viable Counting Methods

Limitations
Spread and pour plate techniques
- Without special staining
- diluted sample of bacteria is
techniques dead cells cannot be
spread over solid agar surface or
distinguished from live cells.
mixed with agar and poured into
- Small cells are difficult to see
Petri plate
under the microscope, and some
- after incubation the numbers of
cells are inevitably missed.
organisms are determined by
- Precision is difficult to achieve.
counting the number of colonies
- A phase-contrast microscope is
multiplied by the dilution factor
required if the sample is not
- results expressed as colony
stained.
forming units (CFU)
- Cell suspensions of low density
(less than about 106 cells/milliliter)
Membrane filter technique (used in our
have few if any bacteria in the
lab during water testing)
microscope field unless a sample
- bacteria from aquatic samples are
is first concentrated and
trapped on membranes
resuspended in a small volume.
- membrane placed on culture
- Motile cells must be immobilized
media
before counting.
- colonies grow on membrane
- Debris in the sample may be
- colony count determines # of
mistaken for microbial cells.
bacteria in sample
If microbe cannot be cultured on plate
media
Dilutions are made and added to suitable
media
Turbidity determined to yield the most
probable number (MPN)
Plate counts can be highly unreliable
when used to assess total cell numbers of
natural samples such as soil and water.
Module 7: Isolation and Cultivation of
Microognasims
Pure Culture (Axenic Culture)
- a culture which contains a single
species of microorganism
- a population of cells arising from a
single cell

Direct microscopic counts of natural Cultivation

samples typically reveal far more - increasing the population of
organisms than are recoverable on plates microorganisms by providing their
of any given culture medium. nutritional and physical
requirements
This is referred to as "the great plate count
anomaly," and it occurs because direct Nutrients
microscopic methods count dead cells - extracellular substances which
whereas viable methods do not, and provide the cell with materials for
different organisms in even a very small building protoplasm and for energy
sample may have vastly different generation
requirements for resources and conditions
in laboratory culture. Culture Medium
- any nutrient material for growth
and cultivation of microorganisms
Measurements of Cell Mass in the laboratory

Dry weight Uses of Culture Media

- time consuming and not very
sensitive
Quantity of a particular cell constituent
- e.g., protein, DNA, ATP, or
chlorophyll
- useful if amount of substance in
each cell is constant
Turbidometric measures (light
scattering)
- quick, easy, and sensitive
Turbidity measurements are an indirect
but very rapid and useful method of
measuring microbial growth. However, to
relate a direct cell count to a turbidity
value, a standard curve must first be
established.
- for growth and maintenance of
microbial cultures
- to favor the production of
particular compounds
- to study microbial action on some
constituents of the medium
Types of Culture Media
According to physical state
- Liquid (Broth) - no solidifying
agent
- Semi-Solid- 0.1 - 0.5% solidifying
agent
- Solid – 1.5 – 2.0% solidifying
agent
- Solidifying agent: agar or gelatin
According to chemical composition
- Synthetic – all components are
chemically defined
- Complex - not all components are
chemically defined
Eg: potato infusion
Beef extract
Yeast extract
Selective
- allows the growth of a specific
type of microorganism only
- with selective agents (ex. salts,
dyes, antibiotics, etc.)
e.g. Bacillus Cereus Agar (BCA)

According to principal function, purpose,

or application

General Purpose
- Can support most or almost type of
species

Eg: Nutrient Agar (NA)

Enrichment
- used to increase the number of
microorganisms with unusual
physiological characteristics
- with special nutrients (ex. blood)

E.g. Blood Agar

Differential
- Distinguishes one type of bacteria
from another
- With special reagents like pH
indicators or dyes

e.g. Eosin Methylene Blue Agar (EMBA)

Assay
- of prescribed composition used for
assay of vitamins, amino acids and
antibiotics
- used to determine qualitative/
quantitative production of such a
compound by an organism
 Enrichment Culture
Isolation Techniques
- isolation of specific types of
Plating
microorganisms by a combination
- Colony: a macroscopically visible
of nutrient and physical conditions
(surface or subsurface) growth or
- used for the isolation of unusual
cluster of microorganisms on a
physiological types of
solid medium
microorganisms which are present
- Streak Plating
in small numbers and which grow
slowly

- Spread Plating

Serial Dilution
- used if the desired microorganism
is present at a higher level than
any other microorganism
- outcome is 10-fold reduction of
cells/cfus in every transfer.

- Pour Plating
 - Transfer
- Verify the purity
- Make stock cultures

Culture Preservations

Objective
- to retain the viability of the stock
culture for a long period of time
while maintaining its purity and trait
of being “true-to-type”

Period Transfer to Fresh Media

Considerations
- Time interval of transfers
- Proper medium
- Proper storage temperature

Overlaying cultures with mineral oil

Aim
- Limit the availability of O2
Advantages
- simple
Single-Cell Isolation Technique
- enables one to remove some
- uses a micropipette or a
growth under the oil and inoculate
microprobe to physically pick a
it in a fresh medium and still
single cell and transfer it on an
preserve the initial culture
agar medium

Disadvantage
Membrane Filter Technique
- viability of microorganisms varies
- for samples with low population
with species
- uses a sterile membrane filter
having a pore size that retains
Freeze-drying (lyphilization)
microorganism
Temperature = -70 C
Employs
- rapid drying in frozen state
- dry ice in alcohol
Advantages
- long-term survival
- less opportunity for changes in the
characteristics of culture
- smallness of storage containers

Freezing with liquid nitrogen

Temperature = -196 C
Considerations
Steps in Preparing Pure Cultures - cryoprotective agent (glycerol)
- Isolation - liquid-nitrogen refs
Drying
Drying Temperature = 45 C
Limitation
- for spore- and cyst-
formers

Banking Microbes

Culture Collections
- organizations which maintain
authenticpure cultures of
microorganisms
- provide ‘type’ strains to
microbiologists throughout the
world

Examples
- American Type Culture Collection
(ATCC) (Maryland)
- National Collection of Type
Cultures (NCTC) (London)
- Japanese Type Culture Collection
(JTCC) (Japan)
- Philippine National Collection of
Microorganisms (PNCM)
(BIOTECH-UPLB)