Microbial Diversity
Part 1: Acellular and Prokaryotic Microbes
Microbiology is the study of microbes, most of which
are too small to be seen by the naked eye.
2 Categories of microbes: Cellular and Accellular
 Cellular - bacteria, archaea, algae, protozoa, fungi
o Prokaryotic – bacteria and archaea
o Eukaryotic – algae, protozoa, and fungi
 Accellular - viruses, viroids, and prions
o Not considered as living organisms
o more correctly referred to as acellular microbes
or infectious particles
ACELLULAR MICROBES
Viruses
 complete virus particles, called virions, are very
small and simple in structure
 extremely small, observed using electron microscope
 10 to 300 nm in diameter, although some—like
Ebola virus—can be up to 1 micrometer in length
 smallest virus is about the size of the large
hemoglobin molecule of a red blood cell
 oncogenic viruses or oncoviruses—cause specific
types of cancer
 viruses are not alive – to replicate, viruses must
invade live host cells because there are no ribosomes
for protein synthesis or sites of energy production
 lack most of the basic features of cells; thus, they
consider viruses to be non-living entities.
 How to distinguish virus from living cells
o The vast majority of viruses possess either DNA
or RNA, unlike living cells, which possess both.
o They are unable to replicate (multiply) on their
own; their replication is directed by the viral
nucleic acid once it has been introduced into a
host cell
o Unlike cells, they do not divide by binary
fission, mitosis, or meiosis.
o They lack the genes and enzymes necessary for
energy production.
o They depend on the ribosomes, enzymes, and
metabolites (“building blocks”) of the host cell
for protein and nucleic acid production
 Typical virion consists of a genome of either DNA
or RNA, surrounded by a capsid (protein coat),
which is composed of many small protein units
called capsomeres (or capsomers).
 nucleic acid + capsid = nucleocapsid
 Some viruses (enveloped viruses) have an outer
envelope composed of lipids and polysaccharides
 Bacterial viruses may have a tail, sheath, tail fibers.
Viruses are classified by the following characteristics:
a) type of genetic material (either DNA or RNA),
b) shape of the capsid,
c) number of capsomeres,
d) size of the capsid,
e) presence or absence of an envelope,
f) type of host that it infects,
g) type of disease it produces,
h) target cell, and
i) immunologic or antigenic properties
 its genome is mostly either double-stranded DNA or
single-stranded RNA,
 few viruses possess single-stranded DNA or double-
stranded RNA
 viral genomes are usually circular molecules, but
some are linear (having two ends)
 may be polyhedral (many sided – 20 sides,
icosahedron), helical (coiled tubes), bullet shaped,
spherical, or a complex combination of these shapes
 Each facet (side) consists of several capsomeres;
thus, the size of the virus is determined by the size of
each facet and the number of capsomeres in each
 the envelope around the capsid makes the virus
appear spherical or irregular in shape in electron
micrographs
 The envelope is acquired by certain animal viruses
as they escape from the nucleus or cytoplasm of the
host cell by budding
 Budding is a form of viral shedding where viruses
acquire their external envelope from either the host
cell’s nuclear membrane or cell membrane
 viruses are then able to alter these membranes by
adding protein fibers, spikes, and knobs that enable
the virus to recognize the next host cell to be
invaded
Viruses have to infect a host cell to access the machinery
that it uses to replicate it DNA, and then hijack that
machinery in order to reproduce. The host cell is forced
to manufacture new viruses, which then leave and look
for new hosts to infect.
Origin of Viruses
 existed for as long as bacteria/archaea have existed
 Coevolution Theory
o Viruses originated in the primordial soup and
coevolved with bacteria and archaea.
o Primordial soup generic term that describes the
aqueous solution of organic compounds that
accumulated in primitive water bodies
o This hypothesis has few supporters

 Retrograde Evolution Theory
o viruses evolved from free-living prokaryotes that
invaded other living organisms, and gradually
lost functions which were provided by host cell
o Viruses arose from cellular organisms, most
likely a bacterium. the cellular organisms
formed viruses by inquiring the ability to
gradually discard their genes until they become
simpler organisms which are the viruses we see
today
o This theory has little support
 Escaped Gene Theory
o viruses are pieces of host cell RNA or DNA that
have escaped from living cells, and are no longer
under cellular control
o most widely accepted explanation for the origin
of viruses
Bacteriophages (phages)
 viruses that infect bacteria
 obligate intracellular pathogens, they must enter a
cell to replicate
 3 categories, based on their shape
o Icosahedron bacteriophages: almost spherical
shape, with 20 triangular facets; smallest
icosahedron phages are 25 nm in diameter
o Filamentous bacteriophages: long tubes
formed by capsid proteins assembled into a
helical structure; they can be up to about 900 nm
long
o Complex bacteriophages: icosahedral heads
attached to helical tails; they may also possess
base plates and tail fibers
 There are single-stranded DNA phages, double-
stranded DNA phages, single-stranded RNA phages,
and double-stranded RNA phages
 Virulent Bacteriophages
o do not actually enter the bacterial cell—rather,
they inject their nucleic acid into the cell
o always cause the lytic cycle, which ends with the
destruction (lysis) of the bacterial cell
o the whole process (from attachment to lysis)
takes less than 1 hour
o once it enters a host cell, it always initiates the
lytic cycle, which results in the destruction of
cell
o Lytic Cycle Steps
1. Attachment
 Adsorption of the phage to the surface of the
bacterial cell.
 Phage attaches to a protein or
polysaccharide molecule (receptor) on the
surface of bacterial cell
 The phage can only attach to bacterial cells
that possess the appropriate receptor—a
protein or polysaccharide molecule on the
surface of the cell that is recognized by a
molecule on the surface of the phage
 Most bacteriophages are species- and strain-
specific – they only infect a particular
species or strain of bacteria.
 Those that infect E. coli are called
coliphages.
 Some bacteriophages can attach to more
than one species of bacterium
2. Penetration
 the phage injects its DNA into the bacterial
cell, acting much like a hypodermic needle,
the capsid remains on the outer surface of
cell
 then the phage DNA “dictates” what occurs
within the bacterial cell
 sometimes described as the phage DNA
taking over the host cell’s “machinery”
3. Biosynthesis
 phage genes are expressed, resulting in the
production (biosynthesis) of viral pieces
 host cell’s enzymes (e.g., DNA polymerase
and RNA polymerase), nucleotides, amino
acids, and ribosomes are used to make viral
DNA and viral proteins
4. Assembly
 viral pieces are assembled to produce
complete viral particles (virions)/ complete
phages
 viral DNA is packaged up into capsids
5. Release
 host cell bursts open and all of the new
virions (about 50–1,000) escape from the
cell
 the complete phages escape from the
bacterial cell by lysis of the cell
 lytic cycle ends with lysis (destruction) of
the host cell
 Lysis is caused by an enzyme (referred to as an
endolysin) that is coded for by a phage gene.
 after assembly, the appropriate viral gene is
expressed, the enzyme is produced, and the bacterial
cell wall is destroyed
 With certain bacteriophages, a phage gene codes for
an enzyme that interferes with cell wall synthesis,
leading to weakness and, finally, collapse of the cell
wall
 Temperate Phages (lysogenic phages)
 o do not immediately initiate the lytic cycle,
rather, their DNA remains integrated into the
bacterial cell chromosome, generation after
generation
 Bacteriophages are involved in two (lysogenic
conversion and transduction) of the four major
ways in which bacteria acquire new genetic
information
Animal Viruses
 Viruses that infect humans and animals
 Some are DNA viruses; others are RNA viruses
 may consist solely of nucleic acid surrounded by a
protein coat (capsid), or more complex
 ex; they may be enveloped or they may contain
enzymes that play a role in viral multiplication
within host cells
 Steps in Multiplication of Animal Virus
1. Attachment
 Phage attaches to a protein or polysaccharide
molecule (receptor) on surface of bacterial cell
 Animal viruses can only attach to cells bearing
the appropriate protein or polysaccharide
receptors on their surface
 Why certain viruses cause infections in dogs, but
not in humans or vv? RECEPTORS. Viruses
can only attach to and invade cells that bear a
receptor that they can recognize
2. Penetration
 entire virion usually enters the host cell,
sometimes because the cell phagocytizes
(engulf/ingest) the virus
3. Uncoating
 the viral nucleic acid escapes from the
capsid
 the viral nucleic acid “dictates” what occurs
within the host cell
4. Biosynthesis
 many viral pieces (viral nucleic acid and
viral proteins) are produced
 quite complicated, depending on what type
(1 or 2 D/RNA strand) of virus infected the
cell
 some animal viruses do not initiate
biosynthesis right away, but rather, remain
latent within the host cell for variable
periods
 Latency is the phase in certain viruses' life
cycles in which, after initial infection,
increase of virus particles ends
5. Assembly
 fitting the virus pieces together/assembled to
produce complete virions
6. Release
 After the virus particles are assembled, they
must escape from the cell by lysis/budding
 Some animal viruses escape by destroying
the host cell, leading to cell destruction and
some of the symptoms associated with
infection with that particular virus
 Other viruses escape the cell by a process
known as budding – become surrounded
with pieces of the cell membrane, thus
becoming enveloped viruses.
 Inclusion Bodies
o remnants or collections of viruses
o often seen in infected cells and are used as a
diagnostic tool to identify certain viral disease
o found in the cytoplasm (cytoplasmic inclusion
bodies) or within the nucleus (intranuclear
inclusion bodies), depending on the disease
o cytoplasmic: rabies, AIDS, Guarnieri bodies of
smallpox
o intranuclear: herpes, poliomyelitis
Latent Virus Infections
 ex: herpes, cold sores (fever blisters), shingles
(painful nerve disease caused by a herpesvirus)
 viral infections in which the virus is able to hide
form host’s immune system by entering cells and
remaining dormant
 limited by the defense systems of the human body—
phagocytes and antiviral proteins called interferons
that are produced by virus-infected cells
 once acquired, herpres virus infection never
completely go away
Antiviral Agents
 Antibiotics function by inhibiting certain metabolic
activities within cellular pathogens, and viruses are
not cells.
 But antibiotics may be prescribed in an attempt to
prevent secondary bacterial infections that might
follow the virus infection
 Drugs used to treat viral infections are called
antiviral drugs
 It is important to understand that antibiotics are not
effective against viral infections
 been developed to interfere to virus-specific
enzymes and virus production by either disrupting
critical phases in viral cycles/multiplications or
inhibiting the synthesis of viral DNA, RNA, or
proteins

Oncogenic Viruses (oncoviruses)
 viruses that cause cancers
 cancer; virus
 nasopharyngeal carcinoma, Burkitt lymphoma, B-
cell lymphoma; Epstein-Barr virus
 Kaposi sarcoma; human herpesvirus 8
 Hepatocellular carcinoma; hepa B and C
Human Immunodeficiency Virus
 Cause of AIDS (2 single strand RNA)
 enveloped, 2 single-stranded RNA virus
 Genus: Lentiviruses, Family: Retroviridae
 Can attach to and invade cells bearing receptors that
the virus recognizes
 Each of its 72 surface knobs contains a glycoprotein
(gp120) that can bind to CD4 receptor of host cell
 CD4 is the most important receptors, CD4 cells:
cells that possess CD4 receptors
 T cells: most important CD4 cells
 HIV infections destroy these important cells of the
immune system
 Macrophage, a type of phagocyte, have CD4
receptors, so can be invaded by HIV.
 able to invade certain cells that do not possess CD4
receptors, but do possess other receptors that HIV is
able to recognize
Mimivirus
 large 2-stranded DNA virus recovered from amebas
 named Mimivirus because it “mimics” bacteria
 very large – can be observed using a standard
compound light microscope
 7-nm-thick capsid with a diameter of 750 nm
 array of 80- to 125-nm-long closely packed fibers
project outward from the capsid surface
 consists of a double-strand DNA core, surrounded
by two 4-nm-thick lipid membranes and a protein
capsid
 numerous fibrils extend outward from capsid surface
 genome is at least 10 times larger than that of the
large viruses in the smallpox family and larger than
the genome of some of the smallest bacteria
 possess close to 1,000 genes, some of its genes
codes for functions
 contains several genes for sugar, lipid, and amino
acid metabolism, and some RNA molecules
 reports said that it might be cause of pneumonia
Megavirus (megavirus chilensis)
 Larger than mimivirus, double-stranded DNA virus
 discovered in a water sample collected in 2010 off
the coast of Chile
 has the largest capsid diameter (440 nm) and largest
and most complex genome of all known viruses
 its genome is predicted to encode over 1,000
different proteins; larger than that of some bacteria
Plant Viruses
 more than 1,000 different viruses cause plant
diseases
 usually transmitted via insects (e.g., aphids, leaf
hoppers, and whiteflies); mites; nematodes (round
worms); infected seeds, cuttings, and tubers; and
contaminated tools (e.g., hoes, clippers, and saws)
 Viroids
o even smaller and less complex infectious agents
o consist of short, naked fragments of single-
stranded RNA (about 300–400 nucleotides in
length) that can interfere with the metabolism of
plant cells and prevent the growth of plants,
sometimes killing the plants in the process
o infectious RNA molecules that cause a variety of
plant diseases
o transmitted between plants in the same manner
as viruses
o cause of potato spindle tuber, citrus exocortis, &
diseases in chrysanthemums, palms, tomatoes
 Prions
o smaller and less complex infectious agents
o small infectious proteins that apparently cause
fatal neurological diseases in animals and
humans
o scrapie in sheep and goats; bovine spongiform
encephalopathy (BSE; “mad cow disease”); and
kuru, Creutzfeldt–Jakob (C–J) disease,
Gerstmann–Sträussler–Scheinker (GSS) disease,
and fatal familial insomnia in humans.
o All these diseases are fatal spongiform
encephalopathies, in which the brain becomes
riddled with holes (sponge-like)
o Stanley B. Prusiner, scientist who coined the
term prion and studied the role of these
proteinaceous infectious particles in disease
o believed to be the most resistant to disinfectants
The Domain Bacteria
Characteristics
 2 domains of prokaryotes: Bacteria and Archaea
 23 phyla, 32 classes, 5 subclasses, 77 orders, 14
suborders, 182 family, 871 genera, and 5,007 species
 broadly divided into three phenotypic categories
(i.e., categories based on their physical
characteristics):
(a) those that are Gram-negative and have a cell wall
(b) those that are Gram-positive and have a cell wall
(c) those that lack a cell wall
 its Gram reaction, basic cell shape, and
morphological arrangement of cells are very
important clues to bacterium’s identification
Cell Morphology
 using light microscope, size, shape, and morphologic
arrangement of various bacteria are easily observed
 Bacteria vary greatly in size, usually ranging from
spheres measuring about 0.2 µm in diameter to 10.0-
µm-long spiral-shaped bacteria, to even longer
filamentous bacteria
 Average coccus is about 1 µm in diameter, and the
average bacillus is about 1 µm wide × 3 µm long
 3 basic shapes: round or spherical (cocci, sing:
coccus); rectangular or rod-shaped (bacilli, sing:
bacillus); curved and spiral (spirilla)
o Cocci: pairs (diplococci), chains (streptococci),
clusters (staphylococci), 4s (tetrads), 8s (octads)
o Bacilli: short or long, thick or thin, and pointed
or with curved or blunt ends.
: pairs (diplobacilli); chains (streptobacilli); long
filaments; branched
 Cocobacilli: rods that are quite short,
resembling elongated cocci.
o Curved and spiral: usually occur singly, but
some species may form pairs.
: referred as spirochetes (spayrokits) - vary in
size, length, rigidity, and the number and
amplitude of their coils
 divides by binary fission—one cell splits in half to
become two daughter cells. The time it takes for one
cell to split into two cells is referred to as that
organism’s generation time.
 After binary fission, the daughter cells may separate
completely from each other or may remain
connected, forming various morphologic
arrangements
 may lose their characteristic shape because adverse
growth conditions (e.g., the presence of certain
antibiotics) prevent the production of normal cell
walls (cell wall–deficient bacteria or L-forms)
 CWD bacteria revert to their original shape when
placed in favorable growth conditions
 Bacterial species having cells of different shapes is
pleomorphic (able to assume different forms)
 Ex; Mycoplasmas - do not have cell walls; thus,
when examined microscopically, they appear in
various shapes. Because they have no cell walls,
mycoplasmas are resistant to antibiotics that inhibit
cell wall synthesis.
Staining Procedures
 most bacteria are colorless,transparent, difficult to
see
 staining methods have been devised to enable
scientists to examine bacteria
 bacteria are smeared onto a glass microscope slide
(resulting in what is known as a “smear”), air-dried,
and then “fixed” (securely place/fastened)
 2 methods of fixation: heat and methanol
o Heat fixation
 accomplished by passing the smear through
a Bunsen burner flame
 too much heat can distort the morphology of
the cells
o Methanol Fixation
 accomplished by flooding the smear with
absolute methanol for 30 seconds
 more satisfactory fixation technique
 purpose of fixation
o It kills the organisms.
o It preserves their morphology (shape).
o It anchors the smear to the slide.
 Specific stains and staining techniques are used to
observe bacterial cell morphology (e.g., size, shape,
morphologic arrangement, composition of cell wall,
capsules, flagella, endospores).
 simple stain is sufficient to determine bacterial
shape and morphologic arrangement (pairs, chains,
clusters)
 Structural Staining Procedures: procedures used
to observe bacterial capsules, spores, and flagella
 Gram Stain or Gram Staining Procedure
o Dr. Hans Christian Gram, 1883
o become the most important staining procedure,
because it differentiates between “Grampositive”
and “Gram-negative” bacteria
o Gram-Positive bacteria:
 blue stained
 was not decolorized during the
decolorization
 The thick layer of peptidoglycan in the cell
walls of Gram-positive bacteria makes it
difficult to remove the crystal violet–iodine
complex during the decolorization step
 Peptidoglycan is a polymer consisting of
sugars and amino acids that forms a mesh-
like layer outside the plasma membrane of
most bacteria, forming the cell wall. ..
o Gram-Negative bacteria
 red stained
 crystal violet was removed from the cells
during the decolorization step, then cells
were stained by the safranin (a red dye), they
will be pink to red
 thin layer of peptidoglycan in the cell walls
of Gram-negative bacteria makes it easier to
remove the crystal violet–iodine complex
during decolorization
  the decolorizer dissolves the lipid in the cell
walls of Gram-negative bacteria; this
destroys the integrity of the cell wall and
makes it much easier to remove the crystal
violet–iodine complex
o Gram-variable Bacteria
 Neither blue to purple nor pink to red after
Gram staining
 Ex are members of the genus
Mycobacterium, such as M. tuberculosis and
M. leprae
 Mycobacterium species are more often
identified using a staining procedure called
the acid-fast stain
 Acid-Fast Stain
 Paul Ehrlich, 1882—a German chemist
 carbol fuchsin <fyuksin> (a bright red dye)
is driven into the bacterial cell using heat
(by flooding the smear with carbol fuchsin,
and then holding a Bunsen burner flame
under the slide until steaming of the carbol
fuchsin occurs).
 The heat is necessary because the cell walls
of mycobacteria contain waxes, which
prevent the stain from penetrating the cells,
so heat softens the waxes, enabling the stain
to penetrate.
 Then a colorizing agent (a mixture of acid
and alcohol) is then used in an attempt to
remove the red color from the cells
 Because mycobacteria are not decolorized
by the acid–alcohol mixture (again owing to
the waxes in their cell walls), they are said
to be “acid-fast.”
 Other bacteria are decolorized by the acid–
alcohol treatment; they are non–acid-fast
o Gram reaction serves as an extremely important
“clue” when attempting to learn the identity
(species) of a particular bacterium
o color of the bacteria at the end of the Gram
staining procedure depends on the chemical
composition of their cell wall
 Gram and acid-fast staining procedures are referred
to as differential staining procedures because they
enable microbiologists to differentiate one group of
bacteria from another
Motility
 A bacterium is “motile” if it is able to swim
 A bacterium is “nonmotile” if it is unable to swim
 most often associated with the presence of flagella or
axial filaments. Bacteria do not have cilia
 Most spiral-shaped bacteria and 1/2 of the bacilli are
motile by means of flagella, but cocci are nonmotile
 flagella stain can be used to demonstrate the
presence, number, and location of flagella on
bacterial cells
 can be demonstrated by stabbing the bacteria into a
tube of semisolid agar or by using the hangingdrop
technique
o Hangingdrop: a drop of culture (population of
bacteria grown in the laboratory) is placed on a
coverslip that is encircled with petroleum jelly
(or any sticky material). The coverslip and drop
are then inverted over the well of a depression
slide. The drop hangs from the coverslip, and
petroleum jelly forms a seal that prevents
evaporation. When examined microscopically,
motile bacteria within the “hanging drop” will
be seen moving fast around in every direction.
o Growth (multiplication) of bacteria in semisolid
agar produces turbidity (cloudiness). Nonmotile
organisms will grow only along the stab line (so
turbidity will be seen only along the stab line).
Motile organisms will spread away from the stab
line (thus, producing turbidity throughout the
medium)
Colony Morphology (appearance of colonies)
 single bacterial cell that lands on the surface of a
solid culture medium cannot be seen, but after it
divides over and over again, it produces a mound or
pile of bacteria, known as a bacterial colony
(contains millions of organisms)
 colony varies from one species to another
 includes the size, color, overall shape, elevation, and
the appearance of the edge or margin of the colony
 colony features serve as important “clues” in the
identification of bacteria
 size of colonies is determined by the organism’s rate
of growth (generation time), and is an important
characteristic of a particular bacterial species
 also includes the results of enzymatic activity on
various types of culture media
Atmospheric Requirements
 in microbio lab, it is useful to classify bacteria on the
basis of their relationship to O2 and CO2
 In a liquid medium such as thioglycollate broth
(THIO), the region of the medium in which the
organism grows depends on the oxygen needs of that
particular species
 as per oxygen, a bacterial isolate can be classified
into one of five major groups:
a. obligate aerobes
- require an atmosphere containing molecular
oxygen in concentrations comparable to that
found in room air (i.e., 20%–21% O2) to grow
and multiply
b. microaerophilic aerobes (microaerophiles)
 - requires reduced oxygen concentration (5%)
- ex: Neisseria gonorrhoeae (causes
gonorrhea) and Campylobacter spp. (causes
diarrhea)
c. facultative anaerobes
- capable of surviving in either the presence or
the absence of oxygen; anywhere from 0%
O2 to 20% to 21% O2.
- members of the family Enterobacteriaceae,
most streptococci, and most staphylococci
d. aerotolerant anaerobes
- does not require oxygen, grows better in the
absence of oxygen, but can survive in
atmospheres containing molecular oxygen
(such as air and a CO2 incubator)
e. obligate anaerobe
- will grow where there is 0% O2
Anaerobes
o organisms that do not require oxygen for life and
reproduction
o they vary in their sensitivity to oxygen
o e.g., certain Neisseria, Campylobacter, and
Haemophilus species
 as per CO2, room air contains less than 1% CO2
 Capnophiles (capnophilic organism)
o grow better in the laboratory in the presence of
increased concentrations of CO2.
o e.g., Bacteroides and Fusobacterium species
 In microbio labs, , CO2 incubators are routinely
calibrated to contain between 5% and 10% CO2
Nutritional Requirements
 bacteria need some form of the elements CO, H2O,
O2, sulfur, phosphorus, nitrogen, etc. for growth
 Certain microbes have specific vitamin
requirements, and some need organic substances
secreted by other living microorganisms during their
growth
 Fastidious (fussy): organisms with especially
demanding nutritional requirement
 usually characteristic of a species of bacteria and
serve as important clues when attempting to identify
the organism
Biochemical and Metabolic Activities
 As bacteria grow, they produce many waste products
and secretions, some are enzymes that enable them
to invade their host and cause disease
 pathogenic strains of many bacteria, such as
staphylococci and streptococci, can be tentatively
identified by the enzymes they secrete
 some bacteria are characterized by the production of
certain gases, such as carbon dioxide, hydrogen
sulfide, oxygen, or methane
 bacteria are inoculated (treated) into various
substrates (e.g., carbohydrates and amino acids) to
determine whether they possess the enzymes
necessary to break down those substrates
 when particular organism is able to break down a
certain substrate, it serves as a clue to the identity of
that organism
Pathogenicity
 ability of virus to cause disease
 Many pathogens are able to cause disease because
o they possess capsules, pili, or endotoxins
(biochemical components of the cell walls of
Gramnegative bacteria)
o they secrete exotoxins and exoenzymes that
damage cells and tissues
 tested by injecting the organism in mice or cell
culture
Genetic Composition
 Molecular Diagnostic Procedures - test procedure
that analyzes the organism’s DNA or RNA
 DNA of an organism is unique to each species
 DNA probes
o make it possible to identify an isolate without
relying on phenotypic characteristic
o single-stranded DNA sequence that can be used
to identify an organism by hybridizing with a
unique complementary sequence on the DNA or
rRNA of that organism
 trough the use of 16S rRNA sequencing, the degree
of relatedness between 2 different bacteria can be
determined
Unique Bacteria (rudimentary)
 do not possess all attributes of typical bacterial cells
 they are so small and difficult to isolate, so they
were formerly thought to be viruses
 Rickettsias, chlamydias, and mycoplasmas
Rickettsias, Chlamydias, and Closely Related
Bacteria
 bacteria with a Gram negative–type cell wall
 obligate intracellular pathogens (must live within a
host cell) that cause diseases in humans and other
animals
 to grow in laboratory, they must be inoculated
(treated) into embryonated chicken eggs, laboratory
animals, or cell cultures
Rickettsia
 named after Howard T. Ricketts, a US pathologist
 have no connection to the disease called rickets,
which is the result of vitamin D deficiency
 appear to have leaky cell membranes
 most rickettsias must live inside another cell to
retain all necessary cellular substances
 diseases caused by Rickettsia species are arthropod-
borne, they are transmitted by arthropod carriers
 lice, fleas, and ticks transmit the rickettsias from one
host to another by their bites or waste products
 ex: typhus and typhus-like diseases (e.g., spotted
fever rickettsiosis), involve production of a rash
 bacterias closely related to rickettsia
o Coxiella burnetii - transmitted primarily by
aerosols, but can be transmitted to animals by
ticks
o Bartonella Quintana – associated with trench
fever (a louse-borne disease), cat scratch disease,
bacteremia, and endocarditis
o Ehrlichia spp. and Anaplasma spp.
- cause human tick-borne diseases such as
human monocytic ehrlichiosis (HME) and
human granulocytic anaplasmosis.
- intraleukocytic pathogens, meaning that they
live within certain types of white blood
cells.
Chlamydia (energy parasites)
 refers to Chlamydia spp. and closely related
organisms (such as Chlamydophila spp.)
 Although they can produce ATP molecules (major
energy-storing molecules of cells), they prefer to use
ATP molecules produced by host cells
 obligate intracellular pathogens (must live within a
host cell) that are transferred by inhalation of
aerosols or by direct contact between hosts—not by
arthropods
 Chlamydia trachomatis: cause different diseases,
including trachoma (leading cause of blindness)
 Chlamydophila pneumonia: cause pneumonia
 Chlamydophila psittaci: causes a respiratory disease
called psittacosis
Mycoplasmas (former pleuropneumonia-like organisms)
 smallest of the cellular microbes
 lack cell walls, so they assume many shapes, from
coccoid to filamentous; thus, they appear
pleomorphic (assume many forms) when examined
 may be free-living or parasitic and are pathogenic to
many animals and some plant
 In humans, pathogenic mycoplasmas cause primary
atypical pneumonia and genitourinary infections;
some species can grow intracellularly
 they have no cell wall, so they are resistant to
treatment with penicillin and other antibiotics that
work by inhibiting cell wall synthesis (formation)
 can be cultured on artificial media in the laboratory,
where they produce tiny colonies (called “fried egg
colonies”) that resemble sunny-side-up fried eggs
 absence of a cell wall prevents mycoplasmas from
staining with the Gram staining procedure
Especially Large and Especially Small Bacteria
 size of a typical
o coccus is 1 micrometre (µm) in diameter
o bacillus is 1.0 µm wide × 3.0 µm long, some are
long thin filaments—up to about 12 µm in
length or even longer, but still 1 micrometre
wide
 most bacteria are microscopic, requiring the use of a
microscope to be seen
 Thiomargarita namibiensis
o Largest bacteria, can be seen with unaided eye
o colorless, marine, sulfide-oxidizing bacterium
o its single spherical cells are 100 to 300 µm, but
may be as large as 750 µm (0.75 mm)
o do not form filaments
 Epulopiscium fishelsonii
o Also a large bacterium
o isolated from the intestines of the reef
surgeonfish
o a bacillus that is about 80 µm wide x 3600 µm
(0.6 mm) long.
o Its cells are about five times longer than
eukaryotic Paramecium cell
o Its volume is about a million times greater than
the volume of a typical bacterial cell
 Nanobacteria – Tiny bacteria
o sizes are expressed in nanometers because these
bacteria are less than 1 µm in diameter
o have been found in soil, minerals, ocean water,
human and animal blood, human dental calculus
(plaque), arterial plaque, and even rocks
(meteorites) of extra-terrestrial origin
o scientists believe that they were formed by
geological, rather than biological, processes
Photosynthetic Bacteria
 include purple bacteria, green bacteria, and
cyanobacteria (blue-green algae)
 use light as an energy source, but they do not all
carry out photosynthesis in the same way
 capable of converting light energy to chemical
energy, ex: cyanobacteria
 oxygenic photosynthesis
o photosynthesis that produce oxygen
o cyanobacteria
 anoxygenic photosynthesis
 o photosynthesis that does not produce oxygen
o purple and green bacteria
 In photosynthetic eukaryotes (algae and plants),
photosynthesis takes place in plastids
 Cyanobacteria
o Exists 3.3 to 3.5 billion years ago
o vary widely in shape; some are cocci, some are
bacilli, and others form long filaments.
o In cyanobacteria, photosynthesis takes place on
intracellular membranes known as thylakoids
o Thylakoids are attached to the cell membrane at
various points and are thought to represent
invaginations of the cell membrane
 the action or process of being turned inside
out or folded back on itself to form a cavity
or pouch
o Attached to the thylakoids, in orderly rows, are
numerous phycobilisomes—complex protein
pigment aggregates where light harvesting
occurs
o Believed by scientists believe as the first
organisms capable of carrying out oxygenic
photosynthesis and, thus, played a major part in
the oxygenation of the atmosphere
o If certain conditions exist, Cyanobacteria in
pond or lake water overgrow, then it creates a
water bloom—a “pond scum” that resembles a
thick layer of bluish green (turquoise) oil paint.
o Conditions include
 mild or no wind
 balmy water temperature (15°–30°C)
 water pH of 6 to 9
 abundance of the nutrients nitrogen and
phosphorous in the water
o cyanobacteria are able to convert nitrogen gas
(N2) from the air into ammonium ions (NH4 +)
in the soil or water (nitrogen fixation)
o Some cyanobacteria produce toxins
(cyanotoxins) that can cause disease and even
death in wildlife species and humans that
consume contaminated water), such as:
 neurotoxins (affect central nervous system)
 hepatotoxins (liver)
 cytotoxins (other types of cell)
The Domain Archaea
 archaean prokaryotes were discovered in 1977
 more closely related to eukaryotes than they are to
bacteria; some possess genes otherwise found only
in eukaryotes
 contains 2 phyla, 8 classes, 12 orders, 21 families,
69 genera, and 217 species
 vary widely in shape; some are cocci, bacilli, and
others form long filament
 Many, but not all, archaea are “extremophiles,”
they live in extreme environments, such as
extremely acidic, alkaline, hot, cold, or salty
environments, or environments where there is
extremely high pressure
 Other archaea, called methanogens, produce
methane, which is a flammable gas
 all archaea possess cell walls, but their cell walls
contain no peptidoglycan (all bacteria contains)
 16S rRNA sequence data suggest that archaea are
more closely related to eukaryotes than they are to
bacteria
GRAM STAINING PROCEDURE - SIMPLIFIED
 the gram stain is the most widely employed staining
in microbiology. it is a differential stain because it
divides bacteria into two classes - gram positive and
gram negative.
 in the first step of the gram stain procedure, cells
from a fresh culture are transferred to a clean slide
and allow it to air dry. the cell should form a thin
barely visible film that is achieved by smearing the
cells obtained from the culture. fresh cultures must
be used because as cells age they lose their ability to
retain stain.
 the cells are then fixed to the slide, so using the heat
fixations, pass the smear slightly above the flame of
a bunsen burner but not too much because too much
heat can distort the morphology of the cells
 the fixed cells are then stained with a crystal violet
dye for 30 to 40 seconds. Then rinse the slide using
water to remove excess stain. at this point, all cell
appears purple under the microscope.
 next, a solution of gram iodine is added and retained
in the slide for about 1 minute. iodine combines
with crystal violet to form di-iodine complex, and
that decrease its solubility within the cell. at this
point, the cell still appear as purple.
 then, the cells are decolorized by washing with
ethanol or acetone. this is the differential step.
gram positive bacteria retain the crystal violet. gram
negative bacteria do not. the ethanol or acetone
should be added dropwise with the slide tilted at an
angle until the drop coming off from the slide started
to become colorless. even gram-positive cells can
lose the violet iodine complex during prolonged
excessive decolorization.
 excess ethanol is then washed off with water. when
viewed under the microscope, gram- positive cells
appear purple and gram-negative cells are colorless.

 finally, the rinsed cells are covered with the

counter stain safranin for 20 to 30 seconds. this
stains the gram negative bacteria red or pink.

 after rinsing with water, the slide is dried by a filter

paper. when viewed microscopically. the gram-
positive bacteria are purple to blue and the gram
negative bacteria are pink to red

Gram-Positive bacteria:
 was not decolorized during the decolorization
 The thick layer of peptidoglycan in the cell walls of
Gram-positive bacteria makes it difficult to remove
the crystal violet–iodine complex during the
decolorization step
 Peptidoglycan is a polymer consisting of sugars and
amino acids that forms a mesh-like layer outside the
plasma membrane of most bacteria, forming the cell
wall. ..

Gram-Negative
 thin layer of peptidoglycan in the cell walls of
Gram-negative bacteria makes it easier to remove
the crystal violet–iodine complex during
decolorization
 the decolorizer dissolves the lipid in the cell walls of
Gram-negative bacteria; this destroys the integrity of
the cell wall and makes it much easier to remove the
crystal violet–iodine complex