Microbiological Techniques (Note 1)
MCB 324
300 Level
BACTERIOLOGY
Bacteriology is the study of bacteria.
This subdivision of microbiology involves the identification,
classification, and characterization of bacterial species. A person who studies
bacteriology is a bacteriologist.
Bacteria
Bacteria (singular: bacterium) constitute a large domain
(or kingdom) of prokaryotic
microorganisms.
Typically a few micrometres in length, bacteria have a wide range of shapes,
ranging from spheres
to rods and spirals. Bacteria were among the first life forms to appear on Earth, and are present in
most habitats
on the planet, growing in soil, water, acidic hot
springs, radioactive waste, and deep in the Earth's crust,
as well as in organic matter and the live bodies of plants and animals,
providing outstanding examples of mutualism in the digestive tracts of humans, termites
and cockroaches.
There are typically 40 million bacterial cells
in a gram of soil and a million bacterial cells in a millilitre of fresh water;
in all, there are approximately 5×1030 bacteria on Earth, forming a biomass
that exceeds that of all plants and animals. Bacteria are vital in recycling
nutrients, with many steps in nutrient
cycles depending on these organisms, such as the fixation of
nitrogen from the atmosphere and putrefaction.
In the biological communities surrounding hydrothermal vents and cold seeps,
bacteria provide the nutrients needed to sustain life by converting dissolved
compounds such as hydrogen sulphide and methane.
On 17 March 2013, researchers reported data that suggested bacterial life forms
thrive in the Mariana Trench, the deepest spot on the Earth.
Other researchers reported related studies that microbes thrive inside rocks up
to 1900 feet below the sea floor under 8500 feet of ocean off the coast of the
northwestern United States. According to one of the researchers,"You can
find microbes everywhere — they're extremely adaptable to conditions, and
survive wherever they are."
Most bacteria have not been characterised, and only about half of the phyla of
bacteria have species that can be grown in the laboratory. The study of
bacteria is known as bacteriology, a branch of microbiology.
There are approximately ten times as many bacterial cells in the human flora
as there are human cells in the body, with large numbers of bacteria on the skin and as gut flora.
The vast majority of the bacteria in the body are rendered harmless by the
protective effects of the immune system, and a few are beneficial.
However, a few species of bacteria are pathogenic and cause infectious diseases, including cholera,
syphilis,
anthrax,
leprosy,
and bubonic
plague. The most common fatal bacterial diseases are respiratory infections, with tuberculosis
alone killing about 2 million people a year, mostly in sub-Saharan Africa. In developed
countries, antibiotics are used to treat bacterial
infections and in agriculture, so antibiotic resistance is becoming common.
In industry, bacteria are important in sewage
treatment and the breakdown of oil spills,
the production of cheese
and yogurt
through fermentation, the recovery of gold,
palladium, copper and other metals in the mining sector, as well as in biotechnology,
and the manufacture of antibiotics and other chemicals.
Once regarded as plants
constituting the class Schizomycetes, bacteria are now classified as prokaryotes.
Unlike cells of animals and other eukaryotes,
bacterial cells do not contain a nucleus
and rarely harbour membrane-bound organelles.
Although the term bacteria traditionally included all prokaryotes, the scientific classification changed after
the discovery in the 1990s that prokaryotes consist of two very different
groups of organisms that evolved from an ancient common ancestor. These evolutionary
domains are called Bacteria and Archaea.
Origin and early evolution
The ancestors of modern bacteria were single-celled microorganisms that
were the first forms
of life to appear on Earth, about 4 billion years ago. For about 3
billion years, all organisms were microscopic, and bacteria and archaea were
the dominant forms of life. Although bacterial fossils exist,
such as stromatolites,
their lack of distinctive morphology prevents them from being used
to examine the history of bacterial evolution, or to date the time of origin of
a particular bacterial species. However, gene sequences can be used to
reconstruct the bacterial phylogeny, and these studies indicate that
bacteria diverged first from the archaeal/eukaryotic lineage.
Bacteria were also involved in the second great evolutionary divergence,
that of the archaea and eukaryotes. Here, eukaryotes resulted from ancient
bacteria entering into endosymbiotic associations with the ancestors
of eukaryotic cells, which were themselves possibly related to the Archaea.
This involved the engulfment by proto-eukaryotic cells of alpha-proteobacterial
symbionts to form either mitochondria or hydrogenosomes,
which are still found in all known Eukarya (sometimes in highly reduced form,
e.g. in ancient "amitochondrial" protozoa). Later on, some eukaryotes
that already contained mitochondria also engulfed cyanobacterial-like organisms.
This led to the formation of chloroplasts in algae and plants. There are
also some algae that originated from even later endosymbiotic events. Here,
eukaryotes engulfed a eukaryotic algae that developed into a
"second-generation" plastid. This is known as secondary endosymbiosis.
Morphology
Bacteria display many
cell morphologies and arrangements
Bacteria display a wide diversity of shapes and sizes, called morphologies. Bacterial cells are
about one tenth the size of eukaryotic cells and are typically 0.5–5.0 micrometres
in length. However, a few species — for example, Thiomargarita namibiensis and Epulopiscium fishelsoni — are up
to half a millimetre long and are visible to the unaided eye; E. fishelsoni
reaches 0.7 mm. Among the smallest bacteria are members of the genus Mycoplasma,
which measure only 0.3 micrometres, as small as the largest viruses. Some bacteria may
be even smaller, but these ultramicrobacteria are not well-studied.
Most bacterial species are either spherical, called cocci (sing.
coccus), or rod-shaped, called bacilli (sing. bacillus). Elongation is associated with
swimming. Some rod-shaped bacteria, called vibrio, are
slightly curved or comma-shaped; others can be spiral-shaped, called spirilla,
or tightly coiled, called spirochaetes. A small number of species even
have tetrahedral or cuboidal shapes. More recently, bacteria were discovered
deep under the Earth's crust that grow as long rods with a star-shaped
cross-section. The large surface area to volume ratio of this morphology may
give these bacteria an advantage in nutrient-poor environments. This wide
variety of shapes is determined by the bacterial cell wall
and cytoskeleton,
and is important because it can influence the ability of bacteria to acquire
nutrients, attach to surfaces, swim through liquids and escape predators.
Many bacterial species exist simply as single cells, others associate in
characteristic patterns: Neisseria form diploids (pairs), Streptococcus
form chains, and Staphylococcus group together in
"bunch of grapes" clusters. Bacteria can also be elongated to form
filaments, for example the Actinobacteria. Filamentous bacteria are
often surrounded by a sheath that contains many individual cells. Certain
types, such as species of the genus Nocardia,
even form complex, branched filaments, similar in appearance to fungal mycelia.
Bacteria often attach to surfaces and form dense aggregations called biofilms
or bacterial
mats. These films can range from a few micrometers in thickness to
up to half a meter in depth, and may contain multiple species of bacteria, protists
and archaea.
Bacteria living in biofilms display a complex arrangement of cells and
extracellular components, forming secondary structures such as microcolonies,
through which there are networks of channels to enable better diffusion of
nutrients. In natural environments, such as soil or the surfaces of plants, the
majority of bacteria are bound to surfaces in biofilms. Biofilms are also
important in medicine, as these structures are often present during chronic
bacterial infections or in infections of implanted medical
devices, and bacteria protected within biofilms are much harder to
kill than individual isolated bacteria.
Even more complex morphological changes are sometimes possible. For
example, when starved of amino acids, Myxobacteria
detect surrounding cells in a process known as quorum
sensing, migrate towards each other, and aggregate to form fruiting
bodies up to 500 micrometres long and containing approximately 100,000
bacterial cells. In these fruiting bodies, the bacteria perform separate tasks;
this type of cooperation is a simple type of multicellular organisation. For example,
about one in 10 cells migrate to the top of these fruiting bodies and differentiate into a specialised dormant state
called myxospores, which are more resistant to drying and other adverse
environmental conditions than are ordinary cells.
Cellular structure
Structure and contents of a typical Gram positive
bacterial cell
Intracellular structures
The bacterial cell is surrounded by a lipid membrane (also known
as a cell membrane
or plasma membrane). This membrane encloses the contents of the cell and acts
as a barrier to hold nutrients, proteins and other essential components of the cytoplasm
within the cell. As they are prokaryotes, bacteria do not usually have membrane-bound organelles
in their cytoplasm, and thus contain few large intracellular structures. They
lack a true nucleus, mitochondria,
chloroplasts
and the other organelles present in eukaryotic cells. Bacteria were once seen
as simple bags of cytoplasm, but structures such as the prokaryotic cytoskeleton and the
localization of proteins to specific locations within the cytoplasm have been
discovered which give bacteria some complexity. These subcellular levels of
organization have been called "bacterial hyperstructures".
Micro-compartments such as carboxysomes
provide a further level of organization; they are compartments within bacteria
that are surrounded by polyhedral protein shells, rather than by lipid membranes.
These "polyhedral organelles" localize and compartmentalize bacterial
metabolism, a function performed by the membrane-bound organelles in
eukaryotes.
Many important biochemical reactions, such as energy generation, use concentration
gradients across membranes. The general lack of internal membranes
in bacteria means reactions such as electron transport occur across the cell
membrane between the cytoplasm and the periplasmic
space. However, in many photosynthetic bacteria the plasma membrane
is highly folded and fills most of the cell with layers of light-gathering
membrane. These light-gathering complexes may even form lipid-enclosed structures
called chlorosomes
in green sulfur bacteria. Other proteins
import nutrients across the cell membrane, or expel undesired molecules from
the cytoplasm.
Most bacteria do not have a membrane-bound nucleus, and their genetic material is
typically a single circular chromosome located in the cytoplasm in an irregularly shaped
body called the nucleoid.
The nucleoid contains the chromosome with its associated proteins and RNA. The order Planctomycetes
are an exception to the general absence of internal membranes in bacteria,
because they have a double membrane around their nucleoids and contain other
membrane-bound cellular structures. Like all living
organisms, bacteria contain ribosomes
for the production of proteins, but the structure of the bacterial ribosome is
different from that of eukaryotes and Archaea.
Some bacteria produce intracellular nutrient storage granules for later
use, such as glycogen,
polyphosphate,
sulfur
or polyhydroxyalkanoates. Certain bacterial
species, such as the photosynthetic Cyanobacteria,
produce internal gas vesicles, which they use to regulate their buoyancy –
allowing them to move up or down into water layers with different light
intensities and nutrient levels.
Extracellular structures
In most bacteria a cell wall is present on the outside of the
cytoplasmic membrane. A common bacterial cell wall material is peptidoglycan
(called murein in older sources), which is made from polysaccharide
chains cross-linked by peptides containing D-amino acids.
Bacterial cell walls are different from the cell walls of plants and fungi, which
are made of cellulose
and chitin,
respectively. The cell wall of bacteria is also distinct from that of Archaea,
which do not contain peptidoglycan. The cell wall is essential to the survival
of many bacteria, and the antibiotic penicillin
is able to kill bacteria by inhibiting a step in the synthesis of peptidoglycan.
There are broadly speaking two different types of cell wall in bacteria,
called Gram-positive and Gram-negative.
The names originate from the reaction of cells to the Gram stain,
a test long-employed for the classification of bacterial species.
Gram-positive bacteria possess a thick cell wall containing many layers of
peptidoglycan and teichoic acids. In contrast, Gram-negative
bacteria have a relatively thin cell wall consisting of a few layers of peptidoglycan
surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins.
Most bacteria have the Gram-negative cell wall, and only the Firmicutes
and Actinobacteria
(previously known as the low G+C and high G+C Gram-positive bacteria,
respectively) have the alternative Gram-positive arrangement. These differences
in structure can produce differences in antibiotic susceptibility; for
instance, vancomycin
can kill only Gram-positive bacteria and is ineffective against Gram-negative pathogens,
such as Haemophilus influenzae or Pseudomonas aeruginosa.
In many bacteria an S-layer of rigidly arrayed protein molecules covers the
outside of the cell. This layer provides chemical and physical protection for
the cell surface and can act as a macromolecular
diffusion barrier. S-layers have diverse but
mostly poorly understood functions, but are known to act as virulence factors
in Campylobacter
and contain surface enzymes
in Bacillus stearothermophilus.
Flagella
are rigid protein structures, about 20 nanometres in diameter and up to
20 micrometres in length, that are used for motility. Flagella are driven
by the energy released by the transfer of ions down an electrochemical gradient across the cell
membrane.
Fimbriae are fine filaments of protein,
just 2–10 nanometres in diameter and up to several micrometers in length.
They are distributed over the surface of the cell, and resemble fine hairs when
seen under the electron microscope. Fimbriae are believed to
be involved in attachment to solid surfaces or to other cells and are essential
for the virulence of some bacterial pathogens. Pili (sing. pilus)
are cellular appendages, slightly larger than fimbriae, that can transfer genetic
material between bacterial cells in a process called conjugation.
Capsules or slime layers are produced by many bacteria to surround their
cells, and vary in structural complexity: ranging from a disorganised slime layer
of extra-cellular polymer,
to a highly structured capsule or glycocalyx.
These structures can protect cells from engulfment by eukaryotic cells, such as
macrophages.
They can also act as antigens and be involved in cell recognition, as well as
aiding attachment to surfaces and the formation of biofilms.
The assembly of these extracellular structures is dependent on bacterial secretion
systems. These transfer proteins from the cytoplasm into the
periplasm or into the environment around the cell. Many types of secretion
systems are known and these structures are often essential for the virulence
of pathogens, so are intensively studied.
Endospores
Certain genera
of Gram-positive bacteria, such as Bacillus,
Clostridium,
Sporohalobacter, Anaerobacter
and Heliobacterium, can form highly resistant,
dormant structures called endospores. In almost all cases, one endospore is formed and
this is not a reproductive process, although Anaerobacter
can make up to seven endospores in a single cell. Endospores have a central
core of cytoplasm
containing DNA
and ribosomes
surrounded by a cortex layer and protected by an impermeable and rigid coat.
Endospores show no detectable metabolism
and can survive extreme physical and chemical stresses, such as high levels of UV light,
gamma
radiation, detergents, disinfectants, heat, freezing, pressure and desiccation.
In this dormant state, these organisms may remain viable for millions of years,
and endospores even allow bacteria to survive exposure to the vacuum
and radiation in space. Endospore-forming bacteria can also cause disease: for
example, anthrax
can be contracted by the inhalation of Bacillus anthracis endospores, and
contamination of deep puncture wounds with Clostridium tetani endospores causes tetanus.
Classification and identification
Classification seeks to describe the
diversity of bacterial species by naming and grouping organisms based on
similarities. Bacteria can be classified on the basis of cell structure, cellular
metabolism or on differences in cell components such as DNA, fatty acids,
pigments, antigens
and quinones.
While these schemes allowed the identification and classification of bacterial
strains, it was unclear whether these differences represented variation between
distinct species or between strains of the same species. This uncertainty was
due to the lack of distinctive structures in most bacteria, as well as lateral gene transfer between unrelated
species. Due to lateral gene transfer, some closely related bacteria can have
very different morphologies and metabolisms. To overcome this uncertainty,
modern bacterial classification emphasizes molecular systematics, using genetic
techniques such as guanine cytosine ratio determination, genome-genome hybridization, as well as sequencing
genes that have not undergone extensive lateral gene transfer, such as the rRNA gene.
Classification of bacteria is determined by publication in the International
Journal of Systematic Bacteriology, and Bergey's Manual of Systematic
Bacteriology. The International
Committee on Systematic Bacteriology (ICSB) maintains international
rules for the naming of bacteria and taxonomic categories and for the ranking
of them in the International Code of Nomenclature of
Bacteria.
The term "bacteria" was traditionally applied to all microscopic,
single-cell prokaryotes. However, molecular systematics showed prokaryotic life
to consist of two separate domains,
originally called Eubacteria and Archaebacteria, but now called Bacteria
and Archaea
that evolved independently from an ancient common ancestor. The archaea and
eukaryotes are more closely related to each other than either is to the
bacteria. These two domains, along with Eukarya, are the basis of the three-domain system, which is currently the
most widely used classification system in microbiolology. However, due to the
relatively recent introduction of molecular systematics and a rapid increase in
the number of genome sequences that are available, bacterial classification
remains a changing and expanding field. For example, a few biologists argue
that the Archaea and Eukaryotes evolved from Gram-positive bacteria.
Identification of bacteria in the laboratory is particularly relevant in medicine,
where the correct treatment is determined by the bacterial species causing an
infection. Consequently, the need to identify human pathogens was a major
impetus for the development of techniques to identify bacteria.
Phylogenetic
tree showing the diversity of bacteria, compared to other organisms.
Eukaryotes
are colored red, archaea
green and bacteria blue.
The Gram stain,
developed in 1884 by Hans Christian Gram, characterises bacteria
based on the structural characteristics of their cell walls. The thick layers
of peptidoglycan in the "Gram-positive" cell wall stain purple, while
the thin "Gram-negative" cell wall appears pink. By combining
morphology and Gram-staining, most bacteria can be classified as belonging to
one of four groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative
cocci and Gram-negative bacilli). Some organisms are best identified by stains
other than the Gram stain, particularly mycobacteria or Nocardia, which
show acid-fastness
on Ziehl–Neelsen or similar stains. Other
organisms may need to be identified by their growth in special media, or by
other techniques, such as serology.
Culture techniques are designed to promote
the growth and identify particular bacteria, while restricting the growth of
the other bacteria in the sample. Often these techniques are designed for
specific specimens; for example, a sputum sample will be treated to identify organisms that cause
pneumonia,
while stool
specimens are cultured on selective media to identify organisms that
cause diarrhoea,
while preventing growth of non-pathogenic bacteria. Specimens that are normally
sterile, such as blood,
urine
or spinal fluid, are cultured under conditions
designed to grow all possible organisms. Once a pathogenic organism has been
isolated, it can be further characterised by its morphology, growth patterns
such as (aerobic or anaerobic growth, patterns of hemolysis) and staining.
As with bacterial classification, identification of bacteria is
increasingly using molecular methods. Diagnostics using such DNA-based tools,
such as polymerase chain reaction, are
increasingly popular due to their specificity and speed, compared to
culture-based methods. These methods also allow the detection and
identification of "viable but nonculturable" cells that
are metabolically active but non-dividing. However, even using these improved
methods, the total number of bacterial species is not known and cannot even be
estimated with any certainty. Following present classification, there are a
little less than 9,300 known species of prokaryotes, which includes bacteria
and archaea but attempts to estimate the true number of bacterial diversity
have ranged from 107 to 109 total species – and even
these diverse estimates may be off by many orders of magnitude.
Interactions with other organisms
Despite their apparent simplicity, bacteria can form complex associations
with other organisms. These symbiotic associations can be divided into parasitism,
mutualism and commensalism.
Due to their small size, commensal bacteria are ubiquitous and grow on animals
and plants exactly as they will grow on any other surface. However, their
growth can be increased by warmth and sweat,
and large populations of these organisms in humans are the cause of body odor.
Predators
Some species of bacteria kill and then consume other microorganisms, these
species called predatory bacteria. These include organisms such as Myxococcus xanthus, which forms swarms of
cells that kill and digest any bacteria they encounter. Other bacterial
predators either attach to their prey in order to digest them and absorb
nutrients, such as Vampirococcus, or invade another cell and
multiply inside the cytosol, such as Daptobacter. These predatory
bacteria are thought to have evolved from saprophages
that consumed dead microorganisms, through adaptations that allowed them to
entrap and kill other organisms.
Mutualists
Certain bacteria form close spatial associations that are essential for
their survival. One such mutualistic association, called interspecies hydrogen
transfer, occurs between clusters of anaerobic bacteria that consume organic acids
such as butyric acid
or propionic
acid and produce hydrogen, and methanogenic Archaea that consume hydrogen. The bacteria in
this association are unable to consume the organic acids as this reaction
produces hydrogen that accumulates in their surroundings. Only the intimate
association with the hydrogen-consuming Archaea keeps the hydrogen
concentration low enough to allow the bacteria to grow.
In soil, microorganisms that reside in the rhizosphere (a zone that includes the root surface and the soil
that adheres to the root after gentle shaking) carry out nitrogen
fixation, converting nitrogen gas to nitrogenous compounds. This
serves to provide an easily absorbable form of nitrogen for many plants, which
cannot fix nitrogen themselves. Many other bacteria are found as symbionts
in humans and other organisms. For
example, the presence of over 1,000 bacterial species in the normal human gut flora
of the intestines
can contribute to gut immunity, synthesise vitamins
such as folic acid,
vitamin K
and biotin,
convert sugars
to lactic acid,
as well as fermenting complex undigestible carbohydrates.
The presence of this gut flora also inhibits the growth of potentially pathogenic
bacteria (usually through competitive exclusion) and these
beneficial bacteria are consequently sold as probiotic
dietary supplements.
Pathogens
If bacteria form a parasitic association with other organisms, they are
classed as pathogens. Pathogenic bacteria are a major cause of human death and
disease and cause infections such as tetanus,
typhoid fever,
diphtheria,
syphilis,
cholera,
foodborne illness, leprosy
and tuberculosis.
A pathogenic cause for a known medical disease may only be discovered many
years after, as was the case with Helicobacter pylori and peptic ulcer
disease. Bacterial diseases are also important in agriculture,
with bacteria causing leaf spot, fire blight
and wilts
in plants, as well as Johne's disease, mastitis,
salmonella
and anthrax
in farm animals.
Each species of pathogen has a characteristic spectrum of interactions with
its human hosts. Some organisms, such as Staphylococcus
or Streptococcus,
can cause skin infections, pneumonia, meningitis and even overwhelming sepsis, a
systemic inflammatory response producing shock, massive vasodilation
and death. Yet these organisms are also part of the normal human flora and
usually exist on the skin or in the nose without causing any disease at all. Other organisms
invariably cause disease in humans, such as the Rickettsia,
which are obligate intracellular parasites
able to grow and reproduce only within the cells of other organisms. One
species of Rickettsia causes typhus, while another causes Rocky Mountain spotted fever. Chlamydia, another phylum of obligate
intracellular parasites, contains species that can cause pneumonia, or urinary tract infection and may be
involved in coronary heart disease. Finally, some
species such as Pseudomonas aeruginosa, Burkholderia cenocepacia, and Mycobacterium avium are opportunistic pathogens and cause disease
mainly in people suffering from immunosuppression
or cystic fibrosis.
Bacterial infections may be treated with antibiotics,
which are classified as bacteriocidal if they kill bacteria, or bacteriostatic
if they just prevent bacterial growth. There are many types of antibiotics and
each class inhibits a process that is different in the
pathogen from that found in the host. An example of how antibiotics produce
selective toxicity are chloramphenicol and puromycin,
which inhibit the bacterial ribosome, but not the structurally different eukaryotic
ribosome. Antibiotics are used both in treating human disease and in intensive
farming to promote animal growth, where they may be contributing to
the rapid development of antibiotic resistance in bacterial
populations. Infections can be prevented by antiseptic
measures such as sterilizing the skin prior to piercing it with the needle of a
syringe, and by proper care of indwelling catheters. Surgical and dental
instruments are also sterilized to prevent contamination by
bacteria. Disinfectants such as bleach are used
to kill bacteria or other pathogens on surfaces to prevent contamination and
further reduce the risk of infection.
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