STRUCTURE OF BACTERIA

STRUCTURE OF BACTERIA

• Smaller and simpler in structure thaneukaryotic cells, with no recognizableorganelles.

• All of the activities performed by organellesalso take place in bacteria, but they are notcarried out by specialized structures.

• The small size, simple design, and broadmetabolic capabilities of bacteria allow themto grow and divide very rapidly and to inhabitand flourish in almost any environment.

STRUCTURE OF BACTERIA

• They were first seen under a microscope by Antonvan Leeuwenhoek in 1676.

• As microscopes have improved, scientists havecome to understand bacterial cell structure better.

Bacterial cell structure

• organized into 3 categories :

• Internal Structures: Cytoplasm, nucleoid, bacterial chromosome, plasmid, ribosomes, and storage granules

• Cell envelope: cell membrane, peptidoglycan cell wall or an outer lipid membrane (only found in Gram-negative cells)

• External structures (appendages & coverings): flagella, fimbriae, sex pilus and glycocalyx

Intracellular structures

• Cytoplasm

• Chromosome

• Plasmid

• Ribosomes

• Inclusion bodies

Cytoplasm• Portion of the cell that lies within the PM• substances within the plasma membrane, excluding

the genetic material. • Gel-like matrix composed of mostly water(4/5 th ),

enzymes, nutrients, wastes, and gases • Contains cell structures - ribosomes, chromosome,

and plasmids , as well as the components necessary for bacterial metabolism.

• It is relatively featureless by electron microscope -although small granules can be seen.

• carries out very important functions for the cell -growth, metabolism, and replication .

Constituents

– Proteins including enzymes

– Vitamins

– Ions

– Nucleic acids and their precursors

– Amino acids and their precursors

– Sugars, carbohydrates and their derivatives

– Fatty acids and their derivatives

Nucleoid

• Unlike the eukaryotic (true) cells, bacteria do not have a membrane enclosed nucleus.

• The nucleoid is a region of cytoplasm where the chromosomal DNA is located.

• It is not a membrane bound nucleus, but simply an area of the cytoplasm where the strands of DNA are found.

Plasmids• small extra-chromosomal DNA

• contain genes for antibiotic resistance or virulence.

• Structure Similar to most bacterial chromosomes, but considerably smaller.

• plasmids are covalently closed circular DNA

• In a few species linear plasmids have been found.

• Size : Chromosomal DNA is typically about 4000 kb,

• plasmid DNA ranges from 1-200 kb.

• Number of plasmids: 1-700 copies of plasmid in a cell.

Plasmid Function• The function of plasmids is not always known, but they

are not normally essential for survival of host, although their presence generally gives the host some advantage.

• Antibiotic resistance - Some plasmids code for proteins that degrade antibiotics-a big advantage for pathogens.

• Some encode for proteins which confer virulencefactors on the host. For example- E. coli plasmid EntP307 codes for an enterotoxin which makes E. colipathogenic.

• Conjugative plasmids - These allow exchange of DNA between bacterial cells.

Plasmids

• Plasmids and the associated traits can be transferred between bacteria, even from one bacterial species to another.

• Plasmids are not involved in reproduction.

• Plasmids replicate independently of the chromosome.

• Plasmids are passed to other bacteria by two means.

• For most plasmid types, copies in the cytoplasm are passed on to daughter cells during binary fission.

Plasmids

• Other types of plasmids ,form tube like structure at thesurface called a pilus that passes copies of the plasmidto other bacteria during conjugation, a process bywhich bacteria exchange genetic information.

• Plasmids have been shown to be instrumental in thetransmission of special properties, such as antibioticdrug resistance, resistance to heavy metals, andvirulence factors necessary for infection of animal orplant hosts.

• The ability to insert specific genes into plasmids havemade them extremely useful tools in the area ofgenetic engineering/RDNA Technology .

Ribosomes- protein synthesis machinery

• Consists of RNA and protein

• Abundant in cytoplasm

• Often grouped in long chains called polyribosomes.

• give the cytoplasm of bacteria a granular appearance in EM.

• smaller than the ribosomes in eukaryotic cells-but have a similar function

• Bacterial ribosomes have sedimentation rate of 70S; their subunits have rates of 30S and 50S.

• The unit used to measure sedimentation velocity is Svedberg

Ribosomes

• They translate the genetic code from the molecular language of nucleicacid to that of amino acids—the building blocks of proteins.

• Bacterial ribosomes are similar to those of eukaryotes, but are smaller andhave a slightly different composition and molecular structure.

• Bacterial ribosomes are never bound to other organelles as theysometimes are bound to the endoplasmic reticulum in eukaryotes, but arefree-standing structures distributed throughout the cytoplasm.

• There are sufficient differences between bacterial ribosomes andeukaryotic ribosomes that some antibiotics will inhibit the functioning ofbacterial ribosomes, but not a eukaryote's, thus killing bacteria but not theeukaryotic organisms they are infecting.

• Streptomycin binds 70S ribosome and stops protein synthesis but it cannot bind 80S ribosome of eukaryotes and thereby eukaryotic cell remainsunaffected.

Bacterial Chromosome - Genophore

• The bacterial chromosome consists of a single, circle of deoxyribonucleic acid.

• DNA is double stranded- two strands line up antiparrallel to each other and the bases are linked together with hydrogen bonds.

• It includes most of the genetic material of the organism .

Bacterial Chromosome

• Unlike the DNA in eukaryotic cells, which resides in the nucleus, DNA in bacterial cells is not sequestered in a membrane-bound organelle but

appears as a long coil distributed through the cytoplasm.

• In many bacteria the DNA is present as a single, circular chromosome and in some cases the DNA is linear rather than circular.

• some bacteria may contain two chromosomes

Bacterial Chromosome

• As in all organisms, bacterial DNA contains the four nitrogenous bases adenine (A), cytosine (C), guanine (G), and t

• The amount of DNA in bacterial chromosomes ranges from 580,000 base pairs in Mycoplasma gallinarum to 4,700,000 base pairs in E. coli to 9,140,000 base pairs in Myxococcus xanthus.

Inclusion bodies

• Inclusion bodies: Bacteria can have within their cytoplasm a variety of small bodies collectively referred to as inclusion bodies.

• Some are called granules and other are called vesicles.

• Inclusions are considered to be nonliving components of the cell that do not possess metabolic activity and are not bounded by membranes.

• The most common inclusions are glycogen, lipid droplets, crystals, and pigments.

Inclusion bodies - Granules• Granules: Densely compacted substances without a

membrane covering.

• Nutrients and reserves may be stored in the cytoplasm in the form of glycogen, lipids, polyphosphate, or in some cases, sulfur or nitrogen for later use.

• Each granule contains specific substances, such as glycogen (glucose polymer) and polyphosphate (phosphate polymer, supplies energy to metabolic processes).

• Sulfur bacteria contains reserve granules of sulfur.

• These granules are depleted in starvation.

Inclusion bodies-vesicles

• Some aquatic photosynthetic bacteria and cyanobacteria have rigid gas-filled vacuoles and it helps in floating at a certain level - allowing them to move up or down into water layers with different light intensities and nutrient levels.

• Some magnetotactic bacterium, eg. Aquaspirilliummagnetotacticum , stores Magnetitite (Ferric oxide). The presence of such magnetic inclusions enables these bacteria to responds to magnetic fields.

Microcompartments• Microcompartments are widespread, membrane-

bound organelles that are made of a protein shell that surrounds and encloses various enzymes.

• Carboxysomes are protein-enclosed bacterial microcompartments that contain enzymes involved in carbon fixation.

• Magnetosomes are bacterial microcompartments, present in magnetotactic bacteria, that contain magnetic crystals.

Cell Envelope

• Plasma Membrane

• Periplasmic Space

• Cell Wall

• Outer membrane

Plasma Membrane

• Phospholipid bilayer surrounding the cytoplasm and regulates the flow of substances in and out of the cell.

• Consists of both lipids and proteins.

• Protects the cell from its surroundings.

• Selectively permeable to ions and organic molecules and controls the movement of substances in and out.

• numerous proteins moving within or upon this layer are primarily responsible for transport of ions, nutrients and waste across the membrane.

Periplasmic space

• Gram-negative bacteria : space between the cytoplasmic membrane and the cell wall and space found between cell wall and the outer membrane

• Periplasm may constitute up to 40% of the total cell volume in G-ve species.

• Gram-positive bacteria : space between the cytoplasmic membrane and the cell wall.

• The periplasm is filled with water and proteins and is reminiscent of the cytoplasm.

Periplasmic Space

• However periplasm contains proteins and other molecules distinct from those in the cytoplasm because the membrane prevents the free exchange between these two compartments.

• Periplasmic proteins have various functions in cellular processes including: transport, degradation, and motility.

• Periplasm controls molecular traffic entering and leaving the cell.

Cell wall

• Outer covering of most cells that protects the bacterial cell and gives it shape (spherical, rod and spiral).

• Composed of peptidoglycan (polysaccharides + protein)

• Mycoplasma are bacteria that have no cell wall and therefore have no definite shape.

Cell wall

• Peptidoglycan - molecule found only in bacterial cell walls.

• The rigid structure of peptidoglycan gives the bacterial cell shape, surrounds the plasma membrane and provides prokaryotes with protection from the environment

• From the peptidoglycan inwards all bacterial cells are similar.

• Going further out, the bacterial world divides into two major classes: Gram-positive and Gram-negative .

• Amount and location of peptidoglycan in the cell wall determines whether a bacterium is G+ve or G-ve

Peptidoglycan = (polysaccharides + protein),

• Peptidoglycan (murein) - huge polymer of interlocking chains composed of similar monomers.

• peptidoglycan is made from polysaccharide chains cross-linked by peptides containing D-amino acids

• The backbone of the peptidoglycan molecule is composed of two derivatives of glucose:

• N-acetylglucosamine (NAG)

• N-acetlymuramic acid (NAM).

• The NAG and NAM strands are connected by inter peptide bridges.

Gram-positive Cells

• G+ve bacteria possess thick cell wall containing many layers of peptidoglycan and teichoic acids.

• In G+ ve cells, peptidoglycan is the outermost structure and makes up as much as 90% of the thick compact cell wall.

Gram-negative

• G-ve bacteria have relatively thin cell wall consisting of few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins

• Peptidoglycan makes up only 5 – 20% of the cell wall and is not the outermost layer, but lies between the plasma membrane and an outer membrane.

Gram Staining

• Developed in 1884 by Danish scientist Christian Gram.

• It is a differential stain.

• In this, bacteria are first stained with crystal violet, then treated with a mordant - a solution that fixes the stain inside the cell.

• Bacteria are then washed with a decolorizing agent, such as alcohol, and counterstained with safranin, a light red dye.

Gram Staining

• Gram-positive bacteria are those that are stained dark blue or violet by Gram staining.

• Gram-negative bacteria cannot retain the crystal violet stain, instead take up the counterstain and appearred or pink.

• The walls of gram-positive bacteria have more peptidoglycans than do gram-negative bacteria. Thus, gram-positive bacteria retain the original violet dye and cannot be counterstained.

Cell wall

• If the bacterial cell wall is entirely removed, it is called a protoplast while if it's partially removed, it is called a spheroplast.

• Antibiotics such as penicillin inhibit the formation of peptidoglycan cross-links in the bacterial cell wall.

• The enzyme lysozyme, found in human tears, also digests the cell wall of bacteria and is the body's main defense against eye infections.

outer membrane• Similar to the plasma membrane, but is less permeable .• This membrane has tiny holes or openings called porins. • Porins block the entrance of harmful chemicals and antibiotics,

making G-ve bacteria much more difficult to treat than G+ve cells. • Composed of lipopolysaccharides (LPS). • LPS is a harmful substance classified as an endotoxin.• Lipopolysaccharides, which acts as an endotoxin, are composed of

polysaccharides and lipid A (responsible for much of the toxicity of G-ve bacteria).

• These differences in structure can produce differences in antibiotic susceptibility

• Ex: vancomycin can kill only Gram +ve bacteria and is ineffective against Gram -ve pathogens, such as Haemophilus influenzae or Pseudomonas aeruginosa.

External structures

• Flagella

• Pili/fimbriae

• Capsule/slime layer

Flagella

• Singular: flagellum• Long, whip-like semi-rigid cylindrical structures that

aids in cellular locomotion• Function much like the propeller on a ship.• about 20 nm in diameter and up to 20 micromts in

length.• Diameter of a prokaryotic flagellum is about 1/10 th

of that of eukaryotic. • Flagella are driven by the energy released by the

transfer of ions down an electrochemical gradient across the cell membrane.

Flagella

• Made up of protein subunits called flagellin.

• Each flagellum is attached to cell membrane with the help of proteins other than flagellin.

• The basal region has a hook like structure and a complex basal body. The basal body consists of a central rod or shaft surrounded by a set of rings.

Flagella

• Bacterial spp differ in the number and arrangement of flagella on their surface.

• Bacteria may have one, a few, or many flagella in different positions on the cell.

• Monotrichous - single flagellum

• amphitrichous a flagellum at each end lophotrichous - clusters of flagella at the poles of the cell

• peritrichous - flagella distributed over the entire surface of the cell .

Arrangement of flagella

Flagella• Motile bacteria are attracted or repelled by certain

stimuli in behaviors called taxis: these include chemotaxis, phototaxis, and magnetotaxis.

• The flagella beat in a propeller-like motion to help the bacterium move toward nutrients; away from toxic chemicals; towards the light (photosynthetic cyanobacteria).

• Prokaryotes exhibit a variety of movements:

move , swim ,tumble ,glide, swarm in response to environmental stimuli.

FIMBRIAE AND PILI

• Hollow, hair like structures made of protein

• Involved in attachment to solid surfaces or to other cells and are essential for the virulence of some bacterial pathogens.

• Fimbriae fine filaments of protein just 2–10 nm 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 AND PILI

• Pili: (sing. pilus) are cellular appendages, slightly larger than fimbriae

• Involved in attachment to surfaces.

• Specialized pili, the sex pili, allows the transfer of genetic material from one bacteria to another in a process called conjugation where they are called conjugation pili or "sex pili".

• type IV pili - generate movement.

• Helps in colonization and pathogenicity.

Glycocalyx

• Glycocalyx : sticky coating produced by many bacteria covering the surface of cell.

• The glycocalyx is composed of polysaccharides (sugars) and proteins.

• The bacterial glycocalyx has 2 forms

• a highly structured rigid capsule

• a disorganised loose slime layer -

• Capsules are found on many pathogenic bacteria

Glycocalyx

• The glycocalyx has several functions including : protection, attachment to surfaces and formation of biofilms.

• The glycocalyx helps protect the bacteria cell by preventing immune cells from attaching to it and destroying it through phagocytosis.

Bacterial reproduction • Cell growth and reproduction by cell division are

tightly linked in unicellular organisms.

• Bacteria grow to a fixed size and then reproducethrough binary fission, a form of asexual reproduction

• Under optimal conditions, bacteria can grow anddivide extremely rapidly, and bacterial populations candouble as quickly as every 9.8 minutes.

• In cell division, two identical clone daughter cells areproduced.

• Budding involves a cell forming a protrusion thatbreaks away and produces a daughter cell

Binary fission

• Most prokaryotes reproduce by a process of binary fission, in which the cell grows in volume until it divides in half to yield two identical daughter cells.

• Each daughter cell can continue to grow at the same rate as its parent.

• For this process to occur, the cell must grow over its entire surface until the time of cell division, when a new hemispherical pole forms at the division septum in the middle of the cell.

• The septum grows inward from the plasma membrane along the midpoint and forms as the side wall which pinches inward, dividing the cell in two.

• In order for the cell to divide in half, the peptidoglycan structure must be different in the hemispherical cap than in the straight portion of the cell wall, and different wall-cross-linking enzymes must be active at the septum than elsewhere.

Binary fission

• Binary fission begins with the single DNA moleculereplicating and both copies attaching to the cellmembrane.

• Next, the cell membrane begins to grow betweenthe two DNA molecules. Once the bacterium justabout doubles its original size, the cell membranebegins to pinch inward.

• A cell wall then forms between the two DNAmolecules dividing the original cell into two identicaldaughter cells

Budding • A group of environmental bacteria reproduces by budding.• In this process a small bud forms at one end of the mother

cell• As growth proceeds, the size of the mother cell remains

about constant, but the bud enlarges.• When the bud is about the same size as the mother cell, it

separates. This type of reproduction is analogous to that inbudding fungi, such as brewer’s yeast (Saccharomycescerevisiae).

• One difference between fission and budding is that, in thelatter, the mother cell often has different properties from theoffspring.

• Ex: In some strains, mother cells have a flagellum and aremotile, whereas the daughter buds lack flagella.

BACTERIAL RECOMBINATION

Three mechanisms of genetic recombination

• Conjugation

• Transformation

• Transduction

CONJUGATION

• Two bacterial cells come together and mate such that a gene transfer occurs between them.

• Can only occur between cells of opposite mating types.

– The donor (or "male") carries a fertility factor (F+).

– The recipient ("female") does not (F−).

• One cell, the donor cell (F+), gives up DNA; and another cell, the recipient cell (F−), receives the DNA.

• The transfer is nonreciprocal, and a special pilus called the sex pilus joins the donor and recipient during the transfer.

• The channel for transfer is usually a special conjugation tube formed during contact between the two cells.

• The DNA most often transferred is a copy of the F factor plasmid.

• The factor moves to the recipient, and when it enters the recipient, it is copied to produce a double-stranded DNA for integration.

BACTERIAL TRANSFORMATION

• Discovered by Frederick Griffith in 1928.

• Many bacteria can acquire new genes by taking up DNA molecules (ex: plasmid) from their surroundings.

• When bacteria undergo lysis, they release considerable amounts of DNA into the environment.

• This DNA may be picked up by a competent cell- one capable of taking up the DNA and undergoing a transformation.

• To be competent, bacteria must be in the logarithmic stage of growth, and a competence factor needed for the transformation must be present.

BACTERIAL TRANSDUCTION

• Bacterial viruses ( bacteriophages) transfer DNA fragments from one bacterium (the donor) to another bacterium (the recipient).

• The viruses involved contain a strand of DNA enclosed in an outer coat of protein.

After a bacteriophage enters a bacterium, it may encourage the bacterium to make copies of the phage.

At the conclusion of the process, the host bacterium undergoes lysis and releases new phages. This cycle is called the lytic cycle.

Under other circumstances, the virus may attach to the bacterial chromosome and integrate its DNA into the bacterial DNA. It may

remain here for a period of time before detaching and continuing its replicative process. This cycle is known as the lysogenic cycle.Under these conditions, the virus does not destroy the host bacterium, but remains in a lysogenic condition with it. The virus is called a temperate phage, also known as a prophage. At a later time, the virus can detach, and the lytic cycle will

ensue. It will express not only its genes, but also the genes acquired

from the donor bacterium.

GROWTH OF BACTERIA

Bacterial Growth

• Growth of Bacteria is the orderly increase of all the chemical constituents of the bacteria.

• Multiplication is the consequence of growth.

• Death of bacteria is the irreversible loss of ability to reproduce.

Generation /doubling time

• Generation time (g) : The time it takes the cells to double.

• The average generative time is about 20-30 minutes in majority of medically important bacteria.

• They are some exceptions among pathogenic bacteria.

• Mycobacterium tuberculosis - 18 hrs.

• Mycobacterium leprae -10-20 days

• Length of generative time is in direct dependence on the length of incubation period of infections.

Growth Kinetics

• Bacterial growth follows four phases.

• lag phase

• log phase

• stationary phase

• death phase

Lag phase

• Immediately following the seeding of a culture medium.

• A period of adaptation for the cells to their new environment

• cells are adapting to the high-nutrient environment and preparing for fast growth.

• The lag phase has high biosynthesis rates, as proteins and metabolic intermediates are built up in adequate quantities for rapid growth & multiplication to proceed.

• New enzymes are synthesized.

• A slight increase in cell mass and volume, but no increase in cell number.

Duration of the lag phase varies with

- the species

- size of inoculum - Prolonged by low inoculum volume, poor inoculum condition (high % of dead cells)

- age of inoculum

- Nature of the culture medium (Prolonged by nutrient-poor medium)

- And environmental factors like temperature, pH etc

Log/Exponential growth phase

• In this phase, the cells have adjusted to their newenvironment and multiply rapidly (exponentially)

• The bacteria will grow and divide at a doubling timecharacteristic of the strains and determined by theconditions during the exponential phase.

• During this phase, the number of bacteria will increase to2n, in which n is the no.of generations.

• Balanced growth –all components of a cell grow at thesame rate.

Deceleration growth phase

Very short phase, during which growth decelerates due to either:

• Depletion of one or more essential nutrients

• The accumulation of toxic by-products of growth (e.g. Ethanol in yeast fermentations)

• Period of unbalanced growth: Cells undergo internal restructuring to increase their chances of survival

Stationary Phase

With the exhaustion of nutrients or build-up of toxic waste substances and secondary metabolic products in the medium , the bacteria stop growing and enter the stationary phase.

- The growth rate equals the death rate – The number of progeny cells formed is just enough to replace the number of cells that die.

- There is no net growth in the organism population – The viable count remains stationary as an equilibrium exists between the dying cells and newly formed cells.

Death Phase

- Phase of decline

- The living organism population decreases with time, due to a lack of nutrients and accumulation of toxic metabolic by-products.

- Cell death may also be caused by autolytic enzymes.

Generation timesBacterium Medium Generation Time (minutes)

Escherichia coli Glucose-salts 17

Bacillus megaterium Sucrose-salts 25

Streptococcus lactis Milk 26

Streptococcus lactis Lactose broth 48

Staphylococcus aureus Heart infusion broth 27-30

Lactobacillus acidophilus Milk 66-87

Rhizobium japonicum Mannitol-salts-yeast extract 344-461

Mycobacterium tuberculosis Synthetic 792-932

Treponema pallidum Rabbit testes 1980

Factors Required for Bacterial Growth

The requirements for bacterial growth are:

(A) Environmental factors

(B) Sources of metabolic energy.

Environmental Factors

Affecting Bacterial

Growth

Nutrients• Nutrients in growth media must contain all the

elements necessary for the synthesis of new organisms.

• Hydrogen donors and acceptors

• Carbon source

• Nitrogen source

• Minerals : sulphur and phosphorus

• Growth factors: amino acids, purines, pyrimidines; vitamins

• Trace elements: Mg, Fe, Mn.

• Microorganisms are sensitive to temperature changes

– Usually unicellular– Enzymes have temperature optima– If temperature is too high, proteins denature, including

enzymes, carriers and structural components

• Temperature ranges are enormous (-20 to 100oC)

Temperature

Temperature

–Organisms exhibit distinct cardinal temperatures (minimal, maximal, and optimal growth temps)

– If an organism has a limited growth temperature range = stenothermal (e.g. N. gonorrhoeae)

– If an organism has a wide growth temperature range = eurythermal (E. faecalis)

Temperature

Psychrophiles can grow well at 0oC, have optimal growth at 15oC or lower, and usually will not grow above 20oC

• Arctic/Antarctic ocean

• Protein synthesis, enzymatic activity and transport systems have evolved to function at low temperatures

• Cell walls contain high levels of unsaturated fatty acids (semi-fluid when cold)

Temperature

– Psychrotrophs can also grow at 0oC, but have growth optima between 20oC and 30oC, and growth maxima at about 35oC

• Many are responsible for food spoilage in refrigerators

– Mesophiles have growth minima of 15 to 20oC, optima of 20 to 45oC, and maxima of about 45oC or lower• Majority of human pathogens

Temperature–Thermophiles have growth minima around

45oC, and optima of 55 to 65oC

• Hot springs, hot water pipes, compost heaps

• Lipids in PM more saturated than mesophiles.

–Hyperthermophiles have growth minima around 55oC and optima of 80 to 110oC

• Sea floor, sulfur vents

Effect of temperature

Temperature optima of bacteria

pH

– pH is the negative logarithm of the hydrogen ion concentration

– Acidophiles grow best between pH 0 and 5.5

– Neutrophiles grow best between pH 5.5 and 8.0

– Alkalophiles grow best between pH 8.5 and 11.5

– Extreme alkalophiles grow best at pH 10.0 or higher

pH

– Sudden pH changes can inactivate enzymes and damage plasma membrane

• Reason for buffering culture medium, usually with a weak acid/conjugate base pair (e.g. KH2PO4/K2HPO4 – monobasic potassium/dibasic potassium)

Bacterial growth at various pH

pH profiles for some prokaryotes

Organism Minimum pH Optimum pH Maximum pH

Thiobacillus thiooxidans 0.5 2.0-2.8 4.0-6.0

Sulfolobus acidocaldarius 1.0 2.0-3.0 5.0

Bacillus acidocaldarius 2.0 4.0 6.0

Zymomonas lindneri 3.5 5.5-6.0 7.5

Lactobacillus acidophilus 4.0-4.6 5.8-6.6 6.8

Staphylococcus aureus 4.2 7.0-7.5 9.3

Escherichia coli 4.4 6.0-7.0 9.0

Clostridium sporogenes 5.0-5.8 6.0-7.6 8.5-9.0

Erwinia caratovora 5.6 7.1 9.3

Pseudomonas aeruginosa 5.6 6.6-7.0 8.0

Thiobacillus novellus 5.7 7.0 9.0

Streptococcus pneumoniae 6.5 7.8 8.3

Nitrobacter sp 6.6 7.6-8.6 10.0

Oxygen concentration

–Obligate aerobes are completely dependent on atmospheric O2 for growth

• Oxygen is used as the terminal electron acceptor for electron transport in aerobic respiration

– Facultative anaerobes do not require O2 for growth, but do grow better in its presence

–Aerotolerant anaerobes ignore O2 and grow equally well whether it is present or not

Oxygen concentration

–Obligate (strict) anaerobes do not tolerate O2 and die in its presence.

–Microaerophiles are damaged by the normal atmospheric level of O2 (20%) but require lower levels (2 to 10%) for growth

Oxygen and growthEnvironment

Group Aerobic Anaerobic O2 Effect

Obligate Aerobe Growth No growth Required (utilized for

aerobic respiration)

MicroaerophileGrowth if

level not

too high

No growthRequired but at levels

below 0.2 atm

Obligate Anaerobe No growth Growth Toxic

Facultative

(An)aerobe

Growth GrowthNot required for growth

but utilized when available

Aerotolerant

Anaerobe

Growth Growth Not required and not

utilized

Anaerobic growth chambers

Water availability• Water is solvent for biomolecules, and its availability is

critical for cellular growth

• The availability of water depends upon its presence in the atmosphere (relative humidity) or its presence in solution or a substance (water activity, (Aw))

• Aw of pure water (100%) is 1.0; affected by dissolved solutes such as salts or sugars.

• Microorganisms live over a range of aW from 1.0 to 0.7. The aW of human blood is 0.99; seawater = 0.98; maple syrup = 0.90; Great Salt Lake = 0.75. Water activities in agricultural soils range between 0.9 and 1.0.

Effect of salt on growth

Pressure–Barotolerant organisms are adversely

affected by increased pressure, but not asseverely as are nontolerant organisms

–Barophilic organisms require, or grow morerapidly in the presence of increasedpressure

–Light: Optimum condition for growth isdarkness.

Radiation-Ultraviolet radiation damages cells by causing

the formation of thymine dimers in DNA.– Ionizing radiation such as X rays or gamma rays

are even more harmful to microorganisms thanultraviolet radiation• Low levels produce mutations and may

indirectly result in death• High levels are directly lethal by direct

damage to cellular macromolecules orthrough the production of oxygen freeradicals

(B) Sources of Metabolic Energy

• Mainly three mechanisms generate metabolic energy. These are

• Fermentation

• Respiration and

• Photosynthesis.

An organism to grow, at least one of these mechanisms must be used.

Continuous Culture

Techniques

• Used to maintain cells in the exponentialgrowth phase at a constant biomassconcentration for extended periods oftime

• Conditions are met by continual provisionof nutrients and removal of wastes =OPEN SYSTEM

• Constant conditions are maintained

•Balanced and Unbalanced

Growth

• Balanced (exponential) growth occurs whenall cellular components are synthesized atconstant rates relative to one another

• Unbalanced growth occurs when the rate ofsynthesis of some components changerelative to the rate of synthesis of othercomponents.

–This usually occurs when the environmentalconditions change

MORPHOLOGICAL CHARACTERISTICS OF BACTERIA

SIZE- SHAPE-ARRANGEMENT

MORPHOLOGY

• Bacteria display a wide diversity of shapes and sizes called morphologies

• Cannot be seen with human eyes (microscopic)• Their presence was only first recognized in 1677, when the Dutch

naturalist Antonie van Leeuwenhoek saw microscopic organisms in a variety of substances with the aid of primitive microscopes.

• Now bacteria are usually examined under light microscopes capable of more than 1,000-fold magnification

• Details of their internal structure can be observed only with the aid of much more powerful transmission electron microscopes.

• Unless special phase-contrast microscopes are used, bacteria have to be stained with a coloured dye so that they will stand out from their background.

Size• Bacteria are the smallest living creatures• Most bacteria are 0.2 um in diameter and 2-8 um in length.• Bacterial cells are about one tenth the size of eukaryotic cells • are typically 0.5 – 5.0 micrometres in length.• Giant bacteria for example, Thiomargarita namibiensis,

Titanospirillum namibiensis and Epulopiscium fishelsoni — are up to half a mm 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 about 0.1 to 0.25 μm in diameter, as small as the largest viruses.

• Some bacteria may be even smaller, but these ultramicrobacteria are not well-studied.

Size• E. coli, a normal inhabitant of the intestinal tract of humans

and animals, is about 2 μm long and 0.5 μm in diameter• spherical cells of Staphylococcus aureus - up to 1 μm in

diameter.• the rod-shaped Bordetella pertussis, causative agent of

whooping cough - 0.2 to 0.5 μm in diameter and 0.5 to 1 μmin length

• corkscrew-shaped Treponema pallidum, causative agent of syphilis averaging only 0.15 μm in diameter but 10 to 13 μmin length.

• Some bacteria are relatively large, such as Azotobacter, which has diameters of 2 to 5 μm or more

• cyanobacterium Synechococcus averages 6 μm by 12 μm• Achromatium, which has a minimum width of 5 μm and a

maximum length of 100 μm, depending on the species.

Cell Shape• Bacteria come in a wide variety of shapes.

• Coccus – are spherical or oval cells.

• Bacillus - are round-ended cylinder shaped cells.

• Vibrios comma shaped ,curved rods and derive the name from their characteristic vibratory motility

• Spirilla – are rigid spiral forms(coil).

• Spirochetes - are long, slender, and flexible spiral forms(from speira meaning coil and chaite meaning hair)

• Filamentous – resembles radiating rays of sun

Cell Shape

• coccobacilli - Some bacilli are so short and fat that they look like cocci and are referred to as coccobacilli.

• A small number of species even have tetrahedralor 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.

Cell Shape• is generally characteristic of a given bacterial species• but can vary depending on growth conditions. • Some bacteria have complex life cycles involving the

production of stalks and appendages (e.g. Caulobacter) and some produce elaborate structures bearing reproductive spores (e.g. Myxococcus, Streptomyces).

• Bacteria generally form distinctive cell morphologies when examined by light microscopy and distinct colony morphologies when grown on Petri plates.

• These are often the first characteristics observed by a microbiologist to determine the identity of an unknown bacterial culture

Cell Shape

• This wide variety of shapes is determined bythe bacterial cell wall and cytoskeleton

• Shape of the cell is important because it caninfluence the ability of bacteria to acquirenutrients, attach to surfaces, swim throughliquids and escape predators

Arrangement of cells

• Cellular arrangements occur singularly, in pairs, in chains and in clusters.

Bacilli

• Diplobacilli (2 cells), tetrad (4 cells), palisade (two cells arranged parallel) or sterptobacilli(chain arrangement)e.g E.Coli and Salmonella.

Cocci

Bacilli

Other shapes

CULTURAL CHARACTERISTICS OF

BACTERIA

• Culture techniques are designed to promote the growth and identify particular bacteria,whilerestricting the growth of the other bacteria in the sample.

• In the laboratory, bacteria are usually grown using solid growth media such as agar plates or liquid media such as broth.

• Solid, agar-based media can be used to identify colonial characteristics (shape, size, elevation, margin type) and to isolate pure cultures of a bacterial strain

• liquid growth media are used when measurement of growth or large volumes of cells are required.

• Growth in stirred liquid media occurs as an even cell suspension, making the cultures easy to divide and transfer

• isolating single bacteria from liquid media is difficult.• The use of selective media (media with specific

nutrients added or deficient, or with antibiotics added) can help identify specific org’s.

• Most laboratory techniques for growing bacteria use high levels of nutrients to produce large amounts of cells cheaply and quickly.

• However, in natural environments nutrients are limited, meaning that bacteria cannot continue to reproduce indefinitely

Cultural characteristics

Basic conditions for cultivation• Optimum environmental moisture. It is possible to

cultivate bacteria in liquid media or solid media with a gelling agent (agar) binding about 90% of water.

• Optimum temperature for cultivation of bacteria of medical importance is about 370C. Saprophytic bacteria are able to grow at lower temperatures.

• Optimum pH of culture media is usually 7.2-7.4 Lactobacillus spp need acid pH and vibrio cholera needs alkaline pH for the growth.

• Optimum constituents of bacteriological culture media.• All culture media share a number of common constituents

necessary to enable bacteria to grow in vitro.

Optimum Quantity of oxygen in cultivation environment.

• Bacteria obtain energy either by oxidation or by fermentation i.e., oxidation – reduction procedure without oxygen.

• Bacteria are classified into four basic groups according to their relation to atmospheric oxygen:

• Obligate aerobes: Reproduce only in the presence of oxygen• Facultative anaerobes : reproduce in both aerobic and anaerobic

environments. Their complete enzymatic equipment allows them to live and grow in the presence or absence of oxygen.

• Obligate anaerobes: grow only in the absence of free oxygen (i,eunable to grow and reproduce in the presence of oxygen). Some species are so sensitive that they die if exposed to oxygen.

• Anaerobic Aerotolerant: microbes do not need oxygen for their growth and it is not fatal for them

Colony morphology

• Form - the basic shape of the colony ex: circular, filamentous etc.

• Size – The diameter of the colony. • Elevation - This describes the side view of a colony.

Turn the Petri dish on end.• Margin/border - magnified shape of the edge of the

colony• Surface - colony appearance

ex: smooth, glistening, rough, wrinkled or dull.• Opacity - ex transparent (clear), opaque, translucent

(like looking through frosted glass), etc.• Colour (pigmentation) ex: white, buff, red, purple, etc.

Colony morphology

• Colony morphology is a method that scientists use to describe the characteristics of an individual colony of bacteria growing on agar in a Petri dish. It can be used to help to identify them.

• Each distinct colony represents an individual bacterial cell or group that has divided repeatedly. Being kept in one place, the resulting cells have accumulated to form a visible patch.

• Most bacterial colonies appear white or a creamy yellow in colour, and are fairly circular in shape.

Effect of media• different types of media, which contain different

nutrients can affect the cultural characteristics of bacteria.

• Some types of media are much more nutritive and will encourage hearty growth. Some types of media may restrict growth.

• Colonial morphology may also be affected by the temperature at which the bacteria is incubated. Some bacteria grow better at body temperature and grow weakly at room temperature, or vice versa.

• Some bacteria express certain characteristics, such as the formation of pigment, more strongly at some temperatures than at others.

CLASSIFICATION OF BACTERIA

Bacterial Classification Based on Shapes

• Bacilli: Rod shaped bacteria. • Diplobacilli, tetrad , palisade (two cells arranged parallel) or

sterptobacilli (chain arrangement). e.g. E.Coli and Salmonella

• Coccus: Spherical or oval cells shaped bacteria which is further classified as monococcus, diplococci, streptococci ,Staphylcocci

e.g. Staphylococcus and Streptococcus

• Spiral: Spiral shaped bacteria are called spirillae.g. Treponema and Borellia

sub divided into spirilla (rigid spiral forms) and spirochetes(flexible spiral forms).

• Comma shaped: Vibrio• Branching filamentous forms : Actinomycetes

Bacterial Classification Based on Staining Methods

• Gram positive bacteria - take up crystal violet dye and retain their blue or violet color.Gram negative bacteria - do not take up crystal violet dye, and thus appear red or pink.

Classification Based on Respiration

• Aerobic Respiration : sugars are broken down in the presence of oxygen to produce carbon dioxide, water, and energy.

• Anaerobic Respiration : anaerobic respiration breaks down sugars and releases energy in the absence of oxygen.

• anaerobic respiration is typically slower and less efficient than aerobic respiration.

• anaerobic respiration involves chemicals other than oxygen and carbondioxide.

Classification Based on Respiration

• Facultative Anaerobic Respiration : Facultative Anaerobes are able to perform either aerobic / anaerobic respiration depending on the oxygen content of their environment.

• Ex: Coliform bacteria

• Microaerophiles : sugars are broken down in the presence of minute amounts of oxygen to produce energy.

Classification Based on Environment

• Mesophiles - which require moderate temp to survive.Neutrophiles - require moderate conditions to survive.Extremophiles - can survive in extreme conditions.Acidophiles - which can tolerate low pH conditions.Alkaliphiles - which can tolerate high pH conditions.Thermophiles - which can resist high temperature.Psychrophiles - can survive extremely cold conditions.Halophiles - can survive in highly saline conditions.Osmophiles - can survive in high sugar osmotic conditions.

Classification Based on Flagella

• Atrichous (no flagella),

• monotrichous (uni flagella)

• amphitrichous (bi flagella)

• polytrichous (more flagella)

Classification Based on Spore Formation

• spore forming

• non-spore forming

Classification Based on their association with host

• Beneficial

• Pathogenic

• Harmless

Nutritional Source

• bacteria are also classified based on the type of energy source utilized by them for survival.

• Autotrophs: obtain the carbon it requires from carbon dioxide

• Photoautotrophs: directly use sunlight in order to produce sugar from carbon dioxide.

• Chemoautotrophs : depend on various chemical reactions.

• use inorganic energy sources, such as hydrogen sulfide, elemental sulfur, ferrous iron, molecular hydrogen, and ammonia.

Nutritional Source

• Heterotrophs : Heterotrophic bacteria obtain sugar from the environment they are in (ex: the living cells or organisms they are in).

• symbiotic

• saprophytes

• parasite

Capsule

• Capsulated

• Encapsulated

Classification of bacteria

• With over millions of bacteria present in the planet, it is not an easy job to identify, isolate and study a particular species or particular bacteria as such.

• Microbiologists categorized bacteria based on basic and important factors making all the bacteria fall under any one of the categories and thus making the process of isolation and identification much easier.

• Bacteria are classified based on various factors like shape (morphology), Cell wall structure, Respiration (metabolism), type of nutritional source, characteristic and environmental factor.

Bacteria are classified based on various factors

• shape (morphology)

• Cell wall structure

• Respiration (metabolism)

• type of nutritional source

• characteristic

• environmental factor etc.

• Chemostat–A continuous culture device that maintains a

constant growth rate by:

• supplying a medium containing a limited amount of an essential nutrient at a fixed rate

• removing medium that contains microorganisms at the same rate

–As fresh media is added to the chamber, bacteria are removed

– Limiting nutrients control growth rates

– Cell density depends on nutrient concentration

• Turbidostat

A continuous culture device that regulates the flow rate of media through the vessel in order to maintain a predetermined turbidity or cell density

• There is no limiting nutrient

• Absorbance is measured by a photocell (optical sensing device)