M1-Introduction to Microorganisms Flashcards
What is microbiology?
Study of microscopic living organisms
What are the importance of microorganisms?
- Oldest form of life
- Largest mass of living material
- Carry out major processes for biogeochemical cycles
What are the key structures of a bacterial cell?
- Cytoplasmic membrane
- Cytoplasm
- Nucleus or nucleoid and ribosomes
- Cell wall
What characteristics are in all bacterial cells?
- Metabolism
- Growth
- Evolution
What characteristics are in some bacterial cells only?
- Differentiation
- Communication
- Genetic exchange
- Motility
What contributions did Robert Hooke make?
- First description of microbes
- Illustrated the fruiting structures of molds
What contributions did Antoni van Leeuwenhoek make?
- First to describe bacteria
- Observed in teeth scrapings, rain water and other specimens
What contributions did Louis Pasteur make?
- Showed that microbes are responsible for fermentation
- Demonstrated that bacteria which caused the spoilage of wine could be killed by heat that was not hot enough to evaporate the alcohol in wine
- Disproved theory of spontaneous generation
- Developed vaccines for anthrax, fowl cholera, and rabies
What contributions did Robert Koch make?
- Definitely demonstrated the link between microbes and infectious diseases
- Identified causative agents of anthrax and tuberculosis
- Koch’s postulates
- Developed techniques (solid media) for obtaining pure cultures of microbes
What are Koch’s postulates?
- Bacteria must be present in every case of the disease
- Bacteria must be isolated from the host with the disease and grown in pure culture
- Specific disease must be reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host
- Bacteria must be recoverable from the experimentally infected host
What characteristics does cytoplasmic membrane have?
- Phospholipid bilayer
- Contains embedded proteins
- Functions as a highly selective permeability barrier
- Does not confer rigidity
What is the difference between bacterial and archaeal cytoplasmic membrane?
- Bacterial membrane lipids contain fatty acids joined to glycerol via ester linkages
- Archaeal membrane lipids contain repeating isoprene units joined to glycerol via ether linkages
What is the difference between gram-positive and gram-negative cell walls?
- Structure of gram-positive cell walls: peptidoglycan + cytoplasmic membrane
- Structure of gram-negative cell walls: outer membrane + peptidoglycan + cytoplasmic membrane
What is peptidoglycan?
- Rigid structural layer that provides strength to the cell wall
- Made of repeating polysaccharide unit (G+M)
What is the difference between the peptidoglycan in gram-positive and gram-negative cell walls?
- Thinner and hidden in gram-negative cell walls
- Thicker and exposed in gram-positive cell walls
What is the crosslinking of the repeating G+M unit of peptidoglycan in gram-positives?
-Occurs via formation of peptide interbridge
What is the crosslinking of the repeating G+M unit of peptidoglycan in gram-negatives?
-NH2 group of DAP of one glycan chain to COOH group of D-alanine on adjacent chain
How the repeating unit of G+M unit of peptidoglycan provides structural integrity?
-Crosslinking between peptidoglycan chains by peptide bonds, provides strength in X and Y directions
What does the gram-positive cell wall contain?
- Up to 90% peptidoglycan
- Teichoic acids embedded
- Lipoteichoic acids: teichoic acids covalently bound to membrane lipids
What does the gram-negative cell wall contain?
- Outer membrane: lipopolysaccharide (LPS), phospholipid
- Periplasm: peptidoglycan
What does the outer membrane in gram-negative cell wall contain?
-lipopolysaccharide (LPS) layer: core polysaccharide, O-polysaccharide and lipid A (endotoxin)
What characteristics does gram-negative lipopolypeptide (LPS) have?
- Comprises 50% of the mass of the outer membrane
- Structural composition of LPS lipid A and core polysaccharide is generally conserved while the O-antigen varies
- Highly toxic for humans
- Toxic properties associated with LPS are mainly caused by endotoxin (lipid A)
- Endotoxins cause fever and systemic toxic effects by stimulating the release of cytokines and other host inflammatory mediators from immune cells
What is periplasm in the gram-negative cell wall?
- Space located between cytoplasmic and outer membranes
- Have gel-like consistency
- Houses many proteins
What is gram stain test?
- Differentiate between gram-positive and gram-negative bacteria
- Due to differences between cell walls of gram-positive and gram-negative bacteria
What is the procedure of gram stain test?
- Flood the heat-fixed smear with crystal violet for 1 min
- Add iodine solution for 1 min
- Decolourize with alcohol briefly
- Counterstain with safranin for 1-2 min
What are the results of the gram stain test?
- Gram-positive: purple
- Gram-negative: red/pink
What bacteria cannot gram stain?
- Genus Mycobacterium
- -*Acid fast bacteria: stained red using acid fast stain
What is S layer?
- Most common cell wall type among Archaea
- Consist of interlocking protein or glycoprotein arranged as a paracrystalline surface structure
- Provides structural rigidity, barrier to diffusion of large molecules
- Also found in some bacteria
What is the difference between archaeal cell walls and bacterial cell walls?
-Archaea do not contain peptidoglycan or an outer membrane in the cell wall
What is pseudomurein?
=Polysaccharide similar to peptidoglycan
- Composed of N-acetylglucosamine and N-acetylalosaminuronic acid
- Found in some methanogenic Archaea
What are capsules?
=Polysaccharide layers
- Assist in attachment to surfaces (biofilm formation)
- Aid in evasion of immune system
- Resist desiccation
- Occurs in both gram-positives and gram-negatives
Give an example of capsule and its function.
-Streptococcus pneumoniae: capsule allows the organism to avoid immune recognition
What is fimbriae?
=Filamentous bacterial surface appendages
- Involved in adherence
- Primary function: adhesion
- Some bacteria can produce more than one type of fimbriae
What is flagella?
=Rotatable filamentous bacterial surface appendages
- Involved in bacterial locomotion
- Different arrangements: polar, lophotrichous or peritrichous
- Helical in shape
- Filament composed of the protein flagellin
- Moves by rotation
What is the structure of gram-negative flagellum?
- Flagella filament is composed of many copies of a protein flagellin
- Hook consists of a single type of protein and connects the filament to the motor at the base
- Motor (Mot proteins) is anchored in the cytoplasmic membrane and cell wall, which drives the rotation of flagella
- Fli proteins function as the motor switch, reversing the direction of rotation of the flagella in response to intracellular signals
What is the process of flagella biosynthesis?
-Flagellin molecules are synthesized in cytoplasm and move up through a hollow core in the filament
What is the movement made by polar flagellated cells?
- Reversible flagella: rotation changed between clockwise and counter-clockwise
- Unidirectional flagella: rotation unchanged; cell stops and reorients
What is the movement made by peritrichous flagellated cells?
-Flagella pushed apart by tumbling to change direction
What characteristics do archaeal flagella have?
- Most archaea produce flagella
- Capable of swimming motility
- Structure of the archaeal flagella motor = not well known
- Filament of archaeal flagella = often composed of several different flagellin proteins
- Some archaeal flagellins = glycoproteins
- Amino acid sequence of archaeal flagellins is not related to that of bacterial flagellins
What is gliding motility?
- Flagella-independent motility
- Significant slower
- Requires surface contact
What is chemotaxis in peritrichous flagellated cells?
- Bacteria respond to differences in chemical concentration
- Attractants sensed by chemoreceptors
- Increased tumbling as attractant concentration decreases
- Decreased tumbling as attractant concentration increases
How is chemotaxis measured?
-Capillary tube assay
What is cell inclusion?
- Many bacteria can synthesize carbon and energy storage compounds, like lipid or glycogen
- Some bacteria can accumulate inorganic phosphage (PO43-) for nucleic acid, phospholipid and ATP synthesis
- Other bacteria can oxidise H2S either to produce energy or as part of a CO2 fixation process
- Magnetosomes: intracellular particles of magnetite (Fe3O4) that enable bacteria to orientate themselves in a specific arrangement within a magnetic field
What are gas vesicles?
- Confer buoyancy in planktonic cells
- Spindle-shaped gas-filled structures made of protein
- Gas vesicle membrane impermeable to water
- Allows photosynthetic bacteria to optimise position in water column
What are endospores?
- Highly differentiated cells that is resistant to dessication, heat, harsh chemicals, and radiation
- “Dormant” stage of bacterial life cycle
- Ideal for dispersal via wind, water, or animal gut
- Only present in some gram-positive bacteria
What is the structure of endospore?
- Differes markedly from that of the vegetative cell
- Exosporium = outermost covering consisting of protein
- Several spore coats may then be found
- Cortex consists of a peptidoglycan layer
- Core contains the core wall, cytoplasmic membrane, cytoplasm, DNA and ribosomes
What is microbial growth?
=Increase in the number of cells within a population
- Cells reproduce by binary fission
- Cell numbers double every generation
What is growth rate?
-Change in cell number or cell mass per unit time
What is generation time/doubling time?
-Interval for the formation of two cells (from one cell)
What is a batch culture?
-A closed-system microbial culture of fixed volume
What is the typical growth curve for a bacterial population?
What are the growth phases in batch culture?
- Lag phase: period before growth begins at a maximal rate
- Exponential phase: period of growth where the number of cells doubles during a constant time interval
- Stationary phase: period of growth where there is no net increase in cell number
- Death phase: period where cells begin to die
What happens in lag phase?
- Cells adapted to stationary phase transferred to fresh medium
- Cells transferred from rich to minimal medium
- In both cases, induction of new enzymes is required
What happens in exponential phase?
- Increase in cell number is initially rather slow but increases at an ever faster rate
- When cells are growing exponentially, the time taken for all components of the cell to double is the same (=balanced growth)
- Growth is unrestricted as long as the concentration of all nutrients does not limit growth or toxic products do not build up
What two parameters are in the maths of microbial growth?
- Generation time (g): time required for the population to double in size
- Mean growth rate constant (k): mean number of generations (divisions) per hour; =1/g
What equation give the number of cells produced over a given incubation time?
N = N0 2n
What equation give the number of generations required to produce N cells?
n = (log N - log N0) / log 2
What equation give the generation time (g)?
g = t / n (hr)
-Since “n” generations happened over “t” incubation time
What happens in stationary phase?
- Occurs when 1 nutrient is limiting for growth or when inhibitory products accumulate
- Some metabolic activity, other activities slow down
- Bacteria adapt to stationary phase by activating the expression of specific genes
What does the expression of genes involved in adaptation to stationary phase depend on?
A specific RNA polymerase sigma factor, RpoS
(sigma recognizes promoter and initiation site)
- RpoS directs RNA polymerase to transcribe genes involved in stationary phase adaptation
- RpoS binds to a distinct promoter consensus sequence
Give an example of what happens in stationary phase.
- In E. coli:
- Stationary phase, cells smaller, increased resistance
- Contain far more glycogen (carbon and energy storage)
What happens in death phase?
- Cell begins to die
- May be associated with cell lysis
What methods are used to measure growth?
- Total cell count
- Viable count
- Turbidimetric measurements
What is total cell count?
Direct counting of cells under a microscope
- Advantage: Rapid way of estimating cell number
- Disadvantages:
- Dead cells not distinguished from living cells
- Small cells difficult to see under microscope
- Precision sometimes difficult to achieve
- Phase contrast is required when sample is not stained
-Only suitable for bacteria at a density greater than 106 per ml
What is viable count?
- Has two different methods: spread plate method and pour plate method
- Advantages:
- Widely used in food, medical, dairy and aquatic microbiology
- Highly sensitive
- Samples containing few cells can be counted
- Use of selective media allows counting of specific organisms in mixed populations
-Disadvanges:
- Number of colonies that develop may depend on conditions
- Small colonies may be overlooked during counting
- To avoid errors, replicate plates of key dilutions are required
- Cell clumping may reduce counts
What is spread plate method and its assumption?
Assumption: each viable cell produces a single colony
What is pour plate method and its assumption?
- Assumption: each viable cell produces a single colony
- Bacteria must be able to withstand 45oC for a limited time when they are placed in molten agar
What is the procedure for viable counting?
- Serial dilutions
- Cultures usually need to be diluted otherwise the number of colonies developing become too great and makes it impossible to count
What is turbidimetric method?
- Rapid method that uses a spectrophotometer
- Measures light that is not scattered by the bacteria, optical density (OD)
- Accurate measure of cell number
- At high cell densities, backscattering leads to a deviation from linearity
- Without disturbing or destroying a culture
What are the properties of a useful industrial microbe?
- Produces spores or can be easily inoculated
- Grows rapidly on a large scale in inexpensive medium
- Produces desired product quickly
- Should not be pathogenic
- Amenable to genetic manipulation
What are primary metabolites?
-Produced during exponential growth
What are secondary metabolites?
- Produced during stationary phase
- Not essential for growth
- Often significantly over-produced
What is a biofilm?
A structured community of bacterial cells enclosed in a self-produced matrix and adhered to an inert or living surface
What is the development of biofilm?
Why do bacteria form biofilms?
- Biofilms are a means of microbial self defense
- Biofilm formation allows cells to colonize favorable niches
- Biofilms enable bacteria to live together
- Biofilm formation is a survival strategy
What are the importances of biofilms in medicine?
- Bacteria that form biofilms cause persistent (chronic) infections
- Bacteria can withstand the action of the host defense system and the highest deliverable doses of antibiotics
How do biofilms become so resistant to antimicrobials?
- Slow penetration: outer layers absorb damage
- Stress response: inner layers have more time to adapt
- Altered microenvironment: nutrient depletion creates zones of altered activity
Persister cells: cells tolerant to antibiotics due to state of dormancy
-Antimicrobial depletion: adsorption of antibiotics, lack of penetration
What is green fluorescent protein (GFP) and its function?
- Has multiple colours
- Tagging of cells
- Direct monitoring of live cells
- No need to stain
