R.O Lecture 3&4 Flashcards
What is bacterial growth?
Refers to the increase in bacterial cell numbers not cell size
True or Fale, binary fission is the same as mitosis?
False - binary fission is not the same as mitosis
Describe binary fission.
- Duplication of contents and division into 2 cells. Most common method of bacterial cell division
- DNA replication prior to cell division (not a defined cell cycle stage)
- Chromosome attached to cell membrane
- Nucleoids divide
- FtsZ proteins from a Z ring. Seperated cytoplasm
- Transverse septum forms
- 2 daughter cells form.
What are the phases of growth for bacteria?
- Lag phase
- Log Phase
- Stationary Phase
- Decline, or death, phase.
- Lag Phase
- No Significant increase in number
- Metabolically active –Increasing size, ATP production, enzyme synthesis, nutrient uptake
- May use up all storage granules Phase length dependent on species, nutrients and age of culture
2.Log phase
- Organisms have adapted to the media and divide at an exponential/logarithmic rate
- Cells are most active metabolically and most sensitive to adverse conditions (e.g. Antibiotics)
- Do not accumulate inclusion bodies
- Limiting factor: the rate at which ATP can be produced (dependent on nutrient availability, waste levels, space etc.)
inclusion bodies
Inclusion bodies are aggregates of virus particles or virus-induced proteins or special structures characteristic of infection by viruses either in the cytoplasm or the nucleus
Generation time or doubling time (td)
time taken for cells to double their number; usually measured during log phase of growth when cells are growing maximally and most factors are “uniform”: Range 20 min - 20hrs
Chemostat
Device that maintains cells in log phase. Pumps out ‘spent’ medium with high toxin levels, purifies bactera medium.
Achieved by continually replenishing media
Can also be carried out manually
3.Stationary Phase
- Metabolism slows down
Growth rate = Death rate (no overall change in numbers) - Spore-forming bacteria form endospores in this phase
- Granular inclusions and abnormal forms appear in some species
4.Death/Decline Phase
- Media conditions no longer support cell division and cells start to die
- Decreases at a logarithmic rate
- Abnormal cells appear (involution)
- Variable duration (species dependent)
- Endospores may survive, if present
Describe Bacteria biofilms.
- A biofilm consists of a collection of bacteria (and often other microorganisms) that colonise a surface/attach to each other.
- Embedded in a matrix secreted/released by bacteria in the biofilm
- Extracellular polymeric substance (EPS) composition: polysaccharides (capsule/aggregate), proteins, nucleic acids etc.
- Formation -> dispersal
Where are biofilms formed?
-Tooth
-Lung epithelium (CF)
-Medical devices
-Piping
-Natural aquatic habitats
-Plant surfaces
Biofilms - resource capture and digestion
Resource capture and digestion
-Matrix can trap nutrients (accumulation)
-Extracellular digestion by community
Biofilms - Gradients
Gradients:
-localised habitats (pH, oxygen, signalling molecules)
- stabilised by immobilisation of biofilm cells witihin the matrix
What are biofilms resistant to?
- Dessication
- Antibiotics
- Immune system components
Describe the process of Quorum sensing (QS)
- Free swimming cells settle on a surface
- Cells synthesise a viscous matrix that holds them tightly to the substrate
- When only a few cells are present, the concentration of the signaling (induce) molecule is low
- When biofilm grows to a certain desnsity (quorum), the concentraion of the inducer molecules causes expression of specifc genes.
- Genetic Induction - signaling molecules induce expression of a protein product, such as food -digesting enzyme.
- Cells secrete their enzymes in unison to digest food particles.
What are autoinducers? (Als)
- Autoinducers are signaling molecules that are produced in response to changes in cell-population density
- In high cell density populations, Als reach a threshold concentraion, are dected by cells and induce gene expression.
What are the 3 different stages for QS?
Synthesis, Recognition and Response.
- Signals produced by bacteria which induce changes in the population once a concentration threshold is reached.
What enables synchronisation of population response?
Cell-cell communication in bacteria populations
What are some of the processes controlled by QS?
Quorum sensing
Processes controlled by QS include bioluminescence, sporulation, competence, antibiotic production, biofilm formation/dispersal, virulence factor secretion, autoinducer biosynthesis.
Viable cell counts
gauge the numbers of live, metabolically active cells - more informative (Growth dependent tests)
Total counts (viable + dead cells)
include all cells on a bacterial culture. These methods can be quicker to perform and help determine the growth phase of the culture
Serial dilution analysis
To reduce cell numbers to levels allowing accurate estimation
What are the two methods for transferring serial dilutions to agar plates?
- Spread Plate method - Pour 0.1 ml onto surface of pre-poured agar, then spread with a bent rod. Bacterial colonies appear only on surface.
- Pour Plate method - Mix thouroughly and pour entire tube of agar into empty petri dish. Cool to harden and incubate. Some colonies appear on surface; many are below surface.
Adavantage to Pour Plate method - bacteria sensitive to oxygen can still grow below the surface.
How to calculate number of cells from a viable count?
Measuring bacterial titre
Number of cells in Original Sample (CFU/ml)= No. of colonies x Dilution Factor x 10
CFU = Colony forming units.
What method is used to measure bacterial titre of samples which contain too few organisms to give reliable measures?
E.g. Food and water sanitation studies Use the most probable number (MPN) method
-Predicts viable cell count based on results from a dilution series and statistical probability
-Uses a reference table to estimate results
Measuring Bacterial Titre - Filtration
Filtration
* Used to concentrate bacteria from a large dilute sample
* Known volume of air or water are passed through a filter
* Pore size traps bacteria
* Filter placed on solid media and colonies counted
Direct Microscope Count
- Used for a total count
- Petroff-Hausser counter
- Count cells in known volume
- Dilutions may need to be prepared
Turbidity
the quality of being cloudy, opaque, or thick with suspended matter.
- Based on principle of increased OD with higher cell numbers
- Measured with spectrophotometer (550-650 nm)
- Assess concentration/growth phase quickly and without destroying sample
Obligate organisms
must have the specified
environmental conditions
Facultative organisms
able to adjust and live in other
environmental conditions
Do all living organisms require oxygen? Where is it obtained from?
- Oxygen element needed by all living organisms as it is used in
the construction of cellular molecules (nucleic acids, proteins,
carbohydrates, lipids etc.) - Can be obtained from O2 gas, or alternatively from H20
Why are neutralising mechanisms required when it comes to obtaining oxygen?
O2 gas is in a sense toxic (by-products of its metabolism);
Neutralising mechanisms required
What are free radicals?
- Formation of highly reactive free radicals (unpaired electrons)
formed during reduction of O2 gas in respiration - Oxygen gas, superoxide radical, hydrogen peroxide, hydroxyl radical - examples of free radicals
What is produced to break down free radicals?
All organisms that live in the presence of O2 produce enzymes
and other antioxidant systems to break down these free radicals. E.g SOD and Catalase
Obligate aerobes
requires free O2
for respiration (Pseudomonas spp.)
Obligate anaerobes
killed in the presence of free O2
(C. botulinum, C. tetani)
Microaerophiles
grow best in presence of low O2
(Campylobacter spp.)
Facultative anaerobes
Can adjust to presence/absence of free O2
(E. coli,
Staphylococcus spp.)
Aerotolerant anaerobes
Can survive in free O2 but do not use it (Lactobacillus
spp.)
Nitrogen, Sulphur and Phosphorus
N and S in proteins; N and P in nucleotides
Constitute 18% dry weight of cell
Sources depend on the metabolic pathways of the species.
Nitrogen: From amino acids or nitrogen fixation
Sulphur from SO4, H2S or amino acids
Phosphorus: from PO42-
Trace Elements
E.g. Fe, Cu, Mo, Zn, Co, Ca
Tiny amounts needed; Cofactors in Enzymatic Reactions
Present in adequate amounts in tap water or can be added to media
Organic Growth Factors
E.g. Amino acids, Vitamins (most are coenzymes), pyrimidines and
purines.
Organic compounds required by species that cannot self synthesize (e.g. Some pathogenic organisms)
Carbon
Aside from Water, carbon is the most important requirement for
microbial growth
Structural backbone of living matter; required for formation of all
organic compounds; energy source for most microbes
Makes up half the bacterial cell dry weight
Autotrophs
Obtain carbon from carbon dioxide (inorganic)
Heterotrophs
Obrain carbon from organic molecules
Phototrophs
Obtain energy from sunlight
Chemotrophs
Obtain energy from organic or inorganic compounds
look at slide 39
Photoautotrophs
Energy from Light; Carbon from Co2
Photoheterotrophs
Energy from Light; Carbon from non CO2
source
Chemoautotrophs:
Energy from Inorganic Compounds; Carbon from CO2
Chemoheterotrophs
Energy and Carbon from Organic Compounds (usually the same
compound e.g. Glucose)
Includes the vast majority of bacteria, all fungi, protozoans and animals
Can be further classified by source of organic molecules:
- Saprophytes: Live on Dead Organic Material
- Parasites: Feed on a Living Host
Why is moisture a requirement for bacterial growth
- Single-celled organisms are exposed directly to their
environment (don’t have same protections as larger,
multicellular organisms) - Require a water environment in order to carry out metabolism
- Some spores can survive without water but not vegetative cells
Temperature
Most species of bacteria grow over a 30°C temperature range
Minimum and maximum temperatures for different species vary
considerably.
According to growth temperature range, bacteria can be classified
as:
Psychrophiles, Mesophiles or Thermophiles
Psychrophiles:
Cold-loving organisms
Grow best 15-20°C (Some can live at 0°C)
Example of an Obligate pyschrophile
Bacillus globisporus, cannot grow above 20°
Facultative psychrophile
Facultative psychrophile Xanthomonas pharmicola: optimal growth
at 20°C but can grow at higher temperatures.
Cannont live in the human body
Mesophiles
Includes most bacteria
Grow best between 25 – 40 °C
Includes human pathogens (most have optimal growing temperatures near 37°C)
Thermoduric
Some organisms live as mesophiles but can survive short periods at high temperatures (thermoduric)
Thermophoiles
Heat-loving organisms
Grow best between 50 – 60 °C (Some can tolerate 110 °C – deep sea hydrothermal vents)
- Obligate thermophiles: Only grow at temperature > 37°C
- Facultative thermophiles: Can grow above and below 37°C
Organisms usually found in places like compost heaps and hot springs
Example: Bacillus stearothermophilus
How is the temperature range established?
Determined predominantly by the temperature range at which its
enzymes function. 3 critical temperatures within this range:
1. Minimum growth temperature
2. Maximum growth temperature
3. Optimum growth temperature
(Gives shortest generation time)
Optimum growth temperature quite
close to maximum growth temperature
Acidophioles
Low pH/acid-loving organisms
Grow best between pH 0.1 – 5.4
- Lactobacillus spp. (produce lactic acid), tolerates only mild acidity
-Sulphuric acid producing bacteria tolerate conditions of pH 1
Very few bacteria grow below pH 4.0 – principle can be used in
food preservation (pickling etc.
Neutrophiles
Neutral pH-loving organisms
Grow best between pH 5.4 – 8.0
Includes human pathogens
-Optimum pH ranges of Pseudomonas aeruginosa is 6.6-7.0 and
E. coli is 6.0-7.0
**Bacteria can produce compounds in culture conditions that lower the pH of their environment – controlled with buffers
Alkaliphiles:
High pH/Base/Alkali-loving organisms
Can exist between pH 7.0 – 11.5
- Vibrio choleae (causes Asiatic cholera) grows best at pH 9.0
- Agrobacterium (soil-dweller) grows at pH 12.0
Hydrostatic Pressure
Hydrostatic pressure is the pressure exerted by standing water
Proportionate to its depth, doubles every 10 m
Barophiles
Bacteria that live at high pressures but die after a
short time under normal atmospheric pressure. Enzymes and
membranes require high pressures to maintain structure and
function
Osmotic pressure
- osmotic pressure is generated by a difference between levels of
dissolved substances either side of bacterial cell membrane
Hypertonic
Cells in solutions with high concentrations of solutes (hypertonic)
can lose water to their environment – Plasmolysis
Hypotonic
Cells in solutions with low concentrations of solutes (hypotonic)
can take in water from their environment – Become Turgid
What does use of salt as a preservative cause?
Use of salt as a preservative; takes advantage of increasing
osmotic pressure to control the growth of certain microbes
Halophiles
Bacteria adapted to living in high salt environments (Oceans etc.)
Obligate halophiles can require 20-30% salt to grow
Why are endospores produced?
- If requirements for bacterial growth are not met; some bacteria produce endospores in response to starvation Survival technique
- Bacillus subtilis and Clostridium perfringenes are examples of
spore forming bacteria.
