Microbial growth Flashcards
Cultivation of microorganisms on agar
To isolate a pure microbial strain and study its morphology, physiology, bioactivity
Restreaking of microorganisms on agar was developed by Robert Koch, Julius Petri and Fanny Hesse in the 1880s
Streak plate techniques – obtain single colony
Types of culture medium – solid medium (contains agar), liquid medium (does not contain agar), semisolid medium (soft agar), minimal medium, complex/undefined/basal medium, defined medium, transport media, differential medium, selective medium
Microbial growth and cell division
Microbial growth = increase in the number of a population of cells
Microbes mostly divide by binary fission
1. A cell synthesises its constituents (e.g proteins, nucleic acids, carbohydrates and lipids) until it reaches a point (doubling its mass)
2. Cell divides into two cells
3. Cycle starts again
Binary fission = an organism duplicates its genetic material, divides into two parts (cytokinesis), with each new organism receiving one copy of DNA
Can involve invagination of the cell membrane, the cell wall and in the case of the gram-neg bacteria, the outer membrane
Some gram-pos cocci and rods divide without showing cell constriction, these bacteria make a septum between the two daughter cells before division
How microbial cells divide
Prokaryotes make a protein (FtsZ) which can form a constricting ring in the middle of the cell (e.g where the cell paritions) = the z ring
Nearly all bacteria and many archaea have the gene that encodes for FtsZ
The assembly of the z ring requires poylmerisation of FtsZ into short filaments and the association with the cell membrane
Two mechanisms – min system, nucleoid occlusion (NO)
Min system
Consists of three proteins (minC, mind, minE) which patrol the cell interior to prevent the polymerisation of FtsZ at the poles of cells
MinD polymerises at one pole of the cell and binds minC
MinC acts as FtsZ inhibitor that prevents FtsZ poylmerisation (can only inhibit when bound to minD)
MinE forms a ring close to the pole and prevents minCD from poylmerising firther into the middle region
minD dissolves into one pole and polymerises in the opposite pole binding minC (minCD complex)
minE forms another ring close to the other pole
the min system protects the poles of the cells from FtsZ polymerisation
Nucleoid occlusion
As the chromosome replicates in the middle of the cell
Newly replicated regions migrate to the poles so the amount of DNA in the middle of the cell decreases
NO protein inhibits unwanted cell division where the nucleoid is localised
Only when the newly replicated chromosomes are about to split, FtsZ finds the space needed to form the Z ring
Formation of the z ring triggers cell division
Phases of microbial growth
i. Lag phase – time that the cell needs to adapt to the new growth conditions and resume growth
ii. Exponential phase – once growth resumes at a steady rate, the cells grow and divide exponentially
iii. Stationary phase – total number of cells remains the same but ratio of viable cells to dead cells changes
iv. Death phase
Microbes cannot grow continuously because – run out of food, produce toxic by-products, produce growth inhibition products
Nutrition and growth - balanced growth
If a culture is in the exponential phase growth is balanced
Balanced growth is when cells do not exceed a certain cell density
To maintain balanced growth, dilute the culture after a certain number of generations
Diluting the culture with fresh medium provides the cells with more nutrients
Balanced growth maintained in lab
- Chemostat – bioreactor to which fresh medium is continuously added at a constant rate so volume of the culture remains constant
Measure microbial growth
- Turbidity – measure of the cell density using a spectrophotometer which passes a ray of light through the culture, indirect measure of bacterial biomass
- Colony count (colony forming unit method (CFU)) – serial dilutions
- Cell viability assay – luminescence-based assay for determination, uses quantification of ATP as an indicator, ATP changes luciferin to oxyluciferin and light, luminescent signal generated proportional to amount of ATP
- Direct counting – counting chamber (hemocytometer)
- Flow cytometer – uses laser beam to simultaneously count and analyse many physical and chemical characteristics of individual cells
FACS (fluorescence activated cell sorting)
Before entry into the flow cytometer the cells can be labelled to collect information about the genetic or biochemical composition – e.g some types of cells in a suspension may take up one colour of fluorescent dye while may take up another colour
Flow cytometer can be equipped with a sorter to separate cells into subpopulations
Temperature and microbial growth
Microbes need liquid water to grow
Temperatures for liquid water can be maintained – below the freezing point by dissolving solutes and above the boiling point by high pressure
Some microbes can grow at temp below 0 in high salt environment
Other microbes can grow at temperatures above boiling water in hydrothermal vents in the deep sea
Each microbe has an optimum temp that supports its growth – microbes can be classified according to their optimal growth temp
As temp increases growth slows – proteins denatured, chemical reactions slow/stop
Temp deceases growth slows – protein activity slows but does not stop
Thermophiles and hyperthermophiles contain – large amount of chaperone proteins (e.g heat shock proteins stabilising new proteins or help to refold proteins that were damaged by cell stress), thermos-protective DNA binding proteins and pos DNA supercoil
Low temp = not lethal, microbial metabolisms slows down
Rapid changes in temp can kill microbes = cold shock
Freezing kills microbes as there is no available liquid water = microbes die from osmotic shock
Glycerol can be added in the lab as a cryoprotectant – disrupts hydrogen bonding between water molecules so prevents formation of ice crystals
Hydrostatic pressure
High hydrostatic pressure can kill plants and animals but not microbes
Can withstand because – cell envelope = extent of permeability to water means can rapidly balance pressure, more permeable membranes, pressure resistant proteins and chaperone, by negative DNA supercoil = under pressure DNA gets more stable
Osmotic pressure
High osmotic pressure places 2 stresses on microbes
1. Decreases available water for the cells (cells dry out as water molecules move from areas of high to low conc)
2. Reduces cells turgor pressure by decreasing differences in osmotic pressure between inside and outside. Most microbes need high turgor pressure to expand their walls as they grow
2 strategies to maintain osmotic pressure
1. Accumulation of molar concs of potassium and chloride
2. Biosynthesis and/or accumulation of organic osmotic solutes = accumulation of certain solutes (salts) can inactivate proteins therefore microbes accumulate other solutes that are less damaging
Extreme pH
Prokaryotes can grow over a wider range of pH than the proteins can tolerate
In general fungi prefer slightly acidic pH and bacteria slightly alkaline. Archaea have representitives of both extremes
Pumping proteins in and out of cell, keeping consistent internal pH
Microbial metabolism
Prokaryotes grouped on their carbon and energy sources
Source of carbon = any form of inorganic (mainly co2) or organic compound
Source of energy = organic, inorganic and photochemical reactions
