L9 Flashcards
What is the purpose of studying bacteria in the lab?
To isolate a pure microbial strain and study its morphology, physiology, bioactivity
What is the aim of the streak plate technique?
Aim is to obtain a single colony
What is the streak plate technique?
Flame inoculation loop, dip in bacterial suspension, carefully wipe the loop over the plate covered in solid growth media
What are the three types of culture medium?
Solid medium, liquid medium, semisolid medium
What is the solid medium?
Contains agar
What is the liquid medium?
Does not contain agar
What is the semisolid medium?
Contains less agar than solid media
What is minimal medium?
Minimal nutrients essential for growth, salt, hydrogen, water.
What is complex/undefined/basal medium?
Water, a carbon source, salts, and a source of amino acids or nitrogen
What is a defined medium?
When proportions of components are known.
What is transport media?
For transport, does not promote growth. Lacks C and N.
What is the differential medium?
To distinguish one microbe from another
What is the selective medium?
To grow selective microbes
What is microbial growth?
Increase in the number of a population of cells
How do microbes divide and explain the process?
By binary fission, a cell synthesises its constituents until it reaches a point, the cell divides into two cells, the cycle starts again.
What is binary fission?
When an organism duplicates its genetic material, divides into two parts, with each organisms receiving one copy of DNA
What can binary fission involve?
Invagination of the cell membrane, the cell wall, and in the case of gram-negative bacteria, the outer membrane. Some gram-positive cocci and rods divide without showing cell constriction. These bacteria make a septum between the two daughter cells before division.
How do the microbial cells divide?
Prokaryotes make a protein (FtsZ) which can form a constricting ring in the middle of the cell. This is the Z ring, nearly all bacteria and many archaea have the gene that encode for FtsZ. The assembly of the Z ring requires polymerisation of FtsZ into short filaments and the association with the cell membrane.
What two mechanisms does the Z ring use to find the middle of the cell?
Min and nucleoid occlusion (NO)
What is the min system mechanism?
Consists of three proteins (MinC, MinD, MinE) which patrol the cell interior to prevent the polymerisation of FtsZ at the poles of the cells. MinD polymerises at one pole of the cell and binds to MinC. MinC acts as a FtsZ inhibitor that prevents FtsZ polymerisation. Mine forms a ring close to the pole and prevents MinCD from polymerising further into the middle region. MinD dissolves in one pole and polymerise in the opposite pole binding MinC. Mine forms another ring close to the other pole. The min system protects the poles of the cell from FtsZ polymerisation.
What happens in nucleoid occlusion (NO)?
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 proteins inhibits unwanted cell division where the nucleoid is localised. Only when the newly replicated chromosomes are about to split, FtsZ dins the space needed to form the Z ring. Formation of the Z ring triggers cell division.
What does microbial growth and cell division look like in the lab?
Clear and transparent liquid goes cloudy.
What are the phases of microbial growth called?
Lag, exponential, stationary.
What happens in the lag phase?
The time that the cell needs to adapt to the new growth conditions and resume growth
What happens in the exponential phase?
Once growth resumes at a steady rate, the cells growth and divide exponentially.
What happens in the stationary phase?
Total number of cells remains the same but ratio of viable cells to dead cells changes
Why can microbes not grow continuously?
They run out of food, produce toxic byproducts, produce growth inhibition products. Therefore during the stationary phase cells stop dividing.
Nutrition and growth - in what stage is their balanced growth and how is it maintained?
If a culture is in the exponential phase its 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
How is balanced growth maintained in a laboratory?
Chemostat, bioreactor to which fresh medium is continuously added. Fresh medium from a reservoir is fed into culture at constant rate, the volume of the culture is constant by removing the excess through overflow. The rate of addition of fresh medium determines the growth rate in the culture. The cell density in the culture is determined by the concentrations of limiting nutrients.
What five methods measure microbial growth?
Turbidity, colony count, cell viability assay, direct counting, flow cytometer.
How do you measure the turbidity of the culture?
Measure cell density using spectrophotometer which passes a ray of light through the culture. This measures the cultures turbidity which is the absorbande or optical density at a particular wavelength. The measurement does not depend on the absorption of light by the bacteria. Bacterial cells scatter the incident light, reducing the amount of light there is detected. It is an indirect measure of bacterial biomass. During the stationary phase - OD does not increase because total no of cells remains the same.
How does the colony count method work?
Make serial dilutions of the cultures to avoid having too many cells. Spread a potion of each dilution on the surface of the agar to ensure that single cells are separated from each other. Incubate the plates at a set temperature and time. Identify a dilution plate with 200-250 distinct colonies. Count the number of colonies. No of bacteria = no of colonies x reciprocal of dilution
How does the cell viability assay work?
Luminescence-based assay for the determination of the viability of microbial cells. Uses quantification of ATP as an indicator of metabolically active, viable cells. ATP changes Luciferin to oxyluciferin and light. Luminescent signal generated is therefore proportional to the amount of ATP.
How do you measure the cell number with a microscope?
The counting chamber (hemocytometer) consists of a modified slide with a central depression of known depth and a grid of know size. Cell suspension is added to the chamber and the chamber viewed under a microscope.
How do you count cells with a flow cytometer?
A flow cytometer used a laser beam to simultaneously count and analyse many physical and chemical characteristics of individual cells. The sample is focused such that one cell at a time pass through a laser beam. Each cell interrupts the light path, scattered the light - information about each cell. Before entry into the flow cytometer the cells can be labeled to collect information about the genetic or biochemical composition. Some types of cells in a suspension may take up one colour of fluorescent dye. Flow cytometer can be equipped with a sorter to separate cell into subpopulations.
What climates can microbes grow in?
Extreme T, extreme pH, high hydrostatic pressures, high osmolarity.
Microbial growth with temperature?
Bacteria and fungi dominate low T, archaea dominate high T. Microbes need liquid water to grow. Temperatures for liquid water can be maintained below the freezing point by dissolving solutes, above boiling point by high pressure. Microbes can grow at temps below 0 in high salt enviro, other microbes can grow at temps above boiling point in hydrothermal vents in the deep sea. Each microbe has an ideal T that supports its growth.
What is the effect of temperature on microbial growth?
As temp increases growth slows. Proteins are denatured - chemical reactions slow and stop. Temp decreases growth slows. Protein activity slows down but does not stop.
How do microbes cope with high temps?
Thermophiles and hyperthermophiles, contain large amounts of chaperone proteins (heat shock). Thermo-protective DNA binding proteins and positive DNA supercoil.
How do microbes cope with low temps?
Low temps - not lethal, microbial metabolism slows down. Although, rapid changes in temperature can kill microbes, freezing kills microbes as there is no available water - microbes die from osmotic shock. In lab - adding glycerol prevent h bonding in water and stops formation of ice crystals.
How do microbes cope with high hydrostatic P?
High hydrostatic pressures can kill plants and animals but not microbes. Microbes withstand high hydrostatic P because of their cell envelope due to permeability to water meaning they can rapidly balance pressure. More permeable membranes, pressure resistant proteins and chaperones, by negative DNA supercoil - under pressure DNA gets more stable.
How do microbes cope with high osmotic P?
High osmotic pressure places two stresses on microbes. Decreases available water for the cells. Reduces the cells turgor pressure by decreasing the differences in osmotic pressure between the inside and outside. Most microbes need high turgor pressure to expand their walls as they grow. Halophiles are microbes that thrive at high salt concentration.
What are salterns with different halophilic microbes - strategies to maintain osmotic P?
Accumulation of molar concentrations of potassium and chloride. Biosynthesis and/or accumulation of organic osmotic solutes - accumulation of certain solutes can inactivate proteins therefore microbes accumulate other solutes are less damaging.
What is trehalose?
Substance synthesised in response to lack of water so also helps bacteria with freezing. High concentrations means increased ability to withstand desiccation.
How do microbes cope with extreme pHs?
Acidophiles - microbes that thrive in low pH. Alkaliphiles - microbes that thrive in high pH. Prokaryotes can grow over a wider range of pH than the proteins can tolerate. Fungi prefer slight acid pH, bacteria slight alkaline. Archaea have both extremes. They cope with extreme pH by pumping protons in and out of the cell, keeping constant internal pH.
