3. Temperature And Microbial Growth

Question 1

What is important to note about the growth mediums for bacteria? 6

Answer 1

1 Optimum growth conditions include nutrients, temperature and time taken
2 carbon source eg. Glucose or other sugars provide energy. Cells are fifty percent carbon
3. Nitrogen source for protein synthesis eg. Nh4cl/ammonium chloride for conversation to amino acids
4. Sodium, potassium, magnesium and trace elements eg. Cu, co, ni, zn
5. Growth factors eg vitamins and nucleotides
6. Some bacteria, especially pathogens, are grown in a blood agar plate for their growth factors

Question 2

Describe bacterial growth and reproduction. 4

Answer 2

1. Increase in number through binary fission by exponential growth (2-4-8-16-32-64)
2. In addition to replication, bacterial cells also grow
3. In rod like organisms, binary fission occurs when the bacterial cell has doubled in length
4. Growth is necessary before binary fission

Question 3

Describe bacterial growth in the lab. 3

Answer 3

1. We can grow 10% of bacteria in the lab
2. We must aerate culture at optimum temp
3. When culture goes cloudy, we have bacteria

Question 4

What is binary fission? 6

Answer 4

1. Chromosomes are tethered to inner membrane
2. Cell doubles in length
3. Partitioning of dna, ribosomes etc
4. They move apart, septum comes down and separation occurs
5. Dna attached to membrane
6. Mean doubling time (mdt) is time it takes for population to double eg. For E. coli it is twenty minutes

Question 5

How is rod division controlled? 6

Answer 5

1. Formation of the FtsZ ring stimulates cell division
2. Cells always divide in the middle due to FtsZ ring
3. Protein polymerises in the centre of the cell
4. Fluorescent antibodies show that as the organism grows, the FtsZ ring forms in the middle and becomes more prominent
5. As septa meet and cell divides, the ring shrinks
6. The cell always divides where the ring is

Question 6

Describe rod cell growth. 4

Answer 6

1. MreB protein forms helical structure around rod
2. Where it touches the cell wall, it breaks peptidoglycan
3. This allows several points of growth
4. Rods only, not cocci

Question 7

Describe the bacterial growth curve. 6

Answer 7

1. Occurs in batch culture of liquid medium
2. No fresh medium added to closed culture
3. Lag phase - low number as cells aren’t dividing yet, they must adjust
4. Log - actively growing and dividing but can only last for a defined period as c and n run out and to is by products accumulate
5. stationary phase - not growing or dividing but still alive, normal phase most bacteria exist in
6. Death phase, cells fall apart and die

Question 8

Define what happens during the lag phase of a bacterial batch culture. 6

Answer 8

1. When cells are inoculated into fresh medium, there is no initial increase in numbers
2. Cell are making new components
3. Cells grow to twice size
4. Calls may be old/depleted of ATP/ribosomes so must wake up
5. Cells may be injured
6. Cells may be adjusting to new medium eg, synthesising necessary enzymes

Question 9

What happens during the exponential growth/log phase of a bacterial batch culture? 3

Answer 9

1. Plotted logarithmically gives a straight line
2. One bacterium with a twenty minute generation time for 48hours (not including stationary phase)
3. Enough bacteria produced to weigh 4000x the earth

Question 10

Describe what happens in the stationary and death phases of a bacterial batch culture. 6

Answer 10

1. Population growth ceases - some are dying and some are growing, at equilibrium. Culture is very turbid (cloudy)
2. Bacteria reach approx 10^9ml^-1 in lab
3. Division stops due to nutrient limitation eg.glucose and oxygen
4. There is an accumulation of toxic waste eg. Streptococci produce lactic acid
5. All cells stop dividing
6. Death phase begins - cells may die at a constant rate

Question 11

Describe the different types of bacterial growth curves. 6

Answer 11

1. Turbidity/optical density - how cloudy the water is
2. Easier and so usually used
3. Viable count is the most accurate way of documenting bacterial population and growth
4. Bacteria taken out of culture and onto plates on a solid medium
5. One viable cell leads to a colony of millions of cells
6. Count colonies

Question 12

What are cardinal temperatures and how are they related to growth? 5

Answer 12

1. Growth rate = slope of exptential phase
2. Minimum/Tmin, bacteria won’t grow below this temperature
   3 optimum/Topt - most efficient enzyme activity
3. Maximum/Tmax - above this temp, bacteria fall apart and die
4. Topt can vary from four degrees to one hundred and thirteen, eg. The the Topt of most pathogens is thirty seven, same as the human body

Question 13

What are the effects of temperature on microbial growth? 5

Answer 13

1. At/blow Tmin, lipids in membrane gel go hard and tea post processes become so slow that growth cannot occur
2. As the operative increase, enzymatic reactions increase in rate
3. At Topt, enzymatic reactions occur at maximum possible rate
4. At Tmax, proteins denature and cytoplasmic membrane collapses, this is called thermal lysis
5. In gram negative, outer membrane also collapses

Question 14

What temperature classes of bacteria are there? 5

Answer 14

1. Psychrophiles- low Topt, below fifteen degrees, found in oceans
2. Mesophiles have a mid range Topt, about 15-45
3. Thermophiles have a high Topt, 45-80
4. Hyperthermophiles have a very high Topt, 90-100+
5. They are found in host springs, geysers and deep sea hydrothermal vents

Question 15

What are the temperature ranges that different thermal classes of bacteria are known to survive in? 5

Answer 15

1. Psychrophile, -5-20
2. Mesophile 12-45
3. Moderate thermophile 41-69
4. Extreme thermophilic bacteria 67-98
5. Extreme thermophilic Archean 100-120

Question 16

What adaptations do psychcrophiles have? 6

Answer 16

1. Semi fluid membranes at low temps
2. The membranes have unsaturated fatty acids with up to nine double bonds, those which survive at the lowest temps are polyunsaturated
3. The membranes don’t become waxy and non functional at lower temperatures, they maintain membrane fluidity
4. Altered proteins with more alpha helices to give greater flexibility
5. Antifreeze molecules which bind to ice crystals are used in fish and several other eukaryotes, but none found in prokaryotes
6. Mesophiles don’t have double bonds in their fatty acids

Question 17

What adaptations do thermophilic bacteria have? 4

Answer 17

1. Semi fluid membranes at high temps
2. High saturated fatty acids
3. Stay solid and stable at higher temperatures
4. Altered proteins, as some amino acid changes lead to heat resistant folding conformations

Question 18

What adaptations to hyperthermophilic bacteria have? 6

Answer 18

1. No fatty acids in membranes
2. Membranes made of phytane, a 40C hydrocarbon
3. Membrane is a lipid mono layer, not a lipid bilayer
4. Membrane is generated by two phytanes joined covalently head to tail
5. Heating a lipid bilayer too much causes it to dissolve
6. Having a monolayer stabilises the membrane
   2

Question 19

What is sterilisation? 4

Answer 19

1. “Destruction it removal of all living bacteria, spores and viruses.”
2. Sterile objects are totally free of viable cells
3. Used in hospitals, labs and food industry
4. Heat kills bacteria

Question 20

Why is the pattern of microbial death by heating? 5

Answer 20

1. Cells lose viability
2. Cells not all killed instantly
3. They die at a constant rate over a period of time
4. This rate varies depending on the temp heated to
5. Population death is exponential (logarithmic)

Question 21

How do we measure the death of bacteria by heat? 5

Answer 21

1. D value is the time required to kill ninety percent of the bacteria
2. D value varies with temperature
3. On a graph of a semi log plot, the time to drop cell numbers by one log cycle
4. D values are used in the food industry
5. Fine line between killing bacteria and ruining food

Question 22

How does steam sterilisation by autoclaving work? 3

Answer 22

1. High temp (121) and pressure (138kPa)
2. Twenty mins is long enough
3. Kills all known germs including viruses