Biochem Week 9

Question 1

What is a pure culture?

Answer 1

A population of cells arising from a single cell to characterise an individual species.

Question 2

The Streak Plate Technique to obtain a pure clone

Answer 2

Place inoculating loop into Bunsen burner to sterilise. Then place loop into cell culture. Streak the loop onto agar. Four sets of streak. Higher concentration when folds closer together.

Question 3

Colony morphology

Answer 3

Individual species often form colonies of characteristic size and appearance.

Question 4

The growth of colonies at the colony edge

Answer 4

At the colony edge, cells grow at maximum rates

Question 5

The growth of colonies at the centre

Answer 5

Cells are lying, growth is much slower

Question 6

What is the reason for the difference in cell growth at centre and edge of colony?

Answer 6

The colony centre is much thicker than at the edge and they must compete for oxygen and nutrients.

Question 7

What is quorum sensing?

Answer 7

It is the regulation of gene expression in response to fluctuations in cell-population density.

Question 8

How is quorum sensing achieved in gram-negative single species bacteria?

Answer 8

The quorum sensing bacteria produce and release chemical signal molecules called autoinducers. The concentration of autoinducers increases with an increase in cell density. The autoinducer diffuses through the cell membrane, attaches to and activates regulatory protein, regulatory protein binds to DNA, creates new AHL and process repeats.

Question 9

What is a biofilm?

Answer 9

Biofilms form when bacteria adhere to surfaces in aqueous environments and begin to excrete a slimy, glue-like substance that can anchor them to several different types of material.

Question 10

Microbial growth in batch culture.

Answer 10

When microorganisms are grown in a closed system, population growth remains exponential for only a few generations and then enters a stationary phase due to factors such as nutrient limitation and waste accumulation.

Question 11

Microbial growth in continuous culture.

Answer 11

If a population is cultured in an open system with continual nutrient addition and waste removal, the exponential phase can be maintained for long periods.

Question 12

Growth curve: Lag phase

Answer 12

Cells begin to synthesise inducible enzymes and use stored food reserves. There is no immediate increase in cell number because the cell is synthesising new components e.g. ribosomes

Question 13

Growth curve: Exponential/ Logarithmic phase

Answer 13

The rate of multiplication is constant. Microorganisms are growing and dividing at the maximal rate possible. The population is mostly uniform in chemical and physiological properties

Question 14

Growth curve: Stationary phase

Answer 14

Death rate is equal to rate of increase. Population growth ceases and the growth curve becomes horizontal.

Question 15

Growth curve: Death phase

Answer 15

Cells begin to die at a more rapid rate than that of reproduction.

Question 16

What is the length of the lag phase dependent on?

Answer 16

The length of the lag phases varies with the condition of the micro-organisms and the nature of the medium.

Question 17

Reasons for the stationary phase.

Answer 17

1. Nutrient limitation
2. The accumulation of toxic waste products
3. A critical population level is reached.

Question 18

Effects of starvation on bacteria in death phase

Answer 18

1. decrease overall size: protoplast shrinkage, nucleoid condensation
2. produce a variety of starvation proteins
3. Increase cell wall strength and peptidoglycan cross-linking
4. Dps protein protects DNA
5. Chaperones prevent protein denaturation and renature damaged proteins

Question 19

What is the role of microtubules in mitosis?

Answer 19

Microtubules play a role in the migration of chromosomes to opposite ends of a mitosing cell during the anaphase.

Question 20

What is the mechanism of taxol?

Answer 20

Taxol stabilizes microtubules, preventing them from de-polymerising. Binding to tubulin causing it to lose its flexibility, preventing cell division.
Taxol also selectively kills dividing cancer cells.

Question 21

The types of cell division

Answer 21

a. Binary Fission
b. Budding
c. Multiple Fission

Question 22

What is binary fission?

Answer 22

A process in which a cell divides to produce two nearly equal-sized progeny cells. This involves three processes:

1. increase in cell size (cell elongation)
2. DNA replication
3. Cell division

Question 23

What is budding?

Answer 23

Budding is a type of asexual reproduction in which a new organism develops from an outgrowth or bud due to cell division at one particular site. The new organism remains attached as it grows, separating from parent organism only when it is mature.

Question 24

What is multiple fission?

Answer 24

Cell division in which the nucleus first divides into several equal parts and then the cytoplasm divides into as many cells as there are nuclei. It is the most common form of asexual reproduction.

Question 25

How to estimate microbial growth?

Answer 25

Population mass

Population number

Question 26

How to measure cell number?

Answer 26

1. total cell count - direct observation under microscope

2. viable count (plate count or colony count) = spread plate method, pour plate method, membrane filtration procedure

Question 27

Disadvantages of direct counting with the counting chamber

Answer 27

The microbial population must be fairly large for accuracy because such a small volume is small
It is difficult to distinguish between living and dead cells

Question 28

When is an electronic counter (Coulter counter) used?

Answer 28

For large microorganisms such as protozoa, algae, nonfilamentous yeasts

Question 29

The method and problems of viable count (spread plate and pour plate method)

Answer 29

The three basic steps: dilution, plating, incubation

Problems include clumps of cells colony forming units (CFU), employed agar medium cannot support growth of all the viable microorganisms present

Question 30

The membrane filtration procedure

Answer 30

Membranes with different pore sizes are used to trap different microorganisms. Incubation times for membranes vary with medium and microorganism.

Question 31

How to measure cell mass?

Answer 31

1. Dry weight - useful for measuring the growth of fungi. Cells growing in liquid medium are collected by centrifugation, washed, dried in an oven and weighed.
2. Turbidity and microbial mass measurement
3. The amount of substance - total protein or nitrogen,chlorophyll

Question 32

How does a photometer measure cell turbidity?

Answer 32

A bacterial culture is exposed to a light source. The cells will scatter some of the light rays and only a fraction will be incident on the light-sensitive detector. Find the turbidity from a calibrating curve.

Question 33

What is the continuous culture of microorganisms?

Answer 33

Continual provision of nutrients and removal of wastes. A microbial population can be maintained in the exponential growth phase and at a constant biomass concentration for extended periods in a continuous culture system.

Question 34

What is a chemostat?

Answer 34

Chemostat is used for continuous cultures, rate of growth can be controlled either by controlling the rate at which new medium enters the growth chamber or limiting required growth factor in medium.

Question 35

What does a chemostat consist of?

Answer 35

A chemostat consists of a culture into which fresh medium is continuously introduced at a constant rate. The culture volume is kept constant by continuous removal of culture at same rate. The supply of a single nutrient controls growth rate.

Question 36

The main features of a chemostat culture

Answer 36

• volume of culture constant
• time to mix small volume of medium with culture should be small. It should approach perfect mixing.
• environmental conditions should be maintained (pH, temperature)
• specific growth rate can be varied from above zero to just below mu(max)
• Substrate-limited growth maintained indefinitely and this can offer relief of catabolic repression and induction of secondary metabolism at low substrate (glucose) concentration

Question 37

The importance of chemostat cultures

Answer 37

It can be used to select mutants with particular physiological characteristic

Question 38

The dilution rate of chemostats

Answer 38

The dilution rate determines the specific growth rate of the culture.
D=F/V
where D= dilution rate, F= medium flow rate, V = culture volume.

Question 39

How does mu relate to D at steady state?

Answer 39

At steady state, mu=D. Thus mu can be varied in a chemostat from just above 0 to just below mu(max)
At steady state, the biomass concentration and limiting substrate concentration in culture remain constant.

Question 40

The effect of nutrient concentration in batch cultures.

Answer 40

The total growth will increase until limiting nutrients are exhausted or metabolic byproducts accumulate that change environmental conditions inhibit growth (toxicity).

The growth rate will increase with increasing nutrient concentration up to a maximum value, beyond which there is no effect (transporters are saturated with substrate)