15 - Bacterial growth and measurement of growth

Question 1

Generation time (g)

Answer 1

Time for a cell to produce 2 cells or for the population to double in number

Question 2

Septum

Answer 2

forms down the middle of the cell, separating it into two during binary fission

Question 3

Why does bacterial growth stop

Answer 3

• nutrients are used up
• metabolic wastes that may be toxic accumulate
• living space becomes limited
• aerobes may suffer from oxygen depletion

Question 4

Significance of bacterial growth

Answer 4

• Human and animal health implications
• Environmental impacts (blooms)
• Industrial applications (medicine, cheese)

Question 5

Stationary cells

Answer 5

Viable but non dividing and/or non viable cells

Question 6

5 phases of bacterial growth in closed system

Answer 6

1. Lag phase
2. Exponential or log phase
3. Stationary phase
4. Death phase
5. Long term stationary phase

Question 7

Lag phase

Answer 7

• Cells have to adapt to new physiological conditions.
• Synthesis of cellular constituents (especially ribosomes and enzymes)
  begins
• Cell volume increases but no cell division
• Growth rate (k) and generation time (g) = 0
• Different genes are upregulated at different times to make needed components
• cell division and population growth begin at end of lag phase

Question 8

Exponential (log) phase

Answer 8

• Bacteria grow and divide at max rate possible
• Population most uniform in terms of physiology
• k and g are constant
• Continues until limiting factors or toxicity drive culture into stationary phase

Question 9

growth rate calculation

Answer 9

k = 1 / g (generations/hour)
if g = 20 mins (0.333 hr)
k = 1/0.333 = 3 gens/hr

Question 10

Stationary phase of growth

Answer 10

• k = 0
• Cells not dividing because nutrients have been used up, or a toxin has built up and cell death balances cell division
• reached at `10^9 cells/mL

Question 11

Properties of cells in stationary phase

Answer 11

• Tend to be smaller than exponential phase
• Different composition of cellular constituents
• available energy is used for maintenance (endospore formation)

Question 12

Death phase

Answer 12

• Cells begin to die as nutrient deprivation, or toxic, waste buildup causes damage to cells
• k becomes negative
• Death rate usually logarithmic
• Some cells lyse, releasing nutrients to other bacteria

Question 13

Long term stationary phase

Answer 13

• Can last months or years
• Population continually evolves. (actively growing cells use nutrients released by dying cells to tolerate toxins)
• Successive waves of genetically distinct variants

Question 14

biofilms

Answer 14

Aggregations of microbes in complex communities, growing on surfaces and held together by extracellular polymers.

Question 15

3 different methods of measuring bacterial growth

Answer 15

• Measure cell biomass
• Direct measurement of cell number
• Viable counting methods

Question 16

3 types of cell biomass methods

Answer 16

• Dry weight determination
• Wet weight determination
• Spectrophotometry

Question 17

Dry weight determination

Answer 17

• Cells in broth collected by centrifugation, washed, dried and weighed
• Insensitive and very slow
• Used on fungi and filamentous bacteria

Question 18

Wet weight determination

Answer 18

• Weigh pellet after centrifugation
• Result less consistent than dry weight
• Insensitive but rapid

Question 19

Spectrophotometry

Answer 19

• Bacterial cells scatter light which is proportional to biomass of cells
• Increases in cell concentration result in greater turbidity (cloudiness) and less light is transmitted.
• As turbidity increases, absorbance increases
• Sensitive and very rapid
• Inaccurate when cell densities are high unless diluted.

Question 20

3 types of direct measurement methods

Answer 20

• Direct cell counts using counting chamber
• Staining with fluorescent stains
• Flow cytometry

Question 21

Direct cell counts using counting chamber

Answer 21

• Slide creates chamber of known volume
• Cells on grid are counted, cells/mL calculated
• Not very sensitive, rapid
• isn’t affected by bacteria absorbance abilities

Question 22

Staining with fluorescent stains

Answer 22

• Sample filtered and bacteria trapped on membrane
• Stained with nucleic acid fluorescent stains and counted under microscope

Question 23

Flow cytometry

Answer 23

• Provides cell count plus detailed info of organism
• Stream of cells passed through beam of laser light
• Measures the amount of light that gets through and the amount scattered
• sensitive, rapid

Question 24

3 types of viable counting methods

Answer 24

• Viable counts/standard plate counts
• Membrane filtration
• Most probable number (MPN)

Question 25

Viable counts/standard plate counts

Answer 25

• Suspensions are diluted (1ml of previous dilution added to 9mL of water)
• Original sample, 10^-1, 10^-2, 10^-3, etc
• pour plate and spread plate method
• Single colonies then counted after incubation
• counts 30-300 are valid
• Sensitive but slow

Question 26

Membrane filtration method

Answer 26

• Membranes with different pore sizes used to trap microorganisms
• Contain grids so colonies can be counted
• Allows bacteria present in low concentrations of water
• Sensitive but slow

Question 27

Most probable number

Answer 27

• Serial dilutions used
• Tubes examined for turbidity
• One cell would give rise to turbid tube
• If no growth, no cells
• Table provides most probable cells/mL in og
• Useful where microbe cannot be cultured
• Very sensitive, slow

Question 28

Primary metabolites

Answer 28

Produced during active exponential growth (enzymes, amino acids)

Question 29

Secondary metabolites

Answer 29

Compounds not needed for normal cell growth but beneficial. Synthesised in stationary phase

Question 30

Chemostat

Answer 30

Enables cells to be kept in exponential phase at specific growth rate by providing constant supply of one essential nutrient at a limiting concentration