Block B Workshop - Bacterial Growth

Question 1

Why is understanding the mechanism of bacterial growth important?

Answer 1

As it helps us find out how to kill them clinically
(Slide 6)

Question 2

What is bacterial growth by binary fission?

Answer 2

Given supportive conditions, cells grow (increase in mass and volume), chromosomes replicates then segregates, and then the bacterial cell divides
(Slide 7)

Question 3

Binary fission is the main way bacteria divide, what are 3 other ways bacteria divide other than this?

Answer 3

Longitudinal division by a γ-proteobacterial symbiotic bacterium
Cell division by budding
Cell division
Synchronous septation
(Slide 8)

Question 4

What are 4 things needed to grow (culture) bacteria?

Answer 4

Nearly always a pure culture
Aseptic technique
Growth medium or media
Growth on solid media (agar) or in liquid
(Slide 14)

Question 5

What is complex media?

Answer 5

Culture media whose precise chemical composition is not known
(Slide 15)

Question 6

What is defined media?

Answer 6

Media whose exact chemical composition is known
(Slide 15)

Question 7

What are 3 uses of a defined media?

Answer 7

Reproducibility
Investigating growth requirements
Isolation of auxotrophic mutants
(Slide 15)

Question 8

What is an auxotrophic mutant?

Answer 8

An auxotrophic mutant is a specific type of mutant organism that has lost the ability to synthesize a particular compound that is essential for its growth, usually through a mutation
(Slide 15)

Question 9

Why is your choice of medium really essential?

Answer 9

As medium components (and contaminants) affect bacterial gene expression, growth and physiology
(Slide 16)

Question 10

What is a batch culture?

Answer 10

Batch culture refers to a method of growing microorganisms, typically bacteria or yeast, in a closed system where nutrients are initially provided in a limited quantity.
(Slide 17)

Question 11

Why do growth conditions change over time in a batch culture?

Answer 11

Due to growth of cells
(Slide 17)

Question 12

What eventually happens in a batch culture?

Answer 12

One of more nutrients become limiting and / or one or more waste product cause growth stasis or death
(Slide 17)

Question 13

What does a typical bacterial growth curve look like?

Answer 13

No significant population change in the lag phase, followed by an exponential growth in the log phase, followed by no significant population change in the stationary phase before showing a linear decline in the death phase
(Slide 19)

Question 14

What do bacteria exhibit in each stage of the bacterial growth curve?

Answer 14

Very different physiology and characteristics in different parts of the growth curve
(Slide 19)

Question 15

What is the lag phase?

Answer 15

The time required for cells to adapt to a new environment and ready themselves for growth
(Slide 20)

Question 16

What 4 things do bacteria do in the lag phase?

Answer 16

Re-stock catabolites
Re-stock anabolic metabolites
Gene expression and protein synthesis of necessary enzymes
May also be repairing previous damage
(Slide 20)

Question 17

What 2 things does the length of the lag phase depend on?

Answer 17

Condition of inoculum(liquid used for inoculation) and nature of new media
Also varies between species or strains
(Slide 20)

Question 18

What 3 things occur in the log phase?

Answer 18

A constant exponential growth rate
OD600 and viable count rises exponentially
Cells at their “healthiest” and sub-culturing now will minimise subsequent lag phase
(Slide 21)

Question 19

What is OD600?

Answer 19

It stands for optical density and is the measurement of the light that passes through the sample at λ=600
(Slide 21)

Question 20

What 2 things does the slope in the log phase depend on?

Answer 20

The species and the culture conditions
(Slide 21)

Question 21

What are cells in the log phase often used for?

Answer 21

The study of enzymes, gene expression etc
(Slide 21)

Question 22

What 2 things occur in the stationary phase?

Answer 22

Growth and division slow down
OD600 and viable count plateau
(Slide 22)

Question 23

Why do growth and division slow down and OD600 and viable count plateau in the stationary phase of bacterial growth?

Answer 23

Usually due to decreasing amounts of nutrients and / or increasing concentrations of waste
(Slide 22)

Question 24

What do bacteria often enter during the stationary phase?

Answer 24

A state of hibernation where metabolic activity slows down greatly or stops completely
(Slide 22)

Question 25

What is cryptic growth and what phase can it sometimes occur in?

Answer 25

It occurs in the stationary phase, and is when cell division equals cell death
(Slide 22)

Question 26

In what cultures does cryptic growth happen more often in?

Answer 26

Mixed cultures and continuous cultures
(Slide 22)

Question 27

What 2 values decrease during the death phase?

Answer 27

Viable cell count decreases
Usually cell lysis occurs so OD600 also decreases
(Slide 23)

Question 28

What may the death phase be?

Answer 28

A temporary levelling off due to selection of resistant mutants which continue to grow or are still in stationary phase
(Slide 23)

Question 29

What are 3 methods of cell counts?

Answer 29

Direct counting of cells
Viable count
Optical Density
(Slide 25)

Question 30

What are 3 downsides to doing a viable cell count?

Answer 30

Cell clumping is possible
Laborious and time consuming
Uses a lot of plastic and reagents
(Slide 27)

Question 31

How do you measure growth by optical density?

Answer 31

Measure the amount of light (usually at at λ = 600 nm) that is scattered when passed through a culture, it uses a spectrophotometer
(Slide 28)

Question 32

In measuring growth by optical density what is the relationship between scattering and cell density?

Answer 32

Scattering is proportional to cell density
(Slide 28)

Question 33

What are 3 disadvantages of measuring growth by optical density?

Answer 33

Needs ~ 10^5-10^6 cells/ml to work
Doesn’t tell you how many cells are viable
Different bacteria have different light scattering properties
(Slide 28)

Question 34

How is OD600 different from a viable cell count?

Answer 34

Offset between OD and viable cell count due to nonviable cells
At high cell densities, light is scatter away from receiver, resulting in OD being an underestimation of the number of cells.
(Slide 29)

Question 35

What are 3 reasons that bacterial growth must be plotted on a semi-log graph?

Answer 35

It allows direct comparison of cultures in the exponential phase
The gradient of the straight line is the growth rate constant
Reproducibility and clear communication
(Slide 35)

Question 36

What units are usually used for growth rate?

Answer 36

Cell number per unit time (usually expressed as generations/hr)
(Slide 36)

Question 37

What is the generation time/ doubling time?

Answer 37

The time taken for a population of cells to double in number - represented by “g”
(Slide 36)

Question 38

How does generation time / doubling time vary?

Answer 38

For species and culture conditions and can vary from ~10 min to serveral; hours (or years)
(Slide 36)

Question 39

How is generation time calculated?

Answer 39

g = t/n
(Slide 37)