L29 - Bacterial Growth And Replication

Question 1

Where do bacteria form biofilms?

Answer 1

On any surface where there is moisture

Question 2

What do bacteria attach to? What happens afterwards?

Answer 2

• bacteria attach to surface
• grow and become enveloped in extracellular matrix (ECM) (polysacch, proteins and DNA)

Question 3

What are the stages of biofilm formation?

Answer 3

• inital attachment
• irreversible attachment
• maturation 1
• maturation 2
• dispersal

Question 4

What happens during initial attachment?

Answer 4

Individual bacteria attach weakly to a surface

Question 5

What happens during irreversible attachment?

Answer 5

Attachment becomes irreversible using fimbrae and pilli

Question 6

What do bacteria do after attachment?

Answer 6

Multiply, attract other microbes to attach

Question 7

What happens during maturation 1?

Answer 7

• bacteria secrete a sticky, protective ECM
• contiue to join and multiply

Question 8

What happens during maturation 2?

Answer 8

Biofilm grows in size and structure form large 3D colony

Question 9

What happens during dispersal?

Answer 9

Sections of the biofilm break off - cells can go and colonise new areas

Question 10

What do biofilms protect against?

Answer 10

• phagocytosis
• antibiotics
• disinfectants

Question 11

How are biofilms a huge problem in healthcare?

Answer 11

Growth on medical devices

Question 12

What are examples of biofilm in healthcare?

Answer 12

• dental plaque
• urinary catheter
• heart valve - endocarditis, enterococcus sp
• lung tissue - cf, pseudomonas aeruginosa

Question 13

How do nutrients affect growth of biofilm?

Answer 13

Nutrients required for cellular biosynthesis and energy generation
- macroelements - C, H, O, N, water, S, P, K, Ca, Mg, Fe
- trace elements - Mn, Co, Mo, Ni, Cu vits, gf

Question 14

How does iron affect growth of biofilm?

Answer 14

• used for energy generation
• iron in body not available
• withing mammalian cells (90% stored in ferritin/haem group, 8% stored elseqhere)
• outside mammalian cell (1-2% attached to transporters
• non complexed iron exists as Fe3+, insoluble

Question 15

What is the bacterial iron transport system?

Answer 15

Siderophores

Question 16

What are siderophores?

Answer 16

• low MW compounds with high affinity for iron
• produced and exported from some bacteria when iron low
• bind iron and allow uptake into the cell
• -remove iron complexed with transferrin, uptake

Question 17

How does oxygen affect growth of biofilm?

Answer 17

• aerobe (with O) - microaerophile - can grow in low conc
• anaerobe (w/o O) - obligate anaerobe - cannot grow in O, faculative anaerobe - can grow in O if available

Question 18

How does temperature affect growth of biofilm?

Answer 18

• psychrophiles - -40C to 20C, optimum <15C // lysteria monocytogene
• thermophiles - 45 to 100C // thermophilus aquaticus
• mesophiles - 20 to 40C, organisms of med and pharm importance

Question 19

How does pH affact growth of biofilm?

Answer 19

Most organisms or medical importance are neutrophiles - pH6.5-7.5

Question 20

What are the different ways of growing bacterial culture?

Answer 20

• suspension
• colony

Question 21

What are bacterial cultures in suspension? Why?

Answer 21

• Bacteria grown in complex liquid media as batch culture
• to determine growth rate/ effect of antimicrobial agents

Question 22

What are bacterial cultures in colonies ilke? Why?

Answer 22

• bacteria grown on complex media solidified with agar
• to obtain pure culture/ viable count/ assess diversity/ aid identification

Question 23

What is the bacterial replication?

Answer 23

Binary fission - one bacteria cell grows and divides to 2 identical daughter cells

Question 24

How does binary fission happen?

Answer 24

• cell elongage ~ double
• cell copies chromosome
• septum beings to form
• 2 copies of chromosome pulled apart
• septum formation continues until 2 cells formed

Question 25

How do bacteria multiply?

Answer 25

Doubling as fast conditions will allow - exponential growth

Question 26

What is generation time?

Answer 26

Time taken for bacteria to divide, double

Question 27

What is rate of cell division determined by?

Answer 27

• time required for DNA replication
• conditions

Question 28

What are the differnent phases?

Answer 28

• exponential phase
• stationary phase
• death phase

Question 29

What is exponential phase?

Answer 29

• the cells are behaving in a constant predictable
• generation time is constant
• ideal phse to use the bacteria for research

Question 30

What is stationary phase?

Answer 30

• population running out of resources
• no increase/decrease in cell numbers
• some are dividing, some are dying = unpredictably

Question 31

What is death phase?

Answer 31

• decline in cell numbers
• some are persister - dont die - viable but non culturable cells

Question 32

Where can generation time be read from?

Answer 32

Exponential phase of growth curve

Question 33

What is generation time (g) during exponential growth?

Answer 33

NT = N0 x 2^n

N0 - cells initially present
NT - cells at T
n - number of generations

Question 34

How do you calculate generation time?

Answer 34

Generation time = time/no of gens

Question 35

How do you do a direct measurement of bacterial number - viable count?

Answer 35

• dilute sample of bacteria
• spread on agar plate
• incubate overnight @ 37C
• count colonies
• VC expressed as Colony forming units/ml CFU/ml
• best way to measure no. actively dividing cells
• exc clumps/chains of cells
• conditions must be suitable for growth
• requires overnight culture

Question 36

How do you do an indirect measurement of bacterial number - optical censity (OD)?

Answer 36

• OD increases with increasing cell no over time
• cell no directly related to OD
• read off cell no from standard curve of OD vs cell number
• cells must be in exponential phase for OD to represent no dividing cells