bacterial growth - notes and lecture

Question 1

what is the generation time?

Answer 1

growth rate - time required for the population of bacteria to double
can be 20 minutes to 20 hours

Question 2

what factors limit growth?

Answer 2

1: nutrient supplies and key resources
2: accumulation of toxic metabolic processes
3: antibiotics from neighboring microbes (or humans)
4: immune system
5: environmental conditions

Question 3

what are the major nutrient requirements for bacterial growth? (list - 10)

Answer 3

1: carbon - to make all molecules
2: nitrogen - to make DNA and proteins
3: phosphorus - for membrane phospholipids and DNA
4: sulfur - to make proteins
5: iron - for many enzymes, esp those in metabolism
6: glucose
7: other sugars
8: peptides, proteins, and AA
9: lipids
10: organic acids and alcohols

Question 4

what are the two main functions of nutrient sources?

Answer 4

provide energy and provide carbon for the synthesis of cellular components

Question 5

where do many bacteria acquire AA and peptides?

Answer 5

many need to pre-formed AA

secrete proteases to allow for the uptake of extracellular proteins

Question 6

where do bacteria acquire nitrogen?

Answer 6

AA, peptides, and proteins, but also ammonia (NH4+) and nitrate

Question 7

where do microbes acquire their nucleotides?

Answer 7

secrete nucleases to break down available DNA and RNA

Question 8

what is the purpose of phospholipases secreted by microbes?

Answer 8

act o n host cell membranes or lung surfactant

degrade phospholipids to provide microbes with C, N, P

Question 9

what induces phospholipase production by microbes?

Answer 9

low phosphorus or low iron levels - because host cell lysis by phospholipase activity yields iron

Question 10

what is the role of iron in the host-pathogen relationship?

Answer 10

host cell lysis by phospholipase from microbes yields iron
host cells can make iron-binding factors that make iron unavailable to microbes
in response, bacteria and fungi make chelators (siderophores) that extract iron from host stores

Question 11

what creates pathogenesis due to microbes?

Answer 11

results from microbes accessing host nutrients

Question 12

what are bacterial growth factors?

Answer 12

organic (C) compounds that are not metabolized to supply energy but make metabolites that the bacterium can’t synthesize on its own
not all bacteria require these to grow, and different organisms require different ones

Question 13

what environmental conditions affect bacterial growth rates (list)

Answer 13

temperature
pH
osmotic conditions

Question 14

how does temperature affect growth rate of bacteria?

Answer 14

each organism needs optimal temperature (unique)
most pathogens grow best at body temp
most environmental bacteria at room temp

Question 15

how does temperature affect mycobacterium leprae growth?

Answer 15

exception to rule that pathogens prefer body temp - only grow at low temp - why you get external lesions in leprosy cases but not damage to internal organs

Question 16

what is the optimum pH for bacterial growth

Answer 16

usually in the 6-8 range, but most commonly 7.4

Question 17

how does pH affect where clostridium botulinum grows?

Answer 17

botulinum toxin

more of a problem in canned foods with less acidity

Question 18

how do osmotic conditions affect the growth of bacteria?

Answer 18

high salt and sugar (so high osmotic conditions) inhibit growth of many pathogens
but most very flexible about osmotic enviornment requirements since have cell wall to maintain internal osmotic pressure

so jams and jellies can inhibit growth

but most bacteria don’t need to regulate their internal osm very much b/c of their cell wall

Question 19

what is the common growth medium for bacteria?

Answer 19

peptone broth with added glucose

Question 20

what is peptone?

Answer 20

peptic digest of meat - peptides and AA

Question 21

what percentage of pathogens will grow on blood agar?

Answer 21

90%

Question 22

how would you attain pure cultures of bacteria (in terms of medium for growth)?

Answer 22

allows observation of colonial morphology
must grow bacteria on solid medium
add agar to liquid media

Question 23

what can the size of a bacterial infection tell you clinically?

Answer 23

if there is an infection

Question 24

should you see bacteria in clinical samples in a healthy patient?

Answer 24

in some, such as CSF, no

in others, such as urine, yes

Question 25

how would you measure cell number using optical measurements?

Answer 25

use optical measurements of cell mass small particles scatter light in proportion to their cell density (so this is basically just spectroscopy) low cell densities can't be detected can correlate amount of scattered light with cell number

Question 26

what are four ways you could use to measure cell number? (list)

Answer 26

1: optical measurements 2: determination of metabolic activity 3: direct cell count 4: viable or plate counts

Question 27

how would you measure cell number using metabolic activity?

Answer 27

measure rate of production of metabolic products such as CO2 or ATP

Question 28

how would you measure direct cell number?

Answer 28

direct counting in a counting chamber in a microscope includes viable and nonviable cells not used often cause need really high concentrations but even if a few bacteria are seen, indicates infection since so many bacteria are needed to see any

Question 29

how would you measure cell count using viable or plate counts?

Answer 29

single bacterium gives rise to colony on petri dish after incubation plate sample and count number of colonies - expressed as colony forming units (CFU)

Question 30

how is the growth of bacterial cells usually graphed?

Answer 30

on a logarithmic scale as a function of time

Question 31

what are the four phases of bacterial growth? (list)

Answer 31

lag phase log phase stationary phase death phase

Question 32

what occurs during the lag phase of bacterial growth?

Answer 32

metabolic activity, but no increase in numbers | cell mass and size begin to increase, macromolecules needed for growth synthsized

Question 33

what determines the length of the lag time?

Answer 33

kind of bacteria age and size of inoculum nature of the medium from which they were taken nutrients present

Question 34

what occurs during the log/exponential growth phase?

Answer 34

cell numbers increase in a logarithmic manner - constant generation time cell number and mass increase in coordinate manner - can use either paramater to determine generation time

Question 35

what does the rate of cell division during log phase depend on?

Answer 35

type of organisms nature of medium temperature for aerobic organisms, rate of aeration

Question 36

what is balanced growth?

Answer 36

orderly increase in all cellular organisms doubling of biomass is accompanied by a doubling of all other components - after size has doubled, cell divides => two identical cells

Question 37

why does the logarithimic growth phase eventually slow?

Answer 37

accumulation of waste products exhaustion of nutrients change in pH decrease in O2 tension

Question 38

what occurs during the stationary phase?

Answer 38

number of cells remains the same | steady state in which some cells die an others continue to divide

Question 39

what is the typical length of time for the stationary phase?

Answer 39

can last for only a few hours but lasts for days months or years in some species and under some conditions

Question 40

what concentration do almost all pathogenic bacteria grow to during stationary phase in lab culture?

Answer 40

between 5 x 10^8 and 1 x 10^ 9 cells

Question 41

what occurs during death phase?

Answer 41

rate of death exceeds rate of reproduction - number of viable cells declines

Question 42

what is the typical length of time of the death phase?

Answer 42

varies widely between organisms some very short whereas others weeks or years cells often assume unusual shapes - therefore difficult to recognize bacteria in old cultures

Question 43

what is the lag phase?

Answer 43

adaptation to the host environment

Question 44

what is likely specific to exponential phase in chronic disease states?

Answer 44

slow growth may occur

Question 45

what is the consequence for the host of rapid growth during the exponential phase?

Answer 45

population of microbe may grow too fast for host to mount immune defense

Question 46

during which phase are bacteria most resistant to destruction?

Answer 46

during stationary phase, increased resistance to host defenses and antibiotics

Question 47

what are three reasons to study growth characteristics of a microbe?

Answer 47

1: identify ways to kill or inhibit microbial growth 2: to better understand disease 3: to identify and quantify a microbe in the clinical laboratory and research lab

Question 48

what are the carbon sources used by bacteria to synthesize new cells and new cell factors?

Answer 48

``` glucose other sugars proteins, peptides, and AA lipids organic acids and alcohols ammonia and nitrate ```

Question 49

what do bacterial nucleases do?

Answer 49

when P levels are low, these enzymes can degrade surrounding DNA and create more for use in reproduction

Question 50

where do bacteria acquire their phospholipids?

Answer 50

degradation of host cell phospholipids => C, N, P low P levels can induce production of phospholipase - these phospholipids can lyse host cells => Fe

Question 51

where do bacteria acquire their Fe?

Answer 51

on source: bacterial phospholipases can lyse host cells => Fe phospholipase activity therefore often induced by low Fe dietary iron - becomes part of transferrin and lactoferrin ferritin intracellularly proteins including hemoproteins siderophores

Question 52

why is Fe important to bacterial function?

Answer 52

required for generation of ATP via electron transport chain one of the defenses of host cells is to fenestrate Fe away from bacterial invaders

Question 53

what are siderophores?

Answer 53

have very high affinity for Fe make Fe available to bacteria, often via specific receptors on the bacterial cell surface secreted by bacteria - bind Fe that's bound to protein and "steals" it since it has such a high affinity - brings it back to the bacteria

Question 54

what does it mean for an organism to be fastidious?

Answer 54

require growth factors to grow - such as purines, pyramidines, vitamins and several AA

Question 55

what does it mean for an organism to be prototrophic?

Answer 55

can usually synthesize everything it needs to grow - these can usually live outside of the host

Question 56

what is the advantage of agar medium (over liquid)?

Answer 56

allows observation of colony morphology quantification can separate out different types of bacteria

Question 57

why do we count bacteria?

Answer 57

knowing size of bacterial population allows you to determine if there's an infection antibiotic susceptibility testing needs to be done with cultures of a known density

Question 58

how do we count bacteria?

Answer 58

using a spectrometer - optical density = determined by the amount of light absorbed by a suspension of bacteria amount of light absorbed by a cell suspension is measured by a spectrophotometer dyes that indicate ECT activity count cells under microscope - need a lot of bacteria though plate counts - count number of colonies (CFUs = colony forming units)

Question 59

at what point can you see bacteria in culture?

Answer 59

get faint turbidity at 1 x 10^6 | very turbid cultures will have 1 x 10^8 - 1 x 10^8 bacteria/mL

Question 60

what is the procedure to count CFUs?

Answer 60

1: make dilutions of the sample 2: plate these dilutions on a nutrient medium (agar) 3: incubate the plate overnight 4: count the number of colonies that grow up (30-300 colonies per plate is optimal) 5: calculate the number of viable cells (CFUs) that were in each mL of the undiluted sample