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Microbial Physiology and Growth > Week 3 - Bacterial growth and replication, including yield and responses to nutrient availability > Flashcards

Week 3 - Bacterial growth and replication, including yield and responses to nutrient availability Flashcards

1
Q

Energy source = light

Carbon source = carbon dioxide

A

= photoautotroph
• plants, algae, and cyanobacteria
• use H2O to reduce CO2, producing O2 as a side product
• photosynthetic green sulfur and purple sulfur bacteria do not use H20 nor produce O2

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2
Q

Energy source = chemical compounds

Carbon source = carbon dioxide

A

= chemoautotrophs

• hydrogen, sulfur, and nitrifying bacteria

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3
Q

Energy source = light

Carbon source = organic compounds

A

= photoheterotrophs

• green nonsulfur and purple nonsulfur bacteria

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4
Q

Energy source = chemical compounds

Carbon source = organic compounds

A

= chemoheterotrophs
• aerobic respiration - most animals, fungi, and protozoa, and many bacteria
• anaerobic respiration - some animals, protozoa, and bacteria
• fermentation - some bacteria and yeasts

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5
Q

Growth

A

increase the number of cells

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6
Q

Binary fission

A

cell division following enlargement of a cell to twice its minimum size
• duplicates that are identical

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7
Q

Generation time

A

time required for microbial cells to double in number

• depends on many factors

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8
Q

During cell division, each daughter cell

A

receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell

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9
Q

Reproduction in prokaryotes

A
  • binary fission
  • budding
  • conidiospores (actinomycetes)
  • fragmentation of filaments
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10
Q

Growth requirements

A
  • physical

* chemical

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11
Q

Physical growth requirements

A
  • temperature
  • light (energy)
  • pH
  • osmotic pressure
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12
Q

Chemical growth requirements

A 
• gases (CO2)
-not all breathe oxygen (eg sulfur dioxide)
• chemicals
- organic (solids)
- inorganic
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13
Q

Physical requirements - temperature

A
  • maximum growth temperature
  • optimum growth temperature
  • minimum growth temperature
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14
Q

Hyperthermophiles

A

65 - 110 C

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15
Q

Thermophiles

A

40 - 70 C

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16
Q

Mesophiles

A

10-50 C

• environmental pathogens of humans

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17
Q

Psychrotrophs

A

0 - 30 C

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18
Q

Psychrophiles

A

-10 - 20 C

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19
Q

Temperatures in this range destroy most microbes, although most temperatures take more time

A

~62 to 130 C

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20
Q

Very slow bacterial growth

A

~52 - 62C

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21
Q

Rapid growth of bacteria

some may produce toxins

A

15 - 52 C

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22
Q

Many bacteria survive, some may grow

A

~5 - 15 C

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23
Q

Refrigerator temperatures

may allow slow growth of spoilage bacteria, very low pathogens

A

0 - 5 C

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24
Q

No significant growth below freezing

A

-30 - 0 C

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25
Q

Binary fission steps

A
  1. cell elongates and DNA is replicated
  2. cell wall and plasma membrane begin to grow inward
  3. cross-wall forms completely around divided DNA
  4. cells separate
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26
Q

Binary fission description

A

• microbial growth is the increase in number of cells, not cell size
• result of microbial growth is discrete colony - an aggregation of cells arising from single parent cell
• binary division - 1 to 2 to 4 to 8 to 16…
• divisome = when wall starts to appear
- this and proteins prime the cell for division/duplication
- peptidoglycan - for grabbing these and keeping them there
• binary fission = grows, duplicates, splits
• chorin-sensing = bacteria can sense if there’s food, temp –> replicate or not, so must communicate

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27
Q

Fts proteins and cell division

A

Fts = filamentous temperature-sensitive protein
• essential for cell division in all prokaryotes
• interact to form the divisome (cell division apparatus)

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28
Q

FtsZ

A

forms ring around center of cell

• related to tubulin

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29
Q

ZipA

A

anchor that connects FtsZ ring to cytoplasmic membrane

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30
Q

FtsA

A

helps connect FtsZ ring to membrane and also recruits other divisome proteins
• related to actin

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31
Q

Bacterial growth

A

replication usually by binary fission
(2 equal daughter cells)
• cells may not completely separate after division, leading to filaments, patches, or clusters
• some filamentous bacteria bacteria are genuine multicellular organisms
- communication and cooperation between cells
- differentiation into different cell types
- pattern formation

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32
Q

DNA replicates before the

A

FtsZ ring forms
• location of FtsZ ring is facilitated by Min proteins
- MinC, MinD, MinE

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33
Q

FtsK protein

A

mediates separation of chromosomes to daughter cells

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34
Q

Alternatives to binary fission

A
  • budding
  • multiple fission
  • sporulation
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35
Q

Budding

A

cell splits unequally to produce a larger “mother” cell and a smaller “daughter” cell
• the mother cell may go through many rounds of replication to produce numerous daughters
• eg hyphomicrobium - a chemoorganotroph that lives on methanol and other carbon food sources
• mostly fungi
• progenitor cell produces bud that goes off to make another cell
• buds take time to mature

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36
Q

Multiple fission

A

cell becomes greatly enlarged and then divides at many points simultaneously
• eg the filamentous cyanobacterium Anabaena
• mix of budding and replication
• grows big filamentous, split –> separate

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37
Q

Sporulation

A 
eg streptomyces - complex multicellular soil bacteria
1. substrate mycelium
2. aerial hypha
3. partitioning
4. spore  maturation
5. dispersal
6. makes spores
• spores resistant to dehydration, will germinate to form new substrate mycelia in favorable environments
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38
Q

Hyphae

A

individual organism attached into colony

• can break off and grow on its own

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39
Q

Requirements for successful replication

A
  1. the cell needs to grow - biosynthesis of DNA, protein, lipid, carbohydrate
  2. DNA replication must be completed before cell division occurs
  3. the chromosomes must segregate into different parts of the cell
  4. the septum must be formed at an appropriate point in the cell
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40
Q

Growth of the cell

A

important = biosynthesis of cytoplasmic membrane and cell wall
• gram + = lots of peptidoglycan
- live outside at first = need lots of protection
• gram - = little peptidoglycan
- live inside = don’t need a lot of protection
• gram stain separates on basis of bacterial cell wall
• penicillin attacks peptidoglycan

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41
Q

DNA replication and the bacterial cell cycle

A

DNA replication has to be tightly coordinated with cell division
1. DNA replication
2. chromosome partitioning
3. cell division
• a cell is prevented from dividing until it’s long enough, and the chromosomes have partitioned
• microbial replication = weakest part, can’t stop growth (eg contamination)

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42
Q

Partitioning of the chromosome copies

A

attachment of the chromosome to the plasma membrane is important
• DNA is replicated at a membrane-bound replication factory
• the new DNA copies remain attached to the plasma membrane
• somehow the attachment sites are pushed to opposite ends of the cell

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43
Q

Septum formation

A

the FtsZ protein (similar to tubulin in eukaryotes)
plays a crucial role in cell division
• it forms a contractile ring in themiddle of the cell

• location of the septum in the middle of the cell requires the Min proteins

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44
Q

Want to focus on the stage that

A

makes the particular product

45
Q

Growth phases

A
  • lag
  • exponential
  • stationary
  • death
46
Q

Lag phase

A

not duplicating, waiting for food

• food appears = exponential growth (duplication)

47
Q

Stationary

A

waiting for food

• enclosed environment = survive then gradually die off

48
Q

Replication cycle

A

need to known maximum growth and how to control it
• too much nutrients –> lots of waste, not utilizing at maximum rate = reduction of product produced
- so must look for optimal

49
Q

Steady state

A

keeps going forever

• must occasionally remove bacterial load, don’t put too much nutrient in

50
Q

Types of cellular transport

A

passive transport

active transport

51
Q

Passive transport

A 
• cell doesn't use energy
• passive transport is basically absorption
1. diffusion
2. facilitated diffusion
3. osmosis
52
Q

Active transport

A 
• cell doesn't use energy
• energy intake must be efficient
- don't want what you're making to cost more than it makes
1. protein pumps
2. endocytosis
3. exocytosis
53
Q

Chemophysical requirements - pH

A
  • acidophiles grow in acidic environments
  • molds and yeasts grow between pH 5 and 6
  • most bacteria grow between pH 6.5 and 7.5
54
Q

Physical requirements

A

osmotic pressure
• hypertonic environments, increase salt or sugar, cause plasmolysis
• extreme or obligate halophiles require high osmotic pressure
• facultative halophiles tolerate high osmotic pressure

55
Q

Plasmlysed

A

cell membrane shrinks within cell wall (in hypertonic)

56
Q

Hypertonic

A
  • highly concentrated solution
  • water molecules diffuse out of the cell
  • cell shrinks
57
Q

Hypotonic

A

lower concentration of solutes than another solution
• water molecules into cell
• cell expands

58
Q

Isotonic

A

having the same concentration of solutes

59
Q

How bacteria and plants deal with osmotic pressure

A
  • have cell wall that prevents them from over-expanding

* in plants, the pressure exerted on the cell wall is called tugor pressure

60
Q

How protists deal with osmotic pressure

A
  • has contractile vacuoles that collect water flowing in and pump it out to prevent the from over-expanding
  • eg paramecium
61
Q

How water fish deal with osmotic pressure

A

pump salt out of their specialized gills so they don’t dehydrate

62
Q

How animal cells deal with osmotic pressure

A
  • animal cells are bathed in blood

* kidneys keep the blood isotonic by removing excess salts and water

63
Q

Normal cell in isotonic solution

A

NaCl concentration outside cell is 0.85%

• under these conditions the osmotic pressure in the cell is equivalent to a solute concentration of 0.85% NaCl

64
Q

Plasmolysed cell in hypertonic solution

A

NaCl is 10%
• if the concentration of solutes such as NaCl is higher in the surrounding medium than in the cell (the environment is hypertonic), water tends to leave the cell. Growth is inhibited
• plasma membrane shrinks inside the cell wall

65
Q

1 colony doesn’t always represent 1 cell

so

A

estimate colony forming untis (CFU)

66
Q

A dense broth culture could

A

contain 10^9 cells per ml

67
Q

How do you estimate the numbers of bacteria

A
  • look at a very small volume
  • carry out a very large dilution
  • find a way that doesn’t involve counting individual cells - eg using a counting chamber
68
Q

Disadvantages of the counting chamber

A
  • tedious (especially for bacteria)
  • counts both live and dead cells (but this problem can now be solved using fluorescent stains that distinguish live and dead cells)

another approach = serial dilution, followed by plating on agar, then count colonies
• concentration of original culture =
dilution factor x colonies / ml plated
• if cells are in clusters then 1 cluster = 1CFU

  • for high count use serial dilution
  • for very low count concentrate cells by centrifugation or filtrate
69
Q

Other ways to estimate cell numbers

methods that don’t rely on counting individual cells

A

eg optical density using a spectrophotometer
• measure light scattering which is approximately proportional to cell count
• very quick but only works on quite dense cultures

  • dense bacterial cultures look cloudy due to light scattering by the cells
  • this can be quantified in a spectrophotometer - measure apparent absorbance at a suitable wavelength
  • this is approximately proportional to cell concentration
  • need a calibration factor to calculate actual cell numbers
70
Q

Spread-plate method

A
  1. sample is pipetted onto surface of agar plate (0.1 ml or less)
  2. sample is spread evenly over surface of agar using sterile glass spreader
  3. incubation
  4. typical spread-plate results shows surface colonies
71
Q

Pour-plate method

A
  1. sample is pipetted into sterile plate
  2. sterile medium is added and mixed will with inoculum
  3. solidification and incubation
  4. typical pour-plate results show surface and subsurface colinies
  • better than spread-plate because in spread-plate you throw away the bugs on the swab
  • higher count on spread-plate = more accurate of circumstances
72
Q

Grow on different mediums to find the medium that the bug grows best on
then

A 
look at genome for chemical of interest
then 
get genetic material out
then
use a different but that grows better/easier
73
Q

Environmental restrictions

A

keep bacteria from overpopulating

74
Q

With binary fission the population

A

doubles at each division

• under constant conditions, doubling occurs at regular intervals, giving exponential growth

75
Q

Exponential growth gives a straight line if

A

number of cells is plotted against time on a logarithmic scale

76
Q

A simple way to grow cells in the lab

A

batch culture - closed system

  1. growth medium
  2. inoculate with a small quantity of culture
  3. incubate (usually with shaking)
77
Q

Batch culture instead of continuous culture because

A

continuous is very tedious and must always be monitored

• batch = closed, end prodcut si the end phase

78
Q

Growth in a batch culture

A
  1. lag phase - inoculum adapting to new conditions
  2. exponential rate - population doubling at regular intervals (doubling time depends on organism, medium, and conditions)
  3. stationary phase - growth slows, then ceases as nutrients are exhausted and waste products accumulate
  4. death phase - cells start to die (starvation and accumulation of toxic waste products)
79
Q

Doubling time depends on

A

organism, medium, and conditions

80
Q

Continuous culture

A

open-system with a chemostat
• culture maintained in a steady-state of exponential growth
• conditions in the chemostat remain constant

  • sterile air, input rate of gas to maximize organism, culture flows up to top of tube –> collect effluent
  • balance stuff going in to product coming out
  • adjust headspace (gas into headspace, doesn’t go right into liquid)
  • what’s best for bacteria may not be best for product so keep at certain life cycle phase
81
Q

Mean generation times vary widely depending on

A

the organism and the conditions

82
Q

Vibrio natriegens

A

a marine bacterium found in salt marsh mud

• can double in 10 minutes

83
Q

Mycobacterium leprae

A

a skin pathogen, the cause of leprosy

• doubling time of several weeks in vivo

84
Q

E. coli in rich medium can double in

A

about 30 minutes

85
Q

Doubling time depends on

A

uptake of nutrients and time required to replicate DNA, proteins, cell walls, etc

86
Q

Rapid doubling gives an advantage

eg

A

sudden appearance of rich food

87
Q

Effect of temperature on growth rate

A

enzymes

• extreme halophiles make acid to control environment

88
Q

What limits the growth of bacteria in the wild?

A

eg phytoplankton - photoautotrophic microbes growing in the oceans, need only minerals, water, carbon dioxide, and sunlight
• minerals tend to be the limiting factor
(nitrate, phosphate, IRON)
• adding a small amount of iron could make a big difference to phytoplankton growth

89
Q

Example of halotolerant

A

Staphylococcus aureus

90
Q

Example of nonhalophile

A

E. coli

91
Q

Example of halophile

A

Alilvibrio fischeri

92
Q

Example of extreme halophile

A

Halobacterium salinarum

93
Q

Bacteria simulate their own growth, as well as that of their neighbors, by

A 
secreting growth factors into their environments
eg Rpf (resuscitation-promoting factor)
94
Q

Classification of organisms based on oxygen requirements

A
  • aerobes
  • anaerobes
  • facultative anaerobes
  • aerotolerant anaerobes
  • microaerophiles
95
Q

Aerobes

A

undergo aerobic respiration

96
Q

Anaerobes

A

do not use aerobic respiration

97
Q

Facultative anaerobes

A

can maintain life via fermentation or anaerobic respiration or by aerobic respiration
- in a tube, cells tend toward the top but are throughout

98
Q

Aerotolerant aerobes

A

do not use aerobic metabolism but have some enzymes that detoxify oxygen’s poisonous forms
- in a tube, cells are throughout but tend toward bottom

99
Q

Microarophiles

A

aerobes that require oxygen levels from 2-10% and have a limited ability to detoxify hydrogen peroxide and superoxide radicals
- in a tube cells in the middle of solution

100
Q

Obligate aerobes

A

oxygen is essential
• final electron acceptor in ETC
- in a tube, all cells at top of solution

101
Q

Obligate anaerobes

A

oxygen is deadly

- in a tube, all cells at the bottom of solution

102
Q

Sergei Winogradsky

A

discovered chemosynthesis - the process by which organisms metabolise a number of different inorganic substrates to obtain carbon
• previously it was believed that autotrophs obtained their energy solely from light, not the oxidation of inorganic compounds such as H2S and NH4+

103
Q

Winogradsky column

A 
aerobic
^
microaerophilic
^
anaerobic
^
H2S

• gases at bottom feed up to help bugs above grow = multiorganism bioreactor

104
Q

Winogradsky column - water

A

aerobic

• algae, cyanobacteria, aerobic heterotrophs

105
Q

Winogradsky column - red-brown

A

microaerophilic

• purple non-sulfur photoheterotrophs

106
Q

Winogradsky column - red-violet

A

anaerobic –> microaerophilic

• purple sulfur bacteria

107
Q

Winogradsky column - green

A

H2S –> anaerobic

• green sulfur bacteria

108
Q

Winogradsky column - sediment

A

H2S

• sulfate reducers, fermentative heterotrophs

109
Q

Preparation of a Winogradsky column

A

substrates =
• cellulose
• sodium sulphate
• calcium carbonate

  • the substrates are mixed into the sediment and the column is half filled with water
  • the remaining half is filled with river water leaving a small air gap at the top and tightly sealed
  • the column is then left for 2 months in strong sunlight

Microbial Physiology and Growth (5 decks)

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