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Flashcards in Test 3 Chap 4 Deck (60):
1

Growth

Orderly increase of major chemical parts of an organism
Not necessarily mass, as it could just be the increase of chemical reserve materials
Usually results in multiplication, except in multinucleated organisms (coenocytic) ex. Fungi, algae, molds
It is the ultimate goal of microbial cell

2

Multicellular organism

Cellular multiplication of this organism leads to increase in size of individual

3

Unicellular organism

Growth leads to an increase in number of individual
So cellular reproduction=organism reproduction

4

Cell growth depends on

Metabolism- large number of chemical reactions
Fueling-transformation of energy
Biosynthesis- synthesis of small molecules (building blocks, coenzymes, vitamins, ect)
Polymerization- reactions that make macromolecules (chains of building blocks)
Assembly of the macromolecules

5

Binary Fission/ Transverse

Process of cell growth and division in bacteria
Most common way of unicellular microbial reproduction
Equal partitioning of all material

Cell elongates then parts in the middle

6

Septum

Invagination of the cell membrane and cell wall
Cell membrane and grows inwards from opposing directions

7

Filamentous temperature sensitive proteins (FTS)

Essential in cell division, present in ALL bacteria
Mutations in the gene for this protein cause cell to not divide normally
FTS interac and form DIVISOME (protein complex)

8

Divisome

It’s formation begins with attachment of FTsZ (tubulin analog) molecule around center of cell in a ring, this attracts over divisome proteins
This FTsZ ring defines the cell division plane
Other proteins invoved:
ZipA: a host that connects FTsZ to cytoplasmic membrane
FtsA: proteins that helps connect ring to membrane
FTsl: needed to form peptidoglycan
FTsk and other proteins: help pull 2 copies of DNA apart

9

Divisome and cell division

Divisome’s form about 3/4 of the way through the cell division
DNA replicates before the FTsZ ring, then this attaches between duplicated nucleoids
Nucleoids block ring formation before the segregate
Min proteins ensure that the ring only forms in the center
As the cell constricts, and the FTsZ depolymerizes. This triggers invagination.
GTP hydrolysis provided energy for FTsZ activity

10

MinC & MinD

They help dictate the center of the cell by inhibiting the FTsZ ring formation outside the cell center
Prevent cell division at poles because it is most abundant at the poles

MinC-FTsZ Antagonist
MinD-oscillates MinC and helps it attach to cytoplasmic membrane
MinE- same as MinD
Help produce higher MinC at poles

11

MreB & Crescentin

MreB- cytoskeleton protein, its an actin analog (microfilament shape) that winds as a coil through the cell to give it the rod shape. If inactive then cell is a Cocci

Crescentin- found in vibrio shaped bacteria alongside MreB, similar to keratin (cell shape: intermediate filament) helps give the shape to bacteria as well

12

Peptidoglycan synthesis

Because the cell wall is outside the membrane, the precursors for the wall need to be transported through the membrane
Bactoprenol is a C55 lipid (hydrophobic) that binds to these precursors and help transport through membrane. They interact with TRANSGLYCOSYLASE which maker glycosidic bonds
Autolysins cut the preexistí get peptidoglycan by breaking the B(1,4) glycosidic bond and at the same time the precursors are inserted

13

Transpeptidation

Final step of the peptidoglycan synthesis.
FTsL catalyze the transpeptidase reaction in E.Coli and other bacteria
Cells are penicillin sensitive because it binds to the FTsL this prevents cross-linkage in the wall, and without this the wall is weakened and causes osmotic lysis (autolysis)

14

Budding

Type of division that results in unequal distribution of cell material. (Happens in Yeast)

Although yeast can also multiply by binary fission (equal)

15

Measuring cell growth

Measure in #cells/ml or mg cells/ ml

During growth the number #of cells and mass double.

Cell mass is continuous and cell division discontinuous (proved by synchronous cultures, where all the cells are in the same stage of the cycle)

16

Exponential growth

Pattern of population increase, where doubling in the number of cells presents each generation time

17

Synchronous cultures

Because all cell are in the same stage, any measurements done equal 1 single cell

18

Bacterial growth curve

The math/ kinetics of growth dont represent the normal patter of growth.
It shows one portion of the growth curve, mostly the Logarithmic/exponential phase.
During this exponential growth, the cell divide according to their generation time. It does not continue for long periods of times.
This applies to population not single cell

19

Generation time (g)

Cell division intervals

G=duration of growth(t)/ #of generations(n)

20

Phases of bacterial growth

Lag phase: does not always occur and when it does, it can vary (k=0)
Exponential/ Log phase= cant continue indefinitely. Limited (k=positive and reaches max)
Stationary phase= no increase in cell number or mass (k=0)
Death phase: cells die exponentially (k=neg)

K= specific growth rate

21

Growth rate

Change in cell number or cell mass per unit of time
Each time a cell divides a doubling occurs

# of doubling is the # of generations (n)
G=generation time
T=duration of growth (time)
N= number of generations
G=t/n
This is only for exponential phase

22

Lag phase

When you inoculate a microbe (stationary or death phase) into a fresh medium, growth doesnt happen immediately, it happens after a period of time. This is the lag phase
Metabolism subdivision I,II,III, IV occur in lag
There is no increase in cell#
At IV cell gets larger and prepares to divide
When it divides it transitions in to exponential phase

23

When does lag phase occur?

-When inoculate cell in stationary/ death phase into fresh medium
-When Inoculate cell in exponential phase in to medium of different chemical composition because it needs to introduce new medium
-Inoculate cells from a rich to poor culture

It does not occur when inoculation in exponential phase into fresh medium of SAME chemical composition

24

Biphasic growth

Growing with two different carbon and energy sources
Ex. Glucose and lactose
Stationary cells are inoculated into medium with two sources. First the growth uses the most rapidly metabolized carbon (glucose) then a lag then the next one (lactose)
2 extra enzymes need
Lactose permeate to bring in lactose and B-galactosidase to break lactose down into galactose and glucose

25

Exponential phase

Pattern of population increase where cells double
Growth rate max at this point
Cells are at the healthiest
Prokaryotes grow faster than eukaryotes
Smaller faster than larger

26

Relation between n and N0

N0=final number of cells; n=number of generations

N0=2^n

n -> N
0. 1
1. 2
2. 4
3. 8
4. 16
5. 32
6. 64

27

Equations

Final count(Nt)=N0 x 2^n

Log Nt= Log N0 + n Log 2, which can be solve for n and gives us :
(Log Nt- Log N0/0.301)= n
G=t/n -> we can substitute n
G=0.301t/ (LogNt-LogN0)
K=1/g=n/t, and we can substitute with above equation

Keep time in hours

28

Stationary Phase

In a tube or flask with limited growth the exponential phase can occur forever
E. Coli grows for 48 hours with a g=20 min,

Stationary phase occurs when:
The nutrient are all used up
Waste by product of the organism builds up inhibitory level

There is no net increase or decrease in cell number, but many functions continue

29

Cryptic growth

some cell die while some cells double.

Without limits 1 EColi cell could grow to 4,000x the weight of the earth in 48 hours

30

Death Phase

When the cells lack nutrients the can die,
It is also exponential but at a slower rate

All these phases apply to a population of cells not an individual cell

31

Bio films

Microorganism usually dont live a single species. They are usually found embedded in an attached polysaccharide matrix known as biofilm
It can include single species, multiple, or fungi
It has a protective layer (hydrogel), they have a synergistic relationship
They share nutrients, and share genetic info
They attach to hard places or mucous
Starts with free swimming bacteria that attaches, then it forms pillar structure with channels for water, nutrients and waste

32

Quórum sensing

Cell to cell comms

33

Benefits and Downsides of biofilms

They work together:
Benefits:Microbes in cow GI tract help break down cellulose, sewage treatment systems (use multiple microbes)
Downsides:70% of human infections involve biofilm
Difficult to treat infections of medical devices (ex. Heart valves)
Dental caries, contact lenses, Cystic fibrosis (biofilm in lungs) these are all examples of biofilm infections

34

Preventing biofilms

It is important
Can be done by cleaning surfaces with antimicrobials
which can inhibit quorum sensing and sequester of iron (lectoferrin)

35

Methods of measuring bacterial growth

Microscope count
Plate count
Membrane or molecular filters
Turbidimetric measurement
Nitrogen determination
Weight determination
Measurement of biochemical activity

SLIDE 36 more info

36

Measuring Microbial reproduction

A. Direct method WITHOUT incubation: microscope count, electronic counters
B. Direct Method WITH incubation: serial dilution and viable plate counts, Membrane filtration, MPN (most probable number, we used in water study)
C. Indirect methods: Turbidity, Metabolic activity, dry weight, genetic methods

37

Dry weight measuring

N content
Ex. If 14% of dry weight is nitrogen then.,
0.14W=N content
For this we do a nitrogen determination (measure content)
Ex. Of this is the Kjeldahl Method, we solve for W
Protein content
If 20% of dry weight is protein then,
0.20 W= protein content
For this we do a protein determination
Ex. Dumas method, we solve for W

38

Turbidity Methods

This is done using a spectrophotometer and measure the amount of light passing through solution, un scattered and un absorbed
Neplelometer: measure the scattered light
Then we use the Beer-lambert law
Which shows us the linear relation between the concentration of an organism and the absorbance of light

39

Direct microscopic count (DMC)

We use a cell counter slide such as the Petroff-Hausser counting chamber or not

Without the counter: a small portion of bacteria (.01-.02mL) suspension is smeared on the slide in a predetermined area of about 1cm^2
Then we stain it and the # of microorganism per microscopic field is recorded
You want to have about 1-2 MO per field and to count about 50 MF, if we have more than 1-2 MO per field then we dont need to cont as many fields

SLIDE 43 for formula

40

Petroglifos-Hauser Counting Chamber

Special slide that contains known volume
The number of bacteria per mL= number of cells counted in X dilution * 50,000

Ex. 300 cells * 50,000= 1.5x10^7

41

Viable plate count

Count colonies in a plate with 30-300 colonies

To find original count you use this formula

# colonies * dilution factor

Examples

30 colonies * .1ml (which is same as 1/10ml)= 30*10=300cells/ml

42

Serial dilutions

Look at video and slide 47

# colonies * dilution factor

S/S+D= dilution factor

43

Environmental effects on Growth

Rate and amount of growth is affected by physical and chemical environments
Physical:Temp, Hydrostatic pressure, osmotic pressure, surface tension, visible radiation, UV radiation, gravity, adsorption phenomena, viscosity
Chemical:Water activity, water structure, pH, organic and inorganic nutrient quantity and quality, gas quality and quantity, hormones, growth regulations, metabolism, poison, inhibitor and nutrient analogs, oxidation-redox potential

Knowing conditions, help control organism

44

Steriliztion

Treatment that frees object of all living organism including endospore

DEATH: irreversible loss of ability to reproduce
VIABLE: means living and capable of reproduction

45

Temperature effects

Temps can affect in opposing ways
Temp rises, chem and enzyme reactions increase and growth becomes faster

Above certain temp proteins become damaged

46

Cardinal Temperatures

Minimum: below no growth occurs
Optimum: growth most rapid
Maximum:above no growth
Optimum always closer to max

47

Temperature levels

Psychrophile: Optimum temp is 15C or lower max 20C, minimum up to -10C (snow algae, ocean water)
Psychrotolerant: grow at 0C but optimum is 20-40C
Mesophiles: Optimim is from 15C to 40C
Thermophiles: optimum is above 45C
Hyperthermophile: optimum above 80C (mostly archaea)

48

Danger zone for bacterial growth

15-50C, rapid growth and toxins produced

49

Snow algae

Psychrophile that grow on the snow banks of Sierra Nevada, CA
Adapt for this environment by making protein structures changes such as:
More alpha helix , less beta sheets
More polar, less hydrophobic
More unsaturated and shorter fatty acids

50

Hyperthermophiles

Optimum temp exceeds 80
Most are archae, its enzymes and proteins are more heat stable
Cytoplasmic membrane stable, especially mono layer


*in bacteria, saturated acids form a stronger hydrophobic environment*

51

Acidity and pH

PH=-log[H+]
If [H+]=.01 pH=-log[10^-2]=2
[H+]=.001 pH=-log[10^-3]=3
If pH changes 1 unit (2 to 3) then it changes 10 fold
2 to 4 changes for each unit so 10x10=100fold
AS PH INCREASES, H+ DECREASES
Bacteria optimal pH 4-9

52

Osmotic Pressure and Water activity (aw)

Water availability in the cell is expressed as Water activity (aw)
Aw=Psoln./Pwater
Psoln=water vapor pressure above solution
Pwater=water vapor pressure of pure water
Aw always <=1
Common aw in slide 61

BACTERIA GROWS BEST in aw=0.95
Inside solute higher than outside= pos water balance

53

Osmotic pressure limits

Halophiles= requiere salt for growth, absolute requirement for Na+
Osmophiles=grow in environment with high sugar concentration
Xerophiles= grow in very dry low water environment

54

Compatible Solutes

Organism that grow in low water activity environment must drive water in by increasing solute inside cell
This is done by:
-Pumping in solutes from the outside
-Making compatible solutes
COMPATIBLE SOLUTES MUST BE:
-Highly water soluble
-Non-inhibitory
Ex. Staph uses amino acid proline
Glycine betaine is also used by halophilic bacteria and Cyanobacteria

55

Oxygen requirements

Aerobes: species capable of growth at full oxygen tension
Obligate aerobes: require O2 for growth
Microaerophiles: can use O2, only when it is present at levels reduced from that in air
Facultative can grow in aerobe or anaerobic
Facultative aerobes: O2 not required but grow better with it
Anaerobes: dont use oxygen
Facultative anaerobes: tolerate O2 but grow better without
Obligate anaerobes: inhibit or even killed by O2
SLIDE 66 and 67

56

Thioglycolate Tubes

Contains dye resazurin which is pink when oxidized, colorless when reduced

LOOK at SLIDE 68 and 69 for picture! And 74

57

Toxic form of Oxygen

Triplet oxygen 3^O2
A major toxic form is the singlet 1^O2
Highly reactive and carry out spontaneous and undesired oxidation
CAROTENOIDS convert to them into this toxic form
Other toxic forms:
Superoxide anion (O2-) Strong oxidation
Hydrogen peroxide (H2O2) least reactive
Hydroxyl radical (OH-) most reactive
THEY ARE ALL BY PRODUCTS OF O2 to H2O
Even anaerobes can catalyze reduction of O2 to O2- with enzymes

58

Enzyme to remove toxic by product

Catalase
Peroxidase
Superoxide dismutase (Aerotolerant Anaerobes)
Superoxide dismutase/ catalase in combination (obligate and facultative aerobes)
Superoxide reductase (obligate anaerobes)

SLIDE 71-73

59

Culture Conditions

Aerobes need extensive aeration, shaking of flask, bubbling sterilized air through medium

Anaerobes cannot have a single trace of oxygen. It needs a reducing agent such and thioglycollate and H2S
Needs an anoxic jar that uses H2 +CO2+ palladium catalyst to remove oxygen by forming water
Anoxic glove box in which air can be evacuated and the space filled with O2 free gas

60

Nuclear Waste eating microbes

Some fungo and bacteria can sustain radiation levels that would kill us
They could be used to protect us from radiation, clean up nuclear waste, continuous food source for astronauts

They can be found near nuclear reactor in Chernobyl
Ex. Wangiella and Cladosporium (Fungi) and Kineococcus radiotolerans (bacteria)