Test 3 Chap 4

Question 1

Growth

Answer 1

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

Question 2

Multicellular organism

Answer 2

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

Question 3

Unicellular organism

Answer 3

Growth leads to an increase in number of individual

So cellular reproduction=organism reproduction

Question 4

Cell growth depends on

Answer 4

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

Question 5

Binary Fission/ Transverse

Answer 5

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

Question 6

Septum

Answer 6

Invagination of the cell membrane and cell wall

Cell membrane and grows inwards from opposing directions

Question 7

Filamentous temperature sensitive proteins (FTS)

Answer 7

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)

Question 8

Divisome

Answer 8

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

Question 9

Divisome and cell division

Answer 9

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

Question 10

MinC & MinD

Answer 10

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

Question 11

MreB & Crescentin

Answer 11

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

Question 12

Peptidoglycan synthesis

Answer 12

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

Question 13

Transpeptidation

Answer 13

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)

Question 14

Budding

Answer 14

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

Although yeast can also multiply by binary fission (equal)

Question 15

Measuring cell growth

Answer 15

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)

Question 16

Exponential growth

Answer 16

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

Question 17

Synchronous cultures

Answer 17

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

Question 18

Bacterial growth curve

Answer 18

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

Question 19

Generation time (g)

Answer 19

Cell division intervals

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

Question 20

Phases of bacterial growth

Answer 20

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

Question 21

Growth rate

Answer 21

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

Question 22

Lag phase

Answer 22

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

Question 23

When does lag phase occur?

Answer 23

• 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

Question 24

Biphasic growth

Answer 24

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

Question 25

Exponential phase

Answer 25

``` 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 ```

Question 26

Relation between n and N0

Answer 26

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

Question 27

Equations

Answer 27

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

Question 28

Stationary Phase

Answer 28

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

Question 29

Cryptic growth

Answer 29

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

Question 30

Death Phase

Answer 30

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

Question 31

Bio films

Answer 31

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

Question 32

Quórum sensing

Answer 32

Cell to cell comms

Question 33

Benefits and Downsides of biofilms

Answer 33

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

Question 34

Preventing biofilms

Answer 34

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

Question 35

Methods of measuring bacterial growth

Answer 35

``` Microscope count Plate count Membrane or molecular filters Turbidimetric measurement Nitrogen determination Weight determination Measurement of biochemical activity ``` SLIDE 36 more info

Question 36

Measuring Microbial reproduction

Answer 36

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

Question 37

Dry weight measuring

Answer 37

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

Question 38

Turbidity Methods

Answer 38

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

Question 39

Direct microscopic count (DMC)

Answer 39

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

Question 40

Petroglifos-Hauser Counting Chamber

Answer 40

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

Question 41

Viable plate count

Answer 41

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

Question 42

Serial dilutions

Answer 42

Look at video and slide 47 colonies * dilution factor S/S+D= dilution factor

Question 43

Environmental effects on Growth

Answer 43

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

Question 44

Steriliztion

Answer 44

Treatment that frees object of all living organism including endospore DEATH: irreversible loss of ability to reproduce VIABLE: means living and capable of reproduction

Question 45

Temperature effects

Answer 45

Temps can affect in opposing ways Temp rises, chem and enzyme reactions increase and growth becomes faster Above certain temp proteins become damaged

Question 46

Cardinal Temperatures

Answer 46

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

Question 47

Temperature levels

Answer 47

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)

Question 48

Danger zone for bacterial growth

Answer 48

15-50C, rapid growth and toxins produced

Question 49

Snow algae

Answer 49

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

Question 50

Hyperthermophiles

Answer 50

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*

Question 51

Acidity and pH

Answer 51

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

Question 52

Osmotic Pressure and Water activity (aw)

Answer 52

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

Question 53

Osmotic pressure limits

Answer 53

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

Question 54

Compatible Solutes

Answer 54

``` 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 ```

Question 55

Oxygen requirements

Answer 55

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

Question 56

Thioglycolate Tubes

Answer 56

Contains dye resazurin which is pink when oxidized, colorless when reduced LOOK at SLIDE 68 and 69 for picture! And 74

Question 57

Toxic form of Oxygen

Answer 57

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

Question 58

Enzyme to remove toxic by product

Answer 58

Catalase Peroxidase Superoxide dismutase (Aerotolerant Anaerobes) Superoxide dismutase/ catalase in combination (obligate and facultative aerobes) Superoxide reductase (obligate anaerobes) SLIDE 71-73

Question 59

Culture Conditions

Answer 59

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

Question 60

Nuclear Waste eating microbes

Answer 60

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)