BIOMI 2900 Quiz 3

after midterm material (lecture 20- )

requirments of life

liq water

carbon

nutrients

free energy

major atoms in microbes

CONHPS

C 50%

O(not O2) 20%

N 14%

H 8%

P 3%

S 1%

What evolutionary adaptation has the greatest potential to benefit a bacterium living in an environment with very low P?

Reduce ribosome number

Macronutrients

– Required by all cells: C, O, N, H, P, S

– Required by most cells: K, Na, Ca, Mg, Cl

Trace Elements

inorganic micronutrients, they are typically found in enzymes where they are found in Cofactors

– Include: Fe, Se, B, Cu, Mn, Mo, Zn, F, Si, As, Cd, Sr, Ba, V, Co, W, Ni

– Requirements vary from species to species

Growth Factors

organic micronutrients

– Include: vitamins, amino acids, purines, pyrimidines, etc

– Vitamins: often function as Coenzymes (non-protein molecule thatcontributes to enzyme function)

– Requirements vary from species to species

Culture media (plural = media, singular = medium)

– Nutrient solutions used to grow microbes in the laboratory

• Liquid or solid

– Solid media requires solidifying agent (e.g. agar)

• Two broad classes

– Defined media

– Complex media: composed of digests of chemically

undefined substances (e.g., yeast, meat or plant extracts)

Defined media:

precise chemical composition is known

Complex media:

composed of digests of chemically undefined substances (e.g., yeast, meat or plant extracts)

Composition of Defined Media

• C: can be an organic C source (for heterotrophic microbes) can be CO2 (for autotrophic microbes)

• N: NH4+, NO3-, N2, or organic N (such as protein)

• P: phosphate PO42- (or organic P)

• S: sulfate SO42- (or organic S)

• Other macronutrients: in the form of Inorganic salts

• Trace elements

• Growth Factors: typically vitamins

• pH buffer

Diagnostic Media: used to identify certain bacteria

-Selective Media: selects for target bacteria

(e.g. use of inhibitors, specific nutrients)

-Differential Media: have in indicator (e.g. a dye) that

changes color when it interacts with a target organism

Selective Media:

selects for target bacteria (e.g. use of inhibitors, specific nutrients)

Differential Media:

have in indicator (e.g. a dye) that changes color when it interacts with a target organism

Blood agar diagnostic media

differential media

• Identifies bacteria able to lyse blood cells

• Pattern of hemolysis is diagnostic

Bile Salts agar diagnostic media

Selective media

• Selects for bacteria resistant to bile salts

MacConkey agar diagnostic media

Selective & Differential

• targets enteric G- bacteria that ferment lactose

• Crystal violet and bile salts inhibit G+ bacteria (selective)

• Lactose is the only C-source (selective)

• pH indicator reveals acid production (differential)

Enrichment Culture

use a medium that provides a target microbe some selective advantage allowing it to be enriched in abundance over time (results in a mixture of bacteria where one type is common)

Direct Isolation:

use selective and/or differential agar to isolate a single colony (using streak or spread plate technique) – used to obtain a pure culture composed of a single strain

streak plate, direct isolation method

diluting to a single colony by streaking from previous streaks on the plate, sterilizing tool each time

spread plate - direct isolation method

spreading a liq with bacteria of an entire plate to get a spread of colonies

Microscopic Counts

counting bacteria on gridded plate

• Limitations of microscopic counts

• Laborious

• Likelihood of human error, visually cannot distinguish cells vs. random impurity

• can’t tell which ones are living or dead cells

Viable Counts (Plate Counts)

• Measure Colony Forming Units (CFU) (slight underestimate)

• Serial Dilution: Sample must be diluted to obtain a countable number of colonies

• Spread Plating: Cells are spread on a plate, plates are incubated, and then colonies are counted

Assumption: One cell, one colony (assumption violated for filamentous and ‘sticky’ cells)

number of bacteria after dilution?

plate count x dilution factor = 1.59 x 10^5

spectrophotometry

measure turbidity: light scatterying by cells

fast,easy, nondestructive

optical density

Growth Rate:

change in cell number over time

Growth Yield:

mass (or number)of cells relative to an input

Batch Culture:

a closed-system culture of fixed volume

Continuous Culture:

an open-system culture of fixed volume

Biofilm Culture:

a system that favors microbial growth on a surface

Growth Curve

Exponential Growth in Batch Culture

Lag phase:

Physiological changes to prepare for growth in new environment, length varies

Exponential phase:

Balanced exponential growth of all cells

Stationary phase:

Growth terminates when the environment no longer supports growth (e.g. nutrient limitation, pH, waste buildup), period of no net growth, physiological changes to prepare for starvation & dormancy

Death/Decline Phase:

Decline in cell number

how many cellsl in exp growth euqation:

N_t = N₀2ⁿ

n = number of generations

to solve for n:

n = (ln N_t - ln N₀) / 0.693

numbers of generations (n)

generation time = time per gen (t/n)

growth rate constant (k) equation

dN/dt = kN → k = (ln N_t - ln N₀) / t

dN - change in cells over t

k = grwoth rate constant

N = cell number

N_t = cell number at time t

N₀ - initial cell number

division rate

1/g

units for growth rate equations

g: doubling time

t: time

Nt: cells at time t

N0: initial cell number

n: generation number

k: growth rate constant

D: division rate

chemostat

a continuous culture device for growth bacteria

growth of continous culture

in chemostat:

growth rate is controlled by dilution rate

growth yield is controlled by the concentration of limiting nutrients

Aerobes interaction with oxygen

require O2 to grow (typically air, 20% O2)

Microaerophiles interaction with oxygen

grow only at low O2 (often 0.5 – 5%)

Facultative organisms interaction with oxygen

can live with or without O2

Aerotolerant anaerobes interaction with oxygen

can tolerate O2 and grow in its presence even though they cannot use it

Obligate anaerobes interaction with oxygen

killed by exposure to O2

Mesophiles

organisms that have midrange temperature optima; found in

• Microbes associated with warm-blooded animals

• Terrestrial and aquatic environments

• Temperate and tropical latitudes

Extremophiles

Organisms that require very hot or very cold conditions

Psychrophiles

Growth optima < 20°C

• inhabit permanently cold environments

• Life possible as long as there is liquid water (observed to -12°C)

Psychrotolerant

Growth optima > 20°C, but can grow at 0°C

• More common than true psychrophiles

Thermophiles

growth optima > 45°C

• Live in hot environments: composts, solar heated, geothermal

Molecular adaptations that support thermophily

• Changes protein structures for stability

• Changes membrane structure fore stability

• Saturated fatty acids increase membrane stability

• Isoprene lipids and lipid monolayers increase membrane stability in Archaea

peroxidase

H2O2 → H2O + O2

catalase

H2O2 → H2O

types of enzymes for detoxification

Water activity (aw) = water availability

• Water activity is related to solute concentration, water affiliating with solutes is not available to cells

• Solutes include all dissolved molecules (salts, sugars, small molecules, amino acids, etc)

• Water activity is measured as the ratio of the vapor pressure of air in equilibrium with a solution to the vapor pressure of pure water

Molecular adaptations related to pH

• change membrane structure for stability

• pH of the cytoplasm can change

• typically remains close to neutral, but can dip as low as 4.6 in extreme acidophiles, and as high as 9.5 in extreme alkaliphiles

• At very high pH there are few available protons (H+), so use Na+ motive force instead of a pmf

Hyperthermophiles

growth optima > 80°C

• Live in geothermal and hydrothermal environments

optima examples (note)

• No Eukarya can grow at > 65°C,

• No Bacteria can grow at > 95°C

• the theoretical limit for growth is ~150-180°C

Sterilization

complete removal of all microbes

Heat sterilization

routine use, requires >120°C

• Autoclave

Chemical sterilization

• formaldehyde bath

• Bleach bath

• -Ethylene oxide gas – sensitive medical devices

Radiation - sterilization

• Gamma rays/x-rays – medical/food industries

• UV light – for surfaces (low penetration)

Filtration

• 0.2μm removes bacteria (bacteria usually larger than .2)

• HEPA filters – for gases/air (High-Efficiency Particulate Air filter)

• Membrane filters – for solutions

what is an autoclave and what is it used for

sealed device that uses steam under pressure to sterilize items and solutions

• Pressure allows heating water to 121°C without boiling

• not pressure, but high temperature kills microbes

Pasteurization

process of using precisely controlled heat to reduce the microbial load in heat-sensitive liquids

• Does not sterilize (ultra-pasteurization comes close), so it is different from sterilization

heat killing (math)

function of temperature v time

Decimal Reduction Time (D) : time required for 10x reduction in cell number

Decimal Reduction Time (D)

time required for 10x reduction in cell number

Which of the following terms describes a chemical that prevents microbial growth but does not cause cells to die?

D. Bacteriostatic

Bacteriostatic

Inhibit growth, bind weakly to target

Bacteriocidal

Kill cells, bind strongly to or irreversibly damage target

Bacteriolytic

Lyses cells

• causes osmotic shock, attacks cell walls etc.

How to determine the chemical type of antimicrobial

red is microscope data, green is spread plating data

Sterilants

destroy all cells including endospores

• e.g. ethylene oxide, bleach bath, flame

Disinfectants/Sanitizers

kill most cells (including human cells), but not endospores, do not sterilize

• e.g. bleach on surfaces, alcohols, harsh detergents

Antiseptics (germicides)

kill many cells but safe enough to be used on living tissues (not injestion)

• e.g. mild detergents, Betadine (iodine)

antibiotic common traits

• Low toxicity to humans (few or no side effects)

• Target structures unique to bacteria

• Water soluble – need to disseminate in body

• Generally bacteriocidal or bacteriostatic

types of antimicrobials

Antibiotics- typically target bacteria

Antifungals- target fungi

Antivirals- target viruses

Most common targets for antibiotics (structures unique to bacteria),

• Bacterial Cell Wall Synthesis

• Bacterial Protein Synthesis

• Bacterial DNA Replication

• Folate Synthesis

• Bacterial Transcription

• Bacterial Cytoplasmic Membrane

beta-lactam

antibiotic that attacks transpeptidase (helps form peptidoglycan)

sealing

seal food more because of cross contamination, not for preventing bacterial growth

food preservation

Heat

• Hot Holding for cooked foods (>57 °C)

• Pasteurization (~65 °C)

• Canning (boiling)

• Ultrapasteurization (up to 135 °C)

Refrigeration and Freezing

– Bacterial growth slowed at 4 °C, but still grow (e.g. Listeria)

pH for food preservation

acidic pH tends to preserve foods

• Acidic additives: sorbic acid, benzoic acid, propionic acid

• Microbial acid production: lactic acid bacteria (yogurt, cheeses, sausages, saurkraut, pickles, etc...)

Water availability for food preservation

• Drying

• Freeze Drying

• Osmotic Pressure e.g. with salt or sugar

Chemical Preservatives

• Smoking (dries and deposits aldehydes, acids and phenols)

• Nitrites, nitrates

• Sulfites

• Etc . . .

irradiation

food preservative

free energy (G)

energy available to do work

ΔG°′

change in free energy at standard condition (°) and pH 7 (′)

exergonic

if ΔG°′ < 0

endergonic

if ΔG°′ > 0

OIL RIG

oxidation is lose electrons (ox state increase),

reduction is gain electrons (oxidation state is reduced)

Reduction potential (E0′):

– Measured in volts (V)

– We start with ‘half reactions’

– ‘Half reactions’ indicate

oxidized/reduced products

– Transfer of e- from more negative donor

to more positive acceptor is exergonic

– Redox couples are reversible

Redox tower:

O2 at the bottom, great electron acceptor and is beneficial to do so with coupled reactions

ΔG0′ = -nFΔE0’

ΔG0′ change in free energy (std cond, pH7)

n = number of electrons transferred

F = Faraday Constant (96.48 kJ/V)

E0’ change in reduction potential

(accepting couple) – (donor couple)

Glycolysis (aka: Embden–Meyerhof pathway)

• a common pathway for catabolism of glucose

• glucose is the electron donor

• anaerobic (no O2 required)

• used by chemoorganoheterotrophs capable of either fermentation, aerobic respiration, or anaerobic respiration

• ATP is generated by Substrate Level Phosphorylation

• Generates 2 ATP, 2 NADH, and 2 pyruvate (C3H3O3)

Problem:

- Fermentations do not have an external electron acceptor.

- During glycolysis you reduce NAD+ to NADH.

- The electrons need to be removed from NADH so that you can

regenerate NAD+ . If you run out of NAD+ then glycolysis will stop.

Hence, YOU NEED AN ELECTRON ACCEPTOR to get rid of the

excess electrons. If there is no external electron acceptor what can

you do. . .

Solution:

- Put the e- from NADH back onto a breakdown product of the original substrate

2) Fermentation

2 Pyruvate 2 Lactate + 2 H+ 2 NADH 2 NAD+

e.g. Homofermentative lactic acid bacteria

2 Pyruvate 2 Ethanol + CO2

2 NADH 2 NAD+

e.g. Yeast

Citric acid Cycle (a.k.a Krebs Cycle)

• A central and highly conserved

metabolic cycle

• Required by many anabolic processes

as intermediates can be siphoned off

and used in biosynthesis

• Required for catabolism by many

heterotrophs

• Pyruvate (C3) enters

• Generates:

3 CO2, 1 ATP, 1 FADH2, 4NADH

Electron transport in Oxidative Phosphorylation (used in Respiration)

- System of integral membrane proteins that perform redox reactions which oxidize electron donors, shuttle electrons along the membrane, generate a Proton Motive Force, and ultimately reduce a terminal electron acceptor

Electron transport in aerobic respiration

1: electrons enter the chain from primary electron donor: NADH → NAD+

2: H+ pumped out (pmf) and 1e- flows through chain

• flavin takes e-, H+ out → quinone takes both H+ and e- → Iron picks up e-, H+ out

3: electron exits by reducing terminal electron accepter: O2 → H2O