microbiologist

Question 1

The discovery of microorganisms

Answer 1

robert koch-shows microorganisms(bacteria) cause disease

Question 2

Modern methods of studying microorganisms

Answer 2

Growing microorganisms-Only tiny fraction of microorganisms we have discovered can be cultivated in the lab

culture media:
Need to grow microorganisms in a nutrient solution (culture/media/broth)
Requires careful preparation
Chose the right recipe for your microbe
Keep sterile
Plates-Can be solidified with agar to make plate-allows you to pick single colonies
Slopes-Tubes containing solid agar set in a slope
Used for pure growth of a microorganism
Liquid culture-blood culture
Sterility test
Continuous cultures

light microscopy
imaging cells in 3D
electron microscopy(TEM/SEM)

Question 3

Types of microorganisms

Answer 3

bacteria:
Protobacteria-largest phylum of bacteria, e.coli
Can be gram-positive p/gram-negative bacteria
Cyanobacteria-photosynthetic

archaea:
Unique properties, separating them from bacteria
Only two phyla – the Euryarchaeota and the Crenarchaeota
Classification is difficult as the majority have not been isolated in the laboratory
Usually look similar to bacteria, but often have genes and metabolic pathways more similar to eukaryotes

protozoa:
Unicellular eukaryotes
Live in soil, wet sand, fresh and salt waters
Great diversity in shape, mobility and metabolism

algae:
Eukaryotes
Contain chloroplasts
Have cell walls
Both terrestrial and aquatic

Question 4

cell size

Answer 4

Size range for prokaryotes: 0.2 μm to >700 μm in diameter
Size range for eukaryotic cells: 10 μm to >200 μm in diameter

advantages:
Small cells have a higher surface area to volume ratio than large cells:
Faster nutrient exchange per unit cell volume
Therefore grow faster
Support a larger population
Faster evolution:
DNA is replicated as cells divide
During replication, mutations occur
Higher rate of cell division -> higher rate of mutation within population
Mutations are ‘raw material’ for evolution
Allows rapid adaptation to changing environments

disadvantages:
You’ve still got to fit everything in!
*A cell 0.15 μm diameter would only just fit in all the cellular components
*Anything you see down a microscope less than 0.1 μm is unlikely to be a cell

Question 5

Endospores

Answer 5

Highly differentiated cells
Produced by certain species of bacteria
Highly resistant to heat, harsh chemicals and radiation
Survival structures – like a nuclear bunker

endospore structure:
Strongly refractive and impermeable to most dyes
Usually seen as unstained regions within cells

endospore morphology:
terminal endospores
subterminal endospores
central endospores

Question 6

sporulation

Answer 6

An essential nutrient is exhausted e.g. carbon or nitrogen
Vegetative cell stops growing
Endospore develops within vegetative cell and is released
Spore can remain dormant for years
‘Germinates’ into a vegetative cell when conditions are good

Question 7

fimbriae and pili

Answer 7

Filamentous structures composed of protein extending from surface of cell

fimbriae:
Enable cells to stick to surface and each other
Instances where fimbriae assist the disease process:

*Salmonella species (Salmonellosis)
*Neisseria gonorrhoeae (gonorrhoea)
*Bordetella pertussis (whooping chough)

pili:
*Similar to fimbriae, but typically longer & only one or two present
*Best seen under electron microscope when coated with virus particles
Functions of Pili
Two major functions:
1.Conjugation - genetic exchange between cells
2.Adhesion of pathogens to specific host tissues and subsequent invasion
*Can also be involved in mobility

Question 8

flagellum

Answer 8

The flagellum (plural, flagella) rotate to push or pull cell through a liquid
Gram-positive and gram-negative bacteria
Can only be seen with light microscopy after being stained

attachment points:
Polar flagellation – flagella are attached at one or both ends (b & c)
A tuft – a group of flagella attached to one end of the cell (c)
Peritrichous flagellation – flagella inserted at many locations (a)

flagella structure:
Flagella are helical
The wavelength (distance between curves) is characteristic for given species
Filament is composed of many copies of a protein called flagellin
Molecular motor that drives rotation of flagellin filament is embedded in cell membrane
Motor:
Central rod
Passes through a series of rings
Mot proteins – act as stators
The rod and rings rotate while the mot proteins stay still

Question 9

Gliding

Answer 9

Considerably slower than swimming with flagella
Cells must be in contact with a solid surface to glide
Colonies of gliding bacteriahave distinct morphologies

Gliding Mechanism:
Not thoroughly understood
More than one mechanism is responsible
*Polysaccharide slime:
*Connects cell surface with solid surface
*As slime adheres to surface, the cell is pulled along
*‘Twitching motility’:
*Repeated extension and retraction of type IV pili

Question 10

Taxis

Answer 10

Most microbial cells can move under their own power
Enables cells to reach different parts of their environment
Taxis – movement towards something that will aid growth or away from toxins

microbial taxis:
Chemotaxis – response to chemicals
Phototaxis – response to light
Evolutionary advantage to moving to a better growth environment

Question 11

Multicellular structures

Answer 11

The myxobacteria
Form multicellular structure– fruiting bodies
Life cycles indicate intercellular communication

Question 12

Carbon sources used for metabolism

Question 13

Autotroph vs heterotroph

Answer 13

Autotroph:
Use CO2 as their carbon source
Primary producers
Synthesise new organic matter

Heterotroph:
Use organic compounds as their carbon source
Either feed directly on other cells
Or live off products other organisms excrete

Question 14

heterotrophic relationships

Answer 14

Using other organisms as a substrate:
Symbiotic, mutualistic
Cooperative relationship with the host
Parasitic
Antagonistic relationship with the host
Saprotrophic
The host is dead

Question 15

Energy sources used for metabolism- using chemicals(chemotrophy)->organic chemicals=chemoorganotrophs

Answer 15

Thousands of different organic chemicals (carbon containing) available
Oxidation of organic compounds releases energy, stored as ATP
Can be aerobic or anaerobic or both!

Question 16

Energy sources used for metabolism- using chemicals(chemotrophy)->inorganic chemicals=chemolithotrophs

Answer 16

Oxidation of inorganic compounds releases energy, stored as ATP
Only occurs in prokaryotes
Several inorganic compounds can be oxidised
*E.g. H2, H2S (hydrogen sulphide), NH3 (ammonia)
A related group of chemolithotrophs specialises in oxidation of a related group of inorganic compounds
Sulphur bacteria
Iron bacteria

as a good metabolic strategy:
Competition from chemoorganotrophs is not an issue
Many of the inorganic compounds used by Chemolithotrophs are waste products of Chemoorganotrophs
It’s common for species from these two groups to live in close association

Question 17

energy sources used for metabolism->using light(phototrophy)->Phototrophs

Answer 17

2 types:
anoxygenic, oxygenic

photosynthesis:
conversion of light to chemical energy
*Organisms that perform photosynthesis are phototrophs
*Most are also autotrophs (use CO2 to make organic compounds)

Question 18

Nitrogen fixation and nitrification

Answer 18

Some bacteria can ‘fix nitrogen’ – convert atmospheric nitrogen gas into a form that can be used by cells

No known eukaryotes can fix nitrogen

Not all bacteria can fix nitrogen
Two types:
Free-living—>require no host, they live free!
Symbiotic—>Can only exist in association with certain plants
—>Live in root nodules

Question 19

nitrogenase and nitrifying bacteria

Answer 19

Nitrogenase catalyses the following reaction:

A complex of two distinct proteins:
Dinitrogenase – contains iron and molybdenum
Dinitrogenase reductase – contains iron

Nitrification – oxidation of inorganic nitrogen compounds
Nitrifying bacteria are widely distributed in soils and water
Two groups of organisms,
each performing a different oxidation reaction:

Question 20

Cell growth

Answer 20

Multicellular organisms: Growth involves the whole organism getting bigger

Single cells organisms: Growth is defined as an increase number of cells in a population

Question 21

Binary fission

Answer 21

One cell divides into two
Prokaryotes and some Eukaryotes
All bacteria

Generation time – the time required for this process to happen

Highly variable between species
Also variable within species:
Depends on nutritional and environmental factors such as temperature
E. coli in a laboratory culture is about 20 min

Question 22

Population growth

Answer 22

Bacterial growth is an increase in the number of cells in a population
So the dynamics of population growth is what’s measured in a laboratory experiment – not growth of individuals

Question 23

Exponential growth + logarithmic paper

Answer 23

2 different ways of plotting an exponential:
On Y axis-Arithmetic(units of 1)
and logarithmic (power of 10)

semi logarithmic paper:
Can be used to estimate generation time
g =generation time
t=time g=t/n
n= number of generations

Question 24

Growth cycle

Answer 24

lag:
Time between when culture is inoculated into fresh media and significant growth
Length varies – depends on history of the inoculum, nature of the medium and growth conditions

log/exponential:
Cell population doubles at regular intervals
Depends on availability of environmental conditions (temperature, nutrients etc) and genetic characteristic of the organism
The healthiest cell state

stationary:
Essential nutrient in culture medium runs out
Organism’s waste products build up to toxic levels
No net increase or decrease in cell numbers
Growth rate = 0
Cell growth = cell death

death:
Exponential decline of viable cells
*Rate of cell death typically faster than rate of growth
*Viable cells may remain in culture for months or even years

Question 25

Growth in practical microbiology

Question 26

Batch cultures

Question 27

How to measure growth-microscopic counts, viable counts, spectrophotometry

Answer 27

microscopic counts:
Count the number of cells present
Samples dried onto slides or liquid samples
-can be stained to increase contrast between cells and their background
issues:
Without special staining techniques, dead and live cells can’t be distinguished
Imprecise
Small cells difficult to see under the microscope
Motile cells must be killed/immobilised – debris from sample may be mistaken for cells

viable counts:
Viable cell – able to divide and produce offspring
These are usually the cells we’re most interested in
AKA a plate count
Main assumption – each viable cell will divide to form one colony

spectrophotometry:
Cells scatter light
Turbidity can be used to estimate cell mass in a sample
More light scattering = more cell mass = more cells

Question 28

microbial ecology

Answer 28

*Like macroorganisms (animals and plants), microbes have an ecology:

A given species lives in certain places but not others
Environments differ in their abilities to support diverse microbial populations

Question 29

microbes in ecosystems-microenvironment and diffusion importance

Answer 29

*Great metabolic diversity
*Primary catalysts of nutrient cycles
*Very important members of the ecosystem

microenvironment:
Microorganisms are very small so only directly experience a tiny local environment
*For a typical 3 μm bacterium, a distance of 3 mm is like 2 km for a human
*Metabolic activities from nearby microorganisms can alter the conditions
*Numerous micro environments can exist within a given habitat

diffusion:
*If you are small, diffusion often determines the availability of resources

*Use microelectrodes to measure oxygen concentration in a soil particle

*Many microenvironments within very short distance
*Microorganisms near outer edges consume all oxygen before it can diffuse to the centre

*Anaerobic organisms thrive near centre

*Aerobic organisms live in outer layers

Question 30

Habitats : Terrestrial and Aquatic

Answer 30

*Pathogenic/symbiotic associations with plants/animals/humans/each other

Terrestrial
*Soils
*Subsurface

Aquatic
*Freshwater
*Coastal/ocean
*Deep sea

Question 31

soil environment-microbial growth, water and nutrient availability

Answer 31

microbial growth
*Most extensive microbial growth takes places on surfaces of soil particles

*Highly promoted in the rhizosphere – roots exude nutrients which microbes can absorb

water availability:
*Water content of soil highly variable
*Depends on soil composition, rainfall, drainage and plant cover
*Water has minerals dissolved in it = ‘soil solution’
*Water content also affect oxygen levels, waterlogged = low oxygen

nutrient availability:
*Greatest microbial activity in organic-rich soil surface layers
*Especially around rhizosphere
*Numbers and activities of microbes greatly depends on type and amount of nutrient present

Question 32

soil environment-the subsurface

Answer 32

*Groundwater - Water in soils and rocks deep underground

*A little explored microbial habitat

*Microbial life extends down at least 3 Km into the Earth

*May account for as much as 40% of global biomass

deep level microbes:
*Chemolithotrophic and autotrophic bacteria, and archaea found at 3 Km deep in South Africa

*Must survive on a very nutrient poor diet

*Probably use H2 as the electron donor for respiration

highly variable conditions:
*Cell numbers in groundwater vary by several orders of magnitude (10^2 -10^8 per ml)

*Due to nutrient availability – especially dissolved organic carbon

*Generation times vary from days to centuries

Question 33

aquatic habitats

Answer 33

Freshwater vs marine differ in many ways:
*Salinity
*Average temperature
*Depth
*Nutrient content

freshwater:
*Highly variable in resources and conditions
*Both oxygen consuming and oxygen producing organisms present
*The balance controls the cycle of nutrients

oxygenic phototrophs:
Include algae and cyanobacteria
*Primary producers-Energy comes from light
*Planktonic-Floating
*Benthic-Attached to bottom or sides of lake/stream

habitats changes with depth

Question 34

coastal/ocean water

Answer 34

Very low nutrient levels, especially nitrogen, phosphorus and iron
*Water temperatures are cooler & are more constant with seasons than freshwater
*Overall microbial numbers are lower in marine compared to freshwater habitats (106 /ml vs 107 /ml)

microbes in the ocean:
*Very small cells
*Typical characteristic of living in nutrient-poor environment
*Requires less energy for cell maintenance
*Require greater number of transport enzymes relative to cell volume to acquire nutrients from very dilute environment
*Oxygenic photosynthesis in the oceans is a major factor in controlling the Earth’s carbon balance
*so important because the oceans are so large!

growth with depth:
*Bacteria predominate in waters above 1000 m
*Photic zone = where light can penetrate to
*Oceans contain the largest microbial biomass on the surface of the Earth

hydrothermal vents:
*Underwater hot volcanic springs
*Found 1000 m to greater than 4000 m deep

Question 35

Abiotic growth factors

Answer 35

Organisms must be finely tuned to their conditions
1.Nutrient availability
2.Temperature
3.pH
4.Moisture
5.Oxygen
6.Pressure
7.Light

Extremophile- An organism whose growth is dependent on extremes of temperature, salinity, pH, pressure, or radiation, which are generally inhospitable to most forms of life

Question 36

temperature - abiotic growth factor

Answer 36

*Arguably the most important factor affecting growth

*All species have:
Minimum temperature – growth isn’t possible below
Optimum temperature – growth is most rapid
Maximum temperature – growth isn’t possible above
*these are cardinal temps. which are different for every species

Question 37

Temperature tolerance cold

Answer 37

psychrophile
*Optimal growth temperature 15°C or lower
*Max growth temperature 20°C
*Killed by warming, found in constantly cold environments
*Psychromonas – sea ice bacterium – grows at -12°C – lowest known
*But probably can go lower
*Some enzymes found to function at -20°C

Question 38

molecular adaptations to the cold

Answer 38

*Enzymes have optimal activities at low temperatures
*Molecular basis not fully understood

Primary structure:
More polar amino acids
Fewer weak bonds

Secondary structure:
Greater α–helix, less β–pleated sheet
Gives protein greater flexibility (β–pleated sheets are quite rigid)

*Cytoplasmic membranes must remain functional
High content of unsaturated and shorter-chain fatty acids
Helps membrane remain in semifluid state at low temperatures

“Cold-shock” proteins:
*Maintain other proteins’ activity and bind specific mRNAs to facilitate their translation
*Not limited to psychrophiles – e.g. found in E. coli
Cryoprotectants:
*Solutes (e.g. glycerol or sugars) that help prevent the formation of ice-crystals in the cell

Question 39

Temperature tolerance hot

Answer 39

thermophile
*Growth temperature optimum greater than 45°C
*Less extreme than the hyperthermophiles
*Found in a wide range of habitats:
*Edges of hot springs
*Soil surfaces
*Fermenting environments
*Artificial environments e.g. hot water heaters

hyperthermophiles:
Growth temperature optimum greater than 80°C

Found in hot springs

Only prokaryotes

Growth rates often quite high – generation times as short as 1hr have been recorded

The most heat-tolerant example known is Methanopyrus – can grow at 122°C

Question 40

molecular adaptations to high temps.

Answer 40

Heat-stable enzymes and proteins
Increased DNA stability
Heat-stable membranes

hyperthermophile membranes
Do not contain fatty acids
Have C40 hydrocarbons (repeating units of isoprene) bonded to glycerol phosphate by ether (rather than ester) link
Forms a monolayer

Question 41

Tolerance to extremes of pH - acidophiles

Answer 41

Grow best at pH 5.5 or below

Different classes optimised to different pHs

Those with a pH optima of below 1 are very rare

Most cannot grow at pH 7

Question 42

Tolerance to extremes of pH - alkaliphiles

Answer 42

Grow best at pH 8 or above

A few extremophiles have a very high pH optima – as high as pH 11

Found in environments such as soda lakes and high-carbonate soils

Question 43

cytoplasmic pH

Answer 43

MUST REMAIN NEAR NEUTRALITY
Optimal pH for growth refers to extracellular environment only
Intracellular pH must stay near pH 7 to prevent destruction of macromolecules

Question 44

Salt tolerance

Answer 44

Halophiles - Require NaCl for growth

Halotolerant – can tolerate NaCl, but grow best in absence of solute