Microbiology

Question 1

Importance of microbial life

Answer 1

Useful for gaining knowledge of biochemistry, cell growth, genetics, molecular mechanisms of life
A lot of model organisms are microorganisms e.g. E.coli
Impacts our everyday life e.g. gut micro biome critical for health as it impacts immune system and allows digestion
Roles in medicine, food and drink industry, production of antibiotics, agriculture (soil micro biome)

Question 2

History of microbiology

Answer 2

2 domain tree of life, bacteria is one, eukarya comes off archea
1822-1895 Louis Pasteur demonstrated sterilisation, defeated idea of spontaneous generation
If flask got in contact with air after being sterilised, bacteria grew
Further developed germ theory of disease
Proposed principles of fermentation
Introduced sterilisation techniques e.g. steam steriliser
Developed pasteurisation of milk and wine
Developed attenuated vaccines for chicken cholera, ovine anthrax, rabies

Question 3

Koch’s Postulates

Answer 3

Robert Koch
1843-1910
Developed postulates for proving that a specific microorganism causes a specific disease
Postulate 1: suspected pathogenic organism should be present in all cases of the disease and absent from healthy animals
Postulate2: the suspected organism should be grown in pure culture
Postulate 3: cells from pure culture should cause disease in a healthy animal
Postulate 4: the organism should be re isolated and shown to be the same as the original

Question 4

Properties of some bacterial pathogens

Answer 4

Single colonies of bacteria can be grown on agar surfaces
A culture derived from a single colony is a pure culture
All cells within a single colony are genetically identical
Underline if writing genus and species by hand, if not then in italics

Question 5

Medically important gram +ve cocci

Answer 5

Staphylococcus aureus
Groups of cocci
Causes skin infections, respiratory infections, toxic shock syndrome
MRSA
Produces toxins and enzymes e.g. alpha-toxin which lyses host cells, enterotoxin which induces vomiting and diarrhoea, toxic shock toxin that causes systemic shock and organ failure
Streptococcus progenes
Chains of cocci
Causes septic sore throat, scarlet fever, autoimmune disease
Produces toxins that lyse host cells, dissolve fibrin clots, dissolve hylauronic acid in connective tissue and cause scarlet fever rash

Question 6

Medically important gram +ve rod shaped bacteria

Answer 6

Bacillus subtilis
Non pathogenic soil bacterium
Model gram +ve bacteria
Bacillus anthracis
Causes anthrax
Pathogenic due to acquisition of cetertain genes, including ones for anthrax toxin

Question 7

Medically important gram -ve cocci

Answer 7

Neisseria gonorrhoeae
Causes gonorrhoea
Is a diplococcus

Question 8

Medically important gram -ve rod shaped

Answer 8

Escherichia coli
Normal inhabitant of gut
Some strains can cause diarrhoea
Express certain genes that cause virulence
Intimate adherence to gut
Transfer of proteins from bacterium to host cell

Question 9

Energy and nutrition requirements

Answer 9

Major- C,H,O,N
Minor- P,S,K,Mg,Ca,Na
Micronutrients- Fe and other metals (too much is toxic)
70-80% of wet weight is H2O

Question 10

Outline of bacterial growth cycle

Answer 10

DNA replication
cell elongation
Septum formation, initiation of division
Completion of septum formation with distinct walls as septum constricts
Cell separation
Cell division is initiated by synthesis and localisation of FtsZ at the mid cell
FtsZ is a tubulin homologue
DivIVA and MinCDJ build up at poles to stop FtsZ polymerising
NOC binds to DNA around origin and binds to membrane to stop FtsZ forming
FtsZ can only polymerise at mid cell, giving 2 identical daughter cells

Question 11

Methods of measurement of growth

Answer 11

Cell grow exponentially
N=N02^n
N = number of generations
G=t/n
G = generation time

Question 12

Problems with optical density

Answer 12

Doesnt differentiate between live and dead cells, assumes bacteria are same size, can’t compare between different spectrophotometers

Question 13

Viable cell count

Answer 13

Samples of culture taken and plated on agar
Assumption that each viable cell will give rise to one colony
Serial dilutions used to give appropriate number of colonies to count

Question 14

Lag phase

Answer 14

Occurs as cells adapt to new environment
Inoculum usually depleted of certain nutrients
Time is required for re synthesis
Some cells in inoculum may be dead
Time of lag phase varies greatly

Question 15

Exponential phase

Answer 15

Rate of increase of cell numbers constantly rises
Cell numbers increase at same rate as cell constituents
Growth rate of cultures vary and can depend on temp, nutrients, pH and genetic factors
Doubling times can range from 20 mins to many hours
Exponential growth phase still limited by nutrients

Question 16

Stationary phase

Answer 16

Exponential phase can’t carry on indefinitely
Growth limited by lack of nutrients or build up of toxic waste products to an inhibitory level
Cells not dead, no net increase or decrease in cell numbers
Certain ends necessary for survival in stationary phase
Sporulation of certain species
Use of accumulated storage products

Question 17

Effect of temperature on growth processes

Answer 17

Incorrect temp= no growth
Increasing T: rates of chemical and enzyme reaction rates rise, proteins denature at high T, membranes destabilise
Decreasing T: membranes tend to gel, transport through membranes becomes limiting, enzymes become very inflexible

Question 18

Simple stain

Answer 18

Spread culture in thin film over slide
Dry in air
Pass slide through flame to fix
Flood slide with stain, rinse and dry
Place drop of oil on slide, examine with 100x objective
Example stains: safranin

Question 19

Gram stain:

Answer 19

Flood heat-fixed smear with crystal voilet for 1 min
All cells purple
Add iodine solution for 3 min, all cells still purple
Iodine and crystal voilet form insoluble complex on cell walls, complex is larger than the original crystal violet stain
Decolorise with alcohol for 20 seconds, dehydrates peptidoglycan layer, shrinking and tightening it
Gram +ve cells purple, large complex trapped in tighter peptidoglycan layer
Gram -ve colourless, outer membrane degraded, thinner peptidoglycan layer unable to retain complex
Counterstain with safranin for 1-2 min
G+ purple, G- pink

Question 20

Structure of bacterial cell envelope

Answer 20

Mainly composed of phospholipids
G+ has large peptidoglycan cell wall
Gram- has thin peptidoglycan layer between membranes, 2 bilayers
Bilayer contains alpha helical transmembrane proteins, usually 20aa
Peptidoglycan layer forms rigid layer outside cytoplasmic membrane
Composed of 2 sugar derivatives and small number of amino acids

Question 21

Structure of peptidoglycan

Answer 21

2 sugar derivatives, small number of amino acids N-acetyl glucosamine (GlcNac) and N-acetyl muramic acid (MurNac) connected to form repeating Glycan chain
Thin sheet where glycan chains are connected by peptide cross links formed by amino acids
Inter bridge can be direct or contain amino acids ( never branched, aromatic or sulphur containing)
Strength provided by glycan bonds reinforced by peptide bonds
Peptidoglycan has beta 1-4 linkage
Not found in archea
G+ has Gly inter bridge

Question 22

G+ cell envelope

Answer 22

Contains Teichoic acid
Polymers containing glycerophosphate or ribitol phosphate
If Covalently bound to membrane lipids they are lipoteichoic acids
Have structural role
Involved in localisation assembly and activation of cell wall elongation and division machinery
Attracts anions
Indirectly confers resistance to antibiotics

Question 23

G- cell envelope

Answer 23

Asymmetrical bilayer
Outer membrane not permeable to high molecular weight molecules , stops periplasmic enzymes leaking out of cell
Permeable to low molecular weight molecules
Permeability due to porins, transmembrane proteins, trimeric structure forming water filled channels
Permeability can be specific or non-specific
Beta barrels

Question 24

Structure of lipopolysaccharide of G-

Answer 24

In outer membrane
O-specific polysaccharide
Varies greatly between species
Can be very long due to repetition
Repetition makes it a good drug target, specifies antibody recognition and phage recognition
Lipid A forms outer leaflet of bilayer, reposnsible for toxicity of LPS ‘endotoxin’, causes fever and shock in animals and man

Question 25

Structure of flagella

Answer 25

Long, thin and can be polar (at one or both poles) or peritrichous (all around cell surface) or lopotrichous (like tufts of hair)
Structure conserved between prokaryotes
Helical, has a wavelength
Composed of flagellin subunits
Hook- single protein, connects filament to motor
Motor anchored in cell wall and cytoplasmic membrane
Motor proteins drive flagellar motion causing rotation

Question 26

Assembly of flagellum

Answer 26

Sequential assembly
Subunits internally exported through rod and hook and added on to end
Cytoplasmic component has ATPase complex that uses ATP to export subunits

Question 27

Type of motility caused by flagellum

Answer 27

Only occurs in semisolid or liquid
Swimming motility- movement in liquid or low viscosity conditions

Question 28

Bacterial flagellum directional movement

Answer 28

Bacteria can display chemotaxis- move towards and attractant
Capillary tube assay used to measure
Substance inside tube
Control has same substance as bacteria are already in
Cells move into tube if attractant present, away from repellant, same distribution as outside for control
Bacteria too small to detect conc gradient along body
Compare state of immediate environment before and after a time period
Respond to temporal gradient not spatial
Responds to changes in conc not absolute conc of a stimulus
Bacteria use temporal changes in conc to control flagellar rotation
Membrane proteins sense presence of attractants, activate a sensor kinase that phosphorylates response regulator
Triggers cascade that affects flagellar motors

Question 29

Bacterial movement when not chemotaxis

Answer 29

Random movement of tumbles and runs
After a tumble the direction of the next run is random
If attractant detected, runs longer and tumbles less frequent
Flagella rotate counter-clockwise during run
Clockwise stimulates tumble

Question 30

Twitching motility

Answer 30

Pilli
Longer than Fimbriae, fewer per cell, can have several functions
Act as bridge between bacteria during mating
Receptors for certain bacterial viruses
Adherence of pathogens to host tissues
Type IV pilli are multifunctional protein fibres produced on surfaces of a wide variety of bacteria and archea
Assembly and retraction of pilli allows movement
Allows twitching motility
Occurs on solid agar, bacteria move on surface
Type IV pilli and flagella also involved in swarming

Question 31

Fimbriae

Answer 31

Shorter than flagella but many more per cell
Fimbriae can be involved in adherence of bacteria to surfaces e.g. e.coli and salmonella to host cells
Donor strand complementation - donate on subunit to next molecule to be polymerised, links them strongly
Mediate microbial attachment, an early step in establishment of an infection, by binding specifically to sugars present in host tissues