1
Q
Robert Hooke
A
First description microbes
Fruiting structure of moulds
2
Q
Antoni van Leeuwenhoek
A
First description bacteria
3
Q
Louis Pasteur
A
Bacteria --> fermentation Food sterilising Disprove spontaneous generation Develop methods to control growth Vaccines
4
Q
Robert Koch
A
Microbes –> infectious diseases
Koch’s postulates
Develop techniques to culture microbes
5
Q
Koch’s Postulates
A
- X needs to be present in every case of Y
- X must be grown in pure culture
- Cells from a pure culture of X must cause disease in a healthy animal
- X must be reisolated and shown to be the same as the original
6
Q
All bacterial cells
A
Metabolism
Evolution
Growth
7
Q
Some bacterial cells
A
Differentiation
Communication
Genetic exchange
Motility
8
Q
Bacterial cytoplasmic membrane
A
fatty acids joined to glycerol via ester linkages
9
Q
Archaeal cytoplasmic membrane
A
isoprene units joined to glycerol via ether linkages
10
Q
Peptidoglycan
A
peptide bonds (amino acids) and glycosidic bonds (sugars):
- N-acetylglucosamine (G)
- N-acetylmuramic acid (M)
11
Q
Gram + Cell Wall
A
-90% peptidoglycan Teichoic acids Lipoteichoic acids -Peptidoglycan = outer layer -Crosslinking through formation of peptide interbridge
12
Q
Gram - Peptidoglycan crosslinking
A
- 10% peptidoglycan
- Sandwiched
- OM has LPS layer
- LPS: core polysaccharide. O-polysaccharide, lipid A (endotoxin)
- [LPS] = patient outcome
- Crosslinking through NH2 group of DAP of one glycan chain to COOH group of D-alanine on adjacent chain
13
Q
Capsules
A
Polysaccharide Attachment (biofilm) Evasion of immune system Looks wet \+ and -
14
Q
Periplasm
A
- Space between cytoplasmic and OM
- Gel-like
- Contain proteins
15
Q
Gram stain
A
- Crystal violet + peptidoglycan (+) –> purple
- Crystal violet + less peptidoglycan (-) –> clear
- Clear (-) + counterstain –> pink
\+ = purple - = pink
16
Q
Archaea cell walls
A
S layer Interlocking proteins and glycoproteins No OM or peptidoglycan Some have pseudomurein - N-acetylglucosamine (G) - N-acetyltalosaminuronic acid (M)
17
Q
Fimbriae
A
Filamentous, linear projections
Adhesion
Multiple types
Some with adhesive domains along shaft that anchor cell by ‘zippering effect’
18
Q
Bacterial Flagella
A
- Rotatable filamentous bacterial surface appendages involved in bacterial locomotion
- Different arrangements:
- Peritrichous (Starfish)
- Polar (Sperm)
- Lophotrichous (Jellyfish)
- Helical in shape, composed of flagellin
- Hook: single type of protein, connects filament to motor at base
- Motor (Mot proteins): anchored in the membrane and cell wall, drives rotation of flagella
- Fli proteins: motor switch, reversing direction of rotation in response to IC signals
- Around 50 genes: structural, chaperone, regulatory proteins
- Flagellin molecules synthesised in cytoplasm, move up through hollow core in filament
- 20,000 flagellin –> one filament
19
Q
Archaeal Flagella
A
Most
Several different flagellin proteins
Amino acid sequence of archaeal flagellins is not related to bacterial flagellins
20
Q
Gliding motility
A
Flagella-independent
Surface contact, slower
- Excretion of polysaccharide slime (cyanobacteria)
- Type IV pili, twitching motility by repeated extension and retraction (Myxococcus xanthus)
- Gliding-specific membrane proteins (Flavebacterium johnsoniae)
21
Q
Cell motion as a behavioural response
Chemotaxis Phototaxis Aerotaxis Osmotaxis Hydrotaxis
A
- Taxis: directed movement in response to gradients
- Chemotaxis: chemicals
- Phototaxis: light
- Aerotaxis: oxygen
- Osmotaxis: osmolarity
- Hydrotaxis: water
22
Q
Run and tumble
A
Chemoreceptors –> chemical concentration
More attractant –> more directed –> less tumbling, more running
23
Q
Phosphate
A
- Some can accumulate inorganic phosphate PO43- for nucleic acid, phospholipid, ATP synthesis
- Accumulated in P-rich environments, used in limiting environments
24
Q
Sulphur
A
- Some can oxidise H2S to produce energy in fixation of CO2
- Elemental sulphur S stored in sulphur globules in the periplasm
25
Magnetosomes
- Intracellular particles of magnetite (Fe3O4)
- Enable bacteria to orient themselves in a specific arrangement within a magnetic field
- Magnetosomes in aquatic bacteria --> orientation in water column
26
Gas Vesicles
- Confer buoyancy in planktonic cells
- Spindle-shaped gas-filled structures made of protein
- Gas vesicle membrane impermeable to water
- Allows photosynthetic Bacteria to optimise position in water column
27
Endospores
- Highly differentiated cells that is resistant to harsh conditions/environments
- "Dormant" stage of bacterial life cycle
- Ideal for dispersal via wind, water or animal gut (like a seed)
- Only present in some gram-positive bacteria
28
Microbial growth
Increase in number of cells
29
Binary fission
Cell elongation
Septum formation
Cell separation
30
Growth rate
Generation time
Batch culture
Growth curve
```
Growth rate- change in cell number/mass per unit time
Generation time (doubling time)- interval for formation of two cells
Batch culture- closed-system microbial culture of fixed volume
Growth curve- growth as a function of time
```
31
Growth phases in batch culture
Lag
Exponential
Stationary
Death
32
Lag phase
- Cells adapted to stationary phase transferred to fresh medium
- Cells transferred from rich to minimal medium
- In both cases induction of new enzymes is required
33
Exponential Phase
- Number of cells doubles during a constant time interval
- Increase initially slow but increases at an ever faster rate
- Time taken for all components of the cell to double is the same (balanced growth)
- Growth is unrestricted (excess nutrients, no toxic products)
34
Stationary Phase
- 1 limiting nutrient or accumulation of inhibitory products
- Some metabolic activity slow down
- E. coli cells get smaller and contain more glycogen
- Adapt by activation expression of certain genes
- RNA polymerase composed of core enzyme and sigma factor (holoenzyme)
- Holoenzyme recognises promoter and initiates transcription
- After initiation of transcription, sigma factor dissociates
- RpoD is major sigma factor in bacteria
- RpoD directs transcription of most genes in the cell
- RpoD is active in exponential and stationary phase
- RpoS directs RNA polymerase to transcribe genes involved in stationary phase adaptation
- RpoS binds to distinct promoter consensus sequence
- Majority of transcription still depends on RpoD
35
Death phase
-Cells begin to die (cell lysis)
36
Total cell count
- Direct counting of cells under microscope in a chamber of known volume
- Rapid estimate of cell number
- Can't distinguish live and dead cells
- Difficult to see under microscope
- Precision difficult to achieve
- Phase contrast required when not stained
- Suitable for density greater than 106 per ml (accuracy)
37
Viable Count
- Spread plate method
- Assumption that each viable cell with produce a single colony
- 10-fold dilutions to make sure a suitable number is plated
- Number of colonies may depend on conditions
- Small colonies could be overlooked
- Replicate plates (accuracy)
- Cell clumping could reduce counts
- Highly sensitive
38
Turbidimetric Measurements
- Spectrophotometer
- Measures light not scattered by bacteria
- Calibration of OD vs viable cell count
- High cell density --> backscattering --> deviation from linearity
- Rapid measurements without disturbing culture
39
Primary Metabolite
-Produced during exponential growth (alcohol)
40
Secondary Metabolite
- Produced during stationary phase (antibiotics)
- Not essential for growth
- Often significantly over-produced
41
Penicillin Production
- Stationary phase
- Excreted into media
- Extracted using organic solvents
42
Sterilisation
Inhibition
Decontamination
Disinfection
Sterilisation- killing or removal of all viable organisms within a growth medium
Inhibition- effectively limiting microbial growth
Decontamination- treatment of abject to make it safe to handle
Disinfection- directly targets removal of all pathogens, not necessarily all microbes
43
Heat Sterilization
Decimal reduction time
Thermal death time
Autoclave
Pasteurisation
- Most widely used
- High temperatures --> denature macromolecules
- Endospores can survive heat
- Decimal reduction time- time required for 10-fold reduction in viability
- Thermal death time- time it takes to kill all cells at a given temperature
- Autoclave- sealed heating device that uses steam under pressure (moist heat sterilization)
- 121 degrees, 10-15 minutes
- Pasteurisation- precisely controlled heat to reduce the microbial load in heat-sensitive liquids
- Doesn't kill all organisms (not sterilisation)
- Controls pathogens and spoilage organisms
- Milk: 71 degrees for 15 seconds
- UHT milk: 135 degrees for 1-2 seconds
44
Radiation Sterilization
Microwaves, UV, gamma rays, electrons
UV has sufficient energy to cause modifications and breaks in DNA
-decontamination of surfaces
- Cannot penetrate solid surfaces (limited to exposed surfaces)
-Ionising radiation
- Electromagnetic radiation of sufficient energy to produce ions and other relative molecular species
Electrons, hydroxyl radicals, hydride radicals
- Cathode ray tubes, X-rays, radioactive nuclides
- Used in medical and food industry
- Approved by WHO and is used in USA for decontamination of foods
45
Filter Sterilization
-Avoids use of heat for sensitive liquids and gases
- Pores of filter are too small for organisms to pass through
- Large enough to allow liquid or gas to pass through
-Depth filters
- High efficiency particulate air (HEPA) filters
-Membrane filters
Function more like a sieve
46
Chemical Growth Control
-Bacteriostatic, bacteriocidal and bacteriolytic
-MIC
- Synthetic agents (growth factor analogues)
- Sulfanilamide (inhibits production of folic acid)
- Isoniazid (interferes with synthesis of myolic acid, a mycobacterial cell wall component)
- Antibiotics (including semi-synthetic agents)
Microbially produced
47
Antibiotics
- Targets properties not present in humans
- Cell wall synthesis
- Protein synthesis (50S and 30S inhibitors)
48
B-lactam Antibiotics
- Penicillin G active mainly against Gram +
- Chemically modified semi-synthetic antibiotics to change properties
- Transpeptidase enzymes in cell wall bind to penicillin (PBP)
- When PBP bind to penicillin, cross linking does not occur
- Cell wall synthesis continues --> weakened cell wall
- Penicillin-PBP complex stimulates release of autolysins (enzymes that degrade the cell wall) --> degraded cell wall
49
Antimicrobial Resistance Mechanisms
-Lacks structure that the antibiotic inhibits
Mycoplasmas lack a cell wall --> resistant to penicillin
-Impermeable to antibiotic
Most G- bacteria impermeable to penicillin
-Can inactivate antibiotic
B-lactamases cleave B-lactam ring (plasmids)
-Modify target of antibiotic
Mutations in PBPs, ribosomes
-Develop resistant biochemical pathway
Folic acid taken up from environment instead of being synthesised
-Efflux
Tetracycline efflux pathway
50
Staphylococcus aureus
Vancomycin is an antibiotic against S. aureus
- Glycopeptide antibiotic --> binds to D-Ala D-Ala on pentapeptide to block cross linking
- Also targets cell wall --> more difficult to develop resistance
Can develop resistance
-Replaces D-Ala D-Ala with D-Ala D-Lac
Peptidase cleaves D-Ala from D-Ala D-Ala and ligase adds lactate
-D-Ala D-Lac is still recognised for cross linking
MICR2000
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