Microbiology Flashcards
Lectures 1 - 8
1
Q
Who originally came up with the ‘Five Kingdoms’? What are they and what are they also known as?
A
WHITTAKERS came up with this idea. They are Plantae, Fungi, Animalia, Protista, Bacteria. They are also known as the crown taxa.
2
Q
Who came up with the idea of ‘The three domains’. What else could it be known as and why is ‘the five kingdoms’ and ‘the three domains’ wrong?
A
WHITTAKERS came up with the idea. It could also be known as ‘MONERA’. Both concepts are wrong because they are very little of total diversity and the microbial world is a lot more diverse.
3
Q
Which microbes are prokaryotes, and describe them.
A
Bacteria –> mostly 1-6micrometers in size with the largest bacteria being visible to the eye.
Archaea –> once thought to be extremophiles only (can only survive in most extreme environments), grow everywhere. First non-extreme archaeon isolated in 2004, no known pathogenic archaea.
4
Q
Describe eukaryotic microbes and their properties.
A
Fungi –> size varies, largest colony is 10,000Kg.
Protists –> Very diverse, size ranges from 1-150 micrometers.
5
Q
Describe the significance in global biomass.
A
Estimated to be 4-6 X 10^30 cells on earth. Bacteria and archaea are a major portion of biomass on earth and provide nutrients for all life.
6
Q
Why are there so many microorganisms on earth?
A
Rapid growth rate, lots of speciation, lateral gene transfer, evolutionary history of 3.8 billion years.
7
Q
When was the first definitive bacterial cell fossilised?
A
2400 million years ago.
8
Q
Where do phototrophs and chemotrophs get energy from?
A
Phototroph –> Energy from light.
Chemotroph –> Energy from chemical bonds.
9
Q
What do organotrophs and lithotrophs use as electron donors.
A
Organotroph –> Use organic compounds as electron donors.
Lithotroph –> Use inorganic compounds as electron donors.
10
Q
What do autotrophs and heterotrophs use as carbon sources?
A
Autotrophs –> CO2 as carbon source.
Heterotrophs –> Organic carbon as carbon source.
11
Q
What macronutrients and micronutrients are required for growth in labs?
A
Macronutrients: C, H, O, N, S, P, K, Mg, Na, Ca, Fe
Micronutrients: B, Cr, Co, Cu, Fe, Mn, Mo, Ni, Se, W, V, Zn.
12
Q
What are prototrophs?
A
Organisms that synthesise all their own cellular components.
13
Q
What are fastidious bacteria?
A
Organisms that need organic components, such as vitamins, in the media.
14
Q
How do bacteria grow?
A
Asexual reproduction by binary fission or budding. This means the cells double in size then split into 2. They have exponential growth. Incomplete separation produces pairs or larger assemblies of bacteria.
15
Q
What is carrying capacity?
A
The maximum an organism is able to grow, dependant upon its surroundings. It’s limited by space and availability of nutrients.
16
Q
Describe the bacterial growth curve and explain the different sections.
A
Lag phase: bacteria adapt to new conditions.
Log/exponential phase: exponential growth.
Stationary phase: limitation by nutrients, buildup of waste products that inhibit growth.
Death: Organisms start dying off and may lyse.
17
Q
Describe 3 methods to measure growth.
A
Haemocytometry: A glass microscope slide is used with grid lines that have a measured distance for valid comparison.
Turbidimetry: Uses transmission and scattering of light to deduce the concentration of organisms in a solution.
Dilution plating: Making organisms grow at different concentrations.
18
Q
Name some ways to identify microorganisms.
A
Microscopy, growth on media, substrate spectrum, testing enzyme activity
19
Q
What is selective and differential media?
A
Selective media allows only some types of organisms to grow on it, helps to identify pathogens. Differential media allows identification of organisms based on growth and appearance on the medium, helps to determine if a culture is pathogenic.
20
Q
How can we test enzyme activities?
A
ApiZym is optimised towards pathogens.
1. A culture of the organism is grown.
2. Cells resuspended in a buffer.
3. Test wells are innoculated.
4. Resulting colour in test strip is compared against a database.
5. Can help to distinguish between pathogenic and benign strains.
21
Q
What is the surface origin hypothesis? Why is it unlikely to be true?
A
‘Warm Little Pond’
Primordial soup (evidence that organic molecules can form spontaneously)
This hypothesis is unlikely to be true because of hostile conditions on surface.
22
Q
What is the subsurface origin hypothesis?
A
Hydrothermal vents at ocean floor, more stable conditions, constant source of energy (reduced inorganic compounds)
23
Q
What does LUCA stand for?
A
Last Universal Common Ancestor
24
Q
Why was early life probably dependent on H2 and CO2?
A
Bacteria made acetate.
Archaea making methane. Through this process the energy and carbon metabolisms diversify.
25
What are phylogenetic methods?
Allow us to determine how related organisms are to each other.
26
What do fossils show about the origin of eukaryotes?
They arose 2.5 billion years ago.
27
What is the endosymbiont theory?
Mitochondria: Incorporation of aerobic chemo-organotrophic bacteria into a host cell.
Chloroplasts: Incorporation of phototrophic cyanobacteria into a eukaryotic cell.
28
What is the hydrogen hypothesis?
Association of an archaeal host using H2 as an energy source with an aerobic bacterium that produced hydrogen as a 'waste' product.
29
What are methanogens archaea?
Produce methane as a waste product. Are at the bottom of the food chain. Release vast amounts of methane to the atmosphere.
30
What are halophilic archaea?
Organisms that can grow in a saturate salt solution. Can take very strange shapes and colours.
31
What is systematics and what is it good for?
It is the study of the diversity of organisms and their relationships, its good for linking phylogeny with taxonomy.
32
What is taxonomy and why is it good?
Bacterial taxonomy traditionally focuses on phenotypic comparisons. Its good so that we can look at evolutionary relationships between organisms.
33
What does the prefix 'staphylo' indicate?
'bunch of grapes' clusters
34
What does the prefix 'coccus' mean?
Coccus-shaped bacterium. Circular.
35
What does the suffix 'epidermidis' mean?
Isolated from skin
36
What does the prefix bacillus mean?
Rod-shaped bacterium.
37
What does the suffix thermophilus mean?
Grows at high temperature.
38
How can you remember the systematic levels?
Do - Domain
Kings - Kingdom
Play - Phylum
Chess - Class
On - Order
Fat - Family
Girls' - Genus
Stomachs - Species
39
What is the no unified species concept?
A group of strains that show a high degree of overall similarity and differ considerably from related strain groups with respect to many independent characteristics.
40
What do we mean by 'using a polyphasic bacterial taxonomy'?
Its a method for classification. Three types:
phenotypic analysis --> morphological, metabolic, chemical characteristics.
genotypic --> comparative at the gene and genome level.
phylogenetic --> framework of evolutionary relationships.
41
Describe the gram staining procedure.
1. Heat fix: Use a heat source, like a flame, to quickly pass the slide over several times.
2. Stain with crystal violet: Flood the slide with crystal violet and let it sit for at least one minute.
3. Rinse: Rinse the slide with water.
4. Apply iodine: Flood the slide with iodine and let it sit for at least one minute.
5. Decolorize: Flood the slide with decolourizer and rock it back and forth for 3–5 seconds.
6. Rinse again: Rinse the slide with water.
7. Counterstain: Flood the slide with safranin and let it sit for 30 seconds.
8. Rinse again: Rinse the slide with water.
9. Dry: Dry the slide with bibulous paper or absorbent paper and place it upright.
10. Examine: Use a microscope's oil immersion objective to examine the organisms.
42
Describe the decomposition of simple carbohydrates as used for a biochemical test.
Fermentation produces acid from glucose in aerobic/anaerobic conditions. This changes the pH allowing the CO2 to collect in the Durham tube and induce a colour change.
43
What kind of things can inhibit growth of organisms on agar?
Selective media, antibiotics, dyes, toxins.
44
Describe the physiological properties of acidophiles, alkaliphiles, barophiles, halophiles, aerobe, anaerobe, microaerophile, psychrophile, mesophile, thermophile and hyperthermophile.
Acidophile - ph<6
Alkaliphile - ph>8
Barophile - requires high pressure
Halophile - requires very salty conditions
Aerobe - needs oxygen
Anaerobe - doesnt need oxygen
Microaerophile - needs low oxygen concn.
Psychrophile - low temp <15*
Mesophile - 'normal' temperature 15-45*
Thermophile - high temp >50*
Hyperthermophile - very high temp >80*
45
What is FAME?
Fatty acid methyl ester. IT determines the fatty acid profile of membrane lipids. FAME products can be analysed using gas chromatography.
46
What is DNA profiling? Mention AFLP in your answer.
AFLP = Amplified fragment length polymorphism.
PCR targets repetitive elements in bacterial genome and is used to distinguish related strains.
47
What is multilocus sequence typing?
1. New isolate or clinical sample.
2. DNA isolation
3. Amplify 6-7 target genes by PCR
4. Sequencing
5. Determining alleles
6. Compare with other strains of the same species
7. Find the linkage distance between 2 species to find how closely related they are.
48
Describe sanger sequencing.
Started in the late 1980's. It gives the ability to sequence a DNA molecule. It was originally done using gels but is now done using fluorescent dyes and a capillary column. It can compare actual sequences and is able to 'build' genomes rapidly using next generation sequencing.
49
Describe the deposition of type cultures. Give me 3 examples of international culture collections.
Validly describes species have to be deposited with at least 2 culture collections that will be preserved as lyophilized socks (good for 10+ yrs). They can be ordered and shipped worldwide.
NCIMB: National Collection of Industrial, Food and Marine Bacteria (Aberdeen, UK)
ATCC: American Type Culture Collection (Virginia, USA)
DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany)
50
What is Bergey's Manual of Systematic Bacteriology?
Made is 1923, there are descriptions of described bacterial and archaeal species.
51
Describe the outer layers of bacterial cells from the outside to the inside.
Capsule, S-layer.
Gram Positive Bacteria --> Cell Wall
Gram Negative Bacteria --> Outer Membrane
Periplasmic space with peptidoglycan in gram negatives and some gram positives.
Cell Plasma Membrane
52
What are capsules? Describe them in detail.
They can be 'slime layers' or glycocalyx ("sugar coat").
Glycocalyx involved in biofilm formation, this aids in establishing complex consortia of bacteria.
Capsule is an organised, tight matrix which is not easily removed, it excludes small particles.
Slime layer is able to diffuse, unorganised and easily removed. It doesn't exclude small particles and can be difficult to visualise.
Capsules are also a carbon store, the protect against desiccation, may be involved in the capture of nutrients.
53
Describe capsules in pathogens specifically.
Most commonly found in polysaccharides structures. There are glycolipid capsules, protein capsules, extracellular slimes.
54
What are bacterial adhesins?
They are capsular polysaccharides, have extracellular slime, fimbriae and lectins.
55
What is the S-layer?
Paracrystalline outer wall layer composed of protein. The structure is external to cell wall. In some archaea, this is the only cell wall structure. May protect against ion and pH fluctuations, osmotic stress predators. It may protect against hose defences.
56
What is peptidoglycan? Describe its structure properly.
Its made of alternating residues of NAG and NAM. It's arranged in dimers which are cross linked by amino acid side chains that create amide bonds. It's also a mesh-like polymer that retains the Gram stain in Gram positive cells.
It's made of non-protein amino acids (D- amino acids) which protect against degradation by proteases.
57
Describe the overall structure of Gram positive cell wall.
They have a thicker outer layer than Gram negative bacteria. Its up to 90% of cell wall, up to 25 sheets of peptidoglycan. Teichoic acid is found only in Gram positive cells, it's role is unclear and can make the membrane negatively charged (could help binding of Ca 2+ and Mg 2+)
58
Describe the overall structure of the Gram negative cell.
There's little peptidoglycan in Gram negative bacteria. It's typically 10% of total cell wall. It's between the inner and outer membrane of the cell. It is asymmetrical due to insertion of lipopolysaccharide into external layer of the outer membrane.
59
What is a lysozyme?
Its an 'antibacterial' enzyme. It degrades the beta 1, 4-glycosidic bond in peptidoglycan backbone. Loss of peptidoglycan makes cells sensitive to changes in osmotic pressure. It's also an important host defense against bacteria.
60
Describe archaeal cell walls?
Have no peptidoglycan or murein, some methanogens contain pseudomurein.
61
What are sterols or hopanoids?
Hopanoids are found in bacteria, but not found in archaea. Sterols are found in eukaryotes. They are both forms of cholesterol.
62
Describe the 2 ways in which the outer membrane is linked to the cell.
1. Braun's lipoprotein:
- Most abundant protein in outer membrane.
- Covalently linked to peptidoglycan and embedded in outer membrane by hydrophobic end.
2. Adhesion sites where the two membranes adhere.
- Around 400 sites in E. Coli.
- Can be visualised using TEM.
63
Describe archaeal membranes.
Different from bacteria and eukaryotes. The branched chain hydrocarbons attached to glycerol by ether links rather than fatty acids-ester links.
64
What is a lipopolysaccharide? Describe it.
It's a large complex molecules containing lipid and carbohydrate. It's best described from salmonella and is also called endotoxin when free in host. It is able to induce a massive immune response (septic shock).
65
What is the structure of a lipopolysaccharide in a Gram negative bacteria? Describe it in as much detail as possible.
3 sections: O-Specific polysaccharide, Core polysaccharide and Lipid A.
- Lipid A is made of 2 glucosamine residues linked to fatty acids and phosphate, it's integrated into the outer membrane, the remaining lipopolysaccharides projects from the cell surface.
- Core Polysaccharide is also referred to as R-antigen or R-polysaccharide, It has sie chains of NAG, phosphate and ethanolamine.
[The core polysaccharide and lipid A make up the constant region.]
- O side chain is the variable region responsible for the antigenic makeup of bacteria. Lipid A and core polysaccharide are straight and O side chain is flexible.
66
What are the functions of lipopolysaccharide?
Lipid A stabilises the outer membrane structure. The core polysaccharide is charged. It protects against host.
67
What is endotoxin? Give 2 functions of it and explain it. Give 2 tests of endotoxins.
Many pathogens produce endotoxins which are released during cell division or by lysis of bacterial cells. Functions: Can act to prime the immune system against a pathogen, can cause septic shock syndrome which has no direct treatment. Test for endotoxins: Rabbit pyrogen test, LAL assay.
68
Name some important properties of endotoxins.
Heat stable, toxic in nanogram amounts, interacts with innate immune system cells, can trigger the release of cytokines in a cascade, activates transcription factors, can result in inflammation, fever, vasodilation and thrombosis.
69
Describe the outer membrane as a permeability barrier.
Outer membrane is more permeable than inner membrane due to the presence of porins. These are protein channels that allow small molecules upto 600 Da to pass through. Larger molecules will need active transport systems.
70
Describe the porin superfamily.
They are homotrimeric, transmembrane proteins. They have highly conserved structure and form water filled channels in the outer membrane.
71
Describe porin selectivity.
Most are non-specific channels that allow the passage of <600Da hydrophilic molecules. Some porins are selective.
72
Describe what a porin looks like from the top.
High thermal stability. Resistant to protease and detergent degradation which is essential for Gram negative bacteria in harsh environment such as the intestinal tract.
73
Describe the structure of porins.
It has common beta strand structure. It has an unusual structure that consists of a 16-stranded antiparallel beta-barrel; its exceptionally stable. Extra stability from formation of a salt bridge between the N- and C-termini which produces a closed structure. It has a hourglass shape with central constriction.
74
What is periplasmic space?
Compounds diffuse through porins into periplasm. Periplasm is the space between outer membrane and cytoplasmic membrane. Gel-like consistency due to abundant proteins. Removal of cell walls without lysing the cells allows study of the proteins and enzymes present in this space.
75
What enzyme activity occurs in the periplasm?
1. Nutrient acquisition --> Hydrolytic enzymes like alkaline phosphatase.
2. Energy conservation --> Electron Transport Protein
3. Some peptidoglycan synthesis enzymes are periplasmic.
4. Periplasmic binding proteins
5. Chemoreceptors
76
Which are the 2 pathways used to export proteins into the periplasm?
Sec pathway:
- Uses cytoplasmic membrane with a translocase.
- Proteins have an N-terminal signal peptide.
TAT pathways (Twin Arginine translocase):
- Exports fully folded enzymes across cytoplasmic membrane.
- Proteins have a twin arginine in the N terminal region.
77
What are flagella? Describe their structure.
Long think extracellular helical structures made of protein subunits that aid in motility (upto 20 micrometers long).
Flagella are connected to a motor that spins them clock or anticlockwise allowing bacteria to swim.
Structure:
- Complex ring structures anchored into the membrane and cell wall. Rings and hook are rigid and attached.
- Flagellum shaft is easily removed by vigorous shaking.
- Different antigenic properties of shaft, tip of shaft and hook.
78
Describe the stages of flagella synthesis.
1. MS and C rings in cytoplasmic membrane.
2. Motor proteins.
3. P and L ring, hook and cap.
4. Flow of flagellin through hook, growth of filament.
79
How does the motor work in a flagella?
The rotary motion is provided by the basal structures. It's provided by the proton motive force (one rotation requires the translocation of 1000 protons).
80
How are the flagella different in gram positive and negative cells?
In Gram positive, there are no L and P rings. In Gram positive, the anchor in the membrane layer is more complex and most proteins surround the inner ring and their movement provides the force.
81
What are the 4 types of flagella?
Monotrichous --> Sperm looking
Amphitrichous --> Sperm with tails on opposite ends.
Lophotrichous --> Sperm with multiple tails on opposite ends.
Peritrichous --> Looks like head hair on a sperm body.
82
What are the 4 tactic responses to changes in the environment?
Aerotaxis --> Movement usually towards oxygen.
Chemotaxis --> Movement towards nutrients and away from toxins.
Magnetotaxis --> Movement along lines of magnetism
Phototaxis --> Movement towards light
83
What are MCP receptors?
Number of putative chemoreceptor genes in sequenced genome ranges.
Sequences of cytoplasmic domains of all these receptors are highly conserved.
Each receptor binds different ligands and different species have different optimum niches.
84
How can cells move without flagella? Explain this type of motility.
Gliding along a slime extruded on the outside. 3 Gld proteins are components of an ATP-binding-cassette transporter.
5 Gld proteins are lipoproteins in the cytoplasmic membrane or outer membrane.
85
Describe twitching motility of flagella
Its done by the pili extending from the cell surface then being retracted, dragging along the cell surface. It's powered by ATP hydrolysis. The retraction proteins control direction of movement.
86
What is a gas vesicle?
Protein vesicles that contain gas, which gives buoyancy to the cell. It occurs in planktonic bacteria and some archaea. It allows cells to float up to oxygenated water or towards light. Can be involved in vertical migration in aquatic systems like lakes.
87
What are fimbriae/pili?
Surface appendages, multi-subunit proteins which were first recognised by electron microscopy on bacteria over 40 years ago. They are involved in genetic exchange between bacteria.
88
Describe the categories of fimbriae/pili
They are bacterial adhesins, mediate adhesion to surface and other cells.
89
How do fimbria/pili assist adhesion?
Type 4 pili have adhesive tip proteins.
1. Pili establish initial contact to host cells to avoid electrostatic repulsion.
2. The pilus' adhesive tip protein binds to a receptor which is usually a glycolipid or glycoprotein.
3. Pili depolymerise and pulls the bacteria close to the host cell.
4. Additional adhesins bind to the host cell.
Colonisation can begin.
90
Describe Type 1 fimbriae.
Well characterized fimbrial system and is an important virulence factor in a range of pathogens. It's thin (7nm wide) and approx 1-2 micrometers long surface polymer.
91
What is P-Pili?
It's also known as PAP: Pyelonephritis-Associated-Pili. It's a critical virulence factor of E.Coli which causes cystitis - this can be fatal.
It's similar in structure and assembly to Type 1 fimbriae.
92
What is Type 4 pili?
It's widely distributed in Gram negative organisms. Clostridia is a Gram positive organism that has Type 4 pili. Its generally longer than fimbriae (upto 10 micrometers) and utilises twitching motility.
93
What is the species specificity of pathogens?
Many pathogens are species specific which can be determined by the O antigen and K antigen.
94
What are bacterial endospores.
It's a dormant stage in bacterial life cycle. It's called endospores because they form inside bacterial cell. It's a survival stage in which the bacteria become extremely resistant to heat, desiccation and radiation. These endospores form when a vegetative cell becomes distressed, the result is a dormant endospore. These endospores are commonly found in soil bacteria.
95
What is sporulation?
Complex series of cellular differentiation events. Several stages, each controlled by more than 200 genes. The process takes about 8 hours to complete.
96
What are the stages of sporulation?
1. Vegetative cell under stress.
2. DNA organised along cell axis.
3. A genome copy enclosed in forespore septum, forespore produced by membrane invagination.
4. Cell membrane engulfs forespore in a second membrane.
5. Cortex between membranes accumulates calcium and dipicolinic acid, becomes dehydrated to <10%.
6. Complex exosporium layers are produced.
7. Spore matures with complete cortical layers.
8. Original cell lyses releasing spore.
97
Describe the structure of endospores.
Made of many new layers, outermost layer is exosporium (thin layer of proteins). The spores consist of spore-specific proteins. Inside the endospore, there's a core which contains genome, cytoplasm and ribosomes; however, this is metabolically inactive. It can be resistant upto 150*C but autoclaving destroys spores.
98
Describe the history of the Bubonic Plague.
It was one of the most devastating infectious diseases humans have suffered from. There were pandemics in Asia and Europe during mid 14th century. Its believed to have wiped out between 30-60% of the European population in the middle ages.
99
What is 'The Human Microbiome'?
A concept first suggested by Joshua Lederberg. A microbiome is the ecological community of commensal, symbiotic and pathogenic microorganisms that literally share our body space.
100
What are virulence factors?
They are structures that bacteria use to causes disease in the host.
Virulence determined by many factors that aid in adhesion to and entry into cells, production of toxins and immune system evasion.
Conventional virulence factors include bacterial toxins, adhesins and LPS.
Other factors also indirectly contribute to virulence: catalases, genetic regulators.
101
What is MRSA, briefly describe it. Describe the first antibiotic ever.
Methicillin Resistant Staphylococcus Aureus. Limited treatment due to multiple antibiotic resistance. Penicillin was the first natural source found in 1928.
102
What are the 5 major types of antibiotics?
1. Beta-lactams:
- Penicillins
2. Macrolides:
- Inhibit protein synthesis
3. Fluoroquinolones:
- Inhibits DNA gyrase
4. Tetracyclines:
- Inhibit protein synthesis.
5. Aminoglycosides:
- Inhibit protein synthesis.
103
What are the solutions to antimicrobial resistance?
New drugs
New targets
NEw drug combinations.
104
Describe the history of vaccinations.
There's evidence of vaccinations in China over 1000 years ago. In 1796, Edward Jenner invents vaccination.
1879, Louis Pasteur found that chicken cholera became much less virulent after exposure to air for a long time.
1881, Louis Pasteur tested an anthrax vaccine on sheep and it worked.
105
Describe the tetanus toxin.
Tetanoispasmin is a plasmid-encoded 150kDa protein. Its also neurotoxic. This can be released by cell lysis and spreads through blood and lymph. It halts the release of glycine and GABA neurotransmitters.
106
Describe bacterial meningitis.
Inflammation of the protective membranes that cover the brain and spinal chord. It has many different causes: viruses, bacteria, fungi, protozoa.
