Bacteria Flashcards
How can you Classify Bacteria?
- Gram Reaction
- Selective Media
- Serology
- PCR
What is the difference between Gramm negative and gram positive bacteria
- Gram positive have a single peptidoglycan layer
- Gram Negative have an additional lipidopolysaccharide coat
What does PAMP Stand for?
A pathogen associated molecular pattern
What are PAMPs?
Pamps are evolutionary conserved molecules which trigger an immune response
How do LPS coats help bacteria?
- They protect bacteria from denaturation
- make bacteria more resilient in marine environments
What is the Gram staining Protocol
- Heat fix a sample so it sticks to the slide
- Stain all bacteria with Crystal Violet as it associates with peptidoglycan
- Add Iodine to form a complex with crystal violet (CV is +vely charged and Iodine in -vely Charged hence the complex)
- Cells are treated with decolourising solution which is usually a form of alcohol (Washes away excess iodine and CV)
- After decolourising solution gram +ve retain dye and gram negative have no colour
- Gram negative are turned pink by a counter stain - Safranin
What are the two types of Selective Media used?
- TCBS - Thiosulphate Citrate Bile Media
2. Listonella Anguillarum media
What Species turns TCBS Yellow
Green to yellow - vibrio species - or grows dark black colony - hydrogen sulphide
Listonella Anguillarium media does to media?
Dark Blue –> Yellow : Listonella Metabolises Sorbitol
What are the components to TCBS which inhibit bacteria and how?
Thiosulphate, Sodium Citrate and alkalinity inhibit bacterial growth
- Ox Bile and sodium Cholate cause slow growth of enterococci and inhibit growth of gram positive bacteria
What does Vibrio do to TCBS
- Vibrio ferments sucrose, reeking the pH and turning bromthymol blue (green agar) to yellow
- Thiosulphat is a sulphur source and vibrio create black colony by producing ferric acid
What optimal conditions must there be for vibrio to grow on the agar?
- Yeast extract and peptone for nutrients, amino acids and vitamins
- Sodium Chloride for growth and metabolic of halophilic vibrio
- agar is the solidifying agent
What is Serology?
It uses antibodies to detect bacteria or products of bacteria
What is the direct method of Serology?
Single cell step staining - can use multiple antibodies form same host - no signal amplification from secondary antibodies and each primary must be labelled indirectly
What is the Indirect Method of Serology
- Secondary antibody amplify signal with only a few of the labelled secondaries can detect the primary antibodies
- Two step staining which requires antibodies form different hosts
What are the types of serology?
- Liquid/Gel based assay
- ELISA (Enzyme linked immune absorbent assay
What are the steps of ELISA?
- Known antibody is adsorbed to a physical platform
- Antigen of interest is added by washing over the Eliza
- antigen in probed with secondary antibody which has horseradish peroxide of another enzyme that metabolises producing fluorescent signal
What does ELISA/Serology do?
- ELISA quantifies the load in the sample
- Serology Gives history of past infection
Why use PCR instead of Serology
Same serology type could have two different genomes - genetic differences which are not displayed in the serology
What is needed for PCR?
- Template, TAQpolymerase, primer (specific), single stranded DNA/RNA from organism
- Nucleotides, and TAQ buffer
heat up to denature - cool to anneal, heat to extend
all done in a thermocycler
`Run the DNA on a gel - electrophoresis - with wells
What are the Symptoms of Vibrio Parahaemolyticus in Blue Crabs?
White nodules form on the gills and jelly like blood clots in the haemolymph - mortality 24-48 hours after infection
What serotype of V. Parahaemolyticus is a human enteropathogen?
O3:K6 - food poisoning through oysters
What are the symptoms of V. Parahaemolyticus in shrimp
Early mortality syndrome - virulence is a large plasmid coding for PirA/PirB toxins
- 100% 2 - 3 days after infection
- Causes AHPND in shrimp
- immobilises hepatopancreas
- two types of cells - swim/swarm
- Large white membrane grows around HP
- pale/white atrophied HP
- empty stomachs
- lethargy/ slow growth
What does vibrio Harveyi cause in shrimp
luminous vibriosis
What does luminous vibrios do?
- secrets extracellular products (ECP)
- releases enzymes which disrupt shrimp coagulation which inhibits clotting
- no cure
- bioluminescence makes shrimp grow and more likely t be eaten - transferred vector
How does bioluminescence work in normal quorum sensing?
- Luxl-like molecules are responsible for synthesis of HSl
- LuxR-like proteins bind the HSL and activate target gene transcription
- Bound LuxR activates transcription of LuxCDABE operon
- HSL binding to LuxR occurs at ~1-10ug/ml
- Bound LuxR also blocks transcription of the LuxR gene – represents feedback mechanisms on systems
At low cell densities how does QS work in V Harveyi?
- LuxQ and LuxN become kinases by donating their phosphate group
- Donate these phosphate groups to LuxU, then LuxU activates LuxO
- LuxO then regulates the expression of Factor X (a repressor)
- Factor X inhibits the transcription of LuxCDABE regulon
At High cell densities how does QS work in V Harveyi?
- two other proteins, LuxS and LuxLM release auto inducer molecules
- LuxS produces AI-2 and LuxLM produces AI-1
- AI-2 and AI-1 are examples of Acylhomoserine lactones(AHLs) are a major class ofautoinducersignal used by Gram-negative proteobacteria for intraspecies quorum sensing
- These autoinducers bind with LuxP (which is connected to LuxQ) and Lux N
- AI-2 binds with LuxP and AI-1 binds with LuxN
- this makes the LuxQ-LuxP complex and LuxN become phosphateases (take a phosphate away from luxU – reverse of pathway)
- LuxO is de-phoosphorolated
- No Factor X repressor so this promotes LuxR to transcripe the LuxCDABE regulon
- LuxCDABE stimulates bioluminescence
