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Marine Pathogens and Parasites > Bacteria > Flashcards

Bacteria Flashcards

1
Q

How can you Classify Bacteria?

A
  • Gram Reaction
  • Selective Media
  • Serology
  • PCR
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2
Q

What is the difference between Gramm negative and gram positive bacteria

A
  • Gram positive have a single peptidoglycan layer

- Gram Negative have an additional lipidopolysaccharide coat

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3
Q

What does PAMP Stand for?

A

A pathogen associated molecular pattern

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4
Q

What are PAMPs?

A

Pamps are evolutionary conserved molecules which trigger an immune response

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5
Q

How do LPS coats help bacteria?

A
  • They protect bacteria from denaturation

- make bacteria more resilient in marine environments

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6
Q

What is the Gram staining Protocol

A
  1. Heat fix a sample so it sticks to the slide
  2. Stain all bacteria with Crystal Violet as it associates with peptidoglycan
  3. Add Iodine to form a complex with crystal violet (CV is +vely charged and Iodine in -vely Charged hence the complex)
  4. Cells are treated with decolourising solution which is usually a form of alcohol (Washes away excess iodine and CV)
  5. After decolourising solution gram +ve retain dye and gram negative have no colour
  6. Gram negative are turned pink by a counter stain - Safranin
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7
Q

What are the two types of Selective Media used?

A
  1. TCBS - Thiosulphate Citrate Bile Media

2. Listonella Anguillarum media

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8
Q

What Species turns TCBS Yellow

A

Green to yellow - vibrio species - or grows dark black colony - hydrogen sulphide

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9
Q

Listonella Anguillarium media does to media?

A

Dark Blue –> Yellow : Listonella Metabolises Sorbitol

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10
Q

What are the components to TCBS which inhibit bacteria and how?

A

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

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11
Q

What does Vibrio do to TCBS

A
  • 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
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12
Q

What optimal conditions must there be for vibrio to grow on the agar?

A
  • Yeast extract and peptone for nutrients, amino acids and vitamins
  • Sodium Chloride for growth and metabolic of halophilic vibrio
  • agar is the solidifying agent
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13
Q

What is Serology?

A

It uses antibodies to detect bacteria or products of bacteria

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14
Q

What is the direct method of Serology?

A

Single cell step staining - can use multiple antibodies form same host - no signal amplification from secondary antibodies and each primary must be labelled indirectly

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15
Q

What is the Indirect Method of Serology

A
  • 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
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16
Q

What are the types of serology?

A
  • Liquid/Gel based assay

- ELISA (Enzyme linked immune absorbent assay

17
Q

What are the steps of ELISA?

A
  1. Known antibody is adsorbed to a physical platform
  2. Antigen of interest is added by washing over the Eliza
  3. antigen in probed with secondary antibody which has horseradish peroxide of another enzyme that metabolises producing fluorescent signal
18
Q

What does ELISA/Serology do?

A
  • ELISA quantifies the load in the sample

- Serology Gives history of past infection

19
Q

Why use PCR instead of Serology

A

Same serology type could have two different genomes - genetic differences which are not displayed in the serology

20
Q

What is needed for PCR?

A
  • 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
21
Q

What are the Symptoms of Vibrio Parahaemolyticus in Blue Crabs?

A

White nodules form on the gills and jelly like blood clots in the haemolymph - mortality 24-48 hours after infection

22
Q

What serotype of V. Parahaemolyticus is a human enteropathogen?

A

O3:K6 - food poisoning through oysters

23
Q

What are the symptoms of V. Parahaemolyticus in shrimp

A

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
24
Q

What does vibrio Harveyi cause in shrimp

A

luminous vibriosis

25
Q

What does luminous vibrios do?

A
  • 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
26
Q

How does bioluminescence work in normal quorum sensing?

A
  • 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
27
Q

At low cell densities how does QS work in V Harveyi?

A
  • 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
28
Q

At High cell densities how does QS work in V Harveyi?

A
  • 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

Marine Pathogens and Parasites (6 decks)

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