Bacteriophage, starch hydrolysis test

• what is a Bacteriophage

  Noncellular, obligate intracellular parasites, either DNA or RNA present not both, not sensitive to antibiotics
• Identification Bacteriophage

  • Very small, measured in nanometer
  • The genetic material (DNA or RNA) is found within the capsid of the phage
  • Can be visualized by Electron Microscope
• Bacteriophageuses


  Phage that infect bacteria
• Coliphage


  Phage that infect E. coli

  Host range narrow

  •Bacteriophage T4 (Coliphage)
• Bacteriophage Infection pathways


  1. Lysis - death of host cell by taking over the replicative machinery of the cell and use it to make new phages and release them

  2. Lysogeny - The phage can incorporate into genome of the host cell
• Lysogen


  The relationship between the phage and the host cell when the phage incorporates into the host genome
• Prophage


  The genetic material of a bacteriophage incorporated into the genome of a bacterium
• Plaques


  Zone of clearing in bacterial lawn
• Bacteriophage Titer


  Amount of bacteriophage present in a sample

  
  

  #of plaques x 1/dilution factor PFU/ml
• Procedure for diluting and plating Streptomyces phage

  1. Add 90µl of sterile phage buffer to tube 10-2, 10-3, 10-4, control

  2. Add 0.2ml (200µl) of Streptomyces griseus to tubes and mix gently

  3. Incubate all tubes at room temperature for 20 minutes

  4. Incubate all plates at 37°C

  5. Spread plate 100µl
• Starch Hydrolysis test


  • Break down of starch by α-amylase present in bacteria and produce clear area
  • Iodine make a complex with starch and produce brown-black color
  • If no starch, no brown-black color produced after addition of iodine
  • Formation of clear halo around the colonies if α-amylase is present
• Phage therapy


  Therapeutic use of bacteriophagesfor the treatment of pathogenic bacterial infections
• When the cell is in stress


  the phage goes through lyticcycle for reproduction
• Starch hydrolysis -ve result

  E. coli
• Starch hydrolysis +ve result

  Bacillus megaterium
• Developer for starch test

  Iodine
• RESULTS OFBACTERIOPHAGE


  • Titer• PFU• Clear of cloudy colonies? [Lysogenic vs Lytic]• TNTC, TFTC [Too numerous to count, Too few to count]
• RESULTS OF STARCHHYDROLYSIS TEST

  • Flood the starch plate with iodine• Leave plate on table• Look for clear halo for positive result
• Phage used in this experiment

  Streptomyces phages are bacteriophages that infect Streptomyces(Streptomyces griseus), a multicellular bacteria
• What was the purpose of the nutrient agar + tryptophan supplement media used in the bacteriophage experiment?


  tryptophan(an α-amino acid that is used in the biosynthesis of proteins) was added to stimulate the growth of a bacterial lawn of E. coli so that it would be easier to tell where the plaques were formed
• β-Lactam antibiotics

  β-Lactam antibiotics are bactericidal agents that interrupt bacterial cell-wall formation as a result of covalent binding to essential penicillin-binding proteins (PBPs),