microbiology

• Basic shapes of bacteria

  • Cocci (spherical)
  • Bacilli (rod-shaped)
  • Spirilli (spiral)
• Types of bacteria

  • Gram positive
  • Gram negative
• Gram positive bacteria


  Have a thick peptidoglycan layer which retains a crystal violet stain, appear purple
• Gram negative bacteria


  Have a thin peptidoglycan layer, alcohol washes out crystal violet, counterstained with safranin to appear red, have extra lipopolysaccharide layers
• Gram stain procedure


  1. Heat fix smear

  2. Stain with crystal violet

  3. Fix stain with iodine

  4. Decolourise with alcohol

  5. Counterstain with safranin
• Gram positive cells appear purple, Gram negative appear red under the microscope
• Culturing bacteria


  Provide right nutrients and conditions in Petri dish or flask
• Bacteria


  • Require correct pH and temperature for growth, most human pathogens grow at 37°C, safer to culture at 25°C in school labs
• Psychrophiles


  Bacteria that grow at 1-5°C
• Thermophiles


  Bacteria that grow best at higher temperatures, found in hot springs and around volcanic vents
• Selective media


  Media in which only certain bacteria will grow
• Antibiotics


  Can be added to agar to prevent growth of non-resistant bacteria
• Oxygen requirements of bacteria

  • Obligate aerobes
  • Facultative anaerobes
  • Obligate anaerobes
• Obligate aerobes


  Must have oxygen to carry out metabolism and reproduce
• Facultative anaerobes


  Can metabolise and reproduce in presence or absence of oxygen, but reproduce better with oxygen
• Obligate anaerobes


  Must have oxygen-free environment to carry out metabolism and reproduce
• Many bacteria can survive harsh conditions and subsequently reproduce when conditions are restored
• Determining oxygen requirements


  Culture in long tube, obligate aerobes grow at top, facultative anaerobes throughout, obligate anaerobes at bottom
• Aseptic technique


  Procedure to avoid contamination of cultures and personnel
• Aseptic technique before starting

  Wash hands, sterilize benches, wear lab coats and goggles, use Bunsen burner for convection current
• Aseptic technique during transfers

  Hold lids in crook of finger, flame necks of bottles/cultures, sterilize loops
• Aseptic technique after inoculation


  Seal Petri dishes with tape, incubate upside down at 25°C, autoclave plates after observations
• Total count


  Counts all cells present, cannot distinguish live and dead
• Viable count


  Counts only cells capable of reproducing and therefore alive
• Serial dilution


  Dilutes sample so not too many cells to count, prevents continuous lawn of colonies
• Calculating bacterial numbers


  Multiply colony count by dilution factor and adjust for volumes
• Gram negative bacteria are resistant to penicillin
• Gram positive bacteria are stained purple
• Different species of bacilli stain red or purple
• Gram negative bacteria stain red
• Gram negative bacteria have extra layers of lipopolysaccharide
• A doctor would need to know if bacteria is Gram negative or positive before prescribing penicillin
• Psychrophile


  Needs low temperatures to grow well
• Thermophile


  Needs high temperatures to grow well
• Halophile


  Needs salt to grow well
• Human pathogens grow best at human body temperature
• Acidophile


  Needs low pH to grow well
• In a laboratory, a common culture medium is agar
• Obligate aerobe


  Requires oxygen for metabolism and reproduction
• Facultative anaerobe


  Reproduces and metabolises better with oxygen, can do so without oxygen