Microbiology

• There are three basic shapes of bacteria:

  The cocci (singular coccus) are spherical, they are drawn as circles.

  Bacilli (singular bacillus) are rod-shaped, drawn as lozenges.

  Spirilli (singular spirillum) are spiral in shape.
• Gram positive bacteria have a thick peptidoglycan layer which retains a crystal violet stain, so after Gram staining they appear purple down the microscope.
• Gram negative bacteria have a thin peptidoglycan layer. Because of this, alcohol used in the procedure will wash the crystal violet out. The counterstaining with safranin stains the cells red. Gram negative bacteria have extra layers of lipopolysaccharide which protects the cells from lysozyme as well as the action of penicillin and other antibiotics.
• The procedure for Gram stain in outline is as follows:

  • Heat fix a smear of bacteria
  • Stain using crystal violet stain
  • Fix the stain with iodine
  • Decolourise with alcohol
  • Counterstain with safranin.

  When the cells are observed down the microscope Gram positive cells are purple in colour and Gram negative are red.
• Bacteria culture


  Petri dish or flask, given appropriate nutrients and conditions
• Agar


  Most common medium for culturing bacteria, can be jelly-like in a Petri dish or liquid in a flask
• Components of agar


  • Carbon source
  • Nitrogen source
  • Water
  • Vitamins
  • Mineral salts
• Substances added to agar

  • Blood agar for bacteria that thrive on blood
  • Starch agar for bacteria that can digest starch
  • Salty agar for halophiles (salt-loving bacteria)
• Dyes in agar


  Change color based on bacterial metabolism, aiding in differentiation between bacteria
• Conditions for bacterial growth

  • Correct pH
  • Correct temperature
• Temperature for human pathogens

  37˚C, the optimal temperature for their rapid multiplication
• Temperature for school laboratories

  25˚C to avoid growing human pathogens
• Psychrophiles


  Grow at low temperatures (1-5˚C)
• Thermophiles


  Thrive at high temperatures, found in natural environments like hot springs and oceanic volcanic vents
• Selective media


  Allow the growth of only specific bacteria types
• Antibiotics in agar


  Inhibit the growth of non-resistant bacteria
• Obligate anaerobes must have an oxygen-free environment in order to carry out metabolism and reproduce.
• Obligate aerobes must have oxygen in order to carry out metabolism and reproduce.
• Facultative anaerobes can metabolise and reproduce in the presence or absence of oxygen, but reproduce better if oxygen is present.
• Using a Bunsen burner


  1. Use a Bunsen burner on a roaring flame to create a convection current

  2. Work close to this area
• Ensuring equipment is sterile

  • Disposable Petri dishes are pre-sterilized
  • Glass spreaders are sterilized by dipping in alcohol and burning it off
  • Inoculating loops are heated until red hot
  • Other equipment can be sterilized in an autoclave (over 120˚C)
• Transfers


  1. Hold lids of agar bottles or cultures in the crook of the little finger; do not place them on benches

  2. Flame the necks of agar bottles and cultures when opening and before closing

  3. Sterilize and cool loops before inserting into cultures and sterilize again afterward

  4. Tilt, do not lift, Petri dish lids when pouring agar or inoculating plates
• After inoculation


  1. Seal Petri dishes with a cross of tape to discourage human pathogens and allow oxygen into the culture

  2. Incubate plates upside down at 25˚C for 24 hours

  3. Do not re-open plates; autoclave them after observations are made
• Total Count


  Counts all cells (live and dead) using methods like haemocytometer or measuring broth cloudiness with a colorimeter
• Viable Count


  Counts only live cells capable of reproducing (forming colonies) by transferring samples from serial dilution to agar plates and counting colonies after incubation
• Colony Formation


  • A colony forms when a bacterium reproduces on a plate, forming a visible clonal population
• Serial Dilution


  Performed to dilute the bacterial sample for accurate counting and to avoid overlapping colonies on the agar plate
• Counting Bacteria


  • Total Count: Uses haemocytometer or colorimeter
  • Viable Count: Uses samples from serial dilution, incubated on agar plates for 24 hours before counting colonies
• Counting Considerations


  • Use the plate with the largest number of distinct colonies for accuracy
  • Avoid plates with overlapping colonies or too few colonies
  • Each colony is assumed to originate from a single bacterium, but merged colonies can lead to underestimation
• Calculating Original Sample


  Multiply the number of counted colonies by the dilution factor and adjust for the volume plated to determine the number of bacteria in the original sample