(5) Culturing Bacteria

• bacteria colonies can grow at apid rates
• in a culture, very large numbers of bacteria produced within hours
• the rate at which alleles become mutated and natural selection occurs within these bacterial populations is also rapid
• bacteria grow by binary fission
• 2^n = number of divisions
• antibiotic resistance can be used as evidence of evolution by natural selection
• bacteria is often studied by scientist :

  • colonies are grown on a nutrient agar plate or within a nutrient broth
  • idea is to study the effectiveness of antibiotics and antiseptics on a variety of bacteria strains
• bacteria are cultured in a lab to study the effectiveness of antibiotics and antiseptics
• traditional linear scales are difficult to use when studying population of bacteria as it has a large range of very little population to very large population
• due to the large range in bacteria population from small to large, it is hard to find a suitable scale on graph axes using traditional linear scales
• logarithmic scales can be very useful when investigating bacteria
• logarithmic scales allow for a wide range of values to be displayed on a single scale
• log is a scale of measurements in which an increase or decrease of one unit represents a tenfold increase or decrease in the quantity measured
• Exponential Growth :

  • the number of cells is converted to a logarithm before plotting
  • this is easy to spot as the intervals on the y axis aren't equal
• Stages of Bacteria Growth :

  • Lag Phase
  • Log phase
  • stationary phase
  • decline phase
• when growing cultures of bacteria, the culture must be pure - 1 type of bacteria
• to get pure culture, aseptic techniques must be used to avoid contamination of the sample
• contamination can affect the growth of the microorganisms on the culture
• Aseptic Techniques :

  • close windows and doors to prevent disturbing the air
  • disinfect work surfaces regularly-don't let utensils touch the work surface-make sure to disinfect immediately with antibacterial cleaner
  • make sure equipment is sterile and discarded safely once use is finished (autoclave)
  • work near a Bunsun burner when transferring bacteria-allows convection currents to move microbes in the air away from the plate as the hot air rises
  • flame the neck of the glass container as soon as it's opened+ before closing to ensure unwanted organisms don't fall in
  • flame inoculating loop
• sterilise equipment by running through ethanol and through roaring blue Bunsun burner flame
• ethanol is flammable
• have to use a blue flame with Bunsun burner
• Method :

  1. Wash hands+sterilise the surroundings. Open lid at angle to prevent contamination. Pour sterile agar into the petri dish
  2. pass inoculating loop through blue Bunsun burner flame to sterilise
  3. open the culture(quickly). Keep lid in hand(off table). Flame the neck of the culture
  4. dip inoculating loop into the culture
  5. return the lid to the culture quickly to prevent contamination
  6. lift the lid at an angle to prevent contamination-inoculating loop gently over the agar
  7. flame equip. to kill contaminent-seal partially
  8. place discs of diff antiseptics into lawn
  9. incubate at 25 deg.
• incubate ar 25 degree :

  • allows for quick bacterial growth without producing harmful bacteria
• Petri dish is partially sealed :

  • to allow oxygen to still enter for aerobic respiration
  • don't want to grow anaerobic bacteria - don’t seal fully
• 2 types of ways to spread bacteria :

  • streak plate = individual colonies
  • Lawn plate = even spread of bacteria/ growth
• to determine how successful an antimicrobial substance is at killing bacteria, we look at the area of killed bacteria or area where bacteria did not grow around the soaked disk
• Zone of inhibition = the area around the inoculated agar where no growth occurs
• zone of inhibition can be caluculated using area=Pi r^2
• the larger the zone of inhibition, the most effective the antibiotic is at killing bacteria
• must use a control disc - water
• aseptic techniques are used to ensure the microbial being investigated doesn't escape or become contaminated with another unwanted and possibly pathogenic microbe
• Aseptic Techniques (book) :

  • wash hands thoroughly
  • no food or drinks allowed in the lab
  • Disinfect work surfaces with disinfectant or alcohol
  • don't allow the growth of microbial/ microorganisms at body temp
  • use flamed loop/ sterile swabs when transferring cultures
  • wear gloves and goggles
  • flame culture bottle necks to prevent contamination
  • sterilise or dispose of all used equipment
  • use a Bunsun burner (hair tied back)
  • only remove petri dish lid when necessary
• the antibiotic will diffuse outwards from each papeer disc so that a gradient of antibiotics form
• the antibioti is most concentrated where the paper disc is located
• if the bacteria being investigated is vulnerable to an antibiotic ,then a clear area will be visible around the disc
• zone of inhibition ends when the concentration of antibiotic reaches a level that the bacteria are no longer susceptible to - more effective antibiotic require a lower concentration to kill bacteria and so will provide a larger zone of inhibition
• if a bacteria is completely resistant to an antibiotic, then there will be no clear zone around the particular disc
• the minimum inhibitory concentration (MIC) = the lowest concentration of a substance that will inhibit the growth of a microorganism - dosage is carefully controlled when antibiotics are used to treat bacterial infections