Culturing microorganisms in the labratory

Culturing microorganims

• They need food, the right temperature, oxygen and pH
• Food = nutrient medium
• Either in liquid form (broth) or solid form (agar)
• Enriched nutrient medium allows a small number of organisms to multiply rapidly
• The nutrient medium must be kept sterile until its ready for use
• Aseptic techniques are very important
• Once the agar or broth is prepared, the bacteria has to be added via inoculation

Inoculating broth

1. Make a suspension of the bacteria to be grown
2. Mix a known volume with the sterile nutrient broth in the flask
3. Stopper the flask with cotton wool to prevent contamination
4. Incubate at a suitable temperature, shaking regularly to aerate the broth

Inoculating agar

1. A wire inoculating loop must be sterilised by being held in a bunsen burner flame until it glows orange
2. Dip the sterilised loop in the bacteria suspension
3. Remove the lid of the petri dish and make a zigzag streak across the surface of the agar
4. Replace the lid of the petri dish and tape it down without sealing it completely so the bacteria have access to oxygen
5. Incubate at a suitable temperature

The growth of bacterial colonies

• They reproduce rapidly --> asexually dividing every minutes in optimum conditions
• There are four stages to a growth curve:
  1. Lag phase --> bacteria are adapting to their new environment --> they're growing and synthesising enzymes bit mot reproducing at their maximum rate
  2. Log or Exponential phase --> rate of reproduction close to or at its theoretical maximum
  3. Stationary phase --> number of cells formed by binary fission equals the number of cells dying
  4. Decline or Death phase --> reproduction has almost ceased and the death rate of cells is increasing
• Limiting factors that prevent the exponential growth of the bacteria
  • Nutrients available - initially there was plenty of food but as cell numbers increase, the food decreases more rapidly
  • Oxygen levels - as the population rises, so does the demand for oxygen 
  • Temperature - high or low temperatures effect the enzyme-controlled reactions within the cells
  • Build-up of waste - as numbers rise, the build-up of toxic waste does too and can inhibit further growth or be poisonous
  • Change in pH - carbon dioxide produced by the respiration of the cells lowers the pH until it effects enzyme-controlled reactions and inhibits population growth