Microbiology

Microbiology

1. Methods of measuring growth

   • serial dilution
     • known volume of known dilution
     • insufficient dilution means colonies clumped together, inaccurate results
     • the level of dilution which is used is the dilution which has clear, countable colonies
     • A sample is streaked on to an agar plate and incubated at 25 degrees Celsius for 24 hours to discourage pathogen growth
   • viable count
     • only counts living cells which are capable of dividing and reproducing
     • This process assumes one bacteria will reproduce to form one colony
   • total count
     • includes both live and dead cells
       • haemocytometry
         • uses a gridded square to count colonies in an area
       • turbiditry
         • measures the cloudiness of the solution to determine the number of colonies
           • Measured using a colourimeter
         • The more organisms there are the cloudier the solution is, the less light is transmitted through.

2. Fermentation

   • continuous fermentation
     • nutrients added at sufficient rate to sustain exponential phase
       • nutrients are added at same rate as product is harvested
     • main aim is to keep the process going and keep it from entering death phase
   • Batch Fermentation
     • all nutrients are added at the beginning
     • microbes grow rapidly, nutrients don't need to be supplied during the process
     • fermentation is able to take place at lower temperatures and therefore the production cost of such things as penicillin are cheaper
     • the optimum conditions and no competition result in maximum efficiency
     • process continues through to the stationary phase where the product is harvested.
     • this process produces penicillin which is a secondary metabolite.
     • temperature monitoring and controlling is required to maintain optimum conditions
     • Producing Penicillin
       • Penicillin is produced after the growth phase as glucose becomes depleted
       • Mycellium is removed by filtration
       • The antibiotic Penicillin is then purified from the culture medium

3. Aseptic Technique

   • Lift lid just enough to allow inoculating loop in to spread bacteria medium
   • flame innoculating loop until red hot
   • flame mouth of bottle for 2 to 3 seconds, passing it over a roaring blue flame
   • use little finger technique to remove lid of culture bottle, keep hold of lid throughout aseptic technique to prevent contamination
   • use equipment such as sterile inoculating loops to prevent contamination
   • all equipment and media must be sterilised before use

4. classifying bacteria

   • cell wall protects against bursting of cell wall(lysis) due to osmosis
   • cross linking provides strength
   • cell wall
     • consists of a mixture of polysaccharide and polypeptides known as peptidoglycan
   • gram positive bacteria
     • lacks lipopolysaccharides in the cell wall
     • more susceptible to antibiotics
     • retains crystal violet and stains purple
   • gram negative bacteria
     • peptidoglycan supplemented with lipopolysaccharides
     • they have more chemically complex cell walls
     • counterstains red
     • more resistant to antibiotics

5. Bacterial population growth curve

   • Stationary Phase
     • This is the phase where the birth rate is equal to the death rate. The nutrients begin to run out and the competition for nutrients increases.
   • Death Phase
     • In this phase cells are dying faster than they are being produced
   • Exponential Phase
     • Cells are dividing rapidly during this phase, the population doubles per unit time. No limiting factors such as nutrients
   • Lag Phase
     • Bacteria are synthesising enzymes and replicating DNA ready for the next phase