Microbiology

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Classification

  • The three classification of bacteria you need to know.
  • Can also be classified by Gram staining:
    -All bacteria has polysaccharide and polypeptide in there membrane, Gram negative bacteria also have lipopolysaccharide which means penicillin and lysozome don't affect it.
    -Gram staining produces a violet colour for positive and red for negative.
    -Gram negative do not retain the dye and only are shown by the counterstain (red). 
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Conditions For Culturing Bacteria

  • Nutrients - On a agar plate with the glucose and nitrogen containing material for growth.
  • Temperature - The range of 25-45 degrees provides a favourable temperature.
  • pH - Most bacteria prefer slightly alkali conditions, and fungi slightly acidic.
  • Oxygen - Many require O2 for metabolism, some prefer not to, and some cannot survive in it.
    -Obligate aerobes: Respire aerobically and require O2.
    -Facultative aerobes: Prefer O2, but can survive without it.
    -Obligate anaerobes: Cannot survive in presence of O2. 
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Principles Of Aseptic Technique

  • Sterilise all apparatus and media before intitial contamination.
  • Sterilise the work surface with work surface with disinfectant.
  • Hold the culture bottle in the little finger to prevent not putting it on the workplace.
  • Flame the mouth of the bottle for 2-3 seconds.
  • Inoculating loop requires flaming initially.
  • Lift the lid only slightly.
  • Secure the lid with a little tape, do not make anaerobic.
    -This produces harmful anaerobes.
  • Incubate at 25 degrees C.
    -If incubated at 37 degrees C then higher chance of human pathogens.
  • To sterilise afterwards use an autoclave, high temperatures and pressure kills any bacteria. 
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Methods of Measuring Growth

  • Rough estimate can be made with measuring the diameter of the colony, finding the rate is called the dilution plating technique.
  • Serial dilution allows for a colony to be diluted into countable amounts.
    -1cm3 of the solution to be added to added to 9cm3 of sterile saline, doing this several times gives a small sample which can be added to an agar sample and produces countable colonies.
  • More accurate method which determines total number of cells, but not just a viable count.
  • A colorometer measures the turbidity of a solution, which is then is used to work out total count of cells. 
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Batch Fermentation

  • Rapid production of cells, low temperature, and relatively cheap to do on a large scale.
  • The following describes a batch fermenter:
    -A pure culture is needed in a sterile vessel.
    -Must have suitable conditions and a sterile medium.
    -Must be protected from contamination and constantly aerated.
    -Mixing will improve contact with nutrients for growth.
    -Temperature and pH monitors are needed, and a cooling water jacket.
    -Air inlets use spargers to force aeration
  • In the production of penicillin:
    -Fermenter is sterile and cultured with Penicillium; which then grows.
    -Takes 30 hours for production of penicillin to occur as it is a secondary metabolite.
    -The penicillin is extracted after 6 days.
    -This is batch culture as all the nutrients are added at the begining with no input. 
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Metabolite Graphs

(http://dc235.4shared.com/doc/ExLof9an/preview_html_76978fca.png)

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