- 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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- 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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