MICROBIOLOGY

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  • Created by: ava.scott
  • Created on: 17-02-15 14:25

SHAPES

  • Round- coccus
  • Bacillus- rod shaped
  • Spiral - spirrilium

Shape is used alongside metabolic reactions for classification. 

Shape is determined by the rigid cell wall which has a unique structure.

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Gram Staining

Crystal Violet (fixed by Lugol's iodine') >> washed using acetyl-alcohol >> Safranin dye

Used to define whether a bacterium can retain cystal violet (positive) or cannot (negative).

Gram positive

  • Thick peptidogylcan wall
  • Membrane

A gram negative bacterium has a more complex cell wall structure.

  • Thick lipopolysaccahride layer 
  • Thin peptidoglycan layer
  • membrane

Penicillin works by interfering with peptidoglycan layer, causing it to disintegrate. This is why gram negative bacteria are resistant to antiobiotics.

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Conditions for culture

Organisms vary in their requirements and usually grow over a range of tempertaures and optimum conditions.

They require:

Nutrients- agar will have carbon (glucose) nitrogen (organic or inorganic),  and growth factors such as vitamins and mineral salts.

SUITABLE Temperature- the range is usually 25-45 centigrade.

pH- Bacteria like slightly alkaline conditions, whereas fungi like slightly acidic

Oxygen- required for metabolism and respiration.

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ASEPTIC TECHNIQUE

Two main purposes:

  • Prevent contamination of the culture of unwanted organisms.
  • Prevents contamination of personnel and environment by the cultured bacteria.

Equipment must be sterilised using appropriate methods:

  • Heat- e.g. autoclave at 121 centigrade for 15 minutes.
  • Heat-Innoculating loop over bunsen burner flame.
  • Irradiation- plastics can be irraidated using gamma rays
  • Items must be protected from contamination after sterilisation.
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Measuring growth

Direct count

  • Total counts including both dead and living cells.

Viable count

  • Only counts living cells
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Viable count: methods

  • Known volume of organisms is added to a agar plate, incubated and the colonies counted.
  • It is assumed that one cell gives rise to one colony.
  • Doesn't give allowance for clumping of cells, so may give an underestimate of numbers.

FIRST: The original culture must be diluted by 10 fold steps, serial dilution, in order to provide a final number within the countable range.

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Direct cell count: methods

Measure colony clumps diameter at regular intervals to measure growth rate.

Haemocytometer

Uses a specialised microscope slide and gives total count.

Turbidimetry

A colorimeter measures teh cloudiness of the sample and compares it to a graph with cloudiness and known cell numbers.

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BATCH CULTURE FERMENTTION 5 things

Requirements:

  • Pure culture of the organism, so that the product is pure when harvested.
  • Suitable conditions for maximum efficiency.
  • Sterilised vessel and sterile medium.
  • Filters to maintain purity.
  • Aseptic technique to maintain purity.
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Fermenter design

Aeration

  • Forced aeration needed for maximum growth of aerobes.
  • Aeration can also help mix the culture to improve contact with nutrients.
  • air inlets may use spargers to improve aeration.
  • Or a mixer can be used.

Temp

  • Temperature monitoring and contol are require to maintain constant conditions.
  • Water jackets removes excess heat.
  • Comercially sophisticated monitors improve the control of temp and pH.
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Penicillin

The fungus Penicillin notatum is grown in a batch culture.

The antibiotic is produced after the growth phase, when glucose is depleted. This reflecrts the need for the organism to reduce competition when food sources are depleted,

The mycleium is removed by filtration of the culture and the antibiotic purified from the residual liquid.

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LOG PHASE

  • genes are switched on
  • synthesis of enzymes
  • replicating DNA
  • Cells growth and swell with products of digestion
  • getting used to the medium
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DEATH PHASE

  • Waste products are toxic 
  • Nutrients are used up
  • Intra-specific competition increases 
  • Carrying capacity has been exceeded
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