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The Composition of Bacteriological Media

The Composition of Bacteriological Media. Lab 4. Media preparation.

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The Composition of Bacteriological Media

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  1. The Composition of Bacteriological Media Lab 4

  2. Media preparation • This is carried out in the media room. It requires sterile glassware (prepared by autoclaving) and sterile plasticware, a supply of dehydrated ready to use media and supplements and fresh blood purchased from commercial suppliers. • Materials are weighed in a fume hood to prevent exposure to dusts and then dissolved in water to make a suspension. This is then autoclaved in the media room. • Plates are poured in a simple plastic hood to protect them from atmospheric contamination. The hood is maintained free from bacteria by an ultraviolet light switched on when not in use. • Poured plates are allowed to cool and then dried in the incubator at 37°C and stored (at 4°C) until required.

  3. Bacterial cultivation • Bacteria are cultivated in artificial media which contain the nutrients essential for their growth using sterile techniques. The sterile techniques used are essential because: • (a) the environment is full of bacteria which can grow in the media used and which would contaminate cultures of specific bacterial species • (b) many of the species used are pathogens and cause disease in man. • Bacteriological media range in composition from solutions of inorganic salts to the complex media prepared from digested meat, enriched with blood or serum.

  4. Types of media • Liquid media • These allow uniform growth of the organism throughout the solution and are usually dispensed in tubes or screw-cap bottles. Tubes are usually closed with a sterile cotton wool plug. • Solid media • Solid media are usually dispensed as slopes or plates and are usually made by the addition of agar to liquid media before autoclaving.

  5. Solid media – simple media • E.g. nutrient media • Agar is added at 1.5-3% to solidify media • Media commonly dispensed into petri dishes (plates) • Bacteria grow as colonies • Each colony arises from the growth of a single bacterial cell or clump of cells (colony forming unit, CFU) • Clinical samples inoculated on to agar plates to determine the number of different bacteria present and to isolate single colonies for pure culture and subsequent analysis

  6. Enriched media • Blood agar. Agar may be supplemented with 7-10% blood to provide a rich medium. This also detects the production of haemolysis producing clear areas around the colonies. • 10% serum agar. Ten per cent serum is added to encourage the growth of some organisms. • Dorset egg medium. A number of organisms such as Mycobacterium require a lipid and protein rich medium provided by using coagulated egg proteins. • Other enriched media. A number of enriched media exist for the growth of specific bacterial species. The additives include cholesterol (for mycoplasmas) sugars, vitamins, amino acids and lipids.

  7. Selective media • Some media contain substances which inhibit the growth of some species of bacteria and allow others to grow. Examples of this type of medium are MacConkey agar, Desoxycholate Citrate Agar (D.C.A.), Brilliant Green Agar, Salmonella Shigella agar (SS agar) which contain inhibitory substances such as bile salts or citrate which allow the growth of enteric bacteria and often inhibit the growth of others. • Selenite broth inhibit the growth of many bacteria other than Salmonella species and are selective for them.

  8. Differential media • Bacterial species differ in their ability to metabolise carbohydrates and other chemical compounds and these differences are used in the identification of bacterial species. Media in which specific substrates are incorporated accompanied by an indicator of some type which changes in the presence of metabolites from the substrate are known as differential media and are used to distinguish colonies of species with the appropriate biochemical property. • Some commonly used differential media include MacConkey agar in which lactose and neutral red are incorporated and Brilliant Green agar and Salmonella Shigella agar (SS) with phenol red and brilliant green as indicators, Desoxycholate Citrate Agar (DCA) contains lactose and neutral red.

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