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Microbial Methodology

Microbial Methodology. by E. Börje Lindström. This learning object has been funded by the European Commissions FP6 BioMinE project. What characterize Microbiology?. The art of the objects. -very small (microscopic size). The characteristics of the objects:. - rapid growth.

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Microbial Methodology

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  1. Microbial Methodology by E. Börje Lindström This learning object has been funded by the European Commissions FP6 BioMinE project

  2. What characterize Microbiology? • The art of the objects -very small (microscopic size) • The characteristics of the objects: - rapid growth - asexual growth - clone • Methodology: - indirect - statistical - cultivation - aseptic/ sterile

  3. Cultivation Nutrition varies: - amount and chemical form important Main nutrients: • energy • carbon • nitrogen • sulphur • phosphor • hydrogen • oxygen m.o. The nutrient requirement = k/ the biosynthetic capacity

  4. Classification By energy source: • phototropic • chemotropic By carbon source: • autotrophy • heterotrophic

  5. Growth factors Three main classes: • amino acids - building blocks e.g. for proteins • purins/ pyrimidines - building blocks e.g. for nucleic acids • vitamins: - building blocks for co-enzymes

  6. Environmental conditions • pH • every bacterium has a rather narrow pH range for optimal growth acidophiles neutrophiles alkalogens activity pH 0 7 14 • molecular oxygen (O2) - strict aerobes - strict anaerobes - facultative • light - fototrophs

  7. Environmental conditions, cont. • temperature moderate thermophiles psycrophiles extreme thermophiles mesophiles activity Temp, C 0 50 100

  8. Different growth media Synthetic -all components are chemically known Complex - some components are unknown Minimal - a synthetic medium with the fewest nutrients which allows growth Selective - contains a substance which do not allow growth of some m.o. Enriched - contains nutrients which is not needed but increase growth Differentiated - contains a substance that can cause differences in the colony morphology Solidified - a nutrient solution can be solidified by adding e.g. agar, gelatine etc.

  9. Some examples of complex media • Beef extract: - a water solution of carbohydrates, organic N, vitamins and metal salts • Peptones: - hydrolysis (acid or enzymatic) of proteinacious material, containing organic N • Yeast extract: - water extract of yeast cells, containing e.g. B-vitamins

  10. Preparation of a growth medium • Use stock solutions • Add the ingredients according to the recipe • Add the ingredients during stirring • adjust the pH before autoclaving Addition of nutrients stirring

  11. Sterilization • Sterilization – killing of all m.o. Definitions: • Disinfections – reducing m.o. Kinetics of the killing • almost always exponential Let N = amount of bacteria t = time -the change of bacteria can then be written -reorganize -integrate

  12. Sterilization, cont -gives log N – log N0= -kt -which can be plotted as follows logN logN slope = -k = killing rate constant N0 N0 t t One-hit curve Multiple- hit curve

  13. Factors that effects the killing • dose (effects the slope) • temperature (heat) • concentration (concentration) • intensity (irradiation) • time • the starting population (N0) • the growth state of the bacteria

  14. Methods for sterilization • Physical: • heat • irradiation • ultra sound • micro waves • filtration • Chemical: - varying compounds

  15. Heat - most important and used for everything that can resist heat • pasteurisation - a mild method; 71-72 °C –15 sec • boiling - not safe • autoclaving, wet - 121 °C – 15 min • owen, dry - 160 – 180 °C – 2 hr

  16. Irradiation • UV • bactericidal at 240-280 nm • target DNA • air sterilization • low penetration • X-ray - ionisation • g- beams - ionisation

  17. Filtration - using membrane filters of different poor sizes • < 0,45 mm for bacteria • < 0,01 mm for viruses Chemical • Phenols - dissolve fat; denature proteins • Alcohols - dissolve fat; denature proteins • Jodopax - I2 + KI + ethanol + detergent • Ethylenoxid - alkylating

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