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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 - asexual growth - clone • Methodology: - indirect - statistical - cultivation - aseptic/ sterile Cultivation Nutrition varies: Main nutrients: - amount and chemical form important • energy • carbon • nitrogen • sulphur m.o. • phosphor • hydrogen • oxygen The nutrient requirement = k/ the biosynthetic capacity Classification By energy source: • phototropic • chemotropic By carbon source: • autotrophy • heterotrophic 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 Environmental conditions • pH -every bacterium has a rather narrow pH range for optimal growth acidophiles neutrophiles alkalogens pH 0 • molecular oxygen (O2) 7 14 - strict aerobes - strict anaerobes - facultative • light - fototrophs Environmental conditions, cont. • temperature psycrophiles moderate thermophiles mesophiles extreme thermophiles Temp, C 0 50 100 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. 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 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 Sterilization Definitions: • Sterilization – killing of all m.o. • Disinfections – reducing m.o. Kinetics of the killing • almost always exponential Let N = amount of bacteria t = time dN kN dt dN kdt N -the change of bacteria can then be written -reorganize -integrate Sterilization, cont N t dN N0 dt 0 kdt -gives log N – log N0= -kt -which can be plotted as follows logN logN slope = -k = killing rate constant N0 N0 t One-hit curve t Multiple- hit curve 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 Methods for sterilization • Physical: - heat - irradiation - ultra sound - micro waves - filtration • Chemical: - varying compounds 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 Irradiation • UV - bactericidal at 240-280 nm - target DNA - air sterilization - low penetration • X-ray - ionisation • g- beams - ionisation - using membrane filters of different poor sizes Filtration • < 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
