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Culturing bacteria in the laboratory Nishad Matange Raghav Ranjan Jeet kalia Bacteria do fantastic things! Disease Food technology Drug industry Bioremediation Culturing bacteria in the laboratory Why culture bacteria in the laboratory? What do we need to successfully culture bacteria in the laboratory? • Medium • Water • Salts-osmolarity and nutrients • Source of carbon-sugars, alcohols • Source of nitrogen-amino acids, ammonium salts • Source of phosphorus-phosphates, nucleotides • pH • Temperature • Aeration Culturing bacteria in the laboratory Why culture bacteria in the laboratory? What do we need in order to successfully culture bacteria in the laboratory? • Medium • Water • Salts-osmolarity and nutrients • Source of carbon-sugars, alcohols • Source of nitrogen-amino acids, ammonium salts • Source of phosphorus-phosphates, nucleotides • pH • Temperature • Aeration Culturing bacteria in the laboratory Why culture bacteria in the laboratory? What do we need in order to successfully culture bacteria in the laboratory? • Medium • Water • Salts-osmolarity and nutrients • Source of carbon-sugars, alcohols • Source of nitrogen-amino acids, ammonium salts • Source of phosphorus-phosphates, nucleotides • pH • Temperature • Aeration Not all bacteria can be easily cultured in the laboratory!!!! Culturing bacteria in the laboratory Culture medium Consistency Chemical composition Nutrient richness Selective • Allows growth of only certain species of bacteria • MacConkey medium Defined/Synthetic • Chemical composition known • M9 medium Indicator/Differential • Indicates certain biochemical properties of the cultured bacterium • Blood agar Complex • Chemical composition known unknown • Luria-Bertani medium Solid • Gels made of agar/agarose Liquid Purpose Selectivity Minimal • Bare minimum requirement provided, usually as inorganic salts • M9 medium Rich • Replete with nutrients • Terrific broth Storage • Used to store bacterial samples • Glycerol-25-40% Transport • Used when specimen needs to be transported before it can be cultured • Stuart medium Today’s menu… Luria-Bertani medium/Nutrient broth/Lennox medium/Lysogeny broth (LB) Per litre • 10 g tryptone • 5 g yeast/meat extract • 10 g NaCl TryptoneTrypsin hydrolysate of casein protein Yeast extractCell-free extract of yeast/meat Liquid and solid LB medium (PC: Wikipedia) Today’s menu… Luria-Bertani medium/Nutrient broth/Lennox medium/Lysogeny broth (LB) Per litre • 10 g tryptone • 5 g yeast/meat extract • 10 g NaCl TryptoneTrypsin hydrolysate of casein protein Yeast extractCell-free extract of yeast/meat Liquid and solid LB medium (PC: Wikipedia) What kind of medium is LB? • Synthetic/Complex • Minimal/Rich • Selective/Differential Today’s menu… Luria-Bertani medium/Nutrient broth/Lennox medium/Lysogeny broth (LB) Per litre • 10 g tryptone • 5 g yeast/meat extract • 10 g NaCl TryptoneTrypsin hydrolysate of casein protein Yeast extractCell-free extract of yeast/meat Liquid and solid LB medium (PC: Wikipedia) Should we use solid or liquid medium? Ans: Depends on the purpose. If you want: • High biomass use liquid • Isolation/selection of specific bacteria use solid Making LB-agar (NA-agar) • Mix appropriate amounts of desiccated LB medium power (or constituents) in water • Heat to dissolve LB and then add 1.5-2.0 % agar powder • Autoclave the suspension at 121 °C for 15-20 mins at 15 psi • This sterilises the medium and melts the agar • Pour molten agar into ‘petri plates’ under sterile conditions and allow it to set Isolating bacteria • Why do we need to isolate bacteria? • How do we isolate bacteria? Bacteria live in communities • Bacteria are usually found in large multi-species communities • In their natural environment they coexist with other bacteria, fungi, protozoans and viruses • Specific bacteria must be isolated from the community in order to study their properties Scanning electron micrograph of gut microbiota pseudo-coloured to show the consortium of gut microbes PC: Martin Oeggerli • For instance, a specific species that causes disease or produces an antibiotic Isolating bacteria • Why do we need to isolate bacteria? • How do we isolate bacteria? • On solid medium bacteria form discrete colonies • Basic principle of all isolation methods-dilution such that each colony comes from a single parent bacterium • Dilution can be achieved by mechanical separation on the surface of the agar plate or by diluting the inoculum in a large volume • Bacteria that form a single colony are all clones of one another! Isolating bacteria I. Streak plate method • Uses the principle of mechanical separation on the surface of the agar • Convenient and quick • Colonies form on the surface of the agar Colony characteristics help distinguish between organisms II. Pour plate method • Uses the principle dilution of inoculum in a large volume • Inoculum added to molten agar and then poured onto the surface of a nutrient agar plate • Colonies formed both on the surface and in the bulk of the medium
