Culturing bacteria in the laboratory

Nishad Matange

Raghav Ranjan

Jeet kalia

Bacteria do fantastic things!

Food technology

Drug industry

Bioremediation

Disease

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

Chemical composition

Defined/Synthetic • Chemical composition

known • M9 medium

Complex • Chemical composition

known unknown • Luria-Bertani medium

Nutrient richness

Minimal • Bare minimum

requirement provided, usually as inorganic salts

• M9 medium

Rich • Replete with nutrients • Terrific broth

Selective • Allows growth of only

certain species of bacteria

• MacConkey medium

Selectivity

Indicator/Differential • Indicates certain

biochemical properties of the cultured bacterium

• Blood agar

Transport • Used when specimen

needs to be transported before it can be cultured

• Stuart medium

Storage • Used to store bacterial

samples • Glycerol-25-40%

Purpose Consistency

Solid • Gels made of

agar/agarose

Liquid

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

Tryptone- Trypsin hydrolysate of casein protein Yeast extract- Cell-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

Tryptone- Trypsin hydrolysate of casein protein Yeast extract- Cell-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

Tryptone- Trypsin hydrolysate of casein protein Yeast extract- Cell-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

• For instance, a specific species that causes disease or produces an antibiotic

Scanning electron micrograph of gut microbiota pseudo-coloured to show the consortium of gut microbes PC: Martin Oeggerli

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 II. Pour 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

• 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

Colony characteristics help distinguish between organisms