Culturing microrganisms in a lab

What is biotechnology?

Applying biological organisms or enzymes to the synthesis, breakdown or transformation of material in the service of people

What is mixed culture?

Controlled growth of a mixture of microorganisms

What is closed culture?

When growth occurs in a vessel that’s isolated from the external environment - extra nutrients aren’t added and waste products aren’t removed from the vessel during growth

What is a culture?

A growth of microorganisms that has been grown under controlled conditions

What is a pure culture?

A population of one type of microorganism

What is a nutrient medium?

Food provided to microorganisms, can be in the form of a liquid broth or solid agar. Must be kept sterile until it is ready for use, so aseptic techniques are important and minimum the risk of contamination by microorganisms

What is inoculation?

Inoculating is putting the microrganism you want to grow.

Liquid broth inoculation is when a bacteria suspension is made, suspension is mixed with nutrient broth in a flask, add cotton wool to the top of the flask to prevent contamination, incubate and shake regularly to aerate the broth

What is the process to inoculate agar? (1)

Make a suspension of the bacteria, sterilise innoculating loop used to transfer bacteria to the agar, by heating it to red hot in a Bunsen burner and leave to cool

Don’t touch the innoculating loop or let it touch any surfaces or it will be contaminated

What is the process of inoculating agar? (2)

Allow the sterilising loop to cool. Dip it into the bacteria suspension you want grow, then use to make zig zag streaks across the agar surface. Place the inoculating loop almost horizontal and move gently

What is the process of innoculating agar? (3)

Secure the lid of your petri dish with tape to stop microrganisms from the air contaminating your culture and to stop airborne microorganisms contaminating your culture and stopping microbe from your culture escaping into the air. Don’t seal all the way to let O2 in. Place in incubator

Why is preventing contamination of cultures important?

Competition with culture microrganisms reduces yields, may produce toxic chemicals, may destroy microorganisms, may cause spoilage of products, expensive if whole culture needs to be disposed of

Examples of aseptic techniques in industry?

Washing, disinfecting and steam cleaning the fermenter regularly, fermenter surfaces are made of a polished stainless steel which is easy to clean, sterilising nutrient mediums, fine filters on all inlets and outlets on the fermenter to prevent microbe entry

How are aseptic techniques used in the lab?

All apparatus for carrying/moving microrganisms sterilised before and after use, culture kept closed when possible to prevent contamination from the air

What happens in the lag phase?

Bacteria are adapting to their new environment. They are growing, synthesising the enzymes they need and are not reproducing at their maximum rate

What happens in the exponential phase?

When the rate of bacterial reproduction is close to or at its theoretical maximum

What is the stationary phase?

Occurs when the total growth rate is zero - the number of new cells forming by binary fission is cancelled out by the number of cells dying

What is the death stage?

Occurs when reproduction has almost ceased and the death rate of cells is increasing

What are some limiting factors (nutrients) which prevent exponential growth?

Nutrients available - initially there is plenty of food, but as the number of microorganisms multiplies exponentially it is used up. Nutrient level will become insufficient to support further growth and reproduction unless more nutrients are added

How do oxygen levels act as a limiting factor for exponential growth?

As the population rises, so does the demand for respiratory oxygen, so oxygen levels can become limiting

How does temperature act as a limiting factor for exponential growth?

Enzyme controlled reactions within microrganisms are affected by the temp of the culture medium. For most bacteria, a low temp slows down growth and reproduction, and a higher temp speeds it up. If temp gets too high it will denature enzymes and kill microrganisms

How does build up of waste act as a limiting factor for exponential growth?

As bacterial numbers rise, the build up of toxic material may inhibit further growth and can even poison and kill the culture

How does change in pH act as a limiting factor for exponential growth?

As CO2 produced by the respiration of the bacterial cells increases, the pH of the culture falls until a point where the low pH affects enzyme activity and inhibits population growth

What elements do bacteria need to grow?

N for amino acids, P for ATP, O for respiration, C and H, S for proteins

How does a higher temp result in a quicker rate of bacteria growth?

Molecules have more energy, enzymes closer to optimum temp, faster enzyme activity, more competition for nutrients earlier on, resources used up more rapidly, toxic metabolites produced quicker, mineral availability becomes limiting factor