Continuous and Batch Culture

1. Continuous and Batch Culture

2. Culture media: general • For general use in isolating and growing micro-organisms, fairly crude media may be used • These may be based on meat extract, blood, milk, yeast extract, or other nutrient-rich sources, and usually contain a wide range of nutrients such as sugars, amino acids and vitamins. • Nutrient broth and nutrient agar are good examples

3. Culture media: synthetic • For more detailed study of microbial growth and metabolism, microbiologists usually use synthetic media of precisely known composition • Because the contents of synthetic culture media are known and controlled, valid comparisons can be made between the growth of different species, or of the same species under different conditions

4. Culture media: minimal • Biochemists studying metabolic pathways in micro-organisms can use minimal media to supply or withhold specific nutrients and observe the effects • Minimal media provide the bare minimum of nutrients required by the species under investigation: for many heterotrophs, this may consist of the necessary mineral ions, with perhaps a single sugar as the only carbon and energy source • Use of minimal media can quickly establish which sugars a particular organism can and cannot metabolise. Other types may not grow in minimal medium unless specific amino acids or growth factors are added, thus giving important information about their synthetic capabilities.

5. Culture media: selective • The nutrient content of the medium, and its pH, can be adjusted to favour the growth of the particular organism being studied and exclude others. Media like this are called selective or narrow-spectrum media • For example to isolate nitrogen fixing bacteria from soil you would inoculate a small sample of soil into a synthetic medium containing sugars but no nitrogen compounds; nitrogen fixers such as Azotobacter will grow, but bacteria unable to fix nitrogen will not

6. Culture media: selective and indicator • MacConkey agar is commonly used to differentiate between various gram-negative rod-shaped organisms. MacConkey agar is both a selective and an indicator medium, in that • it contains bile salts and favours the growth of the gram-negative rods which inhabit the gut • it can differentiate between lactose fermenters and non-fermenters. Many facultative anaerobes in the intestine are lactose fermenters (e.g., Escherichia coli), while several well-known pathogens are unable to ferment lactose (e.g., Shigella, Salmonella).

7. Culture media: solid or liquid • Solid media usually based on agar, a seaweed extract containing polysaccharides • Nutrients are added to molten agar, and the mixture sets into a jelly on which micro-organisms can grow • Because most micro-organisms are unable to digest agar, it remains solid even when they are growing on the nutrients it contains • Liquid media include various broths or synthetic media • Microbiologists may choose to use solid media or liquid media depending on the purpose of a particular investigation • In the table that follows, state whether you would choose a solid or a liquid medium in each case, and give your reasons.

8. Culture media: solid or liquid Solid Separate colonies allow isolation Total cell count only possible in liquid Liquid Liquid for culture, solid for counting Viable cell count by counting colonies Separate colonies allow measurement Solid Periodic sampling and separation from medium Liquid Bulk growth in liquid (in 3 dimensions) Liquid Safer / more stable Solid Liquid Formation of bubbles

9. Pouring plates • Solid media are usually used in Petri dishes; these may be glass (sterilised by autoclaving), or the disposable plastic variety supplied in packs which are sterile until opened. • Medium is made up in liquid form, and may be dispensed into small screw-top bottles before being autoclaved. • After autoclaving, the medium can be kept liquid until required by placing the bottle in a water bath at 60oC. • Before pouring the plate, the agar should be cooled to 50 - 55oC. • To avoid contamination, the plate should be poured quickly with the lid of the Petri dish held ajar. • While the agar in the dish is still hot, the lid may safely be left ajar as the outflow of steam will prevent airborne organisms from entering: this helps to reduce problems of condensation on the Petri dish lid. • After cooling, the closed dish is kept upside down. If condensation is still a problem, the dish can be kept overnight in a warm incubator to remove it.

10. Culture vessels for liquid media Any piece of glassware that can be sterilised and plugged can be used for growing micro-organisms, but for serious study a fermenter or bioreactor of some kind is commonly used:

11. Culture vessels for liquid media • A fermenter of this kind has a number of advantages over a plain culture tube or bottle: • air can be passed in and out through lines fitted with filters: this provides oxygen for aerobes, and also stirs the culture, improving contact between micro-organisms and nutrients. A sparger on the air inlet produces a large number of very tiny bubbles, increasing the total surface area of air in contact with water and thus the rate of entry of oxygen into solution. • a magnetic stirrer can be used as well, further improving mixing of the culture. • syringes can be fitted for inoculation of the medium with micro-organisms, for the addition of materials during the growth of the culture, or for the taking of samples for analysis or population estimation. • In addition, the culture vessel is usually maintained at constant temperature, either in an incubator or in a thermostatically controlled water bath.

12. Continuous and batch culture • The fermenter shown here is set up for a batch culture: that is, it is ready to be inoculated with a culture of micro-organisms, which will multiply, changing the conditions in the medium by using up the nutrients and producing their own waste products. Eventually conditions in the culture will become too unfavourable for the organisms to survive, and the population will die out. • Batch culture is suitable for most school-based experiments on microbial growth, and is used industrially in many processes that harvest the secondary metabolites of micro-organisms (such as antibiotics). • Secondary metabolites are produced by metabolic processes which are not essential to the organism's short-term survival, and are often not produced in large amounts when the organism is growing at its fastest; industrial production is therefore most efficient if the organisms are allowed to reach maximum population size and stop growing before the product is harvested

13. Continuous and batch culture • Continuous culture, by contrast, aims to keep a culture growing indefinitely. This can be done if: • fresh nutrients are continually supplied • accumulated cells and waste products are removed at the same rate • conditions such as temperature and pH are kept at their optimum values. • A culture vessel designed for continuous culture is called a chemostat: In addition to the features shown, the culture vessel would probably be fitted with temperature and pH probes for monitoring growth conditions. Continuous culture is important in industrial processes that harvest the primary metabolites of micro-organisms as their products. (Primary metabolites are produced in greatest quantities when the organisms are growing at their fastest rate).