Culturing microorganisms

1. Microorganisms are grown, or cultured, in the laboratory so that they can be studied, identified and used to test substances such as antibiotics and antiseptics.
2. They are grown on a culture medium that supplies everything they need to grow.
   1. Carbohydrates, such as glucose, provide a source of energy for respiration.
   2. Proteins or other nitrogen compounds provide the raw material for making new proteins.
   3. Mineral ions and vitamins are needed for healthy growth.
   4. Water is needed for all the chemical reactions inside the cells.
3. The medium can be a liquid called nutrient broth, or a jelly called nutrient agar, which is melted, poured into a Petri dish and left to set.
4. Bacteria reproduce by splitting in two, and in good conditions some bacteria divide as often as once every $20$ minutes.
5. A single bacterium placed on agar divides again and again until a visible colony of millions of identical cells forms on the surface.
6. When a liquid culture is spread evenly across the whole plate, the bacteria grow into a continuous layer called a bacterial lawn.
7. Any culture should contain only the one type of microorganism being studied, and the methods used to keep it that way are called aseptic techniques.

Why aseptic technique is needed

1. Aseptic technique has two separate purposes, and a good answer always gives both.
2. The first purpose is to prevent contamination of the culture by unwanted microorganisms.
   1. Microorganisms are present on hands, benches, equipment and dust in the air, and any that reach the medium will grow alongside the culture.
   2. Unwanted microorganisms compete with the culture for nutrients and space, so the results of the investigation are no longer valid.
3. The second purpose is to protect people from pathogens.
   1. A contaminating microorganism could be a pathogen that grows to large numbers on the plate.
   2. Even a harmless culture could produce a mutated, harmful strain as it divides millions of times.
4. For these reasons school cultures use only harmless bacteria, and every culture is treated as though it could be harmful.

Sterilising equipment and media

1. Before any microorganisms are transferred, every piece of equipment and all of the culture medium must be sterilised so that no living microorganisms are left on or in them.
2. Glassware, culture media and solutions are sterilised in an autoclave.
   1. An autoclave works like a large pressure cooker, heating items with steam at high pressure to about $121{}^{\circ }C$.
   2. Items are usually kept at this temperature for about $15$ minutes.
   3. The high temperature kills all bacteria, fungi and viruses, including the tough spores that some bacteria form, which boiling water at $100{}^{\circ }C$ would not kill.
3. Plastic Petri dishes and pipettes are bought already sterilised, having been treated with radiation in a sealed pack, and they should only be unwrapped just before use.
4. The inoculating loop is sterilised by flaming.
   1. The wire loop is held in the hottest part of a blue Bunsen flame until it glows red hot, which kills any microorganisms on it.
   2. The loop is then held still for a few seconds to cool, because a hot loop would kill the microorganisms it is about to pick up.
5. The neck of a bottle of culture is passed briefly through the flame after its lid is removed and again before the lid is replaced, which kills microorganisms at the opening and creates an upward current of air.
6. Alcohol, such as ethanol, is used to wipe down the bench before and after the work, which kills microorganisms on the surface.
7. A glass spreader is dipped in alcohol and then passed through the flame so the alcohol burns off, which sterilises it without melting it.

Keeping contaminants out

1. Once everything is sterile, the aim is to stop microorganisms from the air and from your body falling onto or into the medium.
2. Work is carried out close to a lit Bunsen burner, because the flame heats the air above it, and the rising current of warm air carries airborne microorganisms upwards and away from the plate.
3. The lid of the Petri dish is lifted only slightly and at an angle, and only for as short a time as possible, so that the lid acts as a shield against falling dust.
4. Lids of bottles are held in the hand rather than put down on the bench, where they would pick up microorganisms.
5. You should not talk, cough or breathe over an open plate, because droplets from the mouth and nose carry bacteria.
6. After inoculation, the lid is secured with two short strips of tape rather than sealed all the way round.
   1. The tape stops the lid being knocked off or lifted, so microorganisms from the air cannot get in and the culture cannot get out.
   2. Leaving gaps lets oxygen in, which stops the growth of harmful anaerobic bacteria that grow without oxygen.
7. The plate is labelled on the base, not the lid, and incubated upside down, so that drops of water condensing on the lid do not fall onto the agar and spread the colonies.
8. In school laboratories plates are incubated at no higher than $25{}^{\circ }C$, because warmer temperatures close to body temperature, $37{}^{\circ }C$, favour the growth of human pathogens.
9. When the work is finished, plates are never opened, and they are sterilised in an autoclave before they are thrown away.

Measuring the effect of antimicrobial substances

1. A bacterial lawn can be used to compare how well different antiseptics or antibiotics kill bacteria.
2. Paper discs soaked in each substance are placed on the lawn before incubation, and the substance spreads out from each disc into the agar.
3. Where the concentration is high enough, the bacteria are killed or cannot grow, leaving a clear ring called a zone of inhibition around the disc.
4. The larger the zone of inhibition, the more effective the substance is at killing or stopping the growth of that bacterium.
5. A zone is roughly circular, so its size is best compared by calculating its area from its radius using the equation $A=\pi r^2$.
6. The radius $r$ is half of the diameter, so the diameter is measured across the widest part of the zone with a ruler and then halved.
7. The same equation can be used to find the area of a single circular colony or of a whole Petri dish.
8. To improve the reliability of the results, each substance is tested on several plates and a mean zone area is calculated.