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EXPERIMENTS TO SHOW THE GROWTH OF BACTERIA - basic techniques


(similar techniques are used to grow fungi such as moulds and yeasts)

Microbiological media

Bacteria will grow on practically any source of organic food which provides carbon compounds to be respired for energy, and nitrogen compounds to be incorporated into proteins for growth. These substances are normally provided dissolved in water. However, in nature, bacteria can break down solid and insoluble substances by releasing enzymes into the substrate in which they are growing. These substances are thus broken down or digested to simpler substances and the process is called extracellular digestion because it takes place outside the bacterial cells.

broth - clear when sterile The two normal media used in bacteriology are a clear soup-like liquid nutrient broth, usually in tubes, and nutrient agar, which is set into a jelly by the addition of a seaweed extract called agar, and when melted poured into glass or plastic Petri dishes - also known as "plates".

Petri dish


A standard carbon source is glucose, and nitrogen is often provided by peptones (partially digested proteins), or inorganic salts. Minerals and vitamins may also be provided, according to the growth requirements of the bacteria. Combinations of chemicals (buffers) may be used to keep the pH stable. Measured amounts of the concentrates are added to water, and dissolved to reconstitute the media.

Sometimes, substances are mixed into media, in order to suppress growth of other types of bacteria. There are many such selective media.

Microbiological techniques

Sterilisation, aseptic techniques, inoculation, incubation

These media must then be sterilised by heating in an autoclave (like a pressure cooker) at 121C (pressure 1 bar or 15 lb/sq. in.) for 15 minutes, which kills all living organisms, including spores.

All apparatus used from this point onwards must be sterilised by heat (glassware - 160 C for 2 hrs) or exposure to radiation.

Aseptic techniques must be used to reduce the likelihood of bacterial contamination. This usually involves disinfection of working areas, minimising possible access by bacteria from the air to exposed media, and use of flames to kill bacteria which might enter vessels as they are opened.

Pouring plates
Bacteria may be introduced to the media (inoculated) by various means. Usually the bacteria e.g. from a drop in a heat-sterilised loop are spread on the surface of (ready set) agar. A similar technique is used with broth cultures.
Inoculation of broth or agar
Sometimes bacteria in a liquid are introduced using a sterile pipette to the Petri dish before the (fairly cool) agar medium is poured on top ("pour plates").

Then the Petri dishes containing agar or tubes containing broth are incubated, i.e. put in a special apparatus at a fixed temperature (usually 37C - human body temperature, for possible pathogens - or 25C for bacteria from the environment). In schools, lower incubation temperatures are used in order to discourage the growth of potential pathogens.

When growing bacteria, it is usual to invert the Petri dishes, so as to prevent condensation droplets from falling onto the surface of the agar. Petri dishes are often "sealed" at this stage to prevent people who handle them from contamination by bacteria, which will multiply greatly. It is normal to use 2 strips of adhesive tape from base to lid rather than attempt seal the circular edge of the Petri dish. This is to guard against the possibility of anaerobic organisms growing due to lack of air. However, it must be borne in mind that any drips from a partially sealed Petri dish are potential sources of infection.

Sealed plate

Invpl2

Results

Cultures are usually examined after 24 hrs incubation.

Liquid media such as broth become cloudy if bacteria are present. This could be the result of only one bacterial cell originally entering the medium, then dividing repeatedly to produce millions! Broth turns cloudy when bacteria grow


Bacteria on agar "plates" become visible as distinct circular colonies; each colony should represent an individual bacterial cell (or group) which has divided repeatedly but, being kept in one place, the resulting cells have accumulated to form a visible patch.

Resul2

By an extension of this method using serial dilutions in sterilised liquids, the number of bacteria in a given amount of sample, e.g. food, can be calculated.


After use, bacterial cultures, etc. must be sterilised by the use of heat, before disposal.







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