Culturing Microbes

Learning intention To describe the main factors that are required to culture microbes

Factors affecting microbial growth Slide to brainstorm with class

Factors affecting microbial growth Contamination Growth media Availability of oxygen Temperature pH List of topics to be covered in the presentation

Preventing contamination Agents that destroy or kill bacteria are called bactericidal agents. However, killing bacteria is not always possible so agents that inhibit bacterial growth are often used. These are called bacteriostatic agents.

Why control microbial growth? This is done in the food industry to preserve food and prevent spoilage. It can prevent the spread of infectious disease, given the rapid growth rate of microbes.

How to prevent contamination Heat sterilisation Pasteurisation Radiation sterilisation Filter sterilisation Using chemical agents Using disinfectants and antiseptics

Heat sterilisation This involves using an autoclave. This sealed device allows the entrance of steam under pressure. The temperature used is 121ºC for 10–15 minutes. It is not the pressure in the autoclave that causes the death of the microorganisms but the high temperature achieved when the steam is placed under pressure. This procedure also ensures that bacterial spores are eliminated. Students will be asked to find out about spores in a task.

Pasteurisation The process used to reduce the microbial population in milk and other heat-sensitive products. Named after Louis Pasteur, who first used heat for controlling the spoilage of wine. Pasteurisation involves passing milk through a heat exchanger. The temperature of the milk is raised to 71°C for 15 seconds. The milk is then rapidly cooled.

Radiation sterilisation The number of microbes can be significantly reduced by using electromagnetic radiation. UV waves, for example, break down the DNA molecules in the microbe, thus causing death. Microwaves use thermal effects to kill bacteria.

Filter sterilisation Membrane and depth filters can be used. This method of sterilisation is used for media that are sensitive to heat.

Chemical agents Chlorine is used in air-conditioning systems to prevent the growth of bacteria such as Legionella.

Disinfectants and antiseptics Disinfectants are chemicals that kill microorganisms. They should be used on inanimate objects. Antiseptics are chemical agents that kill or inhibit the growth of microorganisms but are non-toxic enough to be applied to living tissue.

Nutrients Microbes are grown on culture media. This can be a solid or liquid preparation made to ensure the microbe has all the nutrients it needs for growth. The media is sterilised before use.

Types of media Complex media contain one or more crude sources of nutrients. Often their exact chemical composition and components are unknown. Defined media are also called synthetic media. The components of these media are known and are relatively pure.

Type of medium Example Extra constituents Bacteria grown Complex Nutrient agar Meat extracts, yeast extract Many bacteria will grow on this Defined M9 Escherichia coli Enriched Blood agar Blood Streptococcus pyogenes Selective MacConkey agar Bile salts and crystal violet dye Gram –ve bacteria

Oxygen Not all microbes need oxygen, in fact for some microbes exposure to oxygen can be fatal! A microbe that must have oxygen present in order to extract energy from foodstuffs is called an obligate aerobe. It needs oxygen for respiration. An organism that would die in the presence of oxygen is an obligate anaerobe. This is because these organisms lack the enzymes needed to break down the dangerous chemicals (peroxides) made in aerobic reactions.

Some microbes (especially yeast) can grow in the presence or absence of oxygen. These are called facultative anaerobes. They grow better in the presence of oxygen.

Getting oxygen In fermenters the actual process of delivering oxygen can be quite tricky. Piping may need to be used. Stirrers may be placed in the fermenter to ensure that the oxygen is available to all the microbes.

Temperature All microbes have a range of temperatures within which they are able to grow. However, within this range there will be a temperature at which the organism grows best: its optimum temperature.

Growth occurs due to chemical reactions and since enzymes are often involved, temperature will have to be tightly controlled. Some species of microbes live in extremes of temperature and can survive and grow despite the temperature.

Psychrophiles Found in oceans, at the north or south poles. Adapted to live in very cold temperatures.

Mesophiles Microbes that live at ‘normal’ temperatures, eg room temperature They live on the human body.

Thermophiles Microbes that can live in very hot environments. Examples: volcanic vents, compost heaps, hot springs.

pH As with temperature, microbes have an optimum pH. Fungi tend to prefer a more acidic environment (pH 4–6). Bacteria like a neutral pH. Vinegar, citric acid and other chemicals can be used to preserve food because they stop microbes from growing. Buffers can be used to keep the pH relatively constant.