Culturing Microbes

Learning intention

• To describe the main factors that are required to culture microbes

Factors affecting microbial growth

Factors affecting microbial growth

• Contamination

• Growth media

• Availability of oxygen

• Temperature

• pH

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.

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.