Exam 1: Bacterial growth and culture techniques
There are four main phases of growth:
1. The lag phase, where there is little growth but the cells are taking up water and are carrying out protein synthesis and producing enzymes
2. The exponential or log phase, where the population increases rapidly mainly due to binary fission
3. The stationary phase, where bacterial cells are dying at the same rate as they are produced
4. The death phase (also known as the decline phase), where more bacterial cells are dying than are being produced. (The phases of growth are dealt with in more detail in section 4.5 of this unit).
There are four main phases of growth:
1. The lag phase, where there is little growth but the cells are taking up water and are carrying out protein synthesis and producing enzymes
2. The exponential or log phase, where the population increases rapidly mainly due to binary fission
3. The stationary phase, where bacterial cells are dying at the same rate as they are produced
4. The death phase (also known as the decline phase), where more bacterial cells are dying than are being produced. (The phases of growth are dealt with in more detail in section 4.5 of this unit).
Conditions necessary for bacterial growth
Microorganisms reproduce quickly given a suitable environment. For example, bacteria are able to divide every twenty minutes under optimum conditions. In the laboratory, bacteria can be grown on a wide variety of substrates providing they are supplied with suitable physical conditions, nutrients and water. Microrganisms vary in their requirements and usually grow over a range of temperatures and pH values, with an optimum within the range.
Microorganisms require the following conditions for growth:
• Nutrients:
In the laboratory nutrients are supplied in nutrient media, such as agar, and include carbon, usually in the form of glucose; nitrogen, in organic and inorganic form; growth factors such as vitamins and mineral salts. Nitrogen is needed to produce amino acids during protein synthesis.
• Temperature:
As all growth is normally regulated by enzymes the range of 25-45 oC is favourable for the majority of bacteria. The optimum for mammalian pathogens is around 37 oC.
• pH:
Most bacteria are favoured by slightly alkaline conditions (pH 7.4), whereas fungi prefer neutral to slightly acid conditions.
• Oxygen:
Many microorganisms require oxygen for metabolism and are termed obligate aerobes. Some, while growing better in the presence of oxygen, can nevertheless survive in its absence and are called facultative anaerobes. Others cannot grow in the presence of oxygen and are called obligate anaerobes. Clostridium bacteria are obligate anaerobes that produce toxins or poisons in a wound. These bacteria destroy body tissue and the condition is called moist gangrene.
Microorganisms reproduce quickly given a suitable environment. For example, bacteria are able to divide every twenty minutes under optimum conditions. In the laboratory, bacteria can be grown on a wide variety of substrates providing they are supplied with suitable physical conditions, nutrients and water. Microrganisms vary in their requirements and usually grow over a range of temperatures and pH values, with an optimum within the range.
Microorganisms require the following conditions for growth:
• Nutrients:
In the laboratory nutrients are supplied in nutrient media, such as agar, and include carbon, usually in the form of glucose; nitrogen, in organic and inorganic form; growth factors such as vitamins and mineral salts. Nitrogen is needed to produce amino acids during protein synthesis.
• Temperature:
As all growth is normally regulated by enzymes the range of 25-45 oC is favourable for the majority of bacteria. The optimum for mammalian pathogens is around 37 oC.
• pH:
Most bacteria are favoured by slightly alkaline conditions (pH 7.4), whereas fungi prefer neutral to slightly acid conditions.
• Oxygen:
Many microorganisms require oxygen for metabolism and are termed obligate aerobes. Some, while growing better in the presence of oxygen, can nevertheless survive in its absence and are called facultative anaerobes. Others cannot grow in the presence of oxygen and are called obligate anaerobes. Clostridium bacteria are obligate anaerobes that produce toxins or poisons in a wound. These bacteria destroy body tissue and the condition is called moist gangrene.
Growth and culture techniques
All bacterial cultures are potentially dangerous. Samples should never be taken from surfaces in the laboratory. Cultures purchased from a scientific supplier, milk or yoghurt are normally provided to carry out experimental work. There are two possible problems which must be prevented when working with bacteria. These are the prevention of contamination of the supplied cultures from the environment and the prevention of contamination to the environment. Strict safety precautions should be taken and aseptic techniques used at all times.
Aseptic technique
Bacteria (and fungi) are cultured (grown) on, or in, media that are designed to supply the cell with all its nutritional requirements. Aseptic techniques (also known as sterile techniques) are used in which apparatus and equipment are kept free of microorganisms. This sterile equipment is then used to prevent contamination of bacterial cultures and the surrounding environment.
• To prevent the contamination of pure cultures and apparatus by bacteria FROM THE ENVIRONMENT:
1. Sterilise all apparatus and media before use to prevent initial contamination
2. Handle cultures carefully and use equipment such as sterile loops to prevent subsequent contamination
• To prevent contamination TO THE ENVIRONMENT by the bacteria being used in the experiment:
1. Sterilise the work surface before and after an experiment using a disinfectant, e.g. Lysol used as a 3% solution.
2. Use the correct handling techniques (see below) to prevent the contamination of personnel and the immediate environment by the organisms being cultured:
For example, when carrying out the process of inoculation:
• Grasp the culture bottle in one hand (this contains a sample of pure bacterial culture)
• Remove cap with little finger of other hand – do not place the cap down on the work surface
• Flame the mouth of the culture bottle for 2 or 3 seconds (to sterilise the mouth of culture bottle)
• Pass the inoculating loop through a flame until red hot ƒ to sterilise (and then allow to cool)
• Take a small sample of the pure bacterial culture using the sterilised inoculating loop
• Lift the lid of the petri dish just enough to allow entry of the inoculating loop
• Gently brush the inoculating loop over the nutrient agar dish (or dip into nutrient broth)
• Secure the petri dish lid with adhesive tape. Use two pieces of tape to fasten the lid, but do not seal all the way round as this could create anaerobic conditions and encourage the growth of possible pathogenic microorganisms
• Incubate at around 25 0C (do not culture at 37 0C as this is an ideal temperature for the growth of many pathogens)
• Do not open petri dishes fully after incubation, as this may introduce endospores or other bacteria into the culture from the atmosphere
• In a laboratory the preferred method of sterilisation is to use an autoclave. This is a sealed container in which glass and metal equipment is heated at 121 0C in steam under high pressure for 15-30 minutes after the required pressure has been reached
• This ensures that any resistant endospores* are destroyed (some gram positive bacteria such as Clostridium are resistant to boiling as they can produce endospores when in unfavourable conditions)
• Disposable materials, such as plastic petri dishes, can be sealed inside autoclavable plastic bags which are then autoclaved (using the conditions mentioned above) and disposed of
• Radiation e.g. gamma rays can be used commercially to sterilise plastic equipment.
All bacterial cultures are potentially dangerous. Samples should never be taken from surfaces in the laboratory. Cultures purchased from a scientific supplier, milk or yoghurt are normally provided to carry out experimental work. There are two possible problems which must be prevented when working with bacteria. These are the prevention of contamination of the supplied cultures from the environment and the prevention of contamination to the environment. Strict safety precautions should be taken and aseptic techniques used at all times.
Aseptic technique
Bacteria (and fungi) are cultured (grown) on, or in, media that are designed to supply the cell with all its nutritional requirements. Aseptic techniques (also known as sterile techniques) are used in which apparatus and equipment are kept free of microorganisms. This sterile equipment is then used to prevent contamination of bacterial cultures and the surrounding environment.
• To prevent the contamination of pure cultures and apparatus by bacteria FROM THE ENVIRONMENT:
1. Sterilise all apparatus and media before use to prevent initial contamination
2. Handle cultures carefully and use equipment such as sterile loops to prevent subsequent contamination
• To prevent contamination TO THE ENVIRONMENT by the bacteria being used in the experiment:
1. Sterilise the work surface before and after an experiment using a disinfectant, e.g. Lysol used as a 3% solution.
2. Use the correct handling techniques (see below) to prevent the contamination of personnel and the immediate environment by the organisms being cultured:
For example, when carrying out the process of inoculation:
• Grasp the culture bottle in one hand (this contains a sample of pure bacterial culture)
• Remove cap with little finger of other hand – do not place the cap down on the work surface
• Flame the mouth of the culture bottle for 2 or 3 seconds (to sterilise the mouth of culture bottle)
• Pass the inoculating loop through a flame until red hot ƒ to sterilise (and then allow to cool)
• Take a small sample of the pure bacterial culture using the sterilised inoculating loop
• Lift the lid of the petri dish just enough to allow entry of the inoculating loop
• Gently brush the inoculating loop over the nutrient agar dish (or dip into nutrient broth)
• Secure the petri dish lid with adhesive tape. Use two pieces of tape to fasten the lid, but do not seal all the way round as this could create anaerobic conditions and encourage the growth of possible pathogenic microorganisms
• Incubate at around 25 0C (do not culture at 37 0C as this is an ideal temperature for the growth of many pathogens)
• Do not open petri dishes fully after incubation, as this may introduce endospores or other bacteria into the culture from the atmosphere
• In a laboratory the preferred method of sterilisation is to use an autoclave. This is a sealed container in which glass and metal equipment is heated at 121 0C in steam under high pressure for 15-30 minutes after the required pressure has been reached
• This ensures that any resistant endospores* are destroyed (some gram positive bacteria such as Clostridium are resistant to boiling as they can produce endospores when in unfavourable conditions)
• Disposable materials, such as plastic petri dishes, can be sealed inside autoclavable plastic bags which are then autoclaved (using the conditions mentioned above) and disposed of
• Radiation e.g. gamma rays can be used commercially to sterilise plastic equipment.