2       Cultivation, preservation and inactivation of bacteria

 

Appropriate conditions relating to moisture, pH, temperature, osmotic pressure, atmosphere and nutrients are required for bacterial growth. Bacteria replicate by binary fission. The generation time, the length of time required for a single bacterial cell to yield two daughter cells, ranges from 30 minutes to 20 hours. Long-term preservation of microorganisms usually involves freezing procedures. Heat treatment or chemicals can be used for inactivation of bacteria.

Following inoculation of bacterial cells into fresh broth medium, the growth curve of the culture exhibits lag, exponential and stationary phases and a final decline phase. During the lag phase, bacterial cells are metabolically active but not dividing; binary fission of cells results in an exponential increase in numbers. Procedures which can be used for total cell counting include direct microscopy, electronic methods and real-time quantitative polymerase chain reaction (PCR)-based methods. Viable bacterial numbers can be determined by colony counting and by membrane filtration. Accurate cell counts may be required for specific purposes such as vaccine preparation and for bacteriological testing of water.

Bacteria acquire nutrients from their immediate environment. Nutrient media for the isolation of pathogenic bacteria are formulated to supply particular growth factors for particular groups of organisms. Most bacteria require carbon and nitrogen in relatively large amounts. Trace elements and certain growth factors such as vitamins are also essential for bacterial growth.

In addition to nutritional factors, growth of bacteria is influenced by genetic factors and by chemical, physical and other environmental influences. Growth of bacteria in culture is influenced by temperature, hydrogen ion concentration, availability of moisture, atmospheric composition and osmotic pressure. Most bacteria grow optimally at neutral pH. The majority of pathogenic bacteria can be grown aerobically on a nutrient agar medium at 37°C, close to the body temperature of humans and most domestic species. Bacteria with an optimal incubation temperature of 37°C are termed mesophiles and most pathogenic bacteria belong to this category. Based on their preference for particular levels of oxygen, bacteria can be assigned to four main groups, namely aerobes, anaerobes, facultative anaerobes and microaerophiles. A fifth group, capnophiles, are aerobic bacteria with a requirement for carbon dioxide. Anaerobic bacteria are unable to grow in an atmosphere containing oxygen. Strict anaerobes are cultured in tightly sealed jars in an atmosphere from which free oxygen has been removed.

Subculturing can be used for the short-term preservation of bacteria. Limitations of this procedure include death of some cells and a risk of contamination and mutation. Long-term methods of preservation include freeze-drying (lyophilization), freezing at –70°C and ultra-freezing in liquid nitrogen at –196°C. Freezing organisms in vials containing 20–30 porous polypropylene beads which can be removed and cultured singly is a convenient method of avoiding the need for repeated freezing and thawing of cultures. If properly used, these preservation methods can maintain organisms in a hypobiotic state for more than 30 years and ensure that the organisms remain unchanged and uncontaminated.

Sterilization is the method employed for the destruction of microorganisms on equipment used in microbiological and surgical procedures. Physical and chemical methods can be used for inactivation of microorganisms. Chemicals which inactivate bacteria include disinfectants and other compounds with bactericidal activity. Methods for preventing spoilage or limiting microbial growth in food are presented in Table 2.1. Physical methods for sterilizing equipment or fluids are presented in Table 2.2. Sterilization procedures are effective for the destruction of bacterial, fungal and viral agents. When dealing with bacterial endospores, such as those of Clostridium species, heating at a temperature of 121°C for 15 minutes in moist heat is required for their inactivation.

Table 2.1 Methods for preventing spoilage and limiting microbial growth in food.

Method	Application	Comments
Refrigeration at 4°C	Prevention of growth of spoilage organisms and pathogenic bacteria	Pathogens such as Listeria monocytogenes, Yersinia species and many fungal species can grow at 4°C
Freezing at –20°C	Long-term storage of food. Microbial multiplication prevented	Surviving microorganisms can multiply rapidly when thawed food is left at ambient temperatures
Boiling at 100°C	Inactivation of vegetative bacteria and fungi in food	Many endospores can withstand prolonged boiling
Pasteurization at 72°C for 15 seconds	Inactivation of most vegetative bacteria	Heat treatment should be followed by rapid cooling. If present in high numbers or located intracellularly, some bacteria may survive
Acidification	Adjustment of pH to a low level inhibits bacterial growth	Applicable to a limited range of foods such as vegetables
Increasing osmotic pressure	Inhibition of microbial multiplication; used for preservation of food	Addition of salts or sugars increases osmotic pressure; applicable to a limited range of foods
Vacuum packing	Packaging of meat and other perishable foods	Removal of oxygen prevents the growth of aerobes
Irradiation	Inactivation of spoilage organisms and pathogenic bacteria	Not permitted in some countries

Table 2.2 Physical methods for sterilizing equipment or fluids; some can be used for disposing of contaminated material.

Method	Comments
Moist heat (autoclaving) employing steam under pressure to generate 121°C for 15 minutes or 115°C for 45 minutes	Used for sterilizing culture media, laboratory items and surgical equipment. Inappropriate for heat-sensitive plastics or fluids. Prions are not inactivated by this treatment
Dry heat in a hot-air oven at 160°C for 1–2 hours	Used for sterilizing metal, glass and other solid materials. Unsuitable for rubber and plastics
Incineration at 1,000°C	Used for destruction of infected carcasses and other contaminated material; environmental pollution a possible consequence
Flaming	Used for sterilizing inoculating loops in the naked flame of a Bunsen burner
Gamma irradiation	Ionizing rays used for sterilizing disposable plastic laboratory and surgical equipment. Unsuitable for glass and metal equipment
Ultraviolet (UV) light	Non-ionizing rays with poor penetration. Used in biosafety cabinets
Membrane filtration	Used for removing bacteria from heat-sensitive fluids such as serum and tissue culture media. Pore size of filter should be 0.22 μm or less