Factors affecting the growth of bacteria The growth of bacteria is affected by 5 factors: Temperature Oxygen concentration pH External solute concentration Pressure Factors affecting the growth of bacteria Too much or too little of any of the factors will slow down the growth of bacteria. Factors that slow down a process when they are in short supply are called limiting factors. Temperature The rate of bacterial growth is affected by temperature. Most bacteria grow well between 20°C and 30°C. Some can tolerate much higher temperatures without their enzymes becoming denatured. Low temperatures slow down the rate of reaction of enzymes resulting in slower growth. Anaerobe – does not utilize oxygen obligate anaerobe - lacks the enzymes to detoxify oxygen so cannot survive in an oxygen environment (Clostridium spp., Bacteroides spp.) aerotolerance anaerobes – do no utilize oxygen but can survive and grow in its presence (Streptococcus pyogenes) Moisture and Drying Moisture is very essential for the growth of the bacteria because water is essential ingredient of bacterial protoplasm and hence drying is lethal to cells. pH Bacterial enzymes are designed to work at a specific pH. If a bacterium is placed in an unsuitable pH its enzymes will become denatured. Some bacteria can tolerate very low (acidic) (e.g. Helicobacter bacteria) pH and some can tolerate very high (alkaline) pH. Light Darkness provides a favorable condition for growth and viability of bacteria. Bacteria are sensitive to ultraviolet light and other radiations as ultraviolet rays from direct sunlight or a mercury lamp are bactericidal. Bacteria are also killed by ionizing radiations. Exposure to light may influence pigment production. Photochromogenic mycobacteria form pigment only on exposure to light. Osmotic Effect Tolerance to osmotic variation: Bacteria are more tolerant to osmotic variation because of the mechanical strength of the cell wall. Except for the mycoplasma and other cell wall defective organisms, the majority of the bacteria are osmotically tolerant. External Solute concentration Bacteria can gain or lose water by osmosis If the external solute concentration is higher than the bacterial cytoplasm water will move out of the bacteria (Dehydration) food preservation techniques are based on this External Solute concentration Bacteria can gain or lose water by osmosis If the external solute concentration is lower than the bacterial cytoplasm solute concentration water will enter the bacteria Cell wall will prevent bursting in most cases Pressure The growth of most bacteria is inhibited by high pressures. Some bacteria can withstand high pressures. Pressure tolerant bacteria for use in bioreactors can be formed by genetic engineering techniques. Mechanical and Sonic Stresses In spite of tough walls of bacteria, they may be ruptured by mechanical stress such as grinding or vigorous shaking with glass beads. Exposure to ultrasonic vibration may also disintegrate bacteria. Lab procedures when growing microorganisms Microorganism (bacteria, fungi & viruses) can only be seen with an electron microscope. Colonies (groups) of bacteria and fungi may be grown in the lab on agar plates Metabolism Results in Reproduction Microbial growth – an increase in a population of microbes rather than an increase in size of an individual Result of microbial growth is discrete colony – an aggregation of cells arising from single parent cell Reproduction results in growth Bacteria multiply in laboratory conditions on special nutrient media, which are (by their consistence): solid media liquid media semisolid media Bacteria growing on solid media form colonies. Each colony represents a clone of cells derived from a single parent cell. In liquid media, growth of bacteria is diffuse. The growth of bacteria in liquid medium may be accompanied by the formation of sediment, film, wall growth, diffuse growth. The multiplication of bacteria in liquid media In the clinical laboratory it is necessary: • isolate bacteria in pure culture; • obtain sufficient growth of bacteria for demonstration their properties such as study of morphological, cultural, biochemical, antigenic and pathogenic properties, bacteriophage and bacteriocin susceptibility; • determine a sensitivity to antibiotics. Methods of laboratory diagnosis 1. 2. 3. 4. 5. 6. Bacterioscopical (Microscopic examination) Bacteriological (Culture method) Detection sensitivity of bacteria to antibiotics Serological Biological DNA-technology test (PCR) Bacteriological (Culture method) A nutrient material prepared for the growth of microorganisms in a laboratory is called a culture medium. When microbes are introduced into a culture medium to initiate growth, they are called an inoculum. The microbes that grow and multiply in or on a culture medium are referred to as a culture. Cultivation is the process of growing microorganisms in culture by taking bacteria from the infection site (i.e. in vivo environment) by some means of specimen collection and growing them in the artificial environment of the laboratory (i.e. the in vitro environment). By appropriate procedures they have to be grown separately (isolated) on culture media and obtained as pure cultures for study. Once grown in culture, most bacterial populations are easily observed without microscopy and are present in sufficient quantities to allow laboratory testing to be performed. MAIN PURPOSES OF BACTERIAL CULTIVATION Bacterial cultivation has three main purposes: To grow and isolate all bacteria present in an infection. Infection and contaminants or colonizers: To determine which of the bacteria that grow are most likely causing infection and which are likely contaminants or colonizers. Identification and characterization: To obtain sufficient growth of clinically relevant bacteria to allow identification and characterization. COMMON INGREDIENTS OF CULTURE MEDIA Water Agar Pepton Yeast extract Malt extract Blood and Serum Classification of Media Nutrient media can be subdivided: 1. Basal media (Simple media) - meat-peptone broth (MPB), meat-peptone agar (MPA) 2. Synthetic media 3. Complex media 4. Special media: a) Enriched media b) Enrichment media; c) Selective media; d) Indicator and differential media; e) Sugar media; f) Transport media. BASAL MEDIA are those that may be used for growth (culture) of bacteria that do not need enrichment of the media. Examples: Nutrient broth, nutrient agar and peptone water. Staphylococcus and Enterobacteriaceae grow in these media. Nutrient broth Nutrient agar Complex media These have added ingredients for special purposes or bringing out certain characteristics or providing special nutrients requiered for the growth of the bacterium under study. A complex medium contains a variety of ingredients such as meat juices and digested proteins. The exact chemical composition of these ingredients can be highly variable although a specific amount of each ingredient is in the medium. Synthetic or chemically defined culture media They are prepared exclusively from pure chemical substances and their exact composition is known. Chemically defined media are used for various experimental purposes. These are used for various special studies such as metabolic requirements of the experimental microorganisms. Simple peptone water medium, 1% peptone with 0.5% NaCl in water, may be considered a semidefined medium since its composition is approximately known. ENRICHED MEDIA. The media are enriched usually by adding blood, serum or egg. Examples: Enriched media are blood agar and Lowenstein-Jensen media. Streptococci grow in blood agar media. blood agar Enrichment culture media When a substance is added to a liquid medium which inhibits the growth of unwanted bacteria and favors the growth of wanted bacteria is known as enrichment medium. This medium for an enrichment culture is usually liquid and provides nutrients and environmental conditions that favor the growth of a particular microbe but not others. The result is an absolute increase in the numbers of the wanted bacterium relative to the other bacteria. SELECTIVE MEDIA These media favour the growth of a particular bacterium by inhibiting the growth of undesired bacteria and allowing growth of desirable bacteria. Examples: MacConkey agar, Lowenstein-Jensen media, tellurite media (Tellurite inhibits the growth of most of the throat organisms except diphtheria bacilli). Antibiotic may be added to a medium for inhibition. TRANSPORT MEDIA These media are used when speciemen cannot be cultured soon after collection. Examples: Cary-Blair medium, Amies medium, Stuart medium. Amies transport medium STORAGE MEDIA Media used for storing the bacteria for a long period of time. Examples: Egg saline medium, chalk cooked meat broth. ANAEROBIC CULTURE MEDIA These media are used to grow anaerobic organisms, and contain reducing substances. These include: Thioglycollate broth. Cooked meat broth. Cooked meat broth
