Microbial Growth, Metabolism, and Control PDF

Microbial Growth and Metabolism Pharmaceutical Microbiology and Parasitology (PHMP 211) Our Lady of Fatima University College of Pharmacy Microbial Growth Microbial Growth, Metabolism and Control Growth Increase in the population of cells is...

Microbial Growth and Metabolism Pharmaceutical Microbiology and Parasitology (PHMP 211) Our Lady of Fatima University College of Pharmacy Microbial Growth Microbial Growth, Metabolism and Control Growth Increase in the population of cells is called a culture. Cell division is by an asexual process called binary fission, and the time it takes to divide (double) is called generation time. Growth of culture goes through four phases with time, when plotted on a graph. Growth Phases 1. Lag Phase Organisms are adjusting to the environment (little or no division). They are synthesizing DNA, ribosomes and enzymes to breakdown nutrients, and to be used for growth. Growth Phases 2. Log or Logarithmic phase Division is at a constant rate (generation time) but varies with species, temperature and media. Cells are most susceptible to inhibitors. Growth Phases 3. Stationary phase Dying and dividing organisms are at an equilibrium. Death is due to reduced nutrients, pH changes, toxic waste and reduced oxygen. Cells are smaller and have fewer ribosomes. In some cases cells do not die but they are not multiplying. Growth Phases 4. Death or Decline phase The population is dying in a geometric fashion so there are more deaths than new cells. Deaths are due to the factors in stationary phase in addition to lytic enzymes that are released when bacteria lyse. Factors That Affect Microbial Growth These environmental factors affect microorganisms and play important roles in the control of microorganisms in laboratory, industrial, and hospital settings. 1. Availability of 5. Osmotic Nutrients Pressure 2. Moisture 6. Barometric 3. Temperature Pressure 4. pH 7. Gaseous Atmosphere Availability of Nutrients All living organisms require nutrients – various chemical compound use to sustain life. ☐Energy sources ☐Sources of basic elements ☐C, O, H, N, P, S ☐Na, K, Cl, Mg, Ca, Fe, I, Zn Other Growth Factors Other growth factors are organized into three categories: 1. Purines and pyrimidines: required for synthesis of nucleic acids (DNA and RNA) 2. Amino acids: required for the synthesis of proteins 3. Vitamins: needed as coenzymes and functional groups of certain enzymes Terms Relating to Energy and Carbon Sources Terms Relating To Terms Relating To Carbon Source Energy Source Autotrophs Heterotrophs (organisms that use (organisms that use CO2 as a carbon organic compounds source) as a carbon source) Phototrophs (organisms that use Photoautotrophs Photoheterotrophs light as an energy source) Chemotrophs (organisms that use Chemoautotrophs Chemoheterotrophs chemical as energy source) Moisture Water is essential to all life on Earth. Cells are composed of between 70—95% water. All living organisms require water to carry out their normal metabolic processes, and most will die in environments containing too little moisture. ☐Desiccation – complete drying process ☐Some microbial stage can survive desiccation. Temperature Optimum growth temperature – the temperature at which the microorganisms grows best ☐Minimum growth temperature – below this, the microorganisms cease to grow ☐Maximum growth temperature – above this, the microorganisms die ☐Temperature range Temperature Thermophiles – microorganisms that grows at temperature of 50-60°C Mesophiles – microorganisms that grow best at moderate temperature of 20-40°C Psychrophiles – prefer cold temperature of 10- 20°C ☐Psychrotrophs – OGT is 4°C (refrigerator temperature) ☐Psychrodurics – can tolerate or endure very cold temperature of less than –4°C pH The term “pH” refers to the hydrogen ion concentration of a solution and, thus, the acidity or alkalinity of the solution. Most microorganisms prefer a neutral or slightly alkaline growth medium (pH 7.0–7.4). ☐Acidophiles ☐Alkaliphiles  Osmotic Pressure Osmotic pressure is the pressure that is exerted on a cell membrane by solutions both inside and outside the cell. ☐Isotonic ☐Hypotonic ☐Hypertonic plasmolysis (shrinkage of plasma membrane away from the cell wall) in hypertonic plasmotypsis (leakage of cytoplasm from the cell) Halophilic – thrives at high salt concentration such as V. cholerae Haloduric – survive at high salt concentration such as S. aureus Barometric Pressure Most bacteria are not affected by minor changes in barometric pressure. Some thrive at normal atmospheric pressure. Gaseous Atmosphere Oxygen serves as electron acceptor in aerobic respiration. Bacteria can be divided into the following groups based on their oxygen requirements: 1. Obligate aerobe Glucose is completely oxidized to CO2 and H2O, requiring 21% oxygen. Gaseous Atmosphere 2. Microaerophilic The metabolic process is comparable to obligate aerobes but requires 1-15 % oxygen. 3. Facultative anaerobe In the absence of O2, glucose undergoes glycolysis to pyruvic acid, and then fermentation takes place. Gaseous Atmosphere 4. Obligate anaerobe Glucose undergoes glycolysis to pyruvic acid, then fermentation or anaerobic respiration in which oxygen is not the final electron acceptor. Some organisms use nitrate, sulfate or carbonate. 5. Capnophile Requires carbon dioxide to survive. Classification of Culture Media A. Culture media according to consistency: 1. Liquid media are used for growth of pure batch cultures. 2. Solidified media contain agar and are used widely for the isolation of pure cultures, for estimating viable bacterial populations, and a variety of other purposes. Classification of Culture Media B. Culture media according to composition. 1. Chemically-defined (synthetic) medium is one in which the exact chemical composition is known. 2. Complex (undefined) medium is one in which the exact chemical constitution of the medium is not known. Also known as basal medium. Classification of Culture Media C. Culture media according to their function and use. 1. Enrichment media contain specific nutrients required for the growth of bacterial pathogens that may be present alone or with other bacterial species in a patient specimen. e.g. Blood, Chocolate agar Classification of Culture Media 2. Supportive media contain nutrients that support growth of most non-fastidious organisms without giving any organism a growth advantage. 3. Selective media contain one or more agents that are inhibitory to all organisms except those being sought. e.g. MacConkey agar, Eosin methylene blue Classification of Culture Media 4. Differential media employ some factors that allows colonies of one bacterial species or type to exhibit certain metabolic or culture characteristics that can be used to distinguish them from other bacteria growing on the same agar plate. Macconkey agar with lactose (left) and non-lactose (right) fermenters Microbial Genetics B. Bacterial Mutation: 1. Spontaneous mutations have no identifiable cause and are rare. 2. Induced mutations are due to chemical or physical mutagens e.g. UV light, nitrous acid, base analogs etc. 3. Other mutations are caused by transposable genetic elements. Microbial Genetics Three major types of transposable elements 1. Insertion sequences They are segments of DNA that encode enzymes for site specific recombination and have distinct nucleotides at their terminals. Microbial Genetics 2. Transposons They are larger than insertion sequences, code for protein synthesis and can induce mutation the same way as insertion sequences. Microbial Genetics 3. Transposable phages (Mu) They can alternate between lytic and lysogenic growth. During lysogeny. They can insert themselves anywhere on the bacterial chromosome, and later transpose from one location to another, inactivating the bacterial gene just as other transposable elements. Microbial Genetics C. Bacterial Recombination 1. Transformation Donor cells lyse, a fragment of DNA is released and passed into a recipient cell. Enzyme dissolves one strand of the fragment, and the other strand displaces a homologous segment of the recipient's DNA. The recipient then has a recombinant DNA. The displaced fragment is dissolved by an enzyme. Only competent cells can be transformed and DNA of both organisms must be similar. Competence is the ability to take up DNA from the environment. Microbial Genetics 2. Conjugation It involves two live bacteria, donor and recipient, with the transfer of genetic material from a plasmid. The donor is F+, has fertility factor and codes for sex pilus. Once there is contact with the F - or recipient, plasmid DNA begins to replicate by the Rolling circle method. 3. Transduction The transfer of genetic material from one organism to another by a transducing phage. Microbial Genetics a. There are two types of transducing phages: 1) Lytic The phage reproduces and causes the bacterium to lyse. This is a virulent phage. 2) Lysogenic The phage codes for a repressor protein which prevents its own replication. This is a temperate phage. Microbial Genetics b. There are two types of transductions: 1.Generalized Transduction ☐A lytic virus uses the bacterial machinery to make viruses. ☐The bacterium lyse and viral parts are released. Microbial Genetics 2. Restricted or Specialized Transduction When a bacterium with an integrated prophage is induced to be lytic, the phage DNA is excised. Sometimes a piece of bacterial DNA adjacent to the phage DNA remains attached to the excised phage DNA. Only genes adjacent to the phage can be transduced. Microbial Control Microbial Growth, Metabolism and Control Physical Methods of Microbial Control A. Dry Heat Sterilization This is applicable for substances unaffected at a temperature of 148ºC to 260ºC in the oven, at an exposure time of 45 minutes. This method kills spores, as well as vegetative forms of microorganisms. This method is ideal for sterilizing glasswares, metalwares, and anhydrous oils. Physical Methods of Microbial Control A. Dry Heat Sterilization This is applicable for substances unaffected at a temperature of 148ºC to 260ºC in the oven, at an exposure time of 45 minutes. This method kills spores, as well as vegetative forms of microorganisms. This method is ideal for sterilizing glasswares, metalwares, and anhydrous oils. Physical Methods of Microbial Control The principle of sterilization involved is the oxidation of microorganisms by heat. Direct flaming – used to sterilize inoculating instruments by the use of alcohol lamp or burner Incineration – burning materials to ashes. Physical Methods of Microbial Control B. Moist Heat Sterilization This is more effective than dry heat method. The principle of sterilization is the coagulation of the cell protein of the microorganism. Physical Methods of Microbial Control 1. Autoclaving (steam under pressure) It is the most effective method of moist heat sterilization. It can destroy the sporeformers. The temperature used is 121ºC with a pressure of 15 psi for 15-30 minutes exposure. Physical Methods of Microbial Control 2. Boiling Temperature used is 100ºC Kills many vegetative cells and viruses within 10 minutes. 3. Pasteurization It uses a high temperature for a short time (72ºC for 15 seconds) to destroy the pathogens without altering the flavor of the food. Physical Methods of Microbial Control 4. Fractional Sterilization Method are effective for vegetative forms of microorganisms, but not for spores. a. Tyndallization It makes use of moist heat at 100 degrees Celsius, using free flowing steam. It is normally performed by 2 to 3 exposures, alternated with intervals at room temperature or incubator temperature. Physical Methods of Microbial Control b. Inspissation It is a fractional method of sterilization at 60 degrees Celsius in an oven, alternated with intervals at room temperature or incubation for 2 to 3 days. Physical Methods of Microbial Control C. Filtration The passage of liquid or gas through a filter with pores enough to retain microbes. Microbes can be removed from air by high- efficiency particulate air filters. Membrane filters composed of nitrocellulose or cellulose acetate are commonly used to filter out bacteria, viruses and even large proteins. Physical Methods of Microbial Control D. Cold The effectiveness of low temperatures depends on the particular microorganisms and the intensity of the application. Microorganisms do not reproduce at ordinary refrigerator temperatures (0º to 7ºC) Many microbes survive (but do not grow) at the subzero temperatures used to store food. Physical Methods of Microbial Control E. Desiccation In the absence of water, microorganisms cannot grow but can remain viable. Viruses and endospores can resist decolorization. Physical Methods of Microbial Control F. Osmotic pressure Microorganisms in high concentrations of salts and sugars undergo plasmolysis. Molds and yeasts are more capable of growing in materials with low moisture or high osmotic pressure than bacteria are. Physical Methods of Microbial Control G. Radiation 1. Ultraviolet radiation This is used to aid reduction of air borne contamination produced by mercury vapor lamps. This method has poor penetration capability. Its effectiveness depends on: a. length of time of exposure b. intensity of radiation c. susceptibility of the microorganism Physical Methods of Microbial Control 2. Ionization Radiation This radiation method makes use of high energy emitted from radioactive isotopes such as cobalt 60 (gamma rays) or by cathode or beta rays (mechanical acceleration of electrons to high velocity and energy). Physical Methods of Microbial Control Gamma rays are more reliable because there is no mechanical breakdown, but it has a disadvantage of rare source and cannot be shut off immediately. Accelerated electrons provide higher and more uniform dose outfit and can destroy organisms by stopping its reproduction. Chemical Methods of Microbial Control Chemical agents are used on living tissue (as antiseptics) and on inanimate objects (as disinfectants). Few chemical agents achieve sterility. A. Principles of Effective Disinfection The presence of organic matter, degree of contact with microorganisms, and temperature should be considered. Chemical Methods of Microbial Control B. Evaluating a Disinfectant In the use-dilution test, bacterial (S. choleraesuis, S. aureus, and P. aeruginosa) survival in the manufacturer’s recommended dilution of a disinfectant is determined. In the filter paper method, a disk of filter paper is soaked with a chemical and placed on an inoculated agar plate; a clear zone of inhibition indicates effectiveness. Chemical Methods of Microbial Control C. Types of Disinfectants 1. Phenol and Phenolics Phenolics exert their action by injuring plasma membranes, inactivating enzymes, and denaturing proteins. Common phenolics are cresols and hexachlorophene. 2. Chlorhexidine Chlorhexihide damages plasma membranes of vegetative cells. Chemical Methods of Microbial Control 3. Halogens Some halogens (iodine and chlorine) are used alone or as components of inorganic or organic solutions. Iodine is available as a tincture (in solution with alcohol) or as an iodophor (combined with an organic molecule). Chemical Methods of Microbial Control The germicidal action of chlorine is based on the formation of hypochlorous acid when chlorine is added to water. Chlorine is used as a disinfectant in gaseous form (CI2) or in the form of a compound, such as calcium hypochlorite, sodium hypochlorite, and chloramines. Chemical Methods of Microbial Control 4. Alcohols Alcohols exert their action by denaturing proteins and dissolving lipids. In tinctures, they enhance the effectiveness of other antimicrobial chemicals. Aqueous ethanol (60% to 90%) and isopropanol are used as disinfectants. Chemical Methods of Microbial Control 5. Heavy Metals and Their Compounds Silver, mercury, copper, and zinc are used as germicidals. They exert their antimicrobial action through oligodynamic action. Chemical Methods of Microbial Control 6. Surface-Active Agents Surface-active agents decrease the tension between molecules that lie on the surface of a liquid; soaps and detergents are examples. Soaps have limited germicidal action but assist in the removal of microorganisms through scrubbing. Acid-anionic detergents are used to clean dairy equipment. Chemical Methods of Microbial Control 7. Quaternary Ammonium Compounds Quats are cationic detergents attached to NH4+. By disrupting plasma membranes, they allow cytoplasmic constituents to leak out of the cell. They are most effective against gram-positive bacteria. Chemical Methods of Microbial Control 8. Organic Acids and Derivatives Sorbic acid, benzoic acid, and propionic acid inhibit fungal metabolism. They are used as food preservatives. Chemical Methods of Microbial Control 9. Aldehydes Aldehydes such as formaldehyde and glutaraldehyde exert their antimicrobial effect by inactivating proteins. They are among the most effective chemical disinfectants. Chemical Methods of Microbial Control 10. Gaseous Chemosterilizers Ethylene oxide is the gas most frequently used for sterilization. It penetrates most material s and kills all microorganisms by protein denaturation. Chemical Methods of Microbial Control 11. Oxidizing Agents Ozone and peroxide are used as antimicrobial agents. They exert their effect by oxidizing molecules inside cells.