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MICROBIOLOGIA GENERALE Microbial growth control and nutrition The control of microbial growth is necessary in many practical situations, and significant advances in agriculture, medicine, and food science have been made through study of this area of microbiology. What does sterilization mean? •Sterilization is the complete destruction or elimination of all viable organisms (in or on an object being sterilized). •There are no degrees of sterilization: an object is either sterile or not. •Sterilization procedures involve the use of heat, radiation or chemicals, or physical removal of cells. Microbial growth control and nutrition: the control of microorganisms by physical factors Methods of Sterilization: Heat •Most important and widely used. •For sterilization always consider type of heat, time of application and temperature •Endospores are considered the most thermoduric of all cells so their destruction guarantees sterility. The effect of temperature on the viability of a mesophilic bacterium The relationship between the temperature and the rate of killing as indicated by the D for two different microorganisms D value: the time (min) at a given temperature, for a population of a bacterium or endospore to fall by 90% (by one unit on the log10 scale) Microorganism D (min) at 100° C, D100 Bacillus cereus 0.8 Bacillus megaterium 2.1 Bacillus anthracis 5 Bacillus licheniformis 24.1 Clostridium botulinum 300 Bacillus stearothermophilus 459 Botulinum cook = 12 times the D for C. botulinum at that temperature D values: food pathogens and environmental effects Microorganism Source D (min) Clostridium botulinum endospores D121=0.204 Clostridium perfringens cell culture D90=3-5 Salmonella spp. chicken D60=0.40 Staphylococcus aureus chicken D60=5-6 Staphylococcus aureus turkey D60=15 Staphylococcus aureus cell culture D60=2.0-2.5 Botulinum cook = 12 times the D for C. botulinum at that temperature Heat sterilization •Incineration: burns organisms and physically destroys them. Used for needles , inoculating wires, glassware, etc. and objects not destroyed in the incineration process. •Autoclaving (steam under pressure): 121o for 15 minutes (1 atm pressure). Good for sterilizing almost any thing, but heat-labile substances will be denatured or destroyed. •Boiling: 100o for 30 minutes. Kills everything except some endospores. To kill endospores, and therefore sterilize the solution, very long or intermittent boiling is required. •Dry heat (hot air oven): 160o/2hours or 170o/1hour. Used for glassware, metal, and objects that won't melt. Recommended use of heat to control bacterial growth Treatment T °C Effectiveness >500o Vaporizes organic material on nonflammable surfaces but may destroy many substances Boiling 100o 30 minutes of boiling kills microbial pathogens and vegetative forms of bacteria but may not kill bacterial endospores Intermittent boiling 100o Three 30-minute intervals of boiling, followed by periods of cooling kills bacterial endospores 121o/15 min Kills all forms of life including bacterial endospores. The substance being sterilized must be maintained at the effective T 160o/2 For materials that must remain dry and which are not destroyed at T between 121o and 170o (glassware, metal, not plastic or rubber items) Incineration Autoclave (steam under pressure) Dry heat (hot air oven) Pasteurization (batch method) Pasteurization (flash method) h 63o/30 min 72o/15 sec Kills most vegetative bacterial cells including pathogens such as streptococci, staphylococci and Mycobacterium tuberculosis Effect on bacterial cells similar to batch method; for milk, this method is more conducive to industry and has fewer undesirable effects on quality Autoclave Use of the autoclave for sterilization Methods of Sterilization: Irradiation •Irradiation: usually destroys or distorts nucleic acids. •Ultraviolet light is usually used (commonly used to sterilize the surfaces of objects). •Gamma-rays are used to sterilize plasticwares Relationship between the survival fraction and the radiation dose Methods of Sterilization: Filtration •Filtration: involves the physical removal (exclusion) of all cells in a liquid or gas. •It is especially important to sterilize solutions which would be denatured by heat (e.g. antibiotics, injectable drugs, amino acids, vitamins, etc.) Filter sterilization Pore sizes: 0,2 mm – 0,4 mm Membrane filter Nucleopore filter Bacteria and algae trapped on a Nucleopore filter. Leptospira interrogans (0.1 x 20 mM) trapped on a Nucleopore filter. Filter system for large volumes Filter system for small volumes: syringe filters Microbial growth control and nutrition: the control of microorganisms by chemical factors Antimicrobial agents Antimicrobial agents are chemicals that kill or inhibit the growth microorganisms. •Antimicrobial agents include chemical preservatives and antiseptics, as well as drugs used in the treatment of infectious diseases. •Antimicrobial agents may be of natural or synthetic origin, and they may have a static or cidal effect on microorganisms. Three types of action of antimicrobial agents. At the time indicated by the arrow, a growthinhibitory concentration was added to the exponentially growing culture. Antiseptics • Antiseptics: microbicidal agents harmless enough to be applied to the skin and mucous membranes. • They should not be taken internally. • • • • • Mercurials Silver nitrate Iodine solution Alcohols Detergents Disinfectants • Disinfectants: Agents that kill microorganisms, but not necessarily their spores. • Not safe for application to living tissues. • They are used on inanimate objects such as tables, floors, utensils, etc. • Chlorine, hypochlorites & chlorine • Copper sulfate. • Quaternary ammonium compounds. Disinfectants and Antiseptics • Disinfectants and antiseptics are distinguished on the basis of whether they are safe for application to mucous membranes. • Often, safety depends on the concentration of the compound. • For example, sodium hypochlorite (chlorine), as added to water is safe for drinking, but "chlorox" (5% hypochlorite), an excellent disinfectant, is hardly safe to drink. Common antiseptics and disinfectants Common antiseptics and disinfectants Chemical Ethanol (50-70%) Isopropanol (50-70%) Formaldehyde (8%) Action Denatures proteins and solubilizes lipids Denatures proteins and solubilizes lipids Reacts with NH2, SH and COOH groups Tincture of Iodine (2% in 70% alcohol) Inactivates proteins Chlorine (Cl2) gas Forms HClO, a strong oxidizing agent Silver nitrate (AgNO3) Mercuric chloride Detergents (e.g. quaternary ammonium compounds) Phenolic compounds (e.g. lysol, hexylresorcinol, hexachlorophene) Ethylene oxide gas Uses Antiseptic used on skin Antiseptic used on skin Disinfectant, kills endospores Antiseptic used on skin Disinfect drinking water; general disinfectant Antiseptic and used in the eyes Precipitates proteins of newborns Inactivates proteins by Disinfectant, occasionally used reacting with SH groups as an antiseptic on skin Disrupts cell Skin antiseptics and membranes disinfectants Denature proteins and disrupt cell membranes Antiseptics at low concentrations; disinfectants at high concentrations Alkylating agent Disinfectant used to sterilize heat-sensitive objects (plastics) Microbial growth control and nutrition: cultivation of microorganisms Photoautothroph Chemioautothroph Chemioheterothroph Elemental and macromolecular composition of a bacterial cell Major elements, their sources and functions in bacterial cells. Element % dry weight Source C 50 organic compounds or CO2 O 20 H2O, organic compounds, CO2, and O2 N 14 NH3, NO3, organic compounds, N2 H 8 H2O, organic compounds, H2 P 3 inorganic phosphates (PO4) S 1 SO4, H2S, So, organic compounds K 1 Potassium salts Mg 0.5 Magnesium salts Ca 0.5 Calcium salts Fe 0.2 Iron salts • Media for bacteria must include source of C, N, P, S, 4 of the 6 major nutrients as well as micronutrients. • The micronutrients are usually present as trace contaminants in water, on glassware, or in chemicals used to make media. • However certain minerals (Ca, Mg, Fe, K) are needed in slightly higher concentrations, and may be added. • Media can be liquid or solid. They can be used for different purposes Liquid media • Easiest to prepare and use. • Good for growing quantities of microbes needed for analysis or experiments. • Unless inoculated with pure culture, cannot separate different organisms. Solid media • Usually made by adding agar, a seaweed extract, to appropriate liquid. 1.5% agar is standard for plates. •Agar melts at 80-90°C, will remain liquid until temperature cools to 40-42 °C. •Very few microbes can degrade agar, so it is normally not a source of C, and acts as inert gelling medium. Solid media • Usually made by adding agar, a seaweed extract, to appropriate liquid. 1.5% agar is standard for plates. •Agar melts at 80-90°C, will remain liquid until temperature cools to 40-42 °C. •Very few microbes can degrade agar, so it is normally not a source of C, and acts as inert gelling medium. Growth on liquid media Growth on solid media Synthetic or Defined Media • They are usually relatively simple media. • All components are known. • Useful for experimental situations such as to select a microbe that can obtain all its nitrogen from atmospheric N2. • You would prepare synthetic medium with sources of C, P, and S, but no N source. Microorganisms would be unable to grow unless they can fix nitrogen from air. Minimal medium for the growth of Bacillus megaterium. An example of a chemically-defined medium for growth of a heterotrophic bacterium Component Amount Function of component sucrose 10.0 g C and energy source K2HPO4 2.5 g pH buffer; P and K source KH2PO4 2.5 g pH buffer; P and K source (NH4)2HPO4 1.0 g pH buffer; N and P source MgSO4 7H2O 0.20 g S and Mg++ source FeSO4 7H2O 0.01 g Fe++ source MnSO4 H2O 0.007 g Mn++ Source water 985 ml pH 7.0 Defined medium (also an enrichment medium) for the growth of Thiobacillus thiooxidans, a lithoautotrophic bacterium Component Amount Function of component NH4Cl 0.52 g N source KH2PO4 0.28 g P and K source MgSO4 7H2O 0.25 g S and Mg++ source CaCl2 2H2O 0.07 g Ca++ source Elemental Sulfur 1.56 g Energy source C02 5%* C source water 1000 ml pH 3.0 Defined medium M63 for the growth of Escherichia coli DH10B strain SW102, a recombineering lab strain Component Amount Function of component (NH4)2SO4 2g N source KH2PO4 13,6 g P and K source FeSO4 7H2O 0.5 mg Fe++ and S source D-biotin 1 mg L-leucine 45 mg D-galactose/glycerol MgSO4 7H2O Agar water 0.2% 1 mM 15 g/l 1000 ml C source and energy source S and Mg++ source pH 7.0 Complex media • The composition of these media is not completely known. • Often made from inexpensive organic materials such as: – slaughterhouse wastes (tryptic digests called tryptone, trypticase, etc.) – soybeans – yeast wastes from brewing (rich source of vitamins) – animal blood, etc. Complex medium (nutrient broth) for the growth of fastidious bacteria Component Amount Function of component Beef extract 1.5 g Source of vitamins and other growth factors Yeast extract 3.0 g Source of vitamins and other growth factors Peptone 6.0 g C and energy source, N, P and S source Glucose 1.0 g C and energy source Agar 15.0 g Inert solidifying agent water 1000 ml pH 6.6 Selective media • Selective Media: media favors the growth of one or more microbes. • Example: bile salts inhibit growth of most gram-positive bacteria and some gramnegative bacteria, but enteric bacteria adapted to life in animal gut can grow well. • Include bile salts in some media such as EMB, MacConkey agar to select for enterics. Differential media •Differential Media: allows distinguishing between different bacteria that grow. •Ex: MacConkey agar has color indicator that distinguishes presence of acid. Bacteria that ferment a particular sugar (e.g., glucose in culture media) will produce acid wastes on plates, turn pH indicator red. Bacteria that cannot ferment the same sugar will grow but not affect pH, so colonies remain white. •It is possible to design a medium that is both selective and differential. Growth of Escherichia coli on differential media MacConkey agar medium for the growth of enteric bacteria Component Amount Function of component Peptone Bile salts 20 g 1.5 g Lactose Sodium chloride Neutral red Crystal violet 10 g 5g 0.03 g 0.001 g C and energy source Inhibit growth of most grampositive bacteria C and energy source Agar water 13,5 g 1000 ml pH 7.1 Na and Cl source pH indicator inhibit growth of most grampositive bacteria Inert solidifying agent MacConkey agar is a widely-used culture medium which is both selective AND differential. Escherichia coli Gram Negative: growth Lactose Fermentation: positive, (pink colonies) Salmonella typhimurium Gram Negative: growth Lactose Fermentation: neg.,(colorless colonies) Mannitol salt agar is both a selective AND differential growth medium. It is used to differentiate pathogenic Staphylococcus species from nonpathogenic members of the genus Micrococcus Staphylococcus aures Staphylococcus epidermidis positive = acid end products turn the phenol red pH indicator from red to yellow negative = prenol red remains red Blood agar is used both as an enriched medium for growing fastidious . bacteria and as a differential medium. Exotoxins called hemolysins cause lysis of the redblood cells. The degree of the hemolysis is an especially useful tool for identification of many of the Gram positive cocci. 1. Beta hemolysis completely lyse the red blood cells and hemoglobin; this results in complete clearing around colonies. 2. Alpha hemolysis refers to the partial lysis of RBC's and hemoglobin and produces a greenish discoloration of the blood agar around the colonies. 3. No hemolysis, called gamma hemolysis results in no change of the medium b a g