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(Microbial Growth) Bacterial Division Most bacteria divide by binary fission. (Microbial Growth) Binary fission is when cell increases in length and divides into two cells (Microbial Growth) The DNA has replicated so each new cell has it's own nucleoid region (Cellular Nutrition) Heterotroph obtains its carbon from organic material (Cellular Nutrition) Autotroph (self feeder) - obtains its carbon from inorganic material (CO2) Autotroph and Heterotroph Can be further subdivided based on their energy source Phototrophs gain energy from sunlight hemotrophs gain energy from chemical compounds (metabolism) What kind of nutrients Carbon, Nitrogen , Oxygen , Hydrogen , Sulfur , Other Most nutrients come from proteins, carbohydrates, lipids, nucleic acids How do nutrients get into the cell Passive Transport, Active Transport, Generation Time (Passive Transport) Diffusion random entry (Passive Transport) Facilitated diffusion molecule binds a receptor in membrane and is carried across (Active Transport) Carrier-mediated molecules moved using a pump (Active Transport) Group translocation moved across membrane and used metabolically (Active Transport) Bulk transport mass transport by engulfment Generation Time is the time required for a cell to divide, or a given population to double Generation Time Varies among bacteria and is influenced by culture conditions. To calculate the # of cells produced: Number of cells x 2number of generations = final concentration of cells Calculating cell example 4 (cells) x 2 8(number if gernerations) = 4 x 256 = 1024 (Calculating the number of generations) Convert the number of cells to a log (Calculating the number of generations) Log number of final conc. of cells log number of beginning conc. of cells Calculating the number of generations 0.301 (log of 2, each cell divides into 2) Bacterial growth can be charted on a growth curve and divided into 4 phases of growth (4 Phases of Growth) Lag phase very little change in number of cells. Metabolic activity occurring. (4 Phases of Growth) Log phase rapid logarithmic growth. Also called exponential growth phase. High reproduction activity, high metabolic activity. Very sensitive to antimicrobial agents (4 Phases of Growth) Stationary phase metabolic and reproductive activity slows until the number of newly produced cells equals the number of dying cells. Growth slows due to decrease in nutrients, environment becomes unfavorable (4 Phases of Growth) Death phase Number of dying cells exceeds the number of living cells. Continues until only a few cells remain or all cells are dead. Direct Count 1 Plate counts Direct Count 2 Filtration Direct Count 3 Most Probable Number Direct Count 4 Direct microscopic count Methods of Counting Bacteria Plate counts, Pour Plate Dilution, Spread plate technique, Filtration Plate counts type pour plate or streak plate Plate counts pour plate or streak plate Must dilute the bacteria so only 25-250 colonies will grow Plate counts Count the number of colonies as plate forming units (PFU) Pour Plate step 1 A dilution of bacteria is poured into an empty Petri dish Pour Plate step 2 Add liquid agar Pour Plate step 3 Mix plate, solidify, incubate Pour Plate step 4 Count PFU's multiply by dilution factor Spread plate technique 1 Serial dilute the bacteria (1:10, 1:100, 1:1000) Spread plate technique 2 Spread each dilution onto its own plate Spread plate technique 3 Incubate Spread plate technique 4 Count the number of PFU and multiply by dilution factor (Filtration) Only when bacterial concentration is very small (Filtration) Used in water quality testing Filtration Filter 1 100mls of water through a special membrane Filtration Transfer 2 membrane to plate Filtration step 3 Incubate Filtration 4 Count PFU MPN Greater number of bacteria in a sample the more dilutions needed until no bacteria grow MPN Used when bugs won't grow on plates (solid) only broth MPN Compare the number of tubes with growth to the MPN table to determine the probable range of bacteria (Direct microscopic count) a measured volume of a sample is placed in a defined area of a counting chamber slide. (Direct microscopic count) Slide contains a grid, count the number of bacteria inside the grid (Direct microscopic count) Multiply by dilution factor = bacteria/ml Indirect measurement Turbidity, Metabolic activity, Dry weight Turbidity Use a spectrophotometer to determine the amount of transmittance of light through a liquid bacterial sample Turbidity Must have enough bacteria to do technique Turbidity Amount of light transmitted through is inversely proportional to the number of bacteria Turbidity Useful for monitoring bacterial growth Metabolic activity Assume that the amount of a metabolic by-product, such as CO2 or acid is directly proportional to the number of bacteria Dry Weight, If an organism is filamentous like mold or fungus, other measurements don't work well. Dry Weight Removes fungus from growth medium, place in bottle, and dry. Dry Weight Weigh bottle with sample Controlling Microbial Growth Sterilization, Commercial sterilization, Disinfection, Sanitization Sterilization Destruction of ALL microbial growth including endopspores by Autoclave and Radiation Commercial sterilization Limited heating of a product so as to not damage the product. Commercial sterilization Food canning destroys endospores of Clostridium Commercial sterilization Food is not completely sterile Disinfection Aimed only at destroying pathogenic vegetative bugs Disinfection Use UV light, boiling, chemicals, steam Disinfection Chemicals are called disinfectants Disinfection When treating living tissue the chemicals are called antiseptics , Procedure is antisepsis Sanitization Lowers the number of bugs to within health department guidelines Sanitization Minimizes the chance of spreading disease Sanitization Done in restaurants for glass and table wear Sanitization High temperature washing and/or chemicals How anti-microbial agents work Changes in plasma membrane integrity, Damage to protein synthesis, Denaturation or proteins and nucleic acids, Cell Wall, (anti-microbial agents) Changes in plasma membrane integrity Causes leakage or weakening by actions on the plasma membrane components (anti-microbial agents) Damage to protein synthesis Chemicals (antibiotics) target and attack ribosomes and prevent protein biosynthesis (anti-microbial agents) Damage to protein Agents can bind to DNA and prevent transcription (anti-microbial agents) b/c Denaturation or proteins and nucleic acids, Cell Wall (anti-microbial agents) b/c Denaturation or proteins and nucleic acids Chemicals and heat can break chemical bonds in proteins and nucliec acids, causing denaturation (anti-microbial agents) b/c Denaturation or proteins and nucleic acids Can use strong organic solvents (alcohols, acids) and phenol compounds (anti-microbial agents) b/c Cell Wall Agents can block the synthesis of the cell wall, digest it, or break down the surface Physical Methods for controlling growth Heat, Filtration, Low Temperatures, Dessication , Osmotic Pressure, Radiation Heat Used in food preservation, preparation, and sterilization Heat, Some bugs are more resistant than others Heat Can be moist Boiling of water, Autoclaving Dry heat Incineration of biohazardous materials, flaming of loop in lab Pasteurization Controlling bacterial growth with heat Pasteurization, Various foods have their own set temperature and time for pasteurization Pasteurization, Pathogenic bugs are killed but food quality is maintained Pasteurization, For milk, wine and fruit juices is 72°C for 15 seconds For milk, wine and fruit juices High-temperature Short-time (HTST) High-temperature Short-time (HTST) Pasteurization Kills pathogens, lowers bacteria, extends shelf life. Ultrahigh temperature pasteurization (UHT) 134°C for 1-2 seconds, marketed as "sterile" but is not, Shelf life of 3-6 months (Filtration) Passing a liquid or gas through a screen with micropores (Filtration) Large particles are held behind, while other substances can pass through (Filtration) HEPA filters are high-efficiency particualte air filters HEPA filters Used in lab hoods, operating rooms, vacuum cleaners HEPA filters Removes almost all bugs that are larger than 0.3mm HEPA filters Can use filters in the lab for "sterilizing" gases and liquids, Cut off is .22mm and 0.45mm. Physical Methods of Control Low temperatures, Dessication, Osmotic pressure (Physical Methods of Control) Low temperatures Reduces metabolic rate of bugs, Only bacteristatic control, Temp depends on bug (Physical Methods of Control) Dessication Removal of water to slow bacterial growth, Used in food products, Resistance depends on bugs some can remain viable for months. (Physical Methods of Control) in food products Lyophilization - freeze drying EX Coffee, cereal, jerky (Physical Methods of Control) Osmotic pressure Increase in salt or sugar concentration to slow bacterial growth (Physical Methods of Control) Osmotic pressure Used in food preservation EX Ham, bacon (Physical Methods of Control) Osmotic pressure Effects osmotic pressure of bug and can cause dessication (Physical Methods of Control) Osmotic pressure Molds and yeasts are less effected. Radiation Causes disruption in the DNA structure. Common form is UV light Causes thymine dimers to form in DNA , EX Used in hospital nurseries, OR, cafeterias Radiation is Not very penetrating Food exposed to various types of radiation can be sterile for months and years (Chemical methods) of microbial control Phenols, Halogens, Alcohols, Surface-active agents, Chemical food preservatives, Antibiotics, Aldehydes , Gaseous Agents (Chemical Methods) Phenols First used as a disinfectant on patients by Lister, Now used in cough drops mainly for , anesthetic quality, Not used on patients anymore (Chemical Methods) Halogens - Iodine and Cholrine Iodine can be used in aqueous solution Chlorine is a disinfectant, used as a gas for water purification or with other chemicals EX Chlorox (Chemical Control) Alcohols Antiseptic, but doesn't kill endospores or some viruses, Some mechanical removal (Chemical Control) Surface-active agents Decreases surface tension of liquid molecues , Disrupts plasma membranes, Agents include soap and detergents, Some mechanical removal (Chemical Control)Chemical food preservatives Sulfur dioxide, used in wine making, a disinfectant. (Chemical Control) Chemical food preservatives Sodium benzoate prevents growth of molds by interfering with metabolism or plasma membrane. (Chemical Control) Chemical food preservatives Sodium nitrite and nitrate used in meats, prevents germination of Clostridium endospores. (Chemical Control) Chemical food preservatives Ingestion should be limited due to risk of nitrosamines, carcinogenic. (Chemical Control) Aldehydes One of the most effective antimicrobials, Formaldehyde is a disinfectant, Glutaraldehyde is a disinfectant used to disinfect hospital instruments. (Chemical Control) Gaseous agents Sterilize in a closed container (Chemical Control) Gaseous agents Sterilize Ethylene oxide used to sterilize plastic wear PFU is Plate forming units MPN is Most Probable Number UHT is Ultrahigh Temperature Pasteurization HTST is Hight-Temp Short-Time