Microbial Growth.pdf

Microbial Growth Requirement for growth Physical requirements Temperature pH Osmotic pressure Chemical requirements Carbon Nitrogen, sulfur, and phosphorous Trace elements Oxygen Organic growth factors Temperature Minimum growth temperature:...

Microbial Growth Requirement for growth Physical requirements Temperature pH Osmotic pressure Chemical requirements Carbon Nitrogen, sulfur, and phosphorous Trace elements Oxygen Organic growth factors Temperature Minimum growth temperature: lowest temperature at which the species will grow Optimum growth temperature: temperature at which the species grows best Maximum growth temperature: the highest temperature at which growth is possible Temperature Psychrophiles Cold-loving Grow -20 ° C to 10 ° C These cold-loving organisms are often found in environments such as: Antarctic and Arctic Regions: Including ice and snow. Deep Ocean: Where temperatures are consistently near freezing. Permafrost: Soil that remains frozen for long periods. Psychrophiles have adapted to these cold conditions with specialized enzymes and cellular membranes that remain fluid and functional at low temperatures. They play a crucial role in biogeochemical cycles in cold environments and are of interest in various applications, including biotechnology, where their enzymes can be used in cold-adapted industrial processes. Temperature Psychrotrophs Cold-loving Grow 0 ° C to 30 ° C Psychrotrophs are more versatile and can be found in a variety of environments, including refrigerated foods and other settings where temperatures are not extremely cold but are still lower than typical room temperature. Cause food spoilage Temperature Mesophiles moderate-temperature-loving They typically grow best between 20°C and 45°C (68°F to 113°F). Most mesophiles prefer temperatures around 30°C to 40°C (86°F to 104°F) the average temperature of the human body and many common environments. These conditions are neither too hot nor too cold, making mesophiles well-suited for environments like: Soil and Water: Where temperatures are often within their preferred range. Human Body: Many mesophiles are pathogenic bacteria that grow best at around 37°C (98.6°F), the normal body temperature. Compost Piles: Where temperatures can be moderately elevated due to microbial activity. Mesophiles are important in various biological processes, including fermentation, digestion, and decomposition. Their enzymes and metabolic processes are adapted to function optimally within this temperature range. Temperature Thermophiles heat-loving Optimum growth temperature of 50 to 60C Adapted to heat and can be found in various high- temperature environments such as: Hot Springs: Natural springs with temperatures that can be quite high. Geothermal Areas: Locations with significant volcanic activity, such as geysers and fumaroles. Compost Heaps: Where microbial activity generates heat. These organisms have specialized enzymes and cellular structures that enable them to maintain stability and functionality at high temperatures Making them valuable in industrial processes that require high-temperature conditions, such as in the production of biofuels and other biotechnological applications. Temperature Hyperthermophiles Optimum growth temperature >80C176°F). Some can even survive and grow at temperatures exceeding 100°C (212°F) These organisms are often found in extreme environments such as hydrothermal vents on the ocean floor, hot springs, and geysers. Their enzymes and cellular structures are highly adapted to withstand and function optimally at these extreme temperatures. *Hyperthermophiles and thermophiles are both types of heat-loving microorganisms, but they differ in the temperatures at which they thrive pH The symbol for hydrogen ion (H+) concentration; a measure of the relative acidity or alkalinity of a solution Acid Base A substance that dissociates A substance that dissociates into one or more hydrogen ions into one or more hydroxide ions (H+) and one or more negative (OH-) and one or more positive ions ions Proton donor Proton acceptor pH 0-6 pH 8-14 Coffee Bleach Vinegar Baking soda pH Most bacteria grow between pH 6.5 and 7.5 Acidophiles grow in acidic environments Molds and yeasts grow between pH 5 and 6 Alkaliphiles grow in basic environments Bacillus alcalophilus grow in highly alkaline conditions and is often found in soda lakes. Osmotic Pressure Hypertonic environments (higher in solutes than inside the cell) cause plasmolysis due to high osmotic pressure Extreme or obligate halophiles require high osmotic pressure (high salt) Facultative halophiles tolerate high osmotic pressure Carbon Structural backbone of organic molecules Chemoheterotrophs use organic molecules as energy Autotrophs use Nitrogen Component of proteins, DNA, and ATP Most bacteria decompose protein material for the nitrogen source Some bacteria use NH4+ or NO3- from organic material A few bacteria use N2 in nitrogen fixation Sulfur Used in amino acids, thiamine, and biotin Most bacteria decompose protein for the sulfur source Some bacteria use SO42 or H2S Phosphorus Used in DNA, RNA, and ATP Found in membranes PO43 is a source of phosphorus Oxygen Singlet oxygen: (1O2-) boosted to a higher-energy state and is reactive Superoxide radicals: O2 O−2 + O−2 + 2H + → H 2O2 + O2 Superoxide radicals are formed by the addition of an extra electron to molecular oxygen, resulting in a negatively charged radical Peroxide anion: O2 2 – 2H 2 O 2 → 2H 2 O + O 2 The peroxide anion is a negatively charged form of hydrogen peroxide Hydroxyl radical (OH ) H2O2 + 2H+ → 2H2O The hydroxyl radical is one of the most reactive and damaging ROS. It is a highly reactive species with an unpaired electron. Organic and Inorganic Trace elements Inorganic elements required in small amounts Usually as enzyme cofactors Include iron, copper, molybdenum, and zinc Organic Growth Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines Biofilms Microbial communities Form slime or hydrogels that adhere to surfaces – Bacteria communicate cell- to-cell via quorum sensing – Bacteria secrete an inducer (signaling chemical) to attract other bacterial cells Share nutrients Shelter bacteria from harmful environmental factors Biofilm Involved in 70% of infections – Catheters, heart valves, contact lenses, dental caries 1000x resistant to microbicides Found in digestive system and sewage treatment systems can clog pipes Found on algae Hot tubs Culture Media Culture medium: nutrients prepared for microbial growth Sterile: no living microbes Inoculation: introduction of microbes into a medium Culture: microbes growing in or on a culture medium Aseptic Technique Louis Pasteur Methods to prevent contamination, proving that microorganisms come from other microorganisms, not spontaneously from nonliving matter. Aseptic techniques are crucial for maintaining sterility and preventing contamination. Essential tools in aseptic technique Autoclave - sterilizer Biological Safety Hood - controlled, contaminant-free environment Bunsen burner Practical applications Laboratory clinical setting Biological Safety Hood Culture Media Agar Complex polysaccharide Used as a solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100C Solidifies at ~40C Chemically Defined Media exact chemical composition is known – Fastidious organisms are those that require many growth factors provided in chemically defined media – Simmon’s citrate Complex media extracts and digests of yeasts, meat, or plants; chemical composition varies batch to batch o Nutrient broth o Nutrient agar Anaerobic Growth Media dn Methods Special Culture techniques Capnophiles Microbes that require high CO2 conditions CO2 packet Candle jar Biosafety levels ̶ BSL-1: no special precautions; basic teaching labs ̶ BSL-2: lab coat, gloves, eye protection ̶ BSL-3: biosafety cabinets to prevent airborne transmission ̶ BSL-4: sealed, negative pressure; “hot zone” ▪ Exhaust air is filtered twice through HEPA filters Selective and Differential Media Selective media – Suppress unwanted microbes and encourage desired microbes – Contain inhibitors to suppress growth Differential media – Allow distinguishing of colonies of different microbes on the same plate Some media have both selective and differential characteristics Enrichment Culture Encourages the growth of a desired microbe by increasing very small numbers of a desired organism to detectable levels Sabouraud Dextrose Agar Pure Culture A pure culture contains only one species or strain A colony is a population of cells arising from a single cell or spore or from a group of attached cells A colony is often called a colony-forming unit (CFU) The streak plate method is used to isolate pure cultures  Streak plate method Purpose: to get isolated pure colonies Preserving bacterial cultures Deep-freezing: –50 to –95C Cryocultures Lyophilization (freeze- drying): frozen (–54 to –72C) and dehydrated in a vacuum Use media depending on bacteria to rehydrate them. Growth of bacterial cultures Increase in number of cells, not cell size Binary fission: prokaryotic cell reproduction by division into two daughter cells Budding: Asexual reproduction beginning as a protuberance from a parent cell that grows to become a daughter cell Conidiospores (fungi) actinomycetes Fragmentation of filaments Binary Fission Generation Time Time required for a cell to divide – 20 minutes to 24 hours Binary fission doubles the number of cells each generation Total number of cells = 2number of generations Growth curves are represented logarithmically Phases of Growth Lag phase: The time interval in a bacterial growth curve during which there is no growth Log phase: The period of bacterial growth or logarithmic increase in cell numbers Exponential growth phase Stationary phase: The period in a bacterial growth curve when the number of cells dividing equal the number dying. – Bacteria approach the carrying capacity Death phase: The period of logarithmic decrease in a bacterial population Logarithmic decline phase Direct Measure of Microbial Growth Direct measurements–count microbial cells –Plate count –Filtration –Most probable number (MPN) method –Direct microscopic count Plate counts Count colonies on plates that have 30 to 300 colonies (CFUs) To ensure the right number of colonies, the original inoculum must be diluted via serial dilution Counts are performed on bacteria mixed into a dish with agar (pour plate method) or spread on the surface of a plate (spread plate method) Filtration Solution passed through a filter that collects bacteria Filter is transferred to a Petri dish and grows as colonies on the surface Most probable number (MPN) method Multiple tube test Count positive tubes Compare with a statistical table  Direct microscopic count Volume of a bacterial suspension placed on a slide Average number of bacteria per viewing field is calculated Uses a special Petroff-Hausser cell counter Number of cells counted Number of bacteria/ml = Volume of area counted Estimating Bacterial Numbers by indirect methods Turbidity—measurement of cloudiness with a spectrophotometer Metabolic activity—amount of metabolic product is proportional to the number of bacteria Dry weight—bacteria are filtered, dried, and weighed; used for filamentous organisms