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CHE 315: Microbiology Microbial Nutrition and Growth • Growth requirements and classification • Physical parameters that effect growth and classification based on growth patterns • Chemical parameters that effect growth and classification based on growth patterns • Population growth -- growth curve • Population growth -- Methods 9/16/2015 Microbiology CHE 315 AP Wacoo 1 Environmental Effects on Bacterial Growth • Temperature • pH • Osmotic pressure • Oxygen classes 9/16/2015 Microbiology CHE 315 AP Wacoo 2 Temperature and Microbial Growth • Cardinal temperatures – minimum – optimum – maximum • Temperature is a major environmental factor controlling microbial growth. 9/16/2015 Microbiology CHE 315 AP Wacoo 3 Temperature • Minimum Temperature: Temperature below which growth ceases, or lowest temperature at which microbes will grow. • Optimum Temperature: Temperature at which its growth rate is the fastest. • Maximum Temperature: Temperature above which growth ceases, or highest temperature at which microbes will grow. 9/16/2015 Microbiology CHE 315 AP Wacoo 4 Classification of Microorganisms by Temperature Requirements 9/16/2015 Microbiology CHE 315 AP Wacoo 5 Temperature Classes of Organisms • Mesophiles ( 20 – 45C) – Midrange temperature optima – Found in warm-blooded animals and in terrestrial and aquatic environments in temperate and tropical latitudes • Psychrophiles ( 0-20C) – Cold temperature optima – Most extreme representatives inhabit permanently cold environments • Thermophiles ( 50- 80C) – Growth temperature optima between 45ºC and 80ºC • Hyperthermophiles – Optima greater than 80°C – These organisms inhabit hot environments including boiling hot springs, as well as undersea hydrothermal vents that can have temperatures in excess of 100ºC 9/16/2015 Microbiology CHE 315 AP Wacoo 6 pH and Microbial Growth • The (measure of [H+]) acidity or alkalinity of an environment can greatly affect microbial growth. Internal pH regulated by BUFFERS and near neutral adjusted with ion pumps • Most organisms grow best between pH 6 and 8, but some organisms have evolved to grow best at low or high pH. The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic. – Acidophiles (optimum in pH range 1-4) : organisms that grow best at low pH( Helicobacter pylori, Thiobacillus thiooxidans, lactic acid bacteria – 4-7 ) – Alkaliphiles (optimum in pH range 8.5-11): organismsa that grow best at high pH ( Vibrio cholera) Microbiology CHE 315 AP Wacoo – Most of pathogenic bacteria are neutrophiles 9/16/2015 7 9/16/2015 Microbiology CHE 315 AP Wacoo 8 Osmotic Effects on Microbial Growth • Osmotic pressure depends on the surrounding solute concentration and water availability • Water availability is generally expressed in physical terms such as water activity (aw) • Water activity is the ratio of the vapor pressure of the air in equilibrium with a substance or solution to the vapor pressure of pure water ( aw 1.00). aw= P solu P water 9/16/2015 Microbiology CHE 315 AP Wacoo 9 Environmental factors and growth 1. Osmotic Effect and water activity organisms which thrive in high solute – osmophiles organisms which tolerate high solute – osmotolerant organisms which thrive in high salt – halophiles organisms which tolerate high salt – halotolerant organisms which thrive in high pressure – barophiles organisms which tolerate high pressure – barotolerant 9/16/2015 Microbiology CHE 315 AP Wacoo 10 9/16/2015 Microbiology CHE 315 AP Wacoo 11 Microbial Nutrition The hundreds of chemical compounds present inside a living cell are formed from nutrients. • Macronutrients : elements required in fairly large amounts • Micronutrients : metals and organic compounds needed in very small amounts 9/16/2015 Microbiology CHE 315 AP Wacoo 12 Main Macronutrients Carbon: (C, 50% of dry weight) and nitrogen (N, 12% of dry weight) • Virtually all chemical substances in microorganisms contain carbon in some form, whether they are proteins, carbohydrates, or lipids. 50% of a bacterium's dry weight is carbon. Carbon can be obtained from organic materials in the environment, or it may be derived from carbon dioxide. • Autotrophs are able to build all of their cellular organic molecules from carbon dioxide 9/16/2015 Microbiology CHE 315 AP Wacoo 13 Classification of organisms based on sources of C and energy used 9/16/2015 Microbiology CHE 315 AP Wacoo 14 Nitrogen: • is used for the synthesis of proteins, amino acids, DNA, and RNA. Bacteria that obtain nitrogen directly from the atmosphere are called nitrogen-fixing bacteria. They include species of Rhizobium and Azotobacter, both found in the soil. • Most Bacteria can use Ammonia -NH3 and many can also use NO3• Nitrogen fixers can utilize atmospheric nitrogen (N2) 9/16/2015 Microbiology CHE 315 AP Wacoo 15 Classification of organisms based on O2 utilization • Utilization of O2 during metabolism yields toxic by-products including O2-, singlet oxygen (1O2) and/or H2O2. • Toxic O2 products can be converted to harmless substances if the organism has catalase (or peroxidase) and superoxide dismutase (SOD) • SOD converts O2- into H2O2 and O2 • Catalase breaks down H2O2 into H2O and O2 • Any organism that can live in or requires O2 has SOD and catalase (peroxidase) 9/16/2015 Microbiology CHE 315 AP Wacoo 16 Classification of organisms based on O2 utilization • Obligate (strict) aerobes require O2 in order to grow • Obligate (strict) anaerobes cannot survive in O2 • Facultative anaerobes grow better in O2 • Aerotolerant organisms don’t care about O2 •9/16/2015 Microaerophiles require low of O2 Microbiology CHE 315 AP levels Wacoo 17 Toxic Forms of Oxygen and Detoxifying Enzymes Hydrogen peroxide Superoxide 9/16/2015 Microbiology CHE 315 AP Wacoo 18 Environmental factors and growth 4. Oxygen anaerobes lack superoxide dismutase and/or catalase anaerobes need high -, something to remove O2 chemical: thioglycollate; pyrogallol + NaOH H2 generator + catalyst physical: removal/replacement 9/16/2015 Microbiology CHE 315 AP Wacoo 19 Special Culture Techniques Candle Jar 9/16/2015 Microbiology CHE 315 AP Wacoo 20 Special Culture Techniques Gas Pack Jar Is Used for Anaerobic Growth 9/16/2015 Microbiology CHE 315 AP Wacoo 21 Other Macronutrients • Phosphate (P), sulfur (S), potassium (K), magnesium (Mg), calcium (Ca), sodium (Na), iron (Fe) • Iron plays a major role in cellular respiration, being a key component of cytochromes and iron-sulfur proteins involved in electron transport. • Siderophores : Iron-binding agents that cells produce to obtain iron from various insoluble minerals. 9/16/2015 Microbiology CHE 315 AP Wacoo 22 Microbial growth • Microbes grow via binary fission, resulting in exponential increases in numbers • The number of cell arising from a single cell is 2n after n generations • Generation time is the time it takes for a single cell to grow and divide 9/16/2015 Microbiology CHE 315 AP Wacoo 23 Binary Fission 9/16/2015 Microbiology CHE 315 AP Wacoo 24 Rapid Growth of Bacterial Population 9/16/2015 Microbiology CHE 315 AP Wacoo 25 Growth curve • During lag phase, cells are recovering from a period of no growth and are making macromolecules in preparation for growth i.e. • no apparent cell division occurring but the cells are growing in volume or mass, synthesizing enzymes, proteins, RNA e.t.c and increasing in metabolic activity. The duration of lag phase depends on; • Inoculum size • Recovery period of cells from physical damage or shock during transfer to a fresh medium • Time required for synthesis of new inducible enzymes necessary to 9/16/2015 Microbiology CHE 315 AP Wacoo 26 metabolize substrates present in a medium log phase; • cultures are growing maximally • All cells are dividing at a constant rate and the population is growing by geometric progression • Composition of medium and the conditions of incubation determine the rate of cell division • Rate of exponential growth of a bacterial culture is expressed as generation time or doubling time of the bacterial population • Generation time G is defined as the time (t) per generation (nnumber of generations). Hence, G = t/n The Stationary phase; • occurs when nutrients are depleted and wastes accumulate (Growth rate = death rate) • Exhaustion of available nutrients • Accumulation of inhibitory metabolites or end products • Exhaustion of space (biological space) 9/16/2015 Microbiology CHE 315 AP Wacoo 27 In this phase, the rate of cell division = rate of cell death and the bacteria population remains constant. Some bacteria produce secondary metabolites i.e metabolites produced after the active stage of growth (antibiotics and pigments). In spore-forming bacteria, the sporulation process is induced during the stationary phase. Death phase/decline phase:If the incubation continues after the stationary phase, death phase follows. Viable cell population declines. Turbidimetric measurements or microscopic counts cannot detect death phase in culture since living and dead cells are counted. In death phase, number of viable cells decreases geometrically or exponentially. It is the reverse of growth during the log phase. 9/16/2015 Microbiology CHE 315 AP Wacoo 28 Calculation of Generation Time If we start with one cell, when it divides, there are 2 cells in the first generation, 4 cells in the second generation, 8 cells in the third generation, and so on. The generation time is the time interval required for the cells (or population) to divide. G (generation time) = (time, in minutes or hours)/n(number of generations) G = t/n t = time interval in hours or minutes B = number of bacteria at the beginning of a time interval b = number of bacteria at the end of the time interval n = number of generations (number of times the cell population doubles during the time interval) b = B x 2n (This equation is an expression of growth by binary fission) 9/16/2015 Microbiology CHE 315 AP Wacoo 29 Example: What is the generation time of a bacterial population that increases from 10,000 cells to 10,000,000 cells in four hours of growth? 9/16/2015 Microbiology CHE 315 AP Wacoo 30 Methods used to measure microbial growth Growth;-- the formation of new protoplasm from available nutrients involves the increase in cell mass and the number. The time interval for a bacterial cell to divide into 2 new cells or for a population of bacterial cells to double is called generation time. Generation time for bacteria growing under natural conditions ranges from a few minutes to several days. In bacteria and other unicellular organisms, growth can be measured in terms of 2 parameters; a) Changes in cell mass b) Changes in cell numbers 9/16/2015 Microbiology CHE 315 AP Wacoo 31 Measurement of cell mass This may be direct or indirect. • Direct determination of cell dry weight, wet weight or volume of cells is done after centrifugation. This is a physical method. The dry weight of the bacterial mass is directly proportional to their number. • Direct determination of a chemical constituent of the cells e.g. total N, total protein or total DNA content. This is a chemical method. Total N can be determined by the micro-Kjeldahl method. • Indirect measurement of a chemical activity e.g. the rate of respiration using optical oxygen sensors, e.t.c. • Turbidity measurements involve the use of an instrument such as a spectrophotometer to determine the amount of light scattered by cell suspensions. Cells such as bacteria scatter light in proportion to their numbers. Turbidity of a suspension of cells is directly related to the cell mass/cell number. 9/16/2015 Microbiology CHE 315 AP Wacoo 32 Methods used to measure microbial growth • Count colonies on plate or filter (counts live cells) Indirect viable cell counts/plate counts methods. This involves inoculating a known volume of culture on a suitable growth medium in plates. Each viable cell grows into a colony. Colonies are then counted to determine the number of colony forming units (CFUs) and hence the viable number of bacteria/unit volume can be calculated. • Microscopic counts • Flow cytometry (FACS) • Turbitity 9/16/2015 Microbiology CHE 315 AP Wacoo 33 Viable counts • Each colony on plate or filter arises from single live cell • Only counting live cells 9/16/2015 Microbiology CHE 315 AP Wacoo 34 Direct Count Pour Plate 9/16/2015 Microbiology CHE 315 AP Wacoo 35 9/16/2015 Microbiology CHE 315 AP Wacoo 36 Microscopic counts Direct microscopic counts are possible using special slides known as counting chambers e.g. Petroff-Hausser counting chambers. The disadvantage is that both dead and living cells are counted. Only Microbiology CHE 315 AP Wacoo 37 dense9/16/2015 populations can be counted. Turbitity • Cells act like large particles that scatter visible light • A spectrophotometer sends a beam of visible light through a culture and measures how much light is scattered • Scales read in either absorbance or % transmission • Measures both live and dead cells 9/16/2015 Microbiology CHE 315 AP Wacoo 38 Inoculation • Sample is placed on sterile medium providing microbes with the appropriate nutrients to sustain growth. • Selection of the proper medium and sterility of all tools and media is important. • Some microbes may require a live organism or living tissue as the inoculation medium. 9/16/2015 Microbiology CHE 315 AP Wacoo 39 Incubation • An incubator can be used to adjust the proper growth conditions of a sample. • Need to adjust for optimum temperature and gas content. • Incubation produces a culture – the visible growth of the microbe on or in the media 9/16/2015 Microbiology CHE 315 AP Wacoo 40 Isolation • The end result of inoculation and incubation is isolation. • On solid media we may see separate colonies, and in broth growth may be indicated by turbidity. • Sub-culturing for further isolation may be required. 9/16/2015 Microbiology CHE 315 AP Wacoo 41 Inspection • Macroscopically observe cultures to note color, texture, size of colonies, etc. • Microscopically observe stained slides of the culture to assess cell shape, size, and motility. 9/16/2015 Microbiology CHE 315 AP Wacoo 42 Identification • Utilize biochemical tests to differentiate the microbe from similar species and to determine metabolic activities specific to the microbe. 9/16/2015 Microbiology CHE 315 AP Wacoo 43