L4 Microbial Growth PDF - BIO-440 2025

L4: Microbial growth BIO-440 Jan 24, 2025 By Dr. Anny Cárdenas Assistant Professor, Department of Biology 1 Content Topic 1: Culturing microbes and measuring their growth Topic 2: Dynamics of microbial growth Topic 3: Environmental effects on growth: Temperatur...

L4: Microbial growth BIO-440 Jan 24, 2025 By Dr. Anny Cárdenas Assistant Professor, Department of Biology 1 Content Topic 1: Culturing microbes and measuring their growth Topic 2: Dynamics of microbial growth Topic 3: Environmental effects on growth: Temperature, pH, Osmolarity, and oxygen Microbial growth: increase in population size as a result of cell division 2bindfsion Cell nutrition Macronutrients required in large amounts Micronutrients required in minute amounts Average bacterial cell weights 10 -12g and 75% of that mass is water taetognt Based on their dry weight: Average bacterial cell weights 184-15g in which carbon (C), oxygen (O), nitrogen (N), hydrogen (H), phosphorus (P), and sulfur (S) account for Eintages about 96% of the mass etoiiiti.io The next 3.7% of a cell’s mass is composed of potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), chlorine (Cl), and iron (Fe) Cell nutrition Cell nutrition longoncentrations in found Macronutrients antics TAP Micronutrients: Trace metals and growth factors Trace elements: metals needed in very small amounts to perform essential biological functions large amounts Iron (Fe): hemoglobin cytochromes and several other respiratory enzymes Cobalt (Co): Vitamin B12 Copper (Cu): Respiration, cytochrome oxidase, superoxidase dismutase Manganese (Mn): superoxidase dismutase, carbohydrate metabolism Micronutrients: organic micronutrients rather than metals Vitamins Amino acids Purines Growth media and laboratory culture Culture medium: A nutrient solution required for growing a laboratory culture of a specific microorganism. Defined: The exact chemical composition is known (e.g., minimal media for genetic studies). Complex: Contains unknown quantities of nutrients, often derived from natural sources like yeast extract or peptone (e.g., nutrient agar, tryptic soy broth). we Source of C and N, vitamins and growth factors, trace elements, minerals etc. Defined vs complex medium Growth media and laboratory culture Culture medium: A nutrient solution required for growing a laboratory culture of a specific microorganism. Selective: Inhibit the growth of unwanted organisms while allowing the target organism to grow (e.g., agar for Salmonella). Differential: Distinguish between organisms based on specific biochemical reactions (e.g., blood agar for hemolysis). Enrichment: Contain additional nutrients to support the growth of fastidious organisms. Laboratory culture Liquid: Broths used for growing large numbers of organisms. Solid: Contain agar to solidify the medium, used for isolating and observing colonies. Semi-solid: Contain lower agar concentrations, useful for motility studies. Laboratory culture Colonies: A visible cluster of microorganism that originates from a single microbial cell growing on a solid medium Laboratory culture Plates inoculated from a mixed culture (such as a natural sample) or from a contaminated pure culture will typically contain more than one colony type. Assessing microbial numbers Microscopic counts of microbial cell numbers 1 -Total cell count Viable counting of microbial cell numbers 2 -Spread-plate 3 -Pour-plate Turbidimetric measures of microbial cell numbers 4 Optical density Microscopic counts of microbial cell numbers 1 Total cell count: enumerating the cells present by a microscopic cell count Viable counting of microbial cell numbers 2 Spread-plate: a volume of an appropriately diluted culture is spread over the surface of agar plate. Each viable cell will grow and divide to yield a single colony, and hence, colony numbers are taken as a measure of cell numbers. Viable counting of microbial cell numbers 3 Pour-plate: a known volume of media is pipetted into an empty sterile Petri plate. Molten agar medium, tempered to just above gelling temperature is then added and gently mixed before allowing the agar to solidify. Considerations/Assumptions for 2 and 3 One cell forms one colony The number of colonies developing on or in the medium not be too many or too few (plates should contain between 30 and 300 colonies.) Diluting the sample Colony Forming Units (CFUs) represent individual cells or groups of cells capable of forming a visible colony on a solid growth medium under specific conditions. The great plate count anomaly Direct microscopic counts of natural samples typically reveal far more microbes than are recoverable on any single culture medium. … why? Turbidimetric measures of microbial cell numbers 4 Turbidity: Microbial cells scatter light, making a suspension appear cloudy (turbid). The turbidity increases with the number of cells, as more light is scattered when more cells are present. Turbidimetric measures of microbial cell numbers 4 Turbidity: Microbial cells scatter light, making a suspension appear cloudy (turbid). The turbidity increases with the number of cells, as more light is scattered when more cells are present. iiiiiiii.it Limitations Some bacteria form clumps Some form biofilms piition Dynamics of microbial growth Growth is the result of cell division and is defined as an increase in the number of cells. Binary fission: cell division following enlargement of a cell to twice its size Generation time: The time required for a population of microbial cells to double (10 min to months) Septum Dynamics of microbial growth Growth is the result of cell division and is defined as an increase in the number of cells. The septum that separates dividing cells of the Bacillus subtilis is clearly visible in these fluorescent micrographs Growth cycle Batch culture: a closed system microbial culture of fixed volume Limited nutrients! Growth profile called the microbial growth curve The microbial growth curve Is composed of four phases: lag, exponential, stationary, and decline The microbial growth curve 1 Lag phase: the period prior to the onset of growth ngtnode.EE factor many The microbial growth curve 2 Exponential phase: the period when the growing cell population doubles at regular intervals The microbial growth curve 3 Stationary phase: the period in which the rate of growth and death are approximately equal The microbial growth curve 4 Decline phase: the period in which the rate of cell death exceeds the rate of cell growth Quantitative aspects of microbial growth Nt is the cell number at time t, N0 is the initial cell number, n is the number of generations during the period of exponential growth After 3 generations, starting with one cell: showof 4avantative howmuchthey medium likethat Continuous culture Continuous culture device (chemostat) A chemostat enables control over both the specific growth rate and growth yield of a microbial culture c Biofilms: is a population of cells enmeshed in a polysaccharide matrix that is attached to a surface Biofilm formation 1 beginning with the attachment of planktonic cells to a surface. Attachment is often mediated by flagella, fimbriae, or pili Biofilm formation colonies micro forming 2 Colonization of the surface starts when microbes begin to grow and produce sticky extracellular polysaccharides (EPS). Biofilm formation 3 During development, cells in the biofilm begin to change their metabolism. Mushroom-like columns and channels Metabolic differentiation of microbes Biofilm formation 4 Finally, dispersal of cells from a mature biofilm allows microbes to colonize new sites Biofilms and infectious diseases Biofilms often associated with chronic infections and increased resistance to antimicrobial treatments Biofilms can form on medical devices such as catheters, prosthetic joints, and pacemakers Biofilms and bioreactors biofilms are used in bioreactors to efficiently break down organic pollutants and remove contaminants thatpromoteattachmentofbacteria devices it water niti Iclean Microorganisms within the biofilm adhere to surfaces, such as plastic media or rotating discs Environmental effects on growth: Temperature Cardinal temperatures are the minimum, maximum, and optimum growth temperatures for bacterial optimal rate growth a given organism. Temperature classes of organisms mornin everything Psychrophiles (optimal temp < 15ºC) Diatoms and other algae Polaromonas Molecular adaptations to life in the cold Cold-active enzymes tend to have more α-helix (more flexible) and less β-sheets secondary structure Greater polar and lesser hydrophobic amino acid content Membranes have higher content of unsaturated and shorter- chain fatty acids (helps remain in a semifluid state) Cold shock proteins: Molecular chaperones Cryopreservants to prevent the formation of crystals Slime tstohandle bat Most bacteria can be cryopreserved Thermophiles (optimal temp < 45ºC) Hydrothermal vents Methanopyrus: 122ºC Archaea from a boiling spring at Yellowstone National Park Molecular adaptations at high temperatures Heat-stable enzymes have different amino acid sequence from the corresponding mesophilic enzyme More ionic bonding and highly hydrophobic interiors (prevent unfolding) Solutes such as di-inositol phosphate, diglycerol phosphate (stabilize proteins) Membranes have higher contents of long-chain and saturated fatty acids (higher melting point) Lipid monolayers Taq polymerase from Thermus aquaticus. First isolated from Mushroom Spring in the Lower Geyser Basin of Yellowstone National Park, Effect of pH on microbial growth Neutrophiles (5.5-8) Acidophiles Picrophilus oshimae grows optimally at pH 0.7 and 60ºC. Above pH 4, cells of P. oshimae spontaneously lyse. P. oshimae inhabits thermal soils associated with volcanic activity. Alkaliphiles Certain alkaliphiles have commercial uses because they excrete hydrolytic enzymes such as proteases and lipases that maintain their activities at alkaline pH. Bacillus natronophilus is an alkaliphilic bacterium that has been isolated from soda lakes (lakes with high concentrations of carbonate salts) Molecular adaptations under high pH Sodium (Na+) rather than H+ to drive transport reactions (e.g. flagella) -> Sodium motive force Their cytoplasm are neutral! Is Helicobacter pylori an acidophile? NO H. pylori survives in the stomach's acidic environment by producing urease, an enzyme that breaks down urea into ammonia and carbon dioxide. Ammonia neutralizes the acid in its immediate surroundings, creating a buffer zoner around the bacterial cells. Effect of osmolarity on microbial growth Halophiles: require NaCl for growth Marine microorganism accumulate compatible solutes in their cells Compatible solutes are highly water-soluble organic molecules and include sugars, alcohols, and amino acid derivatives. Glycine betaine and dimethylsulfoniopropionate (DMSP), are widely distributed among halophilic bacteria Effect of oxygen on microbial growth Aerobes: Organisms that use O2 in respiration Microaerophiles: An aerobic organisms that can use O 2 only when it is present at levels reduced from that in air (microoxic conditions) Obligate aerobe: An aerobic organisms that can use O 2 only when it is present at levels reduced from that in air (microoxic conditions) Facultative: An organisms that can grow in either the presence or absence of O2 Effect of oxygen on microbial growth Anaerobe: An organism that cannot use O2 in respiration and whose growth is typically inhibited by O 2 Aerotolerant anaerobe: An organisms unable to respire O2 but whose growth is unaffected by it Obligate anaerobe: An organisms that cannot grow in the presence of O2 Incubation under anoxic conditions Anoxic Jar Anoxic glove bag Why is oxygen toxic? Molecular oxygen (O2) per se is not toxic but can be converted to toxic oxygen by-products (Reactive Oxygen Species, ROS), and it is these that can harm or kill cells not able to deal with them. ROS scavenging enzymes E.EEYan Aerobic and aerotolerant cells have enzymes that destroy ROS Catalase test