Ch9-Microbial Growth MA post.pdf

Chapter 9: Microbial Nutrition and Growth Microbial growth – an increase in a population of microbes rather than an increase in size of an individual Colony – an aggregation of cells arising (hopefully) from single cell Biofilm—collection of...

Chapter 9: Microbial Nutrition and Growth Microbial growth – an increase in a population of microbes rather than an increase in size of an individual Colony – an aggregation of cells arising (hopefully) from single cell Biofilm—collection of microbes living on a surface in a complex community Growth Requirements: both physical and chemical.; use a variety of nutrients for their energy needs and to build organic molecules and cellular structures This Photo by Unknown Author is licensed under CC BY © 2018 Pearson Education, Inc.  Growth by Binary fission. Binary fission – process by which most bacteria divide. DNA replicates as the cell elongates. A division septum forms in the center of the cell. Two daughter cells of similar size form and separate, each receiving a copy of the original chromosome. © 2018 Pearson Education, Inc.  The Requirements for Growth Physical requirements – Temperature – pH – Osmotic pressure – Hydrostatic pressure Chemical requirements –Carbon –Nitrogen, sulfur, and phosphorous –Trace elements –Oxygen –Organic growth factors © 2018 Pearson Education, Inc. Physical Growth Requirements: Temperature Effect on proteins- 3D shape Effect on membranes of cells and organelles -If too low, = rigid and fragile -If too high, = too fluid & cannot contain the cell or organelles This Photo by Unknown Author is licensed under CC BY © 2018 Pearson Education, Inc.  Temperature Figure 6.4 Five categories of microbes based on Psychrophiles: temperature ranges for growth. – cold-loving Mesophiles: – moderate-temperature- loving Thermophiles – heat-loving – Optimum growth temperature of 50° to 60°C – Found in hot springs and organic compost Hyperthermophiles – Optimum growth temperature >80°C Psychrotrophs – Grow between 0°C and 20° - 30°C – Cause food spoilage What organisms would grow in your refrigerator? © 2018 Pearson Education, Inc.  Temperature Psychrotrophs – Grow between 0°C and 20° - 30°C – Cause food spoilage  growth temperature between mesophilic & psychrophile range.  Listeria monocytogenes common food pathogen. Gram+ rod. It causes a type of meningitis (~20% mortality)  affects iimmunocompromised people and the elderly  Fetus of pregnant women. Pathogen can cross the placenta resulting in miscarriage, stillbirth, or fatal neonatal infection. Pregnant women advised to avoid consumption of soft cheeses, refrigerated cold cuts, smoked seafood, and unpasteurized dairy products. © 2018 Pearson Education, Inc.  Food Preservation Temperatures Psychotrophs do not grow well at low temperatures; but overtime they are able to degrade food:  mold mycelium, slime on food surfaces, or off- tastes or off-colors in foods. © 2018 Pearson Education, Inc.  pH Extreme pH affects the structure of all macromolecules and cellular functions  Molecules held by hydrogen bonds (ex. Proteins); cellular respiration (chemiosmosis) important for the preservation of food (control microbial growth) and to microorganisms’ survival in the stomach (human microbiota). Neutrophiles- Most bacteria grow between pH 6.5 - 7.5  intestinal pathogens (ex. Salmonella spp.) are resistant to stomach acid Acidophiles grow in acidic environments  Lactobacillus (vagina microbiota) contribute to acidity; inhibits other less tolerant microbes  Molds and yeasts grow between pH 5 and 6 Alkalinophiles– grow best between pH 8-11.5 © 2018 Pearson Education, Inc.  pH Surviving Low pH of Stomach: (Ch. 9.3) Helicobacter pylori – gram-negative, flagellated, helical bacterium; common cause of stomach ulcers (peptic ulcers) H. pylori is a neutrophile. How does it survive in the stomach? H. pylori creates a microenvironment in which the pH is nearly neutral. Produces large amounts of urease, which breaks down urea to form NH4+ and CO2. The ammonium ion raises the pH of the immediate environment. https://www.mayoclinic.org/diseases-conditions/h-pylori/symptoms-causes/syc-20356171 This Photo by Unknown Author is licensed under © 2018 Pearson Education, Inc. CC BY-ND  Water Requirements Microbes require water to live: dissolve enzymes and nutrients required in metabolism reactant in many metabolic reactions Most cells die in absence of water -Some have cell walls that retain water -Endospores and cysts cease most metabolic activity in a dry environment for years Two physical effects of water: -Osmotic pressure -Hydrostatic pressure © 2018 Pearson Education, Inc.  Osmotic Pressure The pressure exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane; related to concentration of dissolved molecules and ions in a solution Hypotonic---water moves into cell. Cell may swell and burst (lysis) in absence of cell walls Hypertonic -----water moves out of cell. Crenation occurs. -This effect helps preserve some foods: brines, salting meat/fish Restricts organisms to certain environments -Obligate halophiles – grow in up to 30% salt -Facultative halophiles – can tolerate high salt concentrations © 2018 Pearson Education, Inc.  Osmotic Pressure Hypertonic environments (higher in solutes than inside the cell) cause plasmolysis the shrinking of the protoplasm away from the intact cell wall) and cell death.  Used to control preserve food (control microbial growth): brines, salting meat/fish Cell wall protects from osmotic pressures- within tight range, otherwise they will not tolerate it and will die. © 2018 Pearson Education, Inc.  Halophiles in salt ponds Halophiles- “salt loving” organisms Extreme or obligate halophiles require high osmotic pressure (high salt) Facultative halophiles tolerate high osmotic pressure staphylococci, micrococci, Colorful halophilic algae and archaea and corynebacteria are thrive in these ponds near San halotolerant microorganisms Francisco. Used for commercial salt that colonize our skin production, the ponds contain water that is five to six times as salty as seawater. © 2018 Pearson Education, Inc.  Hydrostatic Pressure Water exerts pressure in proportion to its depth -For every additional 10 m of depth, water pressure increases 1 atm Organisms that live under extreme pressure are barophiles -Their membranes and enzymes depend on this pressure to maintain their 3-D, functional shape. © 2018 Pearson Education, Inc.  The Requirements for Growth Physical requirements – Temperature – pH – Osmotic pressure – Hydrostatic pressure Chemical requirements Prokaryotes –Carbon participate in many stages of –Nitrogen, sulfur, and phosphorous chemical –Trace elements cycles –Oxygen –Organic growth factors © 2018 Pearson Education, Inc.  Growth Requirements (terms to understand) -Carbon, Energy and electron source [INSERT FIGURE 6.1] https://youtu.be/f8G7IulYxiA?si=RNIsEx6vhOU_qwqH © 2018 Pearson Education, Inc.  Chemical Requirements Carbon – Structural backbone of organic molecules – Chemoheterotrophs use organic molecules as energy – Autotrophs use CO2 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 © 2018 Pearson Education, Inc.  Figure 8.24 Carbon cycle This figure summarizes the carbon cycle. Eukaryotes participate in aerobic respiration, fermentation, and oxygenic photosynthesis. Prokaryotes participate in all the steps shown. (credit: modification of work by NOAA)  Nitrogen Requirements N atoms needed for proteins and nucleotides Anabolism often ceases due to insufficient nitrogen. -Often limiting factor The reduction of nitrogen gas to ammonia (nitrogen fixation) by certain bacteria is essential to life on Earth -Nitrogen sources from organic & inorganic nutrients specific groups of prokaryotes each participate in every step in the cycle. © 2018 Pearson Education, Inc.  Other Chemical Requirements P-- a component of phospholipid membranes, DNA, RNA, ATP, and some proteins - Founding in membranes S-- a component of sulfur-containing amino acids, -disulfide bonds critical to tertiary structure of proteins & in vitamins (thiamin and biotin) – Some bacteria use SO4 or H2S Trace elements -only required in small amounts; usually found in sufficient quantities in tap water Growth factors -necessary organic chemicals that cannot be synthesized by certain organisms (vitamins, certain amino acids, purines, pyrimidines, cholesterol, NADH, and heme) © 2018 Pearson Education, Inc.  Table 6.1 Some Growth Factors of Microorganisms and Their Functions © 2018 Pearson Education, Inc.  Oxygen Requirements Oxygen is essential for obligate aerobes (final e- acceptor in ETC) Oxygen is deadly for obligate anaerobes How can this be true? Neither gaseous O2 nor oxygen covalently bound in compounds is poisonous forms of oxygen that are toxic are highly reactive & oxidizing agents oxidations causes irreparable damage -to proteins and lipids © 2018 Pearson Education, Inc.  Oxygen Requirements for molecular oxygen used to classify microorganisms: Obligate aerobes—require oxygen; undergo aerobic respiration Aerotolerant anaerobes—do not use aerobic metabolism, so tolerate but cannot use oxygen; have some enzymes that detoxify oxygen’s poisonous forms Facultative anaerobes—grow via fermentation, aerobic respiration or anaerobic respiration when oxygen is not available Anaerobes—unable to use oxygen and most are harmed by it (oxygen radicals); do not use aerobic metabolism Microaerophiles—require a minimum oxygen concentration (1- 10%), lower than atmospheric air (21%); have a limited ability to detoxify hydrogen peroxide & superoxide radicals © 2018 Pearson Education, Inc. The Effect of Oxygen on the Growth of Various Types of Bacteria deep soil; deep ocean; cow rumen obligate aerobes: M. tuberculosis, Neisseria meningitidis, N. gonorrhoeae © 2018 Pearson Education, Inc.  Figure 9.19-22 Anaerobic environment Anaerobic environments are still common on earth. They include environments like (a) a bog where undisturbed dense sediments are virtually devoid of oxygen, and (b) the rumen (the first compartment of a cow’s stomach), This clinical photo depicts ulcers on the foot which provides an oxygen-free incubator for of a diabetic patient. Dead tissue methanogens and other obligate anaerobic accumulating in ulcers can provide an ideal bacteria. (credit a: modification of work by growth environment for the anaerobe National Park Service; credit b: modification of Clostridium perfringens, a causative agent work by US Department of Agriculture) of gas gangrene. Gram+, endospore former. (credit: Phalinn Ooi / Wikimedia Commons (CC-BY))  Detoxification of Reactive Oxygen Species Oxygen radicals are produced in various ways: cellular respiration & atmospheric oxygen  need to be broken down Three main enzymes break down toxic byproducts:  superoxide dismutase,  peroxidase, and  catalase Superoxide radicals: O2- Superoxide dismutase (SOD) Obligate anaerobes lack all 3 enzymes – Peroxide anion: O22 catalase peroxidase © 2018 Pearson Education, Inc.  Associations and Biofilms Organisms live in association with different species: -Antagonistic relationships -Synergistic relationships -Symbiotic relationships Nitrogen-fixing bacteria © 2018 Pearson Education, Inc.  Biofilms On a contact lens -> Development of extracellular matrix that adheres cells to one another, allows attachment to a substrate, sequesters nutrients, & may protect individuals in the biofilm Slime or hydrogels that form on surfaces (medical devices, mucous membranes of digestive system) often as a result of quorum sensing Many microorganisms more harmful as part of a Biofilm! © 2018 Pearson Education, Inc.  Figure 9.1 Biofilms Found in digestive system and sewage treatment systems; can clog pipes 1000x resistant to microbicides and antibiotics Involved in 70% of infections – Catheters, heart valves, contact lenses, dental caries Medical devices that are inserted into a patient’s body often become contaminated with a thin biofilm of microorganisms enmeshed in the sticky material they secrete. The electron micrograph (left) shows the inside walls of an in-dwelling catheter. Arrows point to the round cells of Staphylococcus aureus bacteria attached to the layers of extracellular substrate. The garbage can (right) served as a rain collector. The arrow points to a green biofilm on the sides of the container. (credit left: modification of work by Centers for Disease Control and Prevention; credit right: modification of work by NASA) © 2018 Pearson Education, Inc.  Figure 6.6 Biofilm development. – quorum sensing: Bacteria cell-to- cell communication – Bacteria secrete an inducer (signaling chemical) to attract other bacterial cells © 2018 Pearson Education, Inc.  Culturing Microorganisms Inoculum -introduced into medium (broth or solid) Culture – refers to act of cultivating microorganisms or the microorganisms that are cultivated © 2018 Pearson Education, Inc.  Obtaining Pure Cultures Cultures composed of cells arising from a single progenitor -Progenitor is termed a CFU Aseptic technique is used to prevent contamination of sterile substances or objects -Very important for diagnostic reasons among others Two common isolation techniques Streak plates Pour plates © 2018 Pearson Education, Inc.  Culturing Microorganisms Obtaining Pure Cultures Other isolation techniques Some fungi isolated with streak and pour plates. Protozoa and motile unicellular algae isolated through dilution of broth cultures Can individually pick single cell of some large microorganisms and use to establish a culture © 2018 Pearson Education, Inc.  Culture Media Majority of prokaryotes have never been grown in culture medium Six types of general culture media: Enriched - contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious organisms Defined media- exact recipe known Complex media- contain extracts and digests of yeasts, meat, or plants; precise chemical composition not known. -Supports growth of a wide variety of microorganisms -Useful when nutritional needs of an organism are unknown Selective media-inhibitory Differential media- tests based on color/media changes Anaerobic media- includes reducing agents Transport media- Used by health care personnel; ensure clinical specimens are not contaminated and to protect people from infection © 2018 Pearson Education, Inc.  Culture Media © 2018 Pearson Education, Inc.  Figure 6.12 An example of the use of a selective medium. Selective media -Contain substances that favor or inhibit growth of particular microorganisms © 2018 Pearson Education, Inc.  Examples of Differential Media Citrate test Blood agar plate- hemolysis This Photo by Unknown Author is licensed under CC BY-SA-NC Media uses key reagents to make it easy to distinguish colonies of different bacteria by a change in the color of the colonies or the color of the medium. © 2018 Pearson Education, Inc.  Selective and Differential Media Some media have both selective and differential characteristics Mannitol Salt Agar (MSA) Differential: bacteria ferment mannitol into acid, causing medium color change Selective: high salt concentration prevents growth of most bacteria © 2018 Pearson Education, Inc.  Special Culture Techniques Techniques developed for culturing microorganisms: Animal and cell culture (ei.viruses) Low-oxygen culture (ei. Anaerobic; some tissues) Enrichment culture Chemostats © 2018 Pearson Education, Inc.  Anaerobic Growth Media and Methods Reducing media ̶ Used for the cultivation of anaerobic bacteria ̶ Contain chemicals (sodium thioglycolate) that combine O2 to deplete it ̶ Heated to drive off O2 (a) An anaerobic jar is pictured that is holding Petri plates supporting cultures. (b) Openings in the side of an anaerobic box are sealed by glove-like sleeves that allow for the handling of cultures inside the box. (credit a: modification of work by Centers for Disease Control and Prevention; credit b: modification of work by NIST) © 2018 Pearson Education, Inc.  Growth of Microbial Populations Generation Time- The time required for a bacterial cell (or population of cells) to grow and divide -Dependent on chemical and physical conditions -varies with species © 2018 Pearson Education, Inc. Figure 9.5 Growth of Microbial Populations The growth curve of a bacterial culture is represented by the logarithm of the number of live cells plotted as a function of time. The graph can be divided into four phases according to the slope, each of which matches events in the cell. The four phases are lag, log, stationary, and death.  Growth of Microbial Populations Measuring Microbial Reproduction Estimating the number of microorganisms is useful Determine severity of certain infections Determine effectiveness of food preservation techniques Measure the degree of contamination of water supplies Evaluate disinfectants and antibiotics © 2018 Pearson Education, Inc.  Measuring Microbial Reproduction Direct cell count methods not requiring incubation o Manual Counting Direct microscopic count Fluorescent staining Electronic counters Coulter counter Counts cells as they interrupt an electrical current Flow cytometry Detects changes in light transmission as cells pass a detector  These methods may not distinguish between viable and not viable cells © 2018 Pearson Education, Inc.  Figure 9.8 (a) A Petroff-Hausser chamber is a special slide designed for counting the bacterial cells in a measured volume of a sample. A grid is etched on the slide to facilitate precision in counting. (b) This diagram illustrates the grid of a Petroff-Hausser chamber, which is made up of squares of known areas. The enlarged view shows the square within which bacteria (red cells) are counted. If the coverslip is 0.2 mm above the grid and the square has an area of 0.04 mm2, then the volume is 0.008 mm3, or 0.000008 mL. Since there are 10 cells inside the square, the density of bacteria is 10 cells/0.000008 mL, which equates to 1,250,000 cells/mL. (credit a: modification of work by Jeffrey M. Vinocur)  Figure 9.9 Fluorescence staining can be used to differentiate between viable and dead bacterial cells in a sample for purposes of counting. Viable cells are stained green, whereas dead cells are stained red. (credit: modification of work by Emerson J, Adams R, Bentancourt Román C, Brooks B, Coil D, Dahlhousen K, Ganz H, et al.)  Measuring Microbial Reproduction Direct methods- requiring incubation: viable counts Membrane filtration Serial dilution and viable plate counts Most probable number © 2018 Pearson Education, Inc.  Direct measurement: filtration method [INSERT FIGURE 6.22] Solution passed through a filter that collects bacteria Filter is transferred to a Petri dish and grows as colonies on the surface Test bodies of water © 2018 Pearson Education, Inc.  Direct measurement: viable plate count [INSERT FIGURE 6.21] © 2018 Pearson Education, Inc.  Indirect Methods of Measuring Microbial Populations Turbidity -Often the more turbid a culture, the greater the bacterial population Metabolic activity Dry weight Molecular methods -Isolate DNA sequences of unculturable prokaryotes © 2018 Pearson Education, Inc.  Figure 9.15 (a)A spectrophotometer is commonly used to measure the turbidity of a bacterial cell suspension as an indirect measure of cell density. (b)A spectrophotometer works by splitting white light from a source into a spectrum. The optical density (turbidity) of the sample will depend on the wavelength, so once one wavelength is chosen, it must be used consistently. The filtered light passes through the sample (or a control with only medium) and the light intensity is measured by a detector. The light passing into a suspension of bacteria is scattered by the cells in such a way that some fraction of it never reaches the detector. This scattering happens to a far lesser degree in the control tube with only the medium. (credit a: modification of work by Hwang HS, Kim MS; credit b “test tube photos”: modification of work by Suzanne Wakim)