Dynamics of Microbial Growth - PDF

Dynamics of Microbial Growth Chapter 4 In this chapter we will study Bacterial cell division Bacterial Growth curve- different stages Growth in nature Growth in the lab Requirements for bacterial growth Viable cell count  Robert Koch- Medical Microbiology German physicia...

Dynamics of Microbial Growth Chapter 4 In this chapter we will study Bacterial cell division Bacterial Growth curve- different stages Growth in nature Growth in the lab Requirements for bacterial growth Viable cell count  Robert Koch- Medical Microbiology German physician Robert Koch (1843 to 1910) Studied disease-causing bacteria; Nobel Prize Developed methods of cultivating bacteria Worked on methods to allow single bacteria to grow and form colonies Tried potatoes, but nutrients limiting for many bacteria Solidifying liquid nutrient media with gelatin helped Limitations: melting temperature, digestible In 1882, Fannie Hess, wife of associate, suggested agar she used to harden jelly Why do we need to grow microbes? Important to grow microbes in pure culture Study single species Medical significance Nutritional, industrial uses Challenges Microorganisms found in severe conditions Ocean depths, volcanic vents, polar regions all harbor thriving microbial species Many scientists believe that if life exists on other planets, it may resemble these organisms Each species grows under limited set of environmental conditions, including specific nutrients 4.1- Principles of Microbial Growth Describe binary fission and how it relates to generation time and exponential growth. Principles of Microbial Growth Figure 4.1 Prokaryotic cells divide by binary fission One cell divides into two, two into four, 48, 816 and so on Exponential growth: population doubles each division Generation time is time it takes for the population to double Varies among species Environmental conditions Microbial growth is defined as an increase in the number of cells in a population Cell Growth- number calculation Nt = number of cells in population at time t N0 = initial number of cells n = number of generations at that point Example: pathogen in potato salad at picnic in sun Assume 10 cells with 20-minute generation time N0 = 10 cells in original population n = 12 (3 divisions per hour for 4 hours) Nt N0 2n 10 212 Nt = 10 × 4,096 Nt = 40,960 cells of pathogen in 4 hours 4.2- Microbial Growth in Nature Describe a biofilm and explain why biofilms are important to humans. Explain why microbes that grow naturally in mixed communities sometimes cannot be grown in pure culture.  Microbial Growth in Nature Changing, complex conditions in nature differ greatly from laboratory conditions Profound effect on microbial growth, behavior Cells adjust to changes in surroundings by sensing various chemicals Produce materials appropriate for the situation Most attach to surfaces and live in polymer-encased communities termed biofilms Biofilms - Figure 4.3 Biofilms Formation and structure Implications Free cells adhere to surface and Dental plaque leads to tooth decay, multiply gum disease Release polymers to which Most infections seem to involve biofilms unrelated cells may attach and Microbes within biofilms often resistant grow to immune system and antibiotics Extra polymeric substances (EPS) Industrial concerns: accumulations in give slimy appearance pipes, drains Nutrients and wastes pass through May be hundreds of times more resistant to disinfectants characteristic channels Cells communicate with one Biofilms can also be helpful another via chemical signals Bioremediation, wastewater treatment Biofilm in oral hygiene Interactions of Mixed Microbial Communities Microorganisms regularly grow in close association with many different species Interactions can be cooperative Can foster growth of species otherwise unable to survive Strict anaerobes can grow in mouth if others consume O2 Metabolic waste of one can serve as nutrient for other Interactions often competitive Some synthesize toxic compounds to inhibit competitors Some Gram-negative bacteria use the needle-like structure to inject toxic compounds directly into competing bacteria, a process called contact-dependent growth inhibition. 4.3- Microbial Growth in Laboratory Conditions Describe how the streak-plate method is used to obtain a pure culture and how the resulting culture can be stored. Describe the stages of a growth curve and compare this closed system to colony growth and continuous culture. Microbial Growth in Laboratory Conditions Pure culture: population of cells derived from a single cell; allows study of a single species Organisms may behave differently than in nature Only about 1% of microorganisms can be cultured Pure culture obtained using aseptic technique Minimizes accidental introduction of other organisms Obtaining a Pure Culture - Figure 4.4 Need culture medium, container, aseptic conditions, method to separate individual cells With correct conditions, single cell will multiply to form a visible colony (approximately 1 million cells easily visible)  Obtaining a Pure Culture- Agar in a petri dish Agar used to solidify medium Growth in Petri dish Few microbes can degrade Two-part covered container of Not destroyed by high glass or plastic temperatures and can be Allows air to enter, but sterilized excludes contaminants Liquefies above 95 degrees Celsius; solidifies below 45 Agar plate is Petri dish with degrees Celsius agar nutrient medium Solid over temperature range for most microbial growth  Upside down incubation of petri dishes- Importance Condensation droplets formed during incubation will not fall on the agar's surface such droplets are potential sources of contamination Petri dishes with media can also be stored for a longer period in an inverted position. The evaporation of water from media can cause media dryness affecting the microbial growth the rate of evaporation decreases that results in proper microbial growth. the lid of the Petri dish may open during handling when incubated in normal position and it may cause contamination from air. Label the Petri dishes at the bottom part because lid may exchange with other Petri dishes creating confusion and inverted position makes it easy to read the labeling Obtaining a Pure Culture- Streak plate Simplest and most commonly used method for isolation of a single colony Reduces number of cells with each series of streaks allowing single cells to separate Streak plate results- note the dilution in each streak Maintaining Stock Cultures A pure culture can be maintained as stock culture  picture Often stored in refrigerator as agar slant Cells can be frozen at -70 degrees Celsius for long-term storage Mixed with glycerol to prevent ice crystal formation Can be freeze-dried The Growth Curve - Figure 4.7  The Growth Curve phases 3. Stationary phase 1. Lag phase Nutrient levels too low to sustain growth Number of cells does not increase Total numbers remain constant Cells begin synthesizing enzymes Some die, releasing nutrients; others required for growth grow Delay depends on conditions Continue to produce secondary metabolites- chemicals- not necessary for 2. Log (logarithmic or exponential) growth Endospore production begins for the phase bacteria that produce them Cells divide at constant rate 4. Prolonged death phase Generation time can be measured Total number of viable cells decreases Cells most sensitive to antibiotics Cells die at constant rate Cells produce primary metabolites Exponential, but usually much slower such as amino acids required for than cell growth Some fraction may survive growth Adapted to tolerate worsened conditions  Continuous Culture Open system: culture to which nutrients are continually added and waste products removed A chemostat provides an open system that can maintain continuous growth Nutrient content and speed of addition can be controlled to achieve constant growth rate and cell density Produces relatively uniform population to study response to different conditions Can maintain cells in log phase of growth to harvest commercially valuable products 4.4-Environmental Factors That Influence Microbial Growth Describe the importance of a microorganism’s requirements for temperature, O2, pH, and water availability, and define the terms that indicate these requirements. Explain the significance of reactive oxygen species, and describe the mechanisms cells use to protect against their effects. Environmental Factors That Influence Microbial Growth As a group, microorganisms inhabit nearly all environments Some live in comfortable habitats favored by humans Extremophiles live in harsh environments; most are archaea Major factors that affect microbial growth Temperature Atmosphere pH Water availability Temperature Requirements Proteins of thermophiles resist denaturing Each species has well-defined temperature Thermostability comes from amino acid sequence range Number and position of bonds that determine Optimum growth usually close to upper end of structure range Temperature and food preservation Psychrophiles: -5 degrees to 15 degrees Celsius Refrigeration (approximately 4 degrees Celsius) Found in Arctic and Antarctic regions slows spoilage by limiting growth of otherwise fast-growing mesophiles Psychrotrophs: 15 degrees to 30 degrees Psychrophiles, psychrotrophs can still grow, but Celsius slowly Important in spoilage of refrigerated Freezing preserves food; not effective at killing microbes foods Temperature and disease Mesophiles: 25 degrees to 45 degrees Celsius Pathogens 35 degrees to 40 degrees Different parts of human body differ in temperature Celsius Thermophiles: 45 degrees to 70 degrees M. leprae grows best in cooler regions so leprosy involves ears, hands, feet, fingers Celsius Temperature Requirements - Figure 4.8  Oxygen (𝐎𝟐 ) Requirements 1 Can be measured in shake tube: Boil nutrient agar to drive off O2; cool to just above solidifying temperature; add microorganisms; gently swirl Solidified agar slows gas diffusion Top of tube is aerobic - O2 is present Bottom of tube is anaerobic - O2 is absent Position of growth indicates organism’s O2 requirements Obligate aerobes: require O2 Facultative aerobes: use O2, but don’t require it Obligate anaerobes: cannot use O2 Microaerophiles: require small amounts of O2 only Aerotolerant anaerobes: obligate fermenters (can grow in O2, but they don’t use it)  Table 4.2-Oxygen (𝐎𝟐) Requirements of Microorganisms Obligate Facultative Obligate Microaerophile Aerotolerant aerobe anaerobe anaerobe anaerobe Pattern of Growth in a Shake Tube Growth Grows only when Grows best Cannot grow Grows only if Grows equally characteristics O2 is available. when O2 is when O2 is small amounts of well with or available, but present. O2 are available. without O2. also grows without it. Use of O2 in Requires O2 for Uses O2 for Does not use O2. Requires O2 for Does not use O2. energy- respiration. respiration, if respiration. harvesting available. processes Typical Produces Produces Does not Produces some Produces mechanisms to superoxide superoxide produce superoxide superoxide protect dismutase and dismutase and superoxide dismutase and dismutase but Oxygen (O2) Requirements – dealing with oxygen Reactive oxygen species (ROS): harmful by-products of using O 2 in aerobic respiration Include superoxide (O2-) and hydrogen peroxide (H2O2) Damaging to cellular components Cells must have protective mechanisms; obligate anaerobes typically do not Almost all organisms growing in presence of oxygen produce enzyme superoxide dismutase Inactivates superoxide by converting it to O2 and H2O2 Almost all also produce catalase Converts H2O2 to O2 and H2O Exception is aerotolerant anaerobes; makes for useful test  pH requirements Bacteria survive a range of pH; have optimum Most maintain constant internal pH, typically near neutral Pump out protons Bring in protons (H+)if in alkaline environment Most microbes are neutrophiles Range of pH 5 to 8; optimum near pH 7 Food can be preserved by increasing acidity H. pylori grows in stomach; produces urease to split urea into CO2 and ammonia to decrease acidity of surroundings Acidophiles grow optimally at pH below 5.5 Picrophilus oshimae has optimum pH of less than 1! Alkaliphiles grow optimally at pH above 8.5  Water Availability - Figure 4.9- Tonicity All microorganisms require water for growth Dissolved salts, sugars make water unavailable to cell If solute concentration is higher outside of cell, water diffuses out (osmosis) Salt, sugar used to preserve food Some microbes withstand or even require high salt Halotolerant: withstand up to 10% Staphylococcus on dry salty skin Halophiles: require high salt levels  Review-Table 4.3-Environmental Factors That Influence Microbial Growth Environmental Characteristics Factor/Descriptive Terms Temperature Thermostability appears to be due to protein structure. Psychrophile Optimum temperature between -50C and 150C. Psychrotroph Optimum temperature between 150C and 300C but grows well at refrigeration temperatures. Mesophile Optimum temperature between 250C and 450C. Thermophile Optimum temperature between 450C and 700C. Hyperthermophile Optimum temperature of 700C or greater. Oxygen (O2) Availability Oxygen (O2) requirement/tolerance reflects the organism’s energy-harvesting mechanisms and its ability to inactivate reactive oxygen species. Obligate aerobe Requires O2. Facultative anaerobe Grows best if O2 is present but can also grow without it. Obligate anaerobe Cannot grow in the presence of O2. Microaerophile Requires small amounts of O2, but higher concentrations are inhibitory Review-Table 4.3-Environmental Factors That Influence Microbial Growth- continued Environmental Characteristics Factor/Descriptive Terms pH Prokaryotes that live in pH extremes maintain a near-neutral internal pH by pumping protons out of or into the cell. Neutrophile Multiplies in the range of pH 5 to 8. Acidophile Grows optimally at a pH below 5.5. Alkalophile Grows optimally at a pH above 8.5. Water Availability Prokaryotes that can grow in high-solute solutions maintain the availability of water in the cell by increasing their internal solute concentration. Halotolerant Can grow in relatively high-salt solutions, up to approximately 10% NaCl. Halophile Requires high levels of sodium chloride. 4.5- Nutritional Factors that Influence Microbial Growth List the required elements and give examples of common sources of required elements. Explain the significance of a limiting nutrient. Explain why fastidious microbes require growth factors. Describe the energy and carbon sources used by photoautotrophs, chemo-lithoautotrophs, photoheterotrophs, and chemo-organoheterotrophs. Nutritional Factors That Influence Microbial Growth Carbon source distinguishes different groups Heterotrophs use organic carbon Autotrophs use inorganic carbon as CO2 Carbon fixation converts inorganic carbon to organic form Nitrogen required for amino acids, nucleic acids Nitrogen fixation: converting N2 gas to ammonia and then incorporating it into organic compounds Many use ammonia (some convert nitrate to ammonia) Phosphorus and iron are often limiting nutrients Available at lowest concentration relative to need Dictate maximum level of microbial growth Table 4.4-Representative Functions of the Major Elements Chemical Function Nutrients required to Carbon, oxygen, and Component of amino synthesize cell components hydrogen acids, lipids, nucleic acids, and sugars Prokaryotes can use Nitrogen Component of amino diverse sources to acquire acids and nucleic various elements acids Major elements: carbon, Sulfur Component of some oxygen, hydrogen, nitrogen, amino acids sulfur, phosphorus, Phosphorus Component of nucleic potassium, magnesium, acids, membrane calcium, and iron lipids, and ATP Trace elements: cobalt, zinc, Potassium, Required for the copper, molybdenum, magnesium, functioning of certain manganese and more and calcium enzymes; additional functions as well Iron Part of certain  Growth factors Growth factors- organic molecules that an organism cannot synthesize; must be present in the environment Growth factor requirements reflect biosynthetic capabilities Most E. coli strains synthesize all cellular components from glucose; no growth factors needed Neisseria species are fastidious; they require numerous growth factors, including vitamins and amino acids Fastidious species can be used to measure the quantity of vitamins in food products Energy sources Two main types of energy sources Sunlight (Photo-), chemical compounds (Chemo-) Phototrophs obtain energy from sunlight Plants, algae, photosynthetic bacteria Chemotrophs extract energy from chemicals Mammalian cells, fungi, many types of prokaryotes extract energy from organic molecules Sugars, amino acids, fatty acids are common sources Some prokaryotes extract energy from inorganic chemicals Hydrogen sulfide, hydrogen gas, nitrates etc. Energy and Carbon Sources Used by Different Groups of Microorganisms Photoautotrophs: energy from sunlight; carbon from CO2 Photoheterotrophs: energy from sunlight; carbon from organic compounds Chemolithoautotrophs (also termed chemoautotrophs, chemolithotrophs): energy from inorganic compounds; carbon from CO2 Chemoorganoheterotrophs (also termed chemoheterotrophs, chemoorganotrophs): energy and carbon from organic compounds 4.6- Cultivating Microorganisms in The Laboratory Compare and contrast complex, chemically defined, selective, and differential media. Explain how aerobic, microaerophilic, and anaerobic conditions can be provided. Describe the purpose of an enrichment culture. Types of culture media Requires a suitable growth medium and an appropriate atmosphere. General Categories of Culture Media Complex media contain a variety of ingredients Exact composition is highly variable Chemically defined media composed of exact amounts of pure chemicals Typically, slower growth as cells must synthesize components Table 4.6- Ingredients in Two Types of Media That Support the Growth of E. coli Nutrient Broth Glucose-Salts Broth E. coli can grow in (Complex Medium) (Chemically complex media or Defined Medium) chemically defined media Peptone Glucose Example in the table Beef extract Dipotassium phosphate Water Monopotassium phosphate Magnesium sulfate Ammonium sulfate Calcium chloride Iron sulfate Water Special Types of Culture Media - Figure 4.12 Special Types of Culture Media- selective Selective media inhibit growth of certain species in a mixed sample, while allowing growth of species of interest Mannitol- salt agar (MSA) Blocks growth of gram- negative bacteria Allows to grow some species of Staphylococci and select Staphylococcus aureus Special Types of Culture Media- Differential Differential media contain substance that microbes change in identifiable way Allows to differentiate between groups of bacteria Blood agar Beta hemolysis produces clear zone Alpha hemolysis produces zone of greenish partial clearing  Differential media Blood agar EMB agar MacConkey agar Beta hemolysis produces clear Blocks growth of gram- Blocks growth of gram- zone positive bacteria positive bacteria Alpha hemolysis produces zone of greenish partial clearing  Providing Appropriate Atmospheric Conditions Aerobic Anaerobic: obligate anaerobes Most obligate aerobes and facultative sensitive to O2 anaerobes can be incubated in air Anaerobe containers useful if (approximately 20% O2) microbe can tolerate brief O2 Broth cultures shaken to provide maximum exposures; can also use semisolid aeration culture medium containing reducing Many medically important bacteria (for agent such as sodium thioglycolate) example, Neisseria, Haemophilus) grow Reduce O2 to water best with increased CO2 Anaerobic chamber provides more Some are capnophiles, meaning require stringent approach increased CO2 One method is to incubate in candle jar Microaerophilic Require lower O2 concentrations than achieved by candle jar Can incubate in gas-tight container with Providing Appropriate Atmospheric Conditions - Figures 4.13 and 4.14 4.7- Methods to Detect and Measure Microbial Growth Viable cell counts and detecting cell products. Viable cell count Viable cell count- 2 Viable cell counts- cells capable of multiplying Can use selective, differential media for a particular species Plate counts: single cell gives rise to colony Number of colonies reflects how many cells were in sample Plate dilution series to obtain 30 to 300 colonies  Pour and Spread plate methods- Figure 4.19 Samples added to agar plate by 2 methods Spread plate method Pour plate methods Plate counts determine colony-forming units (CFUs) Cells often attach to one another and form a single colony  MPN method Most Probable Number (MPN) Method Estimates cell concentration using dilution series Sets of tubes are incubated; results are recorded and compared to table to give statistical estimate of cell concentration In this chapter we covered Bacterial cell division Bacterial Growth curve- different stages Bacterial Growth in nature Bacterial Growth in the lab Requirements for bacterial growth Temperature Oxygen pH Carbon and energy sources Viable cell count methods