Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Dynamics of Prokaryotic Growth Chapter 4 Preview • • • • • Principles of bacteria growth. Bacteria growth in nature. Bacteria growth in laboratory. Factors affect bacteria growth. Detecting bacteria growth Principles of Bacterial Growth • Prokaryotic cells divide by binary fission – One cell divides into two – Cell growth is exponential • population double with each cell division. • Cell divide at constant pace – Generation time • Time it takes for population to double • A.k.a doubling time • Varies among species Practical Problem • 100 E. coli in potato salad. • How many bacteria are there in salad 2 hrs later (if the double time is 20 min)? • Why do we need to store the salad in cooler? Principles of Bacterial Growth • Growth can be calculated – Nt = N0 x 2n • • • • (Nt ) number of cells in population (N0 ) original number of cells in the population (n) number of divisions Example – N0 = 10 cells in original population – n = 12 » 4 hours assuming 20 minute generation time – Nt = 10 x 212 – Nt = 10 x 4,096 – Nt = 40,960 Bacterial Growth in Nature • Conditions in nature have profound effect on microbial growth – Synthesize compounds useful for growth – produce multicellular associations to increase survivability Biofilm layer • Biofilms: a community formed by a group of bacteria and their secreted slimes. Biofilm – Can cause disease • Difficult to kill – Architecture resist immune response and antimicrobial drugs – Can be beneficial • biofilm Bacterial Growth in Nature – Prokaryotes live in mixed communities • Many interactions are cooperative • Some cells compete for nutrient Bacteria growth in Laboratory • Culture media – broth media – Solid media is broth media with addition of agar • Agar marine algae extract • Liquefies above 95°C • Solidifies at 45°C – Remains solid at room temperature and body temperature – Bacteria grow in colonies on solid media surface • Single colony Laboratory Cultivation • Special types of culture media – These are used to detect or isolate particular organisms – Are divided into selective and differential media Laboratory Cultivation • Selective media – Inhibits the growth of unwanted organisms • Allows only sought after organism to grow – Example • Thayer-Martin agar (multiple antimicrobial) – For isolation of Neisseria gonorrhoeae • MacConkey agar (antimicrobial+bile salt) – For isolation of Gram-negative intestinal bacteria Laboratory Cultivation • Differential media – Contains substance that bacteria change in recognizable way – Example • Blood agar – Test for hemolysis • MacConkey agar – pH indicator Obtaining Pure Culture • Pure culture is defined as population of cells derived from single cell – All cells are genetically identical • to study functions of specific species • Obtain pure culture – Aseptic technique Obtaining Pure Culture • Streak-plate method – Simplest and most commonly used in bacterial isolation – Object is to reduce number of cells being spread Bacterial Growth in Laboratory Conditions • Cells in laboratory grown in closed or batch system • Population of cells increase in predictable fashion – Follows a pattern called growth curve Bacterial Growth in Laboratory Conditions • The Growth Curve – Characterized by five distinct stages • Lag stage • Exponential or log stage • Stationary stage • Death stage • Phase of prolonged decline Bacterial Growth in Laboratory Conditions Lag phase – synthesis of cell components and prepare for division • Log phase – exponential growth • Cell divide at constant rate – Produce primary metabolites • Compounds required for growth – Cells enter late log phase • Cell wall and cell membrane component changes • Synthesize secondary metabolites – Used to enhance survival – Antibiotics Bacterial Growth in Labortory Conditions • Stationary phase – Overall population remains relatively stable • Cells exhausted nutrients and build up toxic waste • Cell growth = cell death • Death phase – Total number of viable cells decreases • 99% of cells die at constant rate – Death is exponential • Much slower rate than growth Bacterial Growth in Laboratory Conditions • Phase of prolonged decline – Marked by very gradual decrease in viable population – Phase may last months or years – Most fit cells survive • Each new cell more fit that previous Bacterial Growth in Laboratory Conditions • Continuous culture – Bacterial culture can be maintained • Continuous exponential growth can be sustained by use of chemostat Bacterial Growth in Laboratory Conditions • Colony growth on solid medium – Position within colony determines resource availability • Cells on edge of colony have little competition and significant oxygen stores • Cells in the middle of colony have high cell density – Leads to increased competition and decreased availability of oxygen Questions • What is biofilm? • What is pure culture? • What are the different stages of bacterial growth? • Selective and differential medium. Environmental Factors on Growth • Major conditions that influence growth Temperature Oxygen pH Water availability Environmental Factors on Growth • Psychrophile – Optimum temperature -5°C to 15°C • Found in Arctic and Antarctic regions • Psychrotroph – 20°C to 30°C • Important in food spoilage • Mesophile – 25°C to 45°C • More common • Disease causing • Thermophiles – 45°C to 70°C • Common in hot springs • Hyperthermophiles – 70°C to 110°C • Usually members of Archaea • Found in hydrothermal vents Environmental Factors on Growth • Temperature and food storage – 4C can slow down bacteria growth – Freezing can stop bacteria growth • Temperature and disease – Different pathogen can only grow in different part of body. • Hansen’s disease • Syphilis disease Environmental Factors on Growth • Oxygen – Prokaryotes divided based on oxygen requirements • Obligate aerobes – Absolute requirement for oxygen » Use for energy production » Micrococcus • Obligate anaerobes – No multiplication in presence of oxygen » May cause death » Clostridium Environmental Factors on Growth • Facultative anaerobes – Grow better with oxygen » Use fermentation in absence of oxygen » E coil • Microaerophiles – Require oxygen in lower concentrations » Higher concentration inhibitory » Helicobacter pylori • Aerotolerant anaerobes – Indifferent to oxygen, grow with or without » Does not use oxygen to produce energy » Streptococcus Environmental Factors - O2 availability Decreasing O2 Environmental Factors - O2 availability Environmental Factors on Growth • pH – Bacteria survive within pH range – Neutrophiles • Multiply between pH of 5 to 8 – Maintain optimum near neutral – Acidophiles • Thrive at pH below 5.5 – Maintains neutral internal pH pumping out protons (H+) – Alkalophiles • Grow at pH above 8.5 – Maintain neutral internal pH through sodium ion exchange » Exchange sodium ion for external H+ Environmental Factors on Growth • Water availability – All microorganisms require water for growth – Water not available in all environments • In high salt environments – Bacteria increase internal solute concentration » Synthesize small organic molecules – Osmotolerant bacteria tolerate high salt environments – Bacteria that require high salt for cell growth termed halophiles Nutritional Factors on Growth • Growth of prokaryotes depends on nutritional factors as well as physical environment • Main factors to be considered are: – Required elements – Energy sources – Growth factors Nutritional Factors on Growth • Required elements – Major elements • Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium and iron – Essential components for macromolecules • Organisms classified based on carbon usage – Heterotrophs » Use organic carbon as carbon source – Autotrophs » Use inorganic carbon (CO2) as carbon source – Trace elements • Cobalt, zinc, copper, molybdenum and manganese – Required in minute amounts Nutritional Factors on Growth • Energy Sources – Organisms derive energy from sunlight or chemical compounds • Phototrophs – Derive energy from sunlight • Chemotrophs – Derive energy from chemical compounds – Organisms often grouped according to energy source Nutritional Factors on Growth • Nutritional Diversity – Organisms thrive due to their ability to use diverse sources of carbon and energy – Photoautotrouphs • Use sunlight and atmospheric carbon (CO2) as carbon source – Called primary producers (Plants) – Chemolithoautotrophs • A.k.a chemoautotrophs or chemolitotrophs • Use inorganic carbon for energy and use CO2 as carbon source – Photoheterotrophs • Energy from sunlight, carbon from organic compounds – Chemoorganoheterotrophs • a.k.a chemoheterotrophs or chemoorganotrophs • Use organic compounds for energy and carbon source • Most common among humans and other animals Nutritional Factors on Growth • Growth factors – Some bacteria cannot synthesize some cell constituents • These must be added to growth environment – Referred to as growth factors – Organisms can display wide variety of factor requirements • Some need very few while others require many – These termed fastidious Questions • Major factors that affect bacteria growth • Growth factor • Carbon source and energy source of chemoheterotroph Detecting Bacterial Growth • Variety of techniques to determine growth – Number of cells – Total mass – Detection of cellular products Detecting Bacterial Growth • Direct cell count • Plate count Detecting Bacterial Growth • Direct microscopic count – Number is measured in a know volume – Liquid dispensed in specialized slide • Counting chamber – Viewed under microscope – Cells counted – Limitation • Must have at least 10 million cells per ml to gain accurate estimate Detecting Bacterial Growth • Plate counts – Measures viable cells growing on solid culture media – Count based on assumption the one cell gives rise to one colony • Number of colonies = number of cells in sample – Ideal number to count • Between 30 and 300 colonies – Sample normally diluted in 10-fold increments – Plate count methods • pour-plates • Spread-plates methods