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Elements of Microbial Growth, Nutrition and Environment Do different organisms require specific diets and environments? Why do we care about growth? • To Encourage the microbes we want • • • • • Brewery, winery, food production Vaccine and drug production Microbial fuel cells Bioremediation, Sewage treatment plant, oil spill clean up Resident microbiota-probiotics to aid microbial antagonism and perform other functions • To Discourage the microbes we don’t want • Pathogens What is Growth? • In microbiology, we define growth in relation to the number of cells, not the size of cells. • Concentrate on population growth • Bacterial cells divide via binary fission, not mitosis. Binary fission • The division of a bacterial cell • Parental cell enlarges and duplicates its DNA • Septum formation divides the cell into two separate chambers • Complete division results in two identical cells Generation Time • The time required for a complete division cycle (doubling) • Length of the generation time is a measure of the growth rate • Growth is exponential not arithmetic • Dependent on chemical and physical conditions Generation Time • Average generation time is 30 – 60 minutes • shortest generation times can be 10 – 12 minutes • E. coli GT=20 min. • Mycobacterium leprae has a generation time of 10 – 30 days • 11 million cells (20 generations) in 7 hours • most pathogens have relatively short generation times Which is bacterial growth curve? Four phases of growth in a bacterial culture 1. Lag Phase • Cells are adjusting, enlarging, and synthesizing critical proteins and metabolites • Not doubling at their maximum growth rate 2. Exponential Growth Phase • Maximum exponential growth rate of cell division • Adequate nutrients • Favorable environment • Most sensitive to antibiotics. Why? Exponential Growth Phase • A person actively shedding bacteria in the early and middle stages of infection is more likely to spread it than a person in the later stages. Why? MRSA 3. Stationary Phase • Cell birth and cell death rates are equal • Survival mode – depletion in nutrients, released waste can inhibit growth 4. Death Phase • A majority of cells begin to die exponentially due to lack of nutrients or build up of waste • Slower than the exponential growth phase How do we measure microbial growth? • Direct measurement – Standard Plate counts • most common, need to DILUTE to get individual, countable colonies – Microscopic Count • count with microscope – Filtration • when # microbes small, • water run thru filter and filter applied to TSA plate and incubated – Coulter Counter • Automated cell counter • Indirect (Estimation) – Turbidity – more bacteria, more cloudiness – can measure w/ spectrophotometer or eye – Metabolic Activity – assumes amount of metabolic product is proportional to # – Dry Weight – used for filamentous organisms, like molds – Genetic Probing – Real-time PCR Direct: Standard Plate Counts Direct: Microscopic Count • Advantages – Easy and fast • Disadvantages – Uses special microscope counting slide – Does not differentiate between live and dead bacteria Direct: Membrane Filtration Direct: Coulter Counter Uses an electronic sensor to detect and count the number of cells. Indirect: Turbidity Using Spectrometer The greater the turbidity, the larger the population size. Which culture (left or right) has more bacteria? Indirect: Metabolism Activity • The metabolic output or input of a culture may be used to estimate viable count. • Examples: • Measure how fast gases and/or acids are formed in a culture • Or the rate a substrate such as glucose or oxygen is used up Indirect: Dry Weight • To calculate the dry weight of cells – cells must be separated from the medium – then dried – the resulting mass is then weighed Indirect: Genetic Methods • Use real-time PCR to “count” how many bacterial genes there are in a sample. Which techniques distinguish between live and dead cells? – Standard Plate counts – Direct Microscopic – Filtration – Coulter counter – Turbidity – Metabolic activity – Dry weight – Genetic Probing Which techniques distinguish between live and dead cells? – Standard Plate counts – Direct Microscopic – Filtration – Coulter counter – Turbidity – Metabolic activity – Dry weight – Genetic Probing What are the requirements for microbial growth? Chemical Composition of an Escherichia coli Cell Microbial Nutrition •Macronutrients: - carbon, hydrogen, and oxygen - required in relatively large quantities and play principal roles in cell structure and metabolism •Micronutrients: - present in much smaller amounts - manganese, zinc, nickel •Inorganic nutrients: - Can have carbon OR hydrogen, but not both •Organic nutrients: - Contain carbon and hydrogen Microbial Nutrition • All cells require the following for metabolism and growth: – Carbon source – Energy source • Growth factors (some bacteria are fastidious/picky and require extra supplements) Microbial Nutrition •Heterotroph: Organic carbon is carbon source •Autotroph: inorganic CO2 as its carbon source - has the capacity to convert CO2 into organic compounds - not nutritionally dependent on other living things •Phototroph: microbes that photosynthesize •Chemotroph: microbes that gain energy from injesting chemical compounds Microbial Nutrition: Autotrophs • Photoautotrophs: - Photosynthetic - Produce organic molecules using CO2 - Ex: Cyanobacteria, algea • Chemoautotrophs: - Ingest organic compounds for energy - Produce organic molecules using CO2 Microbial Nutrition: Heterotrophs • Chemoheterotrophs: - organic compounds for both carbon and energy source - derive both carbon and energy from processing these molecules through respiration or fermentation - The vast majority of microbes causing human disease are chemoheterotrophs - Ex: Most bacteria, all, protists, all fungi, and all animals Diffusion: Review •Transport of necessary nutrients occurs across the cell membrane, even in organisms with cell walls •Diffusion: • Atoms or molecules move in a gradient from an area of higher concentration to lower concentration • Diffusion of molecules across the cell membrane is largely determined by the concentration gradient and permeability of the substance Osmosis: Review •Osmosis: the diffusion of water through a selectively permeable membrane •Isotonic: Equal solutes in cell and in environment - parasites living in host tissues are most likely to be living in isotonic habitats -Hypotonic: More solutes in cell than in environment - A slightly hypotonic environment can be favorable to bacteria cells •Hypertonic: Less solutes in cell than in environment • hypertonic solutions such as concentrated salt and sugar solutions act as preservatives for food(salted ham is an example) Osmosis: Review 37 Environmental (Physical) Factors Effecting Bacterial Growth • Temperature • Gas • pH • Osmotic pressure • Other factors • Microbial association Survival in a changing environment is largely a matter of whether the enzyme systems of microorganisms can adapt to alterations in their habitat Environmental Factors: Temperature • Effect of temperature on proteins: –Too high, proteins unfold and denature –Too low, do not work efficiently • Effect of temperature on membranes of cells and organelles: –Too low, membranes become rigid and fragile –Too high, membranes become too fluid Temperature and Bacterial Growth Five categories of microbes based on temperature ranges for growth Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Minimum Maximum Psychrophile Psychrotroph Mesophile Thermophile Extreme thermophile Rate of Growth Optimum -20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 Temperature °C Which category do human pathogens usually fall into? Why? 130 Environmental Factors: Gases • Two gases that most influence microbial growth – Oxygen • O2 has the greatest impact on microbial growth • O2 is an important respiratory gas and a powerful oxidizing agent – Carbon dioxide Oxygen Requirements • As oxygen enters cellular reactions, it is transformed into several toxic products – highly reactive and excellent oxidizing agents • Resulting oxidation causes irreparable damage to cells by attacking enzymes and proteins Oxygen Requirements •As oxygen enters cellular reactions, it is transformed into several toxic products: - singlet oxygen (O) - superoxide ion (O2-) - hydrogen peroxide (H2O2) - hydroxyl radicals (OH-) •Most cells have developed enzymes that scavenge and neutralize reactive oxygen byproducts •Two-step process requires two enzymes: Oxygen Requirements If bacteria do not have superoxide dismutase or catalase they can not tolerate oxygen. Catalase Test Oxygen Requirements • Aerobes • Anaerobes • Facultative anaerobes • Aerotolerant anaerobes • Microaerophiles Oxygen Requirements: Obligate Aerobe • Requires oxygen for metabolism • Have enzymes that neutralize toxic oxygen metabolites • Ex. Most fungi, protozoa, and bacteria, such as Bacillus species and Mycobacterium tuberculosis Oxygen Requirements: Facultative Anaerobe • Does not require oxygen, but can grow in its presence • During minus oxygen states, anaerobic respiration or fermentation occurs • Possess superoxide dismutase and catalase • Ex. Many Gram-negative pathogens Prefer oxygenated environments because more energy is produced during aerobic respiration compared to anaerobic respiration or fermentation Oxygen Requirements: Obligate Anaerobes • Cannot use oxygen for metabolism • Do not possess superoxide dismutase and catalase • The presence of oxygen is toxic to the cell and will kill it • Ex. Many oral bacteria, intestinal bacteria Thioglycollate broth enables the identification of aerobes, facultative anaerobes, and obligate anaerobes. Use of thioglycollate broth to demonstrate oxygen requirements. Culturing Technique for Anaerobes Anaerobes must grow in an oxygen minus environment, because toxic oxygen metabolites cannot be neutralized. Environmental Factors: pH • Most cells grow best between pH 6-8 – strong acids and bases can be damaging to enzymes and other cellular substances • Pathogens like our neutral pH • Yeast & Molds like acidic conditions Environmental Factors: pH • Acidophiles – thrive in acidic environments. – Ex. Helicobacter pylori • Alkalinophiles – thrive in alkaline conditions – Ex. Proteus can create alkaline conditions to neutralize urine and colonize and infect the urinary system Example of the use of a selective medium for pH Bacterial colonies pH 7.3 Fungal colonies pH 5.6 Environmental Factors: Water • Microbes require water to dissolve enzymes and nutrients • Water is important 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 • Two physical effects of water –Osmotic pressure –Hydrostatic pressure Environmental Factors: Water Osmotic pressure: • Halophiles (Salt lovers) – Requires high salt concentrations – Withstands hypertonic conditions • Ex. Halobacterium • Facultative halophiles – Can survive high salt conditions but is not required – Ex. Staphylococcus aureus Other Physical Factors Influencing Microbial Growth • Radiation- UV, infrared • Barophiles – withstand high pressures • Spores and cysts- can survive dry habitats Microbes require different nutrients and different environments specific to survive. They have specialized over the years! Associations Between Organisms – Organisms live in association with different species – Often involve nutritional interactions • Antagonistic relationships • Synergistic relationships • Symbiotic relationships Associations Between Organisms Non symbiotic Symbiotic Organisms live in close nutritional relationships; required by one or both members. Mutualism Obligatory, dependent; both members benefit. Commensalism The commensal benefits; other member not harmed. Parasitism Parasite is dependent and benefits; host harmed. Organisms are free-living; relationships not required for survival. Synergism Members cooperate and share nutrients. Antagonism Some members are inhibited or destroyed by others. Associations Between Organisms •Antagonism: free-living species compete -Antibiosis: the production of inhibitory compounds such as antibiotics -The first microbe has a competitive advantage by increasing the space and nutrients available to it -Remember importance of microflora?! A biocontrol agent on the right (a bacteria) is making a material that is keeping the pathogen on the left (a fungus) from growing. Associations Between Organisms • Synergism: free-living species benefits together but is not necessary for survival • Together the participants cooperate to produce a result that none of them could do alone • Gum disease, dental caries, and some bloodstream infections involve mixed infections of bacteria interacting synergistically Associations and Biofilms – Biofilms • Complex relationships among numerous species of microorganisms • Develop an extracellular matrix – Adheres cells to one another – Allows attachment to a substrate – Sequesters nutrients – May protect individuals in the biofilm • Form on surfaces often as a result of quorum sensing • Many microorganisms more harmful as part of a biofilm Plaque (biofilm) on a human tooth Biofilms: Quorum Sensing •Quorum sensing: used by bacteria to interact with members of the same species as well as members of other species that are close by •Structure of the biofilm -large, complex communities form with different physical and biological characteristics -the bottom may have very different pH and oxygen conditions than the surface -partnership among multiple microbial inhabitants -cannot be eradicated by traditional methods Now that you know more about the nutritional needs of bacteria let’s look at using this information to ID bacteria! Survey of Microbial Diseases •How to identify bacteria in patient specimens or in samples from nature? - phenotypic: considers macroscopic and microscopic morphology, physiology, and biochemistry - immunologic: serological analysis - genotypic: genetic techniques increasingly being used as a sole resource for identifying bacteria •Data from these methods can provide a unique profile for any bacterium Survey of Microbial Diseases: Phenotypic Methods Physiological/Biochemical Characteristics •Traditional mainstay of bacterial identification •Enzyme production and other biochemical properties are reliable ways to ID microbes •Dozens of diagnostic tests exist for determining the presence of specific enzymes and to assess nutritional and metabolic activities - fermentation of sugars - capacity to digest complex polymers - production of gas - sensitivity to antibiotics - nutrient sources Beta-hemolysis Blood agar as a differential medium Alpha-hemolysis No hemolysis (gamma-hemolysis) Survey of Microbial Diseases: Phenotypic Methods Tests for fermentation and gas production Durham tube (inverted tube to trap gas) No fermentation Acid fermentation with gas Phenotypic Methods: Direct Examination of Specimen •Direct observation of fresh or stained specimen •Stains most often used - Gram stain - acid-fast stain Survey of Microbial Diseases: Phenotypic Methods • Isolation Media and Morphological Testing - Selective media: encourage the growth of only the suspected pathogen - Differential media: used to identify definitive characteristics and fermentation patterns MacConkey Agar: Selective and Differential Selects for Gram-negative and tells you if the bacterium ferments lactose Phenotypic Methods: Biochemical Testing • Physiological reactions: indirect evidence of enzymes present in a species. If bacteria tests + for superoxide dismutase (an oxidase) what does that tell you? Phenotypic Methods: Biochemical Testing Unknown microbe + different substrates Results (+/–) DNPG ADH LDC – – + ODC | CIT | H2S + – – URE TDA IND – – + | VP| | GEL | GLU – – + MAN INO SOR – – – RHA SAC MEL – – – AMY ARA – • Enzyme-mediated metabolic reactions often visualized by a color change - microbe is cultured in a medium with a special substrate, then tested for a particular end product - microbial expression of the enzyme is made visible by a colored dye – Flowchart: We will use this to ID our MM! Cocci Gram (+) Gram (–) Catalase (–), pairs, chain arrangement Catalase (+), irregular clusters, tetrads Aerobic, oxidase (+), catalase (+) Anaerobic, oxidase (–), catalase (–) Streptococcus Strictly aerobic Micrococcus Facultative anaerobic Staphylococcus Planococcus Neisseria Branhamella Moraxella Veillonella Phenotypic Methods: Phage Typing - used when morphological and biochemical tests are insufficient. Ex. S. aureus Phage Group I vs. Group II - bacteriophage infect bacteria in a species-specific and strain-specific way, which is useful in identifying some bacteria - a lawn of bacterial cells is inoculated onto agar, mapped off into blocks, and phage are exposed to each block - cleared areas corresponding to lysed cells indicate sensitivity to that phage Determining Clinical Significance of Cultures •Important to rapidly determine if an isolate from a specimen is clinically important or if it is merely a contaminant or normal biota - a few colonies of E. coli in a urine sample can indicate normal biota, but several hundred can mean an active infection - a single colony of a true pathogen such as Mycobacterium tuberculosis in a sputum culture, or an opportunist in a sterile site, is highly suggestive of disease - repeated isolation of a relatively pure culture of any microorganism can mean it is an agent of disease