Microbial Growth Lecture Notes PDF

Ch 6 Microbial Growth Objectives:  Classify microbes into five groups on the basis of preferred temperature range.  Explain the importance of osmotic pressure to microbial growth.  Provide a use for each of the four elements (C, N, S, P) needed in large amounts for microbial gr...

Ch 6 Microbial Growth Objectives:  Classify microbes into five groups on the basis of preferred temperature range.  Explain the importance of osmotic pressure to microbial growth.  Provide a use for each of the four elements (C, N, S, P) needed in large amounts for microbial growth.  Explain how microbes are classified on the basis of O2 needs.  Identify ways in which aerobes avoid damage by toxic forms of O2.  Describe the formation of biofilms and their potential for causing infection.  Distinguish between chemically defined and complex media.  Justify the use of each of the following: anaerobic techniques, living host cells, candle jars, selective, differential, and enrichment media.  Define colony and CFUs and describe how pure cultures can be isolated with streak plates.  Explain how microbes are preserved by deep-freezing and lyophilization.  Distinguish between binary fission and budding.  Define generation time and explain the bacterial growth curve.  Review some direct and indirect methods of measuring bacterial cell growth. Microbial Growth Microbial growth: Increase in cell number, not cell size! Physical Requirements for Growth: Temperature  Minimum growth temperature  Optimum growth temperature  Maximum growth temperature Five groups based on optimum growth temperature 1. Psychrophiles 2. Psychrotrophs 3. Mesophiles 4. Thermophiles 5. Hyperthermophiles Fig. Fig 6.3: Effect of amount of food on its cooling rate  Physical Requirements for Growth: pH and Osmotic Pressure Most bacteria grow best between pH 6.5 and 7.5: Neutrophils Some bacteria are very tolerant of acidity or thrive in it: Acidophiles (preferred pH range 1 to 5) Molds and yeasts grow best between pH 5 and 6 Hypertonic environments (increased salt or sugar) cause plasmolysis Obligate halophiles vs. facultative halophiles Fig 6.4 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings  Chemical Requirements for Growth: Carbon, N, S, P, etc.  Carbon   Half of dry weight  Chemoheterotrophs use organic carbon sources  Nitrogen, Sulfur, Phosphorus  Needed for ? Vit B1  Found in amino acids and proteins (most bacteria decompose proteins)  S in thiamine and biotin Vit B7  Phosphate ions (PO43–)  Also needed K, Mg, Ca, trace elements (as cofactors), and organic growth factors Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Chemical Requirements for Growth: Oxygen O2 requirements vary greatly Table 6.1: The Effects of Oxygen on the Growth of Various Types of Bacteria Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Toxic Forms of Oxygen  Singlet oxygen: O2 boosted to a higher-energy state  Superoxide free radicals: O2–  Peroxide anion: O22–  Hydroxyl radical (OH) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings  Fig 6.5 Biofilms Microbial communities form slime or hydrogels Starts via attachment of planctonic bacterium to surface structure. Bacteria communicate by chemicals via quorum sensing Sheltered from harmful factors (disinfectants etc.) Cause of most nosocomial infections Clinical Focus: Delayed Bloodstream Infection Following Catheterization Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Culture Media  Culture medium: Nutrients prepared for microbial growth  Have to be sterile (not contain living microbes)  Inoculum: Microbes introduced into medium  Culture: Microbes growing in/on culture medium  Chemically defined media: Exact chemical compo- sition is known (for research purposes only)  Complex media: Extracts and digests of yeasts, meat, or plants, e.g.:  Nutrient broth  Nutrient agar  Blood agar Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings  Agar  Complex polysaccharide  Used as solidifying agent for culture media in Petri plates, slants, and deeps  Generally not metabo- lized by microbes  Liquefies at 100°C  Solidifies ~40°C Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Anaerobic Culture Methods  Use reducing media, containing chemicals (e.g.: thioglycollate) that combine with O2  Are heated shortly before use to drive off O2  Use anaerobic jar  Novel method in clinical labs: Add oxyrase to growth media  OxyPlate (no need for anaerobic jar) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Fig 6.5 Capnophiles: Aerobic Bacteria Requiring High CO2  Low oxygen, high CO2 Candle jar conditions resemble those Fig 6.7 found in  intestinal tract  respiratory tract and  other body tissues where pathogens grow  E.g: Campylobacter jejuni  Use candle jar, CO2- generator packets, or CO2 incubators Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings  Selective Media and Differential Media Selective medium: Additives suppress unwanted and encourage desired microbes – e.g. EMB, mannitol salt agar etc. Differential medium: changed in recognizable manner by some bacteria  Make it easy to distinguish colonies of different microbes – e.g.  and  hemolysis on blood agar; MacConkey agar, EMB, mannitol salt agar etc. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Enrichment Media/Culture  Encourages growth of desired microbe  Example: Assume soil sample contains a few phenol- degrading bacteria and thousands of other bacteria  Inoculate phenol-containing culture medium with the soil and incubate  Transfer 1 ml to another flask of the phenol medium and incubate  Transfer 1 ml to another flask of the phenol medium and incubate  Only phenol-metabolizing bacteria will be growing Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings  Pure Cultures Contain only one species or strain. Most patient specimens and environmental samples contain several different kinds of bacteria Streak-plate method is commonly used Colony formation: A population of cells arising from a single cell or spore or from a group of attached cells (also referred to as CFU). Only ~1% of all bacteria can be successfully cultured Aseptic technique critical! Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings  Streak Plate Method 3 or 4 quadrant methods Preserving Bacterial Cultures  Deep-freezing: Rapid cooling of pure culture in suspension liquid to –50°to –95°C. Good for several years.  Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum. Good for many years. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The Growth of Bacterial Cultures Binary fission – exponential growth Budding Generation time – time required for cell to divide (also known as doubling time) Ranges from 20 min (E. coli) to > 24h (M. tuberculosis) Consider reproductive potential of E. coli Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Fig 6.13 Figure 6.12b  Bacterial Growth Curve Illustrates the dynamics of growth Foundation Fig 6.15 Phases of growth  Lag phase  Exponential or logarithmic (log) phase  Stationary phase  Death phase (decline phase) Compare growth in liquid and on solid media Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Bacterial Growth Curve: Arithmetic vs. Exponential Plotting Fig 6.14 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings  Direct Measurements of Microbial Growth Viable cell counts: Plate counts: Serial dilutions put on plates CFUs form colonies Fig 6.16 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Fig 6.17 Figure 6.15, step 1 Additional Direct Measurements 1. Filtration method of choice for low counts 2. Direct microscopic count: Counting chambers (slides) for microscope Fig 6.20 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings  Estimating Bacterial Numbers by Indirect Methods Spectrophotometry to measure turbidity OD is function of cell number Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Measuring Microbial Growth - Overview Direct Methods Indirect Methods  Plate counts  Turbidity  Filtration  Metabolic activity  MPN  Dry weight  Direct microscopic count Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings