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Microbial growth Dr. Hala Al- Daghistani

Microbial growth Dr. Hala Al- Daghistani. 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

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Microbial growth Dr. Hala Al- Daghistani

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  1. Microbial growthDr. Hala Al-Daghistani

  2. 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 • Psychrophiles • Psychrotrophs • Mesophiles • Thermophiles • Hyperthermophiles

  3. 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

  4. Water Activity Quantifies water availability in an environment;decreases with increasing solute concentration.Plasmolysis:Hypertonic solutions; cytoplasm water loss; compatible solutes.Osmotolerant:Grows over a wide range of water activity; fungi > bacteria.Halophile:“Salt-loving”; requires > 0.2M sodium chloride.

  5. Chemical Requirements for Growth: Carbon, N, S, P, etc. • Carbon •  Half of dry weight • heterotrophs use organic carbon sources • Autotrophs use inorganic carbon sources • Nitrogen, Sulfur, Phosphorus • N, S Needed for protein sythesis • N, P used for NA and ATP synthesis • S in thiamine and biotin • Phosphate ions (PO43–) • Also needed K, Mg, Ca, trace elements (as cofactors), and organic growth factors

  6. Nutrient Concentration Effects in Batch Cultures • Total growth will increase until limiting nutrients are exhausted (included oxygen for aerobes) or metabolic by products accumulate that change environmental conditions to inhibit growth (toxicity). • Growth rate will also increase with increasing nutrient concentration up to a some maximum value, beyond which there is no effect (transporters are saturated with there substrate.

  7. Oxygen Requirement Types 2 to 10% atm O2 Super Oxide Dismutase (SOD):Superoxide radicals (O 2 •-), or superoxide anions (OH2¯² . Superoxide radicals go to hydrogen peroxide & O2. Catalase: hydrogen peroxide go to water & O2.

  8. Toxic Forms of Oxygen • Singlet oxygen: O2 boosted to a higher-energy state • Superoxide free radicals: O2– • Peroxide anion: O22– • Hydroxyl radical (OH)

  9. 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

  10. Advantages of biofilms1. Cell-to-cell chemical communication2. Quorum sensing, allows bacteria tocoordinate their activity and group together into communities thatprovide benefits not unlike those of Multicellular organisms.3. Within a biofilm community, the bacteria are able to share nutrients and are sheltered from harmful factors in the environment, such as desiccation, antibiotics, and the body's immune system.4. Facilitating the transfer of genetic information's (conjugation).

  11. 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 composition is known (for research purposes only) • Complex media: Extracts and digests of yeasts, meat, or plants, e.g.: • Nutrient broth • Nutrient agar • Blood agar

  12. Agar • Complex polysaccharide • Used as solidifying agent forculture media in Petri plates, andslants • Generally not metabolized by microbes • Liquefies at 100°C • Solidifies ~40°C

  13. 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)

  14. Capnophiles: Aerobic Bacteria Requiring High CO2 • Low oxygen, high CO2 conditions resemble those 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 Candle jar

  15. Selective Media and Differential Media Selective medium:Additivessuppress 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.

  16. 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

  17. 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!

  18. Streak Plate Method 3 or 4 quadrant methods

  19. 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.

  20. 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

  21. Fig 6.13 Figure 6.12b

  22. Bacterial Growth Curve Phases of growth • Lag phase • Exponential or logarithmic (log) phase • Stationary phase • Death phase (decline phase)

  23. The lag phase- This period of little or no cell division. During this time, however, the cells are not dormant. The microbial population is undergoing a period of intense metabolic activity involving, synthesis ofenzymes and various moleculesThe log phase (exponential phase)- The cells begin to divide and enter a period of growth,or logarithmic increase. Cellular reproduction is most active during this period, The stationary phase- The growth rate slows, the number of microbial deaths balances the number of new cells, and the population stabilizes. (period of equilibrium)

  24. What causes exponential growth to stop is not always clear.1.The exhaustion of nutrients2.The accumulation of waste products, and harmful changes in ph may all play a role.The death phase- The number of deaths eventually exceeds the number of newcells formed, and the population enters the death phase, orlogarithmic decline phase.

  25. Direct Measurements of Microbial Growth Viable cell counts: Plate counts: Serial dilutions put on platesCFUs form colonies

  26. Figure 6.15, step 1

  27. Additional Direct Measurements Direct microscopic count: Counting chambers (slides) for microscope A specially designed slide called a petroff-hausser cell counteris used in direct microscopic counts.-motile bacteria are difficult to count by this method-Dead cells are bout as likely to be counted as live ones.

  28. FILTRATION • Membrane filtersfor fluids. Pore size for bacteria: 0.2 – 0.4 m Pore size for viruses: 0.01 m At least 100 ml of water are passed through a thin membrane filter whose pores are too small to allow bacteria to pass. Filtration is the method of choice for low counts M.O.

  29. Estimating Bacterial Numbers by Indirect Methods Spectrophotometry to measure turbidity OD is function of cell number

  30. The most probable number (MPN) method- Another method for determining the number of bacteria ina sample is the most probable number (MPN) method - This statistical estimating technique is based on the fact that the greater the number of bacteria in a sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to grow in the tubes in a dilution series.- It is useful when the growth of bacteria in a liquid differential medium is used to identify the microbes (such as coliform bacteria, which selectively ferment lactose to acid, in water testing).

  31. Metabolic activity- indirect way to estimate bacterial numbers is to measurea population's metabolic activity ( acid or CO2, is in direct proportion to the number of bacteria present). Dry weight-For filamentous bacteria and molds, the fungus is removed from the growth medium, filtered, and dried in a desiccator. It is then weighed

  32. Indirect Methods Turbidity Metabolic activity Dry weight Measuring Microbial Growth - Overview Direct Methods • Plate counts • MPN • Direct microscopic count • Filtration

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