Topic 6

Topic 6: Culture and Control

Metabolism

  • Growth Conditions

  • Media

  • Counting Microbes

  • Controlling Microbes

Physiological Diversity

  • Phylogenetic diversity is a result of 4 billion years of evolution.

  • Metabolic diversity allows microorganisms to adapt to exploit available niches.

    • Adaptation is limited by the constraints of chemistry and physics.

  • Illustration: Figure 1.12 depicts this diversity.

Metabolism

Catabolism

  • Definition: The metabolic process that releases energy by breaking down complex molecules into simpler ones.

Anabolism

  • Definition: The metabolic process that consumes energy to build up complex molecules from simpler ones.

Cell Metabolism Components

  • Nutrients for Biosynthesis: Essential for cell structure and function.

  • Waste Products: Includes fermentation products such as acids, alcohols, and carbon dioxide (CO₂) along with reduced electron acceptors.

  • Energy Sources for Biosynthesis: Includes both catabolic and anabolic processes.

  • Anabolism Forms: Macromolecules and other cell components.

  • Energy Usage: Energy derived from catabolism is utilized for motility and nutrient transport.

  • Sources of Energy: Depending on the organism, energy may come from chemicals or light.

Nutritional Requirements

Macronutrients

  • Defined as nutrients required by all cells for building macromolecules:

    • Carbon (C)

    • Nitrogen (N)

    • Phosphorus (P)

    • Sulfur (S)

    • Oxygen (O)

    • Hydrogen (H)

Micronutrients

  • Required by some cells, can include:

    • Iron (Fe)

    • Copper (Cu)

    • Sodium (Na)

    • Magnesium (Mg)

    • Manganese (Mn)

Fundamentals of Nutrition

  1. Energy Source:

    • Categories include:

      • Photo: Photosynthetic (uses organic or inorganic electrons).

      • Chemo: Organic or inorganic sources for energy.

        • organo chemicalenergy 

        • litho minaeral - ch4,sulfide 

  2. Electrons Source:

  3. Carbon Source:

    • Fixed organic (C-C bonds) = Heterotroph (derived from Greek meanings of "other" and "nutrition")

    • Gaseous inorganic (CO₂) = Autotroph (derived from Greek meanings of "self" and "nutrition")

Energy and Carbon Sources (Detailed)

  • Electron and Carbon Diagram:

    • Categorization based on types of sources:

    • Photoorganoheterotroph: Obtains energy from light, carbon from organic compounds.

    • Chemolithoautotroph: Obtains energy from inorganic compounds and carbon from CO₂.

Energy Sources Overview

  • Chemoorganotrophs: Energy gained from oxidizing organic compounds.

  • Chemolithotrophs: Energy derived from oxidizing inorganic compounds, specific to prokaryotes.

  • Phototrophs: Utilize light as an energy source, classified as either oxygenic or anoxygenic.

Carbon Requirements in Microbes

  • Autotrophs ('primary producers') fix carbon directly from CO₂.

  • Heterotrophs utilize organic molecules produced by autotrophs.

  • Carbon serves multiple roles:

    • Energy storage and manipulation.

    • Structural purposes in cellular components.

Acquisition of Nitrogen

  • Microorganisms incorporate nitrogen into usable forms.

  • Assimilation Process: Often involves the incorporation of ammonia into glutamate or glutamine.

  • Nitrogen is critical for synthesizing numerous macromolecules:

    • Amino acids

    • Nucleic acids

Nutrient Concentration

  • Growth rate depends on nutrient availability; growth is limited by the key nutrient available in the lowest quantity.

Effects of Oxygen on Microbes

  • Aerobic Growth: Utilization of oxygen for energy production.

    • Obligate Aerobes: Require O₂.

    • Microaerophiles: Prefer lower levels of O₂ for optimal growth.

    Anaerobic growth :Occurs without O2

    • Aerotolerant Anaerobes: Can survive O₂ but do not utilize it.

    • Obligate Anaerobes: Cannot grow in the presence of O₂.

    • Facultative Anaerobes: Can grow without O₂ but prefer its presence.

Toxic Oxygen Species and Cellular Defenses

  • The impact of O₂ on cellular respiration depends on cellular defenses against toxic oxygen species:

    • Types of Toxic Species:

    • Singlet oxygen ({}^1{O}_2 ) - photochemical reaction;products of peroxidase enzyme 

    • Superoxide anion (O_{2}^{-}) - 

    • Hydroxyl radical (OH ) - by productus of reduction of O2 during respiratioin of O2 during respiration and other biochemical redox reactions

    • Hydrogen peroxide (H2O2)

  • Cellular Defenses Against Toxic Species:

    • Includes antioxidants such as carotenoid pigments and enzymes like superoxide dismutase and catalase.

Catalase Test

  • A test to determine the presence of catalase enzyme:

    • Chemical Reaction: H2O2 + H2O2 - 2H2O + O

Effects of pH on Microbial Growth

  • pH affects macromolecular structures and transmembrane electrochemical gradients.

  • Each microbe has an optimal pH range, with the internal pH usually maintained close to neutral despite external variations.

  • Range for intracellular pH may vary from as low as 4.6 to as high as 9.5 under extreme conditions.

Osmotic Pressure and Water Activity

  • Water activity is essential for cellular biochemical reactions and is measured against solute concentrations, affecting water influx and efflux.

  • Water Activity (aw): Defined as aw = rac{VP ext{ of air in equilibrium with substance or solution}}{VP ext{ of air with pure water}}.

  • Typical values: pure water aw = 1.0; seawater aw = 0.98. Most bacteria require a_w > 0.9 for growth.

Water Activity Mechanism

  • Cytoplasm typically maintains a higher solute concentration than the external environment.

  • Balance Mechanism: Employs strategies such as increasing internal solute concentration and pumping inorganic ions.

Temperature's Effects on Microbial Growth

  • Temperature affects macromolecular structure, membrane fluidity, and enzyme function.

  • Each microbe has optimal temperature ranges for growth.

    • Psychrophiles: Optimal growth at temperatures below 15°C.

      • minimum <0

      • maximum <20

      • very senstive to moderate temperatures 

      • enzyme denature

      • higher proportion of unsaturated fatty acids in membrane phospholipids than mesophiles

    • Psychrotolerance 

      • able to grow 0-4 

      • optimal growth in moderate temperatures 

      • 20-40

      • mesophiles capable of low temp growth 

      • found in temperature climates 

      • many soil microorganisms 

      • laurel creek, your house

    • Hyperthermophiles

      • boulder spring, yellowstone national park 

      • supervolcano

      • boiling spring superheated 1-2 C above boiling point 

      • Microcolony growing on glass slide immersed in boiling spring 

  • Mesophiles: Optimal growth between 20-40°C.

  • Hyperthermophiles: Optimal growth at extreme temperatures (1-2°C above boiling).

  • Growth Limits:

    • Minimum: <0°C for some.

    • Maximum: ~20°C for others in specific habitats.

Molecular Adaptations for High Temperature

  • Favor enzymes and proteins that operate optimally at elevated temperatures.

  • Include features that enhance thermal stability through:

    • Critical amino acid substitutions.

    • Increased ionic bonds between residues.

    • Stabilization through specific solutes like di-inositol phosphate.

Media for Microbial Growth

Types of Media

  • Solid Media: Agar plates.

  • Liquid Media: Broths.

    • Agar concentration: 1.5% final concentration, melted at >85°C.

Composition of Culture Media

  • Agar: A polysaccharide derived from algae, serves as a solidifying agent at specific temperatures, generally not degraded by many microbes.

Colony Morphology

  • Variations can be observed in colony form, texture, elevation, and color.

    • Examples:

    • Compact circular colonies.

    • Filamentous colonies.

Complex and Defined Media

  • Complex Media: Unknowable chemical composition.

  • Defined Media: Knowable composition. Example:

    • M9 Minimal Salts Broth components (per liter):

    • Na₂HPO₄: 6 g, NaCl: 5 g, KH₂PO₄: 3 g, MgSO₄: 0.1 mM, etc.

Specialized Media

  • Selective Media: Allow isolation of specific microbes with distinct properties.

  • Differential Media: Recognizes differences among groups based on visual reactions.

  • Enriched Media: Increases populations of microbes with particular attributes.

Examples of Specialized Media

  • Brilliant Green Agar: Identifies Salmonella; selective for gram-negative bacteria.

  • Eosin Methylene Blue Agar (EMB): Differentiates gram-negative enterics like E. coli.

  • MacConkey Agar: Selectivity and differentiation among lactose fermenters and non-fermenters.

Pure Culture Techniques

Obtaining Purity

  • Solid media facilitate the isolation of cells, allowing separation into pure populations with methods such as streak plating, spread plating, and pour plating.

Streak Plate Method

  • A technique for isolating colonies from a mixed culture.

Spread and Pour Plate Methods

  • Usage of diluted bacterial suspensions mixed with agar.

Methods of Quantifying Microbes

Direct Counts

  • Involves loading a known volume onto a slide grid for cell enumeration under light microscopy.

    • Advantages: Inexpensive, quick, easy.

    • Disadvantages: Cannot differentiate viable from dead cells.

Viable Cell Counts

  • Utilizes serial dilutions and colony-forming units (CFUs) counted post-incubation.

Spectrophotometry for Turbidity Measurement

  • A spectrophotometer sends light through culture, allowing for rough density measures based on absorbance.

  • light absorbance can give a rough measure of cell density in the tube 

Microbial Growth Curve Stages

  1. Lag Phase: Microbes prepare for growth.

  2. Exponential Phase: Steady exponential replication.

  3. Stationary Phase: Replication halts; balances with death rate.

  4. Death Phase: Nutrient depletion; high waste levels cause cell mortality.

Can determine ;

  • generation time” the time to double the population in the exponentail phase 

  • growth rate:number of generations/units of time (inverse of the generation time 

  • growth yield: the maximum population density and/or amount of cellular material produced by the culture 

Continuous Culture

  • Maintains microorganisms in exponential growth for product harvesting, mimicking natural environmental conditions using a chemostat.

  • used to keep miccroorganism in a limited but contious flow of nutrients 

    • mimicking environment conditions 

  • a chemostat flows in fresh medium and takes out some old medium 

Control of Microbial Growth

Methods of Control

  • Filtration: Physical removal of microbes using filters.

  • Temperature Manipulation: Heating denatures proteins and nucleic acids.

  • Radiation: UV radiation and ionizing radiation for microbial control.

  • Chemical Control: Utilizing disinfectants and antiseptics.

Filtration

Mechanism and Limitations

  • physical removal of microbes 

    • nylon/Teflon filters with the pore size of 0.2 or 0.45 um 

    • virues can be removed from liquids by ultrafiltraisation methodes - reding pore size 10 to 100nm 

  • problems can result large particles clog filters 

  • viscous liquids don’t filter well 

  • ultrafilrterisation requires high pressure 

  • Removal of particles from liquids and gasses 

    • 0.2um pore size common for sterilization 

    • ideal when material is heat or radiation sensitive 

  • Varying pore sizes 

    • separation or distinguishing organisms 

    • retrieving small cells from a mixture of large cells 

Depth filter 

  • fibrous sheet or mat 

    • randomly overlapping fibers of different substances 

    • paper glass or cotton 

  • used as a “pre-filter”

    • removes suspened particles in a ‘trapping action”

Types of Filters

  • Conventional Membrane Filters:

    • polymer filter (0.45-0.20)

      • Cellulose acetate or cellulose mitrate 

    • Pore diameter variable during production 

      • “Sieve-like action”

Nucleopore filters 

Thin polycarbonate film (10um thick)

  • radiation damage, cracks enlarged by chemical “etching”

  • consistent pore size 

Useful for microscopy 

  • filtered material on a single surface plane

Temperature Control Techniques

Heat 

  • denatures protein and nucleic aids 

  • 100 kills most microbes quickly 

  • an autoclave adds pressure, keeping fluids from evapourating during the high temp 

  • Killing microbes effectively at 100°C; autoclaves enhance efficiency through pressure.

Potential problems

  • hyperthermophiles 

  • endospores 

  • some materials cant be heated 

Autoclave 

  • steam under pressure 

    • 121 degress celcius 

    • 15 psi 

    • pressure cooker 

  • Efficiency determined by 

    • destruction of endospores 

    • vegetative cells 

Pasteurization

  • Reduces microbial loads

    • destroys pathogens 

    • 90-99% kill of other microbes 

    • increse shelf life 

    • does not sterilze 

  • Louis pasteur developped for wine preservation 

    • flavour and bouquet maintained 

  • Common process:high temp short time (HTST)

    • 72 for 15 seconds 

  • other processes

    • UHT:135 for <1 second 

    • ESL:filtration, then lower temp treatment 

  • lower heat 

    • pasteurization reduces microbe numbers 

  • freezing 

    • can damage cells by forming ice crystals 

    • can stop biochemical rxn 

    • good for long term preservation 

Electromagnetic Radiation for Control

Uv radition of 260-280 nm wavelenght 

  • UV radiation can damage DNA by forming thymine dimers.

  • epoloited to control microbial growth on non-living surfaces and in water 

  • Ionizing radiation

    • Protein damage 

    • DNA damage 

      • double strand breaks 

      • stray electrons 

      • hydroxide ions

      • hydride radicals 

    • higher energy (measured in Grays GY), better penetration 

    • limited to large industrial operations in specialized facilities becuse of costs and hazards of equipment 

    • Medical supplies and drugs,disposable labware, food industry, insects deinfestation, even tissye for grafting   

Chemical Agents in Control

Agents target different groups 

  • Microbicial, baacteriocial, fungicidal, algicidal, viricidal 

Agents vary with respect to selective toxicity 

  • non-selective -affects sulfhydryl groups 

  • selective - antiboditics affecting prokaryotic ribosomes ;penicillin

  • selective agents useful for treating dieases 

  • Non-selective agents have uses but not internal use

Chemical control 

Disinfectants : used on non living surfaces to kill potentially infectious microbes 

Antiseptics: used on living tissue to kill potentially infectious microbes 

What a chemical a good microbe killler 

  • kills a wide range of microbes 

  • isnt it corrossive or overly toxic 

  • doesn’t leave a residue or emit fumes 

  • cheap 

  • temperature stable 

Antiimicrobics/ Antibiotics 

  • antimicrobics includes microbial and syntheic agents 

    • antibiotics are antimicrobial agents produced by microbes 

  • may be either cidal, static or lytic 

    • most work by binding to proteins or cellular organells and disruptes essentail funcitions necearry for growth and survival of microorganisms

Measuring Effectiveness of Control Methods

  • Decimal Reduction Time (D Value): Indicates the time to kill 90% of the target organism under specific conditions.

There are many additional tables and figures mentioned but not detailed here due to format constraints.