Lesson 3: Microbial Nutrition PDF

LESSON 3: MICROBIAL NUTRITION  Macronutrients / Macroelements  required by microorganism in relatively large amount  C, H, O, N, S, Ca, P, Fe, K, Mg  Cations: K, Ca, Mg, Fe  K - required for the activity of enzymes, osmoregulation, pH homeostasis, protein synthe...

LESSON 3: MICROBIAL NUTRITION  Macronutrients / Macroelements  required by microorganism in relatively large amount  C, H, O, N, S, Ca, P, Fe, K, Mg  Cations: K, Ca, Mg, Fe  K - required for the activity of enzymes, osmoregulation, pH homeostasis, protein synthesis  Ca - for heat resistance, required in cell division and cell membrane  Mg - stabilizing ribosomes and membrane  Micronutrients / Trace Elements  Required by microorganism in relatively small amount  Normally part of enzymes and cofactors, aid in catalysis of reactions and maintenance of protein structure  Fe2+ & Fe3+ - cofactor of enzymes and electron carrying proteins, part of cytochromes  Vitamins - functions as coenzymes, required growth factor  Manganese - aids enzymes catalyzing the transfer of phosphate groups  Zinc - present at the active site of some enzyme, catalytic subunits of E. coli aspartate carbomoyl transferase  Molybdenum, nickel, copper  Nutritional Types of Microorganisms Carbon Sources  Autotrophs - use CO2 as sole or principal source of carbon, uses light as energy source  Heterotrophs - use reduced pre-formed organic molecules as carbon sources Energy Sources  Phototrophs - use light as energy source  Chemotrophs - obtain energy from the oxidation of chemical compounds (organic / inorganic) Electron Sources  Lithotrophs - use reduced inorganic substances as electron source  Organotrophs - extract electrons from organic compounds Note: They could be combined. Ex:  Photolitotrophs - photo + litho  Photolithotrophic autotrophs - photo + litho + autotrophs  Chemoorganotrophic heterotrophs - chemo + organo + heterotrophs  Requirements for Nitrogen, Phosphorus, and Sulfur  Nitrogen  Needed for the synthesis of amino acids, purines, pyramidines, some carbohydrates and lipids, enzyme cofactors  In most autotrophs and many non photosynthetic microbes (Cyanobacteria Rhizobium) :  Reduce nitrate to ammonia  Incorporate ammonia in assimilatory nitrate reduction  Phosphorus  Present in nucleic acids, phospholipids, ATP, several cofactors, some proteins and cell components  Used by ALL microbes  Sulfur  Needed for synthesis of amino acids (cysteine, methionine), some carbohydrates (biotin, thiamine)  Some microbes require reduced form of sulfur such as cysteine  Growth Factors  Used by microbes that lack one or more enzymes needed for the cell components or precursors of the mentioned components Major Classes:  Amino acids  for protein synthesis  Purines & Pyrimidines  For nucleic acid synthesis  Vitamins  Small organic molecules that make up all or part of enzymes cofactors, only very small amounts sustain growth  Growth Requirements Gaseous Requirements  Aerobic Bacteria  require oxygen for growth  Obligate aerobes to grow only in the presence of oxygen (Pseudomonas aeruginosa)  Anaerobic Bacteria  Grow in absence of oxygen (Clostridium tetani)  Obligate anaerobes - may die on exposure to oxygen  Facultative anaerobes - can grow in presence of oxygen & survive without oxygen via anaerobic respiration or fermentation  Microaerophilic Bacteria  Proliferate (increase in #) in the presence of low oxygen tension Temperature Requirements  Most important factor that can affect growth of microorganisms  Temperature range - must not be above the maximum or below the minimum as it can affect growth  Optimum - growth occurs best (ex: pathogenic bacteria, -37 degrees C) Temperature classes (in degrees C):  Psychrophiles - 15 or lower - max: -10   Psychrotrophs / Facultative Psychrophiles  20 - 30  max: 35  Mesophiles  25 - 40  min: 15-20, max: 45  Thermophiles  55 - 60  min: 45 Other Requirements:  Moisture and drying  Hydrogen ion concentration  Light  Osmotic effect  Mechanical and sonic stress  Growth Media Culture Media  Nutrient solutions tailored to the particular organism to be grown  It must be sterilized before use thru heating of medium under pressure in an autoclave  Prepared selectively or differentially or both 2 classes:  Defined media - exact composition of a defines medium (quali and quanti sense) is known, prepared by adding precise amounts of pure inorganic or organic chemicals to distilled water  Complex media - made from digests of microbial, animal, plant products like milk protein (casein), beef extract, soybeans (tryptic soy broth), yeast cells (yeast extract) Based on Consistency  Solid medium  Contains 2% agar  Used for colony morphology, pigmentation, hemolysis  Ex: nutrient agar, blood agar  Liquid medium  No agar  Used for inoculum preparation, blood culture, isolation of pathogens from a mixture  Becomes cloudy as indication of growth  Ex: nutrient broth  Semi solid medium  For checking motility Based on Nutritional Component 1. Simple media   Aka basal media  Used for non-fastidious (not having complex nutritional requirements) bacteria  Ex: nutrient agar and broth 2. Defined media  All the ingredients are known (quali and quanti) 3. Complex media  Contain undefined ingredients and the exact contents are known  Ex: nutrient broth, tryptic soya broth, MacConkey agar (for Lactobacillus)  May contain undefined components:  Peptones - prepared by partial proteolytic digestion of meat, casein, soyameal, gelatin, and other protein sources  Meat extract - aqueous extracts from lean beef and contain amino acids, peptides, nucleotides, organic acids, minerals, vitamins  Yeast extract - from brewer’s yeast and contain an excellent source of vit B, nitrogen, carbon compounds Based on Functional Use / Application 1. Enriched medium  Broth or solid medium containing a rich supply of special nutrients  Used for fastidious microbes  Ex: blood agar, chocolate agar 2. Indicator media  Contain an indicator which changes color when a bacterium grows in them  Ex: Christensen’s urease medium 3. Transport media  Used for transporting the samples  Delicate organisms may not survive the time taken for transporting specimen w/o it  Ex: buffered glycerol saline 4. Anaerobic media  Used to grow anaerobic organisms  Ex: Robertson’s cooked meat medium 5. Differential medium  One to which an indicator (typically a dye) is added, which reveals by a color change whether a particular metabolic reaction has occurred during growth  Ex: MacConkey agar (for lactose fermentation) 6. Selective media  Contain compounds that inhibit the growth of some microbes but not others  Ex: Eosin Methylene Blue (EMB) - contains dyes that are not for gram positive bacteria and allows growth of gram negative bacilli and enteric bacilli - coliforms and faccal coliforms Isolation of Pure Cultures  A pure culture is needed to characterize and identify an individual species  Culture methods include: 1. Stab culture  Prepared by puncturing a suitable medium - with a long, straight wire  Used to study oxygen requirements 2. Pour plate method  Gives an estimate of the viable bacterial count in a suspension  Used for quantitative urine cultures   Should be avoided when trying to determine viable cell counts of heat-sensitive bacteria isolate 3. Liquid culture  Inoculated by touching with a charged loop or by adding inoculum with pipettes or syringes  Used for blood culture, sterility tests & continuous culture methods 4. Anaerobic culture methods 5. Streak culture  Isolation of bacteria in pure culture from clinical specimens 6. Lawn culture  Provides a uniform surface growth of the bacterium  Can be used for bacteriophage typing, antibiotic sensitivity testing, preparation of bacterial antigens and vaccines  Prepared by flooding the surface of the plate with a liquid suspension of the bacterium 7. Stroke culture  Made in tubes containing agar slope or slant provide a pure growth of bacterium for slide agglutination and other diagnostic tests Laboratory Culture of Microorganisms 1. Agar  Used to solidify the culture media  An algal polysaccharide first used in the classical studies of Robert Koch  Solid media immobilize cells so that as they grow, they accumulate in a pile to form visible, isolated cell masses called colonies 2. Colony morphology  Visible characteristics of a colony  Used to identify microbes but is routinely used to determine if culture is:  Pure - only one microbe  Contaminated - undesired organisms co-occur  Mixed - many microbes present 3. Aseptic technique  Series of steps by which microbes are transferred between growth media without contamination.  Contamination can be introduced from microbes in the air, in liquid droplets, or on surfaces  Streak plate technique - method for obtaining pure cultures that contain a single microbe, and of verifying culture purity using inoculating loop  Microbial Growth Growth  Increase in number of cells (binary fission)  Cell division following enlargement of a cell to twice its minimum size  During cell division, each daughter cell receives a chromosomes and sufficient copies of all other cell constituents to exist as an independent cell Four Phases of Microbial Growth 1. Lag phase  Initial phase, bacteria are metabolically active but not dividing 2. Log phase (exponential phase)  Time of exponential growth  Period when the growing cell population doubles at regular intervals (balanced growth) 3. Stationary phase  Growth reaches plateau as the number of dying cells equals the number of dividing cells  Not net increase or decrease in cell number, thus growth rate=0  Cell prepares for maintenance and survival 4. Death phase  Exponential decrease in number of living cells  Decline phase - total number of cells decreases due to cell death Generation time = time per generation/ number of generations (aka doubling time) - time required for cell to separate N - final cell number No - initial cell number N - number of generations during the period of exponential growth Synchronous Growth  Microbiological culture or a cell culture that contains cells that are all in the same growth stage  Information about growth behaviour of individual bacteria can be obtained by synchronous cultures  Synchronized cultures must be composed of cells which are all at the same stage of the bacterial cell cycle Continuous Culture  The cultures so far discussed for growth of bacterial populations are called batch cultures (describes the growth of microbes in a fixed volume of liquid enclosed within a container such as a test tube or a flask)  Since the nutrients are not renewed, exponential growth is limited to a few generations  Chemostat - most common type of continuous culture device  An open system aiming to attain steady state Diauxic Growth  Used to describe the growth phases of a microbes in batch culture as it metabolizes a mixture of 2 sugars  Resulting in 2 separate growth phases Methods for Measurement of Cell Mass Microscopic cell count - total count of microbial numbers can be observed and enumerated here Viable cell - one that is alive and able to grow Viable count - performed by spreading microbes on solid media and counting colonies, aka plate count (requiring agar plates) Spread-plate & Pour-plate - 2 ways in performing plate count Colony-forming units (CFU) - expressed as the number data from viable counts A. Direct Microscopic Count  A counting chamber consisting of a ruled slide and a coverslip is employed  Constructed in a manner that the coverslip, slide, and ruled lines delimit a known value  The number of bacteria in a small known volume is directly counted microscopically  The number of bacteria in the larger original sample is determined by extrapolation B. Electronic Enumeration of Cells  The microbial suspension is forced through a small hole or orifice in the coulter chamber  An electrical current flow through the hole and electrodes placed on both sides of the orifice measure its electrical resistance C. Plate Count Method  Standard Plate Count (SPC) - number of bacterial colonies that develop on a medium in a petri dish seeded with a known amount of inoculum  Volume (usually 0.1–1.0 ml) of culture is pipetted into an empty sterile Petri plate. Molten agar medium, tempered to just above gelling temperature (50°C), is then added and gently mixed before allowing the agar to solidify. D. Membrane Filter Technique  The number of bacterial in aquatic sample can be determined from direct counts after the bacteria have been trapped on special membrane filters such as nitrocellulose more size filter or a black polycarbonate membrane filter E. Turbidimetric Methods  When bacteria growing in a liquid medium are mixed, the culture appears turbid due to bacterial culture acting as a colloidal suspension that blocks and reflects light passing through the culture F. Determination of Nitrogen Content  The major constituent of cell material is protein, and since nitrogen is a characteristic part of proteins, one can measure a bacterial populations or cell crop in terms of bacterial nitrogen G. Determination of Dry Weight  Measure some quantifiable cell property that increases as a direct result of microbial growth  Simplest technique is to measure the weight of cells in a sample H. Measurement of Specific Chemical Changes  Detects specific changes caused in growth medium as a result of multiplication of cells  Includes detecting activity cell products such as acid and gas production I. Spread-plate Method  A volume of an appropriately diluted culture is spread over the surface of an agar plate using sterile glass spreader