Unit 1 Lessons 1-4 PDF

LESSON 1: FUNCTIONAL ANATOMY 2. Eukaryotic OF PROKARYOTIC AND EUKARYOTIC - Eukarya: algae, protozoa, fungi CELLS - Contains organelles Microorganism 2 microbial cells: - Aka microbes...

LESSON 1: FUNCTIONAL ANATOMY 2. Eukaryotic OF PROKARYOTIC AND EUKARYOTIC - Eukarya: algae, protozoa, fungi CELLS - Contains organelles Microorganism 2 microbial cells: - Aka microbes Prokaryotes - Inhabit environment that supports - Evolved first due to the presence life – microbial communities of RNA first in the planet - Undifferentiated, single celled or - Lacks organelles multicellular - Circular DNA - Essential in sustaining life: - No cationic proteins called provides oxygen histones - Pathogens - microbes causing - No nucleus diseases - Archaea, cyanobacteria, - Bacteria, archaea, algae eubacteria - Microbiology is the study of the - Divides faster: dominant form of life on Earth surface area > volume - Microbial culture - collection of - Horizontal transfers (share to the cells that have been grown on a same generation): conjugation, nutrient medium another bacteria. Then undergoes - Liquid or solid mixture that recombination giving rise to new contains all the nutrients phenotypes needed for a microbe to - Transduction - transfer by grow bacteriophage (a virus) which - Growth refers to increase in cell could be generalized or specialized number and not in cell mass - Conjugation - cell to cell contact forming a colony thru plasmids (circular DNA that - Viruses are not cells, survived only are self replicating) or transposons with the hosts (jumping genes, motile) - No meiosis, only conjugation Microbial Cells - Cytoplasmic membrane - permeability barrier, separates cytoplasm from outside - Cytoplasm - mixture of macromolecules, small organic molecules, inorganic ions, ribosomes Eukaryotes - Ribosomes - protein synthesis - Contain organelles - Cell wall - structural strength - Linear DNA - DNA genome - full set of genes in - Divides slower : a cell// living blueprint surface area < volume (chromosomes) - Animals and plants - Genes - segments of DNA that - Plants and fungi are the only ones encode protein or RNA molecule that have cell wall 2 fundamental cell types: 1. Prokaryotic - Bacteria & Archaea 3 Domain Systems of Classification  1. Domain Archaea - Some are commensals (gets - Prokaryotes, single celled energy from host but does not - Thrive areas with high salinity, affect the host at all) temperature, pressure - Nucleus is vesicular (active (extremophiles) transcription) - Heterotrophs (energy from other organisms) or autotrophs (make Slime molds their own food) - Aka myxomycetes - Composed of acellular mass of 2. Domain Bacteria naked protoplasm - Aka Eubacteria - Saprophytic (feeds on dead - Prokaryotes, single celled animals// recyclers) - Heterotrophs - Lack chitin in cell wall - Most abundant - Unicellular - Cell walls have peptidoglycan: - Vegetative stage has no cell walls - N-acetylmuramic acid and N-acetylglucosamine Water molds linked by short peptides - Diploid nuclei - Oogamous - immobile egg + 3. Domain Eukarya motile sperm - Major groups: fungi and protista - Saprotrophic - Flagellated reproductive cells Fungi - Most thrive in water or moist areas - Unicellular - yeast - Multicellular - lichens (symbiotic Viruses partnership of alga and fungus), - Noncellular mushrooms - Have RNA or DNA - Heterotrophic - Nucleic acid enclosed in protein - Lack motility coat or capsid// could be single or - Cell walls have chitin double stranded - Most fungi have hyphae (feathery filaments for vegetative growth) Protista Algae - Seen using scanning and - Polyphyletic (more than one transmission electron microscope common evolutionary ancestor) - Cannot reproduce by itself - Photoautotrophic (from sunlight) (dependent to host) - Primarily aquatic - Unicellular Archaea vs Bacteria - Thallophytic: No vascular system, - Archaea: least characterized and did not undergo cell differentiation plays major role in recycling - Some are motile (flagella) nitrogen - Component of membranes: Protozoans - ether lipids (archaea) - Unicellular, motile - ester lipids (bacteria) - Free-living - Archaea and bacteria influenced the evolution of eukaryotes Cell morphology gram negative - thin Spherical peptidoglycan, fuchsia pink - Coccus = 1 - Diplococcus = 2 Gram Positive Cell Wall - Streptococcus = straight line - Thick and rigid layers of - Tetracoccus = 4 peptidoglycan - Sarcina = 2 tetra together Teichoic acids: - Staphylococcus = fusion (-) charged, regulate movement of Spiral cations - Vibrio Regulate growth and prevent lysis - Spirillum Used to identify bacteria - Spirochetes Types: Rod-shaped - Lipoteichoic acids - linked to cell - Single bacillus membrane, spans the cell wall - Diplobacilli - Wall teichoic acids - linked to - Streptobacilli peptidoglycan layer - Palisades - Susceptible to antibiotics due to no Other shapes outer membrane - Filamentous - Star shaped - Rectangular - Hyphae BACTERIA Cytoplasmic membrane - Selective permeability - Maintains integrity - separations of inside and outside environment - Hypothetical absence: loss of control, disrupted homeostasis = cell death Cell wall (Cell envelope) - Prevent lysis: protection against Gram Negative Cell Wall osmotic pressure - Thin and complex of peptidoglycan - Shape and rigidity - Has an outer membrane: - Peptidoglycan and LPS - unique - Bonds to lipoproteins: to bacteria structure linked to outer membrane - Sterols in cell wall-less bacteria and periplasmic space (mycoplasma, ureaplasma) - Periplasmic space: - D-type = dextro contains degradative enzymes and transport proteins Gram Staining - Hans Christian Gram Structure of Cell Wall - to differentiate bacteria: 1. Outer membrane gram positive - thick 2. Periplasmic space peptidoglycan, dark purple  3. Peptidoglycan Unique archaeal cell wall compositions 4. Periplasmic space Examples: 5. Cytoplasmic Membrane Methanosarcina sp. - non-sulfated polysaccharides Components of Outer Membrane (OM) Halococcus sp. - sulfated - Phospholipid bilayer Halobacterium sp. - negatively - Porins - membrane protein// charged acidic amino acids to allows passage of small molecules counteract high Na+ environment, - Lipoproteins - structural stability lying at NaCl concentrations below - Lipopolysaccharides (LPS) : 5% Polysaccharides: acts as antigens Methanomicrobium sp. and Lipid A - endotoxin causing fever methanococcus sp. - cell walls and shock made of protein subunits - It is for protection such as evasion and barrier (against antibiotics) Cytoplasmic membrane - Selective permeability Structure of bacterial - Lipid bilayer lipopolysaccharide hydrophobic tail - fatty acid 1. O-specific polysaccharide hydrophilic head - phosphate - O-antigen - present in antibiotics to - Phospholipid molecule - phosphate avoid the host’s immune system group and lipid 2. Core polysaccharide - Hepanoids : prokaryotes - Kdo - for structural support - Sterols : eukaryotes (pentacylic 3. Lipid A sterol-like molecules) - Anchors LPS to membrane - 2 glucosamine Function of prokaryotic cytoplasmic (N-acetylglucosamine) sugar membrane: backbone connected by phosphate 1. Osmotic or permeability barrier groups 2. Transport systems - specific solutes (nutrients and ions) ARCHAEA 3. Energy generation - respiratory Cell walls and photosynthetic electron - No peptidoglycan transport systems, proton motive - Structure: force, transmembranous Pseudomurein in methanogens ATP-synthesizing ATPase (similar to peptidoglycan) 4. Synthesis of membrane lipids and S-layer - paracrystalline, the murein outermost layer 5. Assembly and secretion Structure: glycan composition 6. Specialized enzyme (arrangement of sugar molecules) 7. Coordination - NAT or T : 8. Chemotaxis - movement of cell to N-acetyl-D-galactosamine chemical gradient such as - NAG or G : nutrients and water N-acetyl-D-glucosamine - L-type = levo LESSON: 2 CELL SURFACE Fimbriae, Pili, and Hami STRUCTURES AND INCLUSIONS - Pili Capsules and Slime Layers - Thin filamentous structures - Sticky coat of polysaccharide made of protein extend formed outside the cell envelope from surface of cell - Mediate attachment, protection, - Enable bacterial cells to alter diffusive environment stick to surfaces - When binding to solid surfaces, - Form pellicles in liquid they form a thick layer = biofilm surfaces - Capsule - Form biofilms in solid - organized in tight matrix, surfaces excludes small particles, - All gram-negative bacteria tightly attached to cell produce pili - Visible in light microscopy, Types of Pili: treated with India ink - Conjugative pili - facilitate (stains only the genetic exchange during background, cannot conjugation (cell-to-cell penetrate the capsule) attachment) - Also seen in electron - Electrically conductive microscopy pili - conduct electrons toward or away the cell, plays role in metabolism - Type IV pili - facilitate adhesion, support twitching motility (allows cells to move along solid surface)// assist in infectivity of some pathogens: cholera, gonorrhea, strep throat, scarlet fever - F pilus (sex pilus) - path of entry of genetic material - Microcapsule - if the layer during mating is too thin to be seen by light microscope - Fimbriae - Short pili that mediate - Slime layer attachment - Easily deformed, loosely attached, include particles, more difficult to see, easily detected in colonies, abundant and embedded in common matrix (ex: lactic acid Leuconostoc) - Also stained by india ink  - Hami/ Hamus Polyphosphate, Sulfur, and - Resembles a tiny grappling Carbonate Minerals hook - Polyphosphate - Present in SM1 group - Granules in accumulation (Archaea) of inorganic phosphate - Attach cells to surfaces to - Formed when phosphate is form a networked biofilm in excess (more efficient trap for - Can be a source of scarce nutrients present) phosphate for nucleic acid and to each other and phospholipid biosynthesis CELL INCLUSIONS - Sulfur - Often visible with light microscope - Sulfur bacterias: organisms Carbon Storage Polymers that oxidize reduced sulfur - Poly-β-hydroxybutyric acid compounds (PHB) - Discovered by Sergei - most common inclusion Winogradsky body in prokaryotes, - Also used for energy monomer polymerized by metabolism ester linkage - Carbonate materials - Monomers are usually - Formed by filamentous hydroxybutyrate (C4) cyanobacteria on external - poly-β-hydroxyalkanoate & internal surface of cell (PHA) - more generic term, - Gloeomargarita : forms synthesized when there is granules of bentonite an excess carbon// broken (carbonate material) down for energy (carbon) containing barium, - Glycogen strontium, magnesium - Polymer of glucose - Biomineralization - - Reservoir of carbon and microbiological process of energy forming minerals - Also produced when there is an excess carbon - Resembles starch (storage reserve of plants)  Gas Vesicles - They only grow when conditions - Confer buoyancy allows floating become favorable (high alanine - Allow cells to position themselves and nutrients) in regions of water column best - Easily dispersed by wind, water, suited for their metabolism animal gut (widely distributed) - Conical-shaped - Produced only by 2 groups: - Impermeable to water and solutes, (1) Bacillales (2) Clostridiales permeable to gases - Both are gram positive - Gas vacuoles - clusters of - Causes food spoilage and vesicles foodborne diseases + - Blooms tetanus & botulism - Gas-vesiculate microbes that form massive Formation and Germination accumulations - Sporulation - Common on near lake - process of cellular surfaces where sunlight is differentiation that results in intense endospore formation - Triggered when nutrient Magnetosomes becomes limiting - Allows bacteria to orient - Germination themselves in a magnetic field thru - Conversion of endospore magnetic dipole back to a vegetative cell - Biomineralized particles of: - Triggered by availability of - magnetic iron oxides nutrients like amino acids magnetite or greigite and sugars - Magnetotaxis - process of - Occurs in 3 steps: migrating along Earth's magnetic activation, germination, field lines outgrowth - Spiked-shaped - most common - Could also be square/rectangular Structure and Features - Visible by light microscope as ENDOSPORES strongly refractile structures - Specialized spores - Impermeable to most dyes - Endo = within - Malachite green stain - used - Highly differentiated dormant cells infused to spore with steam that can tolerate harsh conditions - Contains many layers absent from - Dormant stage of bacteria life vegetative cell (seen in electron): cycle: vegetative cell -> endospore - Core - innermost, contains -> vegetative cell DNA and ribosomes, from cytoplasm of vegetative cell - Inner membrane - from cyto membrane of vege cell - Cortex - 2 peptidoglycan layers - Outer membrane - special membrane formed during sporulation  - Endospore coat - Flagellum / Flagella composed of layers of - Provides swimming motility for spore-specific proteins bacterias - Exosporium (not all) - - Long, thin appendages outer proteinaceous layer - Observed by light microscopy or - Dehydration of the core - ultimate electron microscopy reason for endospore toughness// - Tuft - group of flagella caused by accumulation on - Do not rotate in constant speed, it calcium-dipicolinic acid depends on the proton motive - Small acid-soluble spore force proteins (SASPs) - Fastest speed - 60 cell-lengths/s - made during sporulation - Not straight but helical - 2 functions: - In order to change direction, they 1. Bind tightly to DNA also need to change the direction in the core of their rotation (ex: CW - CCW) 2. Protect from - Can be anchored to a cell in damage of UV different locations: radiation, dry heat, Polar flagellation desiccation - attached at one or both ends - they spin around moving from The Sporulation Cycle place to place - tuft at one end is called lophotrichous seen by dark-field or phase contrast microscopy - tuft at both ends is called amphitrichous Peritrichous flagellation - flagella inserted around cell surface - move slowly in a straight line CELL LOCOMOTION Structure of Flagella Motility -Filament - allows cell to reach different parts - composed of many copies of of their environment proteins called flagellin - 2 major types of prokaryotic - hook - wider region at the base, movement: swimming & gliding single type of protein, connects the - Taxis - how motile cells are able to filament to the flagellum motor move towards or away from stimuli - flagellum motor - reversible - Flagella & Archaella - tiny rotating rotating machine, anchored in machines that function to push or cytoplasmic membrane and cell pull the cell through a liquid wall  - Rotor - consists of central rod, L, P, C, MS rings// make up the flagellar basal body - Stator - consists of the mot proteins that surround the rotor and function to generate torque Flagellar Synthesis - Filament grows from tip - MS & C rings are synthesized -> inserted to cyto membrane -> followed by other rings -> early hook -> cap -> late hook -> filament synthesis Central rod - passes thru a series of rings L-ring - outer ring in outer membrane P-ring - in peptidoglycan layer - Flagellin flows through the hook to MS and C Rings - third set of form filament rings, within cytoplasmic - Cap - a protein present at the end membrane and cytoplasm of growing flagellum to guide them Mot proteins - series if proteins into position that surrounds the inner rings, in cyto membrane and Archaellum / Archaella peptidoglycan// sets the proton - Present in archaea flow rate - Rotate same as flagella Fli proteins - set of proteins, - 7-12 genes encode the major functions as motor switch, proteins that make up it reversing the direction of rotation - Usually studied in Halobacterium of the flagella (salt-loving) - The speed is slow due to smaller diameter than flagella (smaller diameter = reduced torque) - Methanocaldococcus can swim at 500 cell lengths per sec - Embedded in archaeal cell wall and cyto membrane - Hydrolysis of ATP drives the rotation (not proton motive force) Flagellum motor has 2 components:  MICROBIAL LOCOMOTION Internal Movement (Cytoplasmic Streaming) - Organelles move within cytoplasm - Governed by actin filaments and cytoskeleton - Facilitates distribution of nutrients and materials inside the cell External Movement (Motility) - Involves specialized organelles for locomotion - Capable of CW and CCW rotations - Pseudopodia - temporary extensions of the cell body CILIA - Cilia - short, haiir-like structures - For cell motility and sweeping food - Flagella - long, whip-like organisms into oral cavity - Energy from ATP - Beating caused by intraciliary SURFACE MOTILITY excitation followed by interciliary - All surface motility are conduction considerably slower than 4 types of ciliary movements swimming motility 1. Pendulus ciliary movement - Carried out in single plane Twitching motility - Occurs in paramecium (those who - Requires type IV pili which extend have rigid cilia) from one pole of the cell, attach to 2. Unciform ciliary movement the surface, then retract to pull the - Hook-like movement cell forward - Metazoan cells - Energy is from ATP hydrolysis 3. Infundibuliform ciliary - Allows cells to move in groups movement - Facilitated by type IV pili and - Occurs due to rotary movement of secretion of extracellular cilium and flagellum polysaccharides 4. Undulant movement - Observed in Pseudomonas & - Waves of contraction myxobacteria - Myxobacteria exhibit 2 forms: CYTOSKELETAL COMPONENTS social motility (caused by Intermediate filaments twitching) & adventurous motility - Rope-like assemblies of fibrous (gliding) polypeptides - Support the nuclear envelope and plasma membrane Microtubules - Small, hollow cylinders made of tubulin protein - Composed of a-tubulin and b-tubulin  Gliding motility Chemotaxis in Polarly - Smooth motion along the axis of Flagellated Bacteria the cell without the aid of external - Similar to peritrichously flagellated propulsive structures cells but they do not tumble but - Continuous form of movement in a instead they swim backwards helical track - Biased random walk - can - Gliding bacterias are typically navigate effectively through their filamentous or rod shaped environments toward conditions - No gliding archaea known that favor growth and away from - Best observed in myxococcus and those that could inhibit growth or flavobacterium otherwise cause harm - Gliding motors - rotary motors driven by proton motive force which are operationally similar to Other Forms of Taxis flagellar motors - Phototaxis - response to light - Observed in filamentous cyanobacteria CHEMOTAXIS - Photoreceptor - senses light - Taxis - directed movement toward - Scotophobotaxis occurs when a a stimuli phototrophic bacterium happens to - Chemotaxis - response to swim into darkness outside the chemicals illuminated field of view of the - Observed in swimming bacterias microscope (E. coli) by observing altering of - mechanism to prevent rotations of flagellum phototrophic cells from swimming away from a Chemotaxis in Peritrichously lighted zone into darkness Flagellated Bacteria - Runs & tumble - Runs - cell is swimming - Osmotaxis - response to ionic forward in a smooth fashion strength - Tumble - when the cell stops and - Hydrotaxis - response to available jiggles about randomly water - In the absence of attractant, the - Aerotaxis - response to O2, ex: cell moves about its environment in Microaerophiles random fashion through a series of - Magnetotaxis - magnetotactic runs and tumbles bacteria found where O2 are low - In the present of attractant, the cell - do not actually exhibit moves toward the attractant but directed motility toward not DIRECTLY but thru a biased magnetic fields but instead random walk are exhibiting aerotaxis - Chemoreceptors - sense attractants and repellents LESSON 3: MICROBIAL NUTRITION Energy Sources - Phototrophs - use light as energy Macronutrients / Macroelements source - required by microorganism in - Chemotrophs - obtain energy relatively large amount from the oxidation of chemical - C, H, O, N, S, Ca, P, Fe, K, Mg compounds (organic / inorganic) - Cations: K, Ca, Mg, Fe - K - required for the activity of Electron Sources enzymes, osmoregulation, pH - Lithotrophs - use reduced homeostasis, protein synthesis inorganic substances as electron - Ca - for heat resistance, required source in cell division and cell membrane - Organotrophs - extract electrons - Mg - stabilizing ribosomes and from organic compounds membrane Note: They could be combined. Ex: Micronutrients / Trace Elements - Photolitotrophs - photo + litho - Required by microorganism in - Photolithotrophic autotrophs - relatively small amount photo + litho + autotrophs - Normally part of enzymes and - Chemoorganotrophic cofactors, aid in catalysis of heterotrophs - chemo + organo + reactions and maintenance of heterotrophs protein structure - etc. - Fe2+ & Fe3+ - cofactor of enzymes and electron carrying Requirements for Nitrogen, proteins, part of cytochromes Phosphorus, and Sulfur - Vitamins - functions as - Nitrogen coenzymes, required growth factor - Needed for the synthesis of amino - Manganese - aids enzymes acids, purines, pyramidines, some catalyzing the transfer of carbohydrates and lipids, enzyme phosphate groups cofactors - Zinc - present at the active site of - In most autotrophs and many non some enzyme, catalytic subunits of photosynthetic microbes E. coli aspartate carbomoyl (Cyanobacteria Rhizobium) : transferase - Reduce nitrate to ammonia - Molybdenum, nickel, copper - Incorporate ammonia in assimilatory nitrate Nutritional Types of reduction Microorganisms - Phosphorus Carbon Sources - Present in nucleic acids, - Autotrophs - use CO2 as sole or phospholipids, ATP, several principal source of carbon, uses cofactors, some proteins and cell light as energy source components - Heterotrophs - use reduced - Used by ALL microbes pre-formed organic molecules as - Sulfur carbon sources - Needed for synthesis of amino acids (cysteine, methionine), some carbohydrates (biotin, thiamine) - Some microbes require reduced Temperature Requirements form of sulfur such as cysteine - Most important factor that can affect growth of microorganisms Growth Factors - Temperature range - must not be - Used by microbes that lack one or above the maximum or below the more enzymes needed for the cell minimum as it can affect growth components or precursors of the - Optimum - growth occurs mentioned components best (ex: pathogenic Major Classes: bacteria, -37 degrees C) - Amino acids Temperature classes (in degrees C): - for protein synthesis - Psychrophiles - Purines & Pyrimidines - 15 or lower - For nucleic acid synthesis - max: -10 - Vitamins - Psychrotrophs / Facultative - Small organic molecules Psychrophiles that make up all or part of - 20 - 30 enzymes cofactors, only - max: 35 very small amounts sustain - Mesophiles growth - 25 - 40 - min: 15-20, Growth Requirements max: 45 Gaseous Requirements - Thermophiles - Aerobic Bacteria - 55 - 60 - require oxygen for growth - min: 45 - Obligate aerobes to grow only in the presence of oxygen Other Requirements: (Pseudomonas aeruginosa) - Moisture and drying - Anaerobic Bacteria - Hydrogen ion concentration - Grow in absence of oxygen - Light (Clostridium tetani) - Osmotic effect - Obligate anaerobes - may - Mechanical and sonic stress die on exposure to oxygen - Facultative anaerobes - Growth Media can grow in presence of Culture Media oxygen & survive without - Nutrient solutions tailored to the oxygen via anaerobic particular organism to be grown respiration or fermentation - It must be sterilized before use thru - Microaerophilic Bacteria heating of medium under pressure - Proliferate (increase in #) in the in an autoclave presence of low oxygen tension - 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 - Meat extract - aqueous digests of microbial, animal, plant extracts from lean beef and products like milk protein (casein), contain amino acids, beef extract, soybeans (tryptic soy peptides, nucleotides, broth), yeast cells (yeast extract) organic acids, minerals, vitamins Based on Consistency - Yeast extract - from - Solid medium brewer’s yeast and contain - Contains 2% agar an excellent source of vit B, - Used for colony morphology, nitrogen, carbon pigmentation, hemolysis compounds - Ex: nutrient agar, blood agar Based on Functional Use / Application - Liquid medium 1. Enriched medium - No agar - Broth or solid medium containing a - Used for inoculum preparation, rich supply of special nutrients blood culture, isolation of - Used for fastidious microbes pathogens from a mixture - Ex: blood agar, chocolate agar - Becomes cloudy as indication of 2. Indicator media growth - Contain an indicator which - Ex: nutrient broth changes color when a bacterium grows in them - Semi solid medium - Ex: Christensen’s urease medium - For checking motility 3. Transport media - Used for transporting the samples Based on Nutritional Component - Delicate organisms may not 1. Simple media survive the time taken for - Aka basal media transporting specimen w/o it - Used for non-fastidious (not having - Ex: buffered glycerol saline complex nutritional requirements) 4. Anaerobic media bacteria - Used to grow anaerobic organisms - Ex: nutrient agar and broth - Ex: Robertson’s cooked meat 2. Defined media medium - All the ingredients are known 5. Differential medium (quali and quanti) - One to which an indicator (typically 3. Complex media a dye) is added, which reveals by a - Contain undefined ingredients and color change whether a particular the exact contents are known metabolic reaction has occurred - Ex: nutrient broth, tryptic soya during growth broth, MacConkey agar (for - Ex: MacConkey agar (for lactose Lactobacillus) fermentation) - May contain undefined 6. Selective media components: - Contain compounds that inhibit the - Peptones - prepared by growth of some microbes but not partial proteolytic digestion others of meat, casein, soyameal, - Ex: Eosin Methylene Blue (EMB) - gelatin, and other protein contains dyes that are not for gram sources positive bacteria and allows growth  of gram negative bacilli and enteric Laboratory Culture of bacilli - coliforms and faccal Microorganisms coliforms 1. Agar - Used to solidify the culture media Isolation of Pure Cultures - An algal polysaccharide first used - A pure culture is needed to in the classical studies of Robert characterize and identify an Koch individual species - Solid media immobilize cells so - Culture methods include: that as they grow, they accumulate 1. Stab culture in a pile to form visible, isolated - Prepared by puncturing a suitable cell masses called colonies medium - with a long, straight wire 2. Colony morphology - Used to study oxygen - Visible characteristics of a colony requirements - Used to identify microbes but is 2. Pour plate method routinely used to determine if - Gives an estimate of the viable culture is: bacterial count in a suspension - Pure - only one microbe - Used for quantitative urine cultures - Contaminated - undesired - Should be avoided when trying to organisms co-occur determine viable cell counts of - Mixed - many microbes heat-sensitive bacteria isolate present 3. Liquid culture 3. Aseptic technique - Inoculated by touching with a - Series of steps by which microbes charged loop or by adding are transferred between growth inoculum with pipettes or syringes media without contamination - 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 - Contamination can be introduced - Can be used for bacteriophage from microbes in the air, in liquid typing, antibiotic sensitivity testing, droplets, or on surfaces preparation of bacterial antigens - Streak plate technique - method and vaccines for obtaining pure cultures that - Prepared by flooding the surface of contain a single microbe, and of the plate with a liquid suspension verifying culture purity using of the bacterium inoculating loop 7. Stroke culture - Made in tubes containing agar Microbial Growth slope or slant provide a pure Growth growth of bacterium for slide - Increase in number of cells (binary agglutination and other diagnostic fission) tests - Cell division following enlargement of a cell to twice its minimum size  - During cell division, each daughter N - final cell number cell receives a chromosomes and No - initial cell number sufficient copies of all other cell N - number of generations during the constituents to exist as an period of exponential growth 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 Synchronous Growth cell number, thus growth rate=0 - Microbiological culture or a cell - Cell prepares for maintenance and culture that contains cells that are survival all in the same growth stage 4. Death phase - Information about growth - Exponential decrease in number of behaviour of individual bacteria living cells can be obtained by synchronous - Decline phase - total number of cultures cells decreases due to cell death - Synchronized cultures must be composed of cells which are all at Generation time = time per generation/ the same stage of the bacterial cell number of generations cycle (aka doubling time) - time required for cell to separate 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 A. Direct Microscopic Count container such as a test tube or a - A counting chamber consisting of a flask) ruled slide and a coverslip is - Since the nutrients are not employed renewed, exponential growth is - Constructed in a manner that the limited to a few generations coverslip, slide, and ruled lines - Chemostat - most common type of delimit a known value continuous culture device - The number of bacteria in a small - An open system aiming to attain known volume is directly counted steady state microscopically - The number of bacteria in the Diauxic Growth larger original sample is - Used to describe the growth determined by extrapolation phases of a microbes in batch culture as it metabolizes a mixture B. Electronic Enumeration of Cells of 2 sugars - The microbial suspension is forced - Resulting in 2 separate growth through a small hole or orifice in phases 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 Methods for Measurement of Cell Mass culture is pipetted into an empty Microscopic cell count - total count of sterile Petri plate. Molten agar microbial numbers can be observed and medium, tempered to just above enumerated here gelling temperature (50°C), is then Viable cell - one that is alive and able to added and gently mixed before grow allowing the agar to solidify. Viable count - performed by spreading microbes on solid media and counting D. Membrane Filter Technique colonies, aka plate count (requiring agar - The number of bacterial in aquatic plates) sample can be determined from Spread-plate & Pour-plate - 2 ways in direct counts after the bacteria performing plate count have been trapped on special Colony-forming units (CFU) - expressed membrane filters such as as the number data from viable counts 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