SIR1001 Fundamental Microbiology 2024-2025 PDF

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University of Malakand

A/P Dr Bong Chui Wei, A/P Dr Lee Choon Weng, Dr Muhamad Afiq Bin Aziz

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microbiology fundamental concepts microorganism biology

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This document outlines a Fundamental Microbiology course, covering the introduction to microbiology concepts, techniques, evolution, structure and function of microorganisms. The course is taught at the University of Malaya.

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SIR1001 Fundamental Microbiology By: A/P Dr Bong Chui Wei [email protected] A/P Dr Lee Choon Weng [email protected] Dr Muhamad Afiq Bin Aziz [email protected] Synopsis of course Introduction...

SIR1001 Fundamental Microbiology By: A/P Dr Bong Chui Wei [email protected] A/P Dr Lee Choon Weng [email protected] Dr Muhamad Afiq Bin Aziz [email protected] Synopsis of course Introduction to concepts and basic techniques of microbiology, evolution, structure and function of prokaryotic, dissemination, control and identification of microorganisms. Exposure to aseptic laboratory techniques for the transfer, isolation and stain of microorganisms. Course outcomes 1.Distinguish various types of microorganisms. 2.Understand the core concepts of microbiology, including the evolution and diversity of microbes; cell structure and function; growth and metabolism. 3.Demonstrate basic practical microbiology skills. Lecture schedule (Wed => 10-11:50 am) Practical (Wed => 2-5 pm, M1 & M2) Week Topic 1 Foundation of Microbiology 2 Microbial evolution 3 Characteristics of Prokaryotes and Eukaryotes Prokaryotic Structure and Function 4 Microbial Transport Systems Microbial Systems Biology 5 Microbial Growth & Reproduction Nutrition and culture 6 Control of microbial growth Basic techniques in Microbiology 7 Techniques for Transfer, Isolation, Culture and Storage of Microorganisms Differential Staining 8 Enumeration of Microorganism 9 Biochemical Tests for Bacterial Identification 10 Overview of the Immune System I Innate and adaptive immunity 11 History of Virology Basic characteristics of viruses/ Comparison of virus and Eukaryotes 12 Diversity and classification of virus Lytic cycle/ Lysogenic cycle (1)/ (2) 13 Microbe-Human Interaction Presentation and Discussion 14 Evaluation structure Importance date 50%Final examination Test 1 10% Test (calculation) Test 2 10% Test (theoretical) 5% Lab report Lab report submission 25% Dr Afiq's part Reference Where to get lecture notes? Talaro, KP. Foundations in Microbiology: Basic Principles, 10th ed, 2018, McGraw-Hill. http://spectrum.um.edu.my/ Ted R. Johnson & Christine L. Case. Laboratory Experiments in Microbiology, Why should I visit the website? 12th ed, Pearson Education Lecture notes Madigan M.T., Martinko J.M., Bender K.S., Practical manual Buckely D.H., Stahl D.A. & Brock T. Brock Obtain and submit online assignments Biology of Microorganisms, 15th ed, 2019, Benjamin Cummings. Online discussion Important announcement Class agreement Contact 1. Be punctual- make sure you scan QR code attendance for classroom and on time for all classes Name A/P Dr Lee Choon Weng 2. Mute yourself during the online classes Room 2.1.4, Blok C, Bangunan Genetik dan 3. Must secure a minimum of 80% attendance to Mikrobiologi secure the eligibility to sit for the final examination E-mail: [email protected] 4. If your absence is due to illness, please provide a medical certificate Ext : 5841 5. 6. What is Microbiology? Microscopic organisms Gems Bacteria Micro biology Archaea Study of living things Viruses Small Fungi Bugs Protozoa The study of all living organisms that are Algae Microbes too small to be visible with the naked eye Microorganisms Their importance and functions Nutrient & energy Production of foods, flow drugs, vaccines Infectious disease Natural processes Potential uses Pathogens Decomposition Bioremediation Microbiology One of the largest & most complex of biological sciences Every aspect of microbes Genetics Physiology Characteristics Interactions Uses Environmental Bacteriology Protozoology Helminthology Phycology microbiology Mycology Virology Parasitology Immunology Molecular biology Diverse disciplines Microbiology Main themes of research Basic/Pure Exploratory & conduct for Apply existing scientific knowledge better understand of basic life of microorganism to develop more processes, fundamental cellular practical applications processes, microbial ecology in Microbial taxonomy deeper theoretical level Bacteriology Phycology Mycology Virology Microbial Parasitology Metabolism Immunology Food & beverage technology Protozoology Genetics Epidemiology Infection control Environmental Pharmaceutical microbiology Ecology Etiology Chemotherapy microbiology Genetic engineering Relation to Organisms Process Disease related Environmental Industrial disease Origins of microorganisms First fossils bacteria like cells existed for at least 3.5 billions years Prokaryotes Characteristics of ancient cells: Simple cell Small Genetic material not bound into Simple a separate compartment called nucleous/karyon Lack specialized internal structure Term assigned>>> for cell functions prokaryotic meaning “before the nucleus” ~1.8 billions years Eukaryotes appeared in fossil record Complex cell Nucleus Organelles Term assigned>>> eukaryotic in reference to their “true” nucleus The Origin & Evolution of Microorganisms Phylogeny Morphological, biochemical, and gene sequence data à all organisms are genetically Relationship between related & genealogical relationships of living all the organisms on things can be represented by a vast Earth that have evolutionary tree descended from a common ancestor, The Tree of Life= the phylogeny of organisms whether they are (the history of organismal lineages as they change through time) extinct or extant Branching pattern of ancestor–descendant Evolution through the ages from ancestral relationships among forms into more derived forms. New lineages generally retain many of their ancestral ‘taxa’ (e.g., species or features, which are then gradually modified their genes) and supplemented with novel traits that help them to survive & thrive Studying phylogeny can help explain similarities Phylogenetic knowledge and differences among macro and micro- organisms relationship The Tree of Life provides a rigorous framework to guide research in all biological subdisciplines, and it is therefore an ideal model for the organization of biological knowledge System of Taxonomy Traditional Whittaker System of Classification Evolutionary model of phylogeny based on: differences in the sequences of nucleotides in the cell’s ribosomal RNAs (rRNA), cell’s membrane lipid structure & its sensitivity to antibiotics Common characteristics of cell What is unicellular? Consisting only single Shapes C ytoplasm cell Aqueous mixture of macromolecules (e.g. proteins, Cubical/spherical/cylindrical Cell lipids, nucleic acids & polysaccharides), maintain turgidity Ribosomes Chromosomes What is multicellular? Protein synthesis/translation Consisting of One or more chromosomes containing DNA numerous cell Cytoplasmic membrane Cell wall* Encases an internal matrix (cytoplasm) from outside Basic unit of Outer layer surrounding cell that located outside the cell membrane Selective permeability Structural & functional of life Provide strength & structural strength to a cell biological activity Prokaryotic cells Eukaryotic cells Few internal structures Animals, plants, fungi & protists Lack nucleus & organelles Contain membrane enclosed organelles that perform useful functions (metabolisms, nutrition, synthesis) Nucleus – most visible organelle Characteristic Prokaryotic Eukaryotic Cell size 0.2-2.0 µm in diameter 10-100 µm in diameter Nucleus No nuclear membrane/nucleoli True nucleus, consisting of nuclear membrane and nucleoli Membrane enclosed organelles Absent Present (e.g: lysosomes, golgi complex, endoplasmic reticulum, mitochondria, chloroplasts) Flagella Consist of two protein building blocks Complex; consist of multiple microtubules Cell type Usually unicellular (some cyanobacteria maybe Usually multicellular multicellular) Cell wall Usually present, chemically complex When present, chemically simple (typical bacterial cell wall includes peptidoglycan) Plasma membrane No carbohydrates and generally lacks sterols Sterols and carbohydrates that serve as receptors present Cytoplasm No cytoskeleton/cytoplasmic streaming Cytoskeleton; cytoplasmic streaming Ribosomes (Large subunit) (Small 70S (50S + 30S) (23S + 5S rRNA) (16S rRNA) 80S (60S + 40S) (25S/28S + 5S + 5.8S rRNA) (18S rRNA) subunit) Chromosome (DNA) Single circular chromosome; lacks histones Multiple linear chromosomes with histones arrangement Cell division Binary fission Mitosis Sexual reproduction No meiosis; transfer of DNA fragments only Involves meiosis Example Bacteria & Archaea Animal, Plant, Fungi & Protist cell Microbial dimension Energy & Nutrient flow Macroscopic Usually in cm & m Range of µm, nm, mm Majority free existence Relatively harmless often beneficial Close associations with other organisms (Parasites, hosts) Lived & evolved for billions of years………. Microscopic Roles??? constitute a major fraction of global biomass (2 x 1030 microbial cells) & key reservoirs of nutrients essential for life Vital components of structure & function of ecosystems Flow of energy & food through the earth’s ecosystems Photosynthesis Atomic Ultramicroscopic Phototrophs : light fueled conversion of CO2 to O2 & organic materials Photosynthetic microorganisms contributing majority O2 to the atmosphere (>50% earth’s photosythesis ) Decomposition & nutrient cycling Breakdown of dead matter into simple compounds & direct back into natural cycles Microorganisms>>main forces that drive the structure & content of soil, water, & atmosphere Bacteria Fungi Algae Unicellular microorganisms Eukaryotic cell Three major shapes: Unicellular/multicellular Eukaryotic cell Lack defined structure, no roots, o Bacillus (rod like) Heterotrophic stems/leaves o Coccus (spherical/ovoid) Unicellular/multicellular/ dimorphic Photosynthetic organisms (O2 & CHO) o Spiral (corkscrew/curved) Thick cell wall (Chitin & glucan) some chemoheterotrophic, others Mostly have peptidoglycan cell wall Mostly produce spores (haploid, mitosis) saprobes & parasites Binary fission Growth as hyphae May possess flagella Reproduction in vegetative No chlorophyll (fragmentation) and both asexual (spore Use wide range of chemical substances Decomposer known as saprotrophs formation, binary fission) & sexual forms for nutrition (fusion) Free living some symbiotic Archaea Protozoa Virus Infectious agent Prokaryotic cell (bacteriophages, Common freshwater plankton mycophages, Single celled organisms- cell bounded by a single Eukaryotic microorganisms virophages) lipid membrane Microscopic unicellular & microscopic Reproduce in living hosts Lack nucleus & organelles Complex internal structure (obligate intracellular Cell wall lack of peptidoglycan Variety of shapes (2-200 µm) parasites) Reproduce asexually (fission/budding) Mostly free living or parasites Acellular (no cytoplasm/cellular organelles) Including: Motile o Methanogens Binary fission (most common), sexual No metabolism using host cell’s metabolic o Halophiles & asexual reproduction machinery o Extreme thermophile Nucleus enclosed with in membrane Structurally simple, a core made of DNA/RNA & Mostly chemoautotrophs (grow on simple Obtain nourishment by absorption or surrounding by protein coat (encased by inorganic chemical), others ingestion envelope) heterotrophs Cannot growth in synthetic culture media Historical Foundations of Microbiology From the very earliest, humans noticed that when certain food spoiled Spoil food Prominent discoveries in Inedible Enhance the past 300 years…… flavor Microbiology techniques Development of medical microbe Human sickness/ disease Black plague Scientific method Smallpox Microscopy Unclear of transmission source & path Spontaneous generation some forms of life could arise from vital forces present in non living/decomposition matter Abiogenesis Simply organisms can arise from non living matter Redi experiment (1665) Jablot experiment (1710) Needham experiment (1745) Francesco Redi, Italy Louis Jablot, French John Needham, England Test on spontaneous generation theory Microbes are present in dust particles Supports spontaneous generation of organisms cells cells Both heated & unheated test containers teemed with microbes Maggots are not spontaneously Only the open vessel developed life had been created from nonlife produced in rotten meat microorganisms “life is necessary to produce life” Pasteur experiment (1859) Disproved spontaneous generation of microbes Louis Pasteur, France microorganisms are everywhere - even in the air Test whether sterile nutrient broth could spontaneously generate microbial life Microbes caused fermentation & spoilage True awareness of widespread distribution of microorganisms and their characteristics was made possible by the development of first microscopes Antonie van Leeuwenhoek Robert Hooke (1632-1723) (1655) first to use a microscope to Dutch microscopist, first observer observe microbes/living things for bacteria & protozoa first used the term “cells” (1665) to Air and dust were the source of microbes Refuted the doctrine of describe the small chambers spontaneous generation within cork Lay foundations for the sciences Laid the groundwork for of bacteriology and protozoology development of “cell theory” Information How do scientists apply scientific method??? Deductive approach taken by scientists to explain Developing a hypothesis a certain natural phenomenon based on existing theory and then designing a General observations of phenomenon to develop research strategy to test explanatory a set of facts to explain the hypothesis Form a hypothesis-a tentative explanation that can be supported/refuted (scientific thought) A valid hypothesis will allow for experimentation & testing & can be shown to be false A lengthy process of experimentation analysis, and testing eventually leads to conclusions predictive >>>support/refute hypothesis Should not be immediately accepted Test & retest Discard/modify to require fit the results of reworking experiment additional tests Does not mean results are invalid To predict what is expected to happen.....under known condition Results must be published & repeated by other investigators If hypothesis is supported by evidence & survives rigorous scrutiny, it moves to next level of confidence-it becomes a theory Theory is a collection of statements. Technologies propositions/concepts that explains/accounts for a natural event Is not the result of a single experiment repeated over & over again but is an entire body of ideas that expresses/interprets many aspects of a phenomenon If evidence of a theory is so compelling that next level of confidence is reached, it becomes a Law/principle Edward Jenner and the introduction of smallpox vaccine Early experiments on the sources of microorganism led to the profound realization that MICROBES are EVERYWHERE Edward Jenner (1749-1823) Father of Immunology Pioneer of vaccination 1796:Inoculation with cowpox gave immunity to smallpox, was an immerse medical breakthrough & saved countless lives 1967: World Health Organization (WHO) began a global vaccination program 1980: The disease was officially declared eradicated Discovery of Spores & Sterilization Development of Aseptic Techniques John Tyndall The human body is a source of infection (1820-1893) Dr. Oliver Wendell Holmes Sr (1809 - 1894) Mothers of home births had fewer Irish scientist infections (puerperal fever) than those who Discovered some bacteria existed in gave birth in hospitals (US) o Heat sensitive form (vegetative cell) o Heat stable form (endospore) Need prolonged/intermittent heating to Dr. Ignaz Semmelweis (1818 – 1865) destroy correlated infections with physicians Resulted a method of sterilizing liquid by coming directly from autopsy room to heating it to boiling point (tyndallization) on maternity ward successive days Ferdinand Cohn Joseph Lister (1827 – 1912) (1828-1898) British surgeon and scientist German botanist introduced aseptic techniques reducing Discovered heat resistant forms of microbes in medical settings and preventing bacteria (Endospore) Bacillus & Clostridium wound infections spp. Sterilization technique requires the Involved disinfection of hands using elimination of all lifeforms including chemicals prior to surgery endospores & virus Laid ground work for bacterial classification Use of heat for sterilization Discovery of Pathogens & Germ Theory of Disease Many diseases are caused by the growth of microbes in the body and not by sins, bad character, or poverty, etc. Two major contributors: Louis Pasteur Robert Koch Robert Koch (1843-1910) Founder of Bacteriology German scientist Demonstrated role of bacteria in causing disease Established Koch’s postulates :a sequence of experimental steps that verified the germ theory Identified cause of anthrax, TB, and cholera Developed pure culture methods Pure cultures and Microbial Taxonomy Pure culture “Suspected pathogen must be isolated and grown away from other microorganisms in laboratory culture” Walther Hesse & Koch When a solid surface incubated in air, masses of microbial cells developed, each having a characteristic shape and colour. Solid media provided an easy way to obtain pure cultures. Richard Petri Developed the transparent double-sided “Petri dish” in 1887, standard tool for obtaining pure cultures Taxonomy Levels of classification Taxonomy: organizing, classifying, and naming living Domain - Archaea, Bacteria, & Eukarya things Kingdom o Formal system originated by Carl von Linné Phylum or Division Class Concerned with: Order o Classification: orderly arrangement of organisms into Family groups (taxa) on the basis of similarities or Genus relationships Species -May applied to existing named taxa or newly described taxa o Nomenclature: assigning names to taxonomic groups Assigning Scientific Names o Identification: determining and recording traits of Binomial (scientific) nomenclature organisms for placement into taxonomic schemes Require knowledge of their morphologic, biochemical, Gives each microbe 2 names: physiological, and genetic characteristics o Genus - capitalized o species - lowercase Importance of microbial taxonomy v Allows scientists to organize huge amounts of knowledge Both italicized or underlined v Allows scientists to make predictions and frame hypotheses o Staphylococcus aureus (S. aureus) about organisms v Places organisms in meaningful, useful groups with precise names, thus facilitating scientific communication Inspiration for names is extremely varied and v Essential for accurate identification of microorganisms often imaginative Cell shape Cell morphology Ø Spherical/ovoid- coccus (cocci) Ø Cylindrically shaped- rod/bacillus Ø Curved/loose spiral shapes – spirilla Ø Long, thin cells/chains of cells-filamentous Ø Tightly coiled/ extensions of cells as long tubes/stalks-spirochete Some Bacteria & Archaea remain together in groups/clusters after cell division Cocci ----------------------------------------------------------------------------------------------------------------- Can be oval/elongated/fattened on one side Diplococci: A pair of attached cocci. Remain attached after dividing some cocci form long chains (Streptococcus) Tetrads: Groups of four. Divide in two planes Three-planes & attached in cubelike groups of eight (Sarcinae) Multiple planes & form grapelike clusters/ broad sheets (Staphylococcus) - Bacilli ------------------------------------------------------------------------------------------------------------------------------------ Varied rod shapes (blocky, spindle shaped, round ended, long & threadlike (filamentous), clubbed/ drumstick shapes) Diplobacilli: A pair of attached bacilli. Remain attached after dividing Streptobacilli: Chain like arrangement Coccobacillus: Intermediate shape between coccus & bacillus. Oval rods Have one or more twists-- Vibrio: comma shaped cell. Look like curved rods Spiral/curvi form shaped bacteria ----------------------------------------------------------------------------------------------------------------------------------- Spirilla: Helical, corkscrew shaped bacteria with rigid bodies, use whip like external flagella to move Spirochetes: Helical bacteria with flexible bodies, use axial filaments (internal flagella) to move Halophilic archaean, Haloquadratum walsbyi, discovered in 1980 by AE Walsby, in a coastal hypersaline pool on the Sinai Peninsula in Egypt. Cultured only in 2004. Thin (0.15 um) square-shaped structure. Cell size Cells of Bacteria & Archaea vary in size (0.2 μm - >700 μm) -rod-shaped species 0.5 & 4 μm wide & < 15 μm long -Epulopiscium fishelsoni (0.6 mm in length) -Thiomargarita namibiensis (750 µm in diameter) Small cells - more surface area relative to cell volume than large cells >>higher surface-to-volume (s/v) ratio -higher S/V ratio of small cells: faster rate of nutrient & waste exchange per unit of cell volume - Free-living smaller cells tend to grow faster than larger cells & for a given amount of resources (nutrients for growth) Prokaryotic cells grow faster & evolve more rapidly than eukaryotic cells Cell membrane Fatty acids point inward toward each other to cytoplasmic membrane (plasma membrane/inner membrane) form hydrophobic region Thin structure lying inside cell wall & enclosing cytoplasm of cell Hydrophilic portion exposed to either “gatekeeper”- for entrance and exit of dissolved substances environment/cytoplasm & interacts with 8–10 nm wide, physically weak [prokaryotes by phospholipids & protein, some cytoplasmic milieu bacteria strengthened by hopanoids, eukaryotes by carbohydrates & sterols This membrane structure is called a lipid (cholesterol)] bilayer/a unit membrane because each Phospholipid [both hydrophobic (water-repelling) & hydrophilic (water attracting) phospholipid “leaf” forms half of the unit components] bilayer containing embedded proteins Bacteria and Eukarya: hydrophobic component-fatty acids & hydrophilic component Proteins embedded in the membrane >>integral membrane proteins of a glycerol molecule containing phosphate & one of several other functional groups (e.g. sugars, ethanolamine/choline) bonded to the phosphate Peripheral membrane proteins are more Archaea: Constructed from either phosphoglycerol diethers [C20 side chains (phytanyl loosely attached group)/ or diphosphoglycerol tetraethers (C40 side chains (biphytanyl group)] Three major functions: Ø cell’s permeability barrier, preventing the passive leakage of solutes into or out of the cell Ø cytoplasmic membrane anchors several proteins that catalyze a suite of key cell functions Ø cytoplasmic membrane of Bacteria and Archaea plays a major role in energy conservation and consumption Peripheral membrane proteins are loosely attached & some are lipoproteins [proteins that contain a hydrophobic lipid tail that anchors the protein into the membrane] Peripheral membrane proteins typically interact with integral membrane proteins that involved in important cellular processes (e.g. energy metabolism and transport) Other functions: Passive Processes Ø Breakdown of nutrients & production of energy Ø Synthesis of cell wall components Simple diffusion Ø Assists with DNA replication Net movement of molecules/ions from high Ø Site of photosynthesis: Photosynthetic bacteria have membrane concentration>>>low concentration. extensions called thylakoids, where photosynthesis occurs. Equilibrium: Net movement stops when molecules are evenly Ø Secretes proteins distributed Ø Contains bases of flagella Used by cells to transport small molecules (O2, CO2) across Ø Responds to chemical substances in the environment their membranes Impermeable Facilitated diffusion large proteins, ions, and most polar molecules–larger than pores in integral Net movement of molecules/ ions from high concentration to proteins that function as channels low concentration Permeable Substance to be transported combines with a carrier protein smaller molecules (water, O2, CO2, simple sugar) easily pass through in plasma membrane Substances that dissolve easily in lipids (O2, CO2, non-polar organic Extracellular enzymes may be used to break down large molecules) enter & exit easily than other substances>>>membrane consist substances before they can be moved into the cell by mostly phospholipids facilitated diffusion Movement of Materials Across Membranes Passive Active Substances cross Move substances membrane from from low high concentration concentration>>low >>high concentration concentration Move with Use energy (ATP) Concentration gradient/different Without any expenditure of energy (ATP) Osmosis Net movement of water (solvent) molecules across a semipermeable membrane from high Active Processes concentration to low concentration Active Transport Osmotic Pressure: Pressure required to prevent the movement of pure water into a solution Requires carrier proteins or pumps in plasma membrane Bacterial cells can be subjected to three different types of osmotic solutions: Isotonic (equal) Ø Concentration of solutes equals that found inside a cell Group Translocation Similar to active transport, but substance transported is chemically altered during Ø Waters leaves & enters cell at the same rate (no net change) Ø Cell’s contents are in equilibrium with solution outside cell wall process After modification, the substance remains inside cell Important for cells to accumulate various substances even though they in low Hypotonic (hypoosmotic, under/less) concentrations outside the cell Ø Concentration of solutes outside the cell is lower than that inside the cell Require energy supplied by high energy phosphate compounds Ø Net movement of water into the cell Ø Most bacteria live in hypotonic environments, swelling is contained by cell wall, G- [phosphoenolpyruvic acid (PEP)] may burst/osmotic lysis>>excessive water intake E.g. glucose is phosphorylated during group translocation in bacterial cells. Endocytosis (phagocytosis, pinocytosis, etc.) does not occur in procaryotic cells Hypertonic (above/more) Ø Solute concentration is higher outside the cell Ø Net movement of water out of the cell Ø Most bacterial cells shrink & collapse/plasmolyze >>water leaves the cells by osmosis Cell wall Semirigid structure that lies outside the cell membrane in almost all bacteria confers structural strength on the cell to keep it from bursting due to osmotic pressure Negative Positive N-acetylmuramic acid (NAM) N-acetylglucosamine (NAG) Cell wall thin (8-12 nm) , wavy Cell wall thick (20-80 nm), smooth Two layer lipid membrane One layer lipid membrane With outer membrane (lipopolysaccharide, LPS) No outer membrane Periplasmic space present in all Periplasmic space present in some Peptidoglycan, lipopolysaccharide, lipoproteins Peptidoglycan, teichoic acid & lipotechoic acid Porins proteins, more lipid No porins proteins, less lipid Less peptidoglycan & less penetrable More peptidoglycan & penetrable More resistance to molecules Less resistance to molecules Gram reaction: pink/red Gram reaction: blue/purple LPS has two components: Archaea O polysaccharides: Antigens, used to identify Cell walls containing: bacteria Polysaccharides Lipid A: Endotoxin causes fever and shock Proteins/glycoproteins or Some mixture of these macromolecules Porins: Membrane proteins that allow the passage of nucleotides, disaccharides, peptides, amino acids Certain methane-producing Archaea (methanogens) contain Gram Stain Mechanism pseudomurein (similar to peptidoglycan) Based on differences in structure of cell wall (G+ & G-) Archaea lack pseudomurein contain other polysaccharides & reaction to various reagents Methanosarcina sp. have thick polysaccharide walls composed of Crystal violet (primary stain) polymers of glucose, glucuronic acid, galactosamine uronic acid, and Dye enters cytoplasm acetate Iodine (mordant) Extremely halophilic (salt-loving) Archaea e.g. Halococcus, contain Forms large crystals with dye that difficult to escape large amounts of sulfate in cell wall through cell wall Alcohol G+:Dehydrate peptidoglycan>> make it more Paracrystalline surface layer (S-layer) S-consist of interlocking impermeable to crystal violet iodine molecules of protein/glycoprotein G-: colourless, dissolve outer membrane & leaves small -Methanocaldococcus jannaschii holes in thin peptidoglycan layer through crystal violet iodine diffuse Functions: Safranin (counterstain) Ø serving as protection from osmotic lysis G->>pink Ø as the interface between the cell and its environment (selective sieve) allowing the passage of low-molecular-weight solutes, *G+ cells----->G- response>>>>cells dead excluding large molecules/structures (viruses/lytic enzymes) Bacillus, Clostridium, Mycobacterium-gram variable Ø Retain proteins near the cell surface that must function outside the cytoplasmic membrane Atypical cell walls No walls/very little wall material Mycoplasmas No cell wall, smallest known bacteria that can grow and reproduce outside of host cells Pass through most bacterial filters Unique plasma membrane contains lipids (sterols) >>protect them from osmotic lysis Mycoplasma pneumoniae is the cause of primary atypical bacterial pneumonia (walking pneumonia) Archaebacteria May lack cell walls/have cell walls without peptidoglycan Composed of protein, polysaccharides/peptidoglycan-like molecules, but never do they contain murein (pseudomurein) * Unique feature distinguishes the bacteria from the Archaea Cytoplasm Substance of cell inside plasma membrane Prokaryotes: major structures Thick, aqueous, semitransparent, elastic Ø Nuclear area (containing DNA) Contains: Ø Ribosomes Ø 80% water Ø Inclusions Ø Proteins Prokaryotic lacks certain features of eukaryotic cytoplasm Ø Carbohydrates Ø Cytoskeleton Ø Lipids Ø Cytoplasmic streaming Ø Inorganic ions (much higher concentration) Ø Low molecular weight compounds Ribosomes The Nuclear Area (nucleoid) Site of protein synthesis (translation) Contains single chromosome, a long circular molecule of Present in all eucaryotic and procaryotic cells. double stranded DNA (bacterial chromosome- not Made up of protein and ribosomal RNA (rRNA). surrounded by nuclear envelope [membrane] & not include Procaryotic ribosomes (70S) are smaller and less dense than histones) eucaryotic ribosomes (80S) Can be spherical/elongated/dumbbell shaped Ø Prokaryotic:small subunit (30S) & large subunit (50S) Chromosome is attached to the plasma membrane, protein Ø Eukaryotic:small subunit (40S) & large subunit (60S) in plasma membrane>>replication of DNA &segregation of S is Svedberg units indicate relative rate of sedimentation new chromosome to daughter cells in cell division during ultra high speed centrifugation [sedimentation rate: Occupy 20% of the intracellular volume of active cells function of size, weight & shape of a particle] Several antibiotics (streptomycin, gentamycin, erythromycin, chloramphenicol) work by inhibiting protein synthesis by Plasmids procaryotic ribosomes, without affecting eukaryotic ribosomes Small, circular, double stranded DNA molecules Extrachromosomal genetic elements that not connected to bacterial chromosome, replicate independently of Inclusions Reserve deposits in the cytoplasm of cells chromosomal DNA Some are common in bacteria, others are limited (serve as a Associated with plasma membrane proteins basis for identification) Contain from 5 -100 genes that are usually not essential for survival of bacterium under normal environment conditions Metachromatic Granules (volutin) Found in many bacterial cells in addition to chromosomal Large inclusions Stain red with blue dyes (e.g. methylene blue) DNA Contain inorganic phosphate (polyphosphate) that can be used Carried genes for: Ø Antibiotic resistance in the synthesis of ATP Ø Tolerance to toxic metals Found in bacteria, algae, protozoa, and fungi Characteristic of Corynebacterium diphtheriae, causative agent Ø Production of toxins of diphtheria Ø Synthesis of enzymes Transferable, plasmid DNA is used for gene manipulation in Useful for identification purposes biotechnology Polysaccharide Granules Magnetosomes Consist of glycogen & starch Contain iron oxide (Fe3O4 ), which acts like a magnet Carbon & energy reserves Formed by several G- bacteria to move downward until reach a suitable Glycogen granules stain reddish brown & starch granules attachment site appear blue with iodine May protect cells against hydrogen peroxide accumulation Magnetite from bacteria can be used in the production of magnetic tapes Lipid inclusions for sound & data recording Common lipid storage material Polymer-β-hydroxybutyric acid-unique to bacteria Revealed by staining cells with fat soluble dyes (Sudan dyes) E.g. Mycobacteria, Bacillus, Azotobacter, Spirillum Sulfur Granules Contain sulfur and sulfur containing compounds. “Sulfur bacteria” (Thiobacillus) obtain energy by oxidizing sulfur and its compounds Carboxysomes Contain enzyme ribulose 1,5-diphosphate carboxylase, necessary for carbon fixation during photosynthesis Found in nitrifying bacteria, cyanobacteria, and thiobacilli Gas vacuoles Hollow cavities found in many aquatic bacteria. Each vacuole consists of row of several individual gas vesicles that are hollow cylinders covered by protein Used to regulate buoyancy so cells can remain at appropriate water depth>>> to receive sufficient O2, light, nutrients Endospores “resting” cells formed by certain G+ bacteria Ø Bacillus, Anoxybacillus, Paenibacillus, Clostridium Ø Sporomusa ovata, Coxiella spp., Acetonema spp. (G-ve) Highly durable dehydrated cells with thick cell walls and additional layers Formed internal to bacterial cell membranes Can survive extreme temperatures, disinfectants, acids, bases, lack of water, toxic chemicals, and radiation Sporulation/sporogenesis: Process of endospore formation within a vegetative (parent) cell take several hours Important for survival during adverse environmental conditions Ø Type of dormant cell, intracellular structures Ø Formed by vegetative cells in response ~ limiting factor Ø Highly resistant to environmental stresses The endospore might be located terminally (at the end), subterminally (near one end), centrally inside the vegetative cell The water present in forespore cytoplasm is eliminated by the time sporulation is complete & endospore do not carry out metabolic reactions Contains only DNA, small amount of RNA, ribosomes, enzymes, few important small molecules, dipicolinic acid, calcium ions Retain viability under appropriate environmental condition Germination: The return of an endospore to its vegetative state, triggered by physical/chemical damage to the endospore’s coat Important in clinical & food industry because they are resistant to processes that kill vegetative cells (heating, freezing, desiccation, chemicals, radiation) Can survive in boiling water for several hours or more, thermophilic bacteria surviving in boiling water for 19 hours Endospores One cell produces one spore 1. Spore septum: Newly replicated DNA is isolated by an ingrowth of the plasma membrane 2. Spore septum becomes a double-layered membrane that surrounds chromosome and cytoplasm (forespore) 3. Thick layers of peptidoglycan are laid down between the two membrane layers of forespore 4. Spore coat forms: Thick layer of protein around the outer membrane. This coat makes endospore resistant to many harsh chemicals 5. Maturation: Cell wall ruptures, endospore is released Flagella Bacterial motility is typically provided by structures known as flagella. The eukaryotic flagellum, which operates as a flexible whip-like tail utilizing microtubules that are powered by ATP. The bacterial flagellum is rigid in nature, operates more like the propeller on a boat, and is powered by energy from the proton motive force. There are three main components to the bacterial flagellum: the filament – a long thin appendage that extends from the cell surface. The filament is composed of the protein flagellin and is hollow. Flagellin proteins are transcribed in the cell cytoplasm and then transported across the cell membrane and cell wall. A bacterial flagellar filament grows from its tip (unlike the hair on your head), adding more and more flagellin units to extend the length until the correct size is reached. The flagellin units are guided into place by a protein cap. the hook – this is a curved coupler that attaches the filament to the flagellar motor. the motor – a rotary motor that spans both the cell membrane and the cell wall, with additional components for the gram negative outer membrane. The motor has two components: the basal body, which provides the rotation, and the stator, which provides the torque necessary for rotation to occur. The basal body consists of a central shaft surrounded by protein rings, two in the gram positive bacteria and four in the gram negative bacteria. The stator consists of Mot proteins that surround the ring(s) embedded within the cell membrane. Rotation of the flagellar basal body occurs due to the proton motive force, where protons that accumulate on the outside of the cell membrane are driven through pores in the Mot proteins, interacting with charges in the ring proteins as they pass across the membrane. The interaction causes the basal body to rotate and turns the filament extending from the cell. Rotation can occur at 200-1000 rpm and result in speeds of 60 cell lengths/second (for comparison, a cheetah moves at a maximum rate of 25 body lengths/second). Flagella long, thin appendages (15–20 nm wide) free at one end & anchored into the cell at the other Gram negative Gram positive end Tiny rotating machines that function to push/pull the cell through a liquid Present in many bacteria [flagellum (plural, flagella)] and Archaea [archaellum (plural, archaella)] A bacterium may have one/several flagella Filament Gram-negative bacteria o Outermost region Ø L ring> anchored in outer membrane o Contains globular protein Ø P ring>anchored in peptidoglycan layer (flagellin) Ø MS & C rings> located within cytoplasmic o Not covered by a sheath like membrane & cytoplasm eucaryotic filaments Gram-positive bacteria Hook Ø Only inner pair of rings present o Wider segment that anchors Ø Surrounding inner ring & anchored in the filament to basal body cytoplasmic membrane & peptidoglycan are a series of proteins called Mot proteins Basal Body o Complex structure with a central o Fli proteins as the motor switch, rod surrounded reversing the direction of rotation of by a set of rings the flagella in response to intracellular signals Bacterial flagella move by rotation from basal body Flagellar movement may be either clockwise (CW)/counterclockwise (CCW) Bacteria may be capable of several patterns of motility Ø Runs/swims: bacterium moves in one direction Ø Tumbles: bacterium changes direction. Caused by reversal of flagellar rotation Some bacteria are motile but lack flagella they move by gliding -motility is a slower & smoother form of movement & typically occurs along the long axis of the cell Taxis Movement of a cell toward or away from various stimuli Chemotaxis: Movement in response to a chemical stimulus Phototaxis: Movement in response to a light stimulus enhance a cell’s access to resources/allow it to avoid harmful substances that could damage or kill it Rotation can occur in a clockwise (CW) or a counterclockwise (CCW) direction, with different The occurrence of tactic behavior provides evidence for the ecological (survival) advantage of flagella in bacteria and other results to the cell. A bacterium will move forward, prokaryotes called a “run,” when there is a CCW rotation, and reorient randomly, called a “tumble,” when there is a CW rotation. Fimbriae Pili Bristle like short fibres (0.03-0.14 µm) occurs on Similar to fimbriae but are typically longer (0.5-2 µm) only the surface of a cell, shorter in length than pili one/few pili are present on surface of a cell Made up of fimbrillin protein Made up of pilin protein Present on both Gram positive & negative Found in all gram-negative bacteria & many gram-positive bacteria bacteria § Salmonella typhimurium § Escherichia coli § Shigella dysenteriae § Pseudomanas § Neisseria gonorrhoeae Enable cells to stick to surfaces including animal tissues in the case of pathogenic bacteria Function: form pellicles (thin sheets of cells on a liquid o facilitating genetic exchange between cells in conjugation surface) (conjugative/sex pili) biofilms on solid surfaces o Enabling adhesion of pathogens to specific host tissues that they subsequently invade (type IV & other pili) Not for motility, not receptor for viruses Can be receptors for certain types of viruses Important virulence factor & pathogenicity Eukaryotic flagella and cilia Used for cellular locomotion/moving substances along the surface of cell Contain cytoplasm and are enclosed by plasma membrane Flagella :projections are few and long in relation to the size of cell Cilia : projections are numerous & short resembling hair Anchored to plasma membrane by a basal body, and both consist of nine pairs of microtubules (doublets) arranged in ring, plus another two microtubules in the center of the ring (9+2 array or 9+0 array) Microtubules: long hollow tubes made up of a protein called tubulin Eukaryotic flagellum moves in wavelike manner Capsule and Slime Layer Many bacteria & Archaea secrete sticky/slimy materials (polysaccharide/protein) on their cell surface § not considered part of the cell wall because not confer significant structural strength on the cell These layers referred as “capsule” & “slime layer” Capsule: layer that organized in a tight matrix that excludes small particles and is tightly attached Slime layer: polysaccharide substance that is loosely attached to the cell wall Function: o assist in the attachment of microorganisms to solid surfaces o development and maintenance of biofilms o acting as virulence factors & preventing dehydration § Bacillus anthracis (anthrax)-thick capsule of protein § Streptococcus pneumoniae (pneumonia)-thick capsule of polysaccharide *Encapsulated cells>>avoid destruction by the host’s immune system o outer surface layers of virtually any type bind water and likely protect the cell from desiccation in periods of dryness Eukaryotic cell wall Group translocation does not occur in eukaryotic cells Most eukaryotic cells have cell wall, much simpler than prokaryotic Can use endocytosis: Process in which plasma membrane encircles particles Ø Algae,plants and some fungi: Cellulose outside of cell and bring it into the cell Ø Fungi: Chitin (polysaccharide) Two important types of endocytosis: Ø Yeasts: Glucan and mannan (polysaccharides) Ø Phagocytosis Glycocalyx -Cellular projections called pseudopods engulf particle and bring them into the A layer of material containing substantial amounts of sticky cell carbohydrates -Used by white blood cells to destroy bacteria & foreign substances Ø Pinocytosis Covalently bonded to proteins & lipids in plasma membrane, forming - Plasma membrane fold inward, bringing extracellular fluid into the cell along glycoproteins and glycolipids that anchor glycocalyx to the cell with whatever substances are dissolved in the fluid Strengthens the cell surface, help attach cells together and contribute to cell cell recognition Plasma (cytoplasmic) membrane Very similar in function & basic structure with procaryotic Have different in the types of membrane proteins Contain carbohydrates that are important for cell-cell recognition and serve as sites for bacterial attachment Contain sterols (not found in prokaryotic plasma membranes) which associated with the ability of membranes to resist lysis resulting from increased osmotic pressure Movement across eucaryotic cell membranes: Ø Simple diffusion Ø facilitated diffusion Ø Osmosis Ø active transport Cytoplasm Encompasses substance inside the plasma membrane and outside the nucleus Has a complex internal structure, consisting microfilament, intermediate filaments & microtubules >>>cytoskeleton: a complex network of thread and tube-like structures, which provides support, shape, and movement. Helps distribute nutrients and move the cell over surface (cytoplasmic streaming) Many important enzymes found in cytoplasmic fluid of prokaryotes are sequestered in organelles of eukaryotes Nucleus Usually spherical/oval frequently the largest structure & contain DNA Surrounded by nuclear envelope Nuclear pores-tinny channels in the membrane Ø allow nucleus to communicate with cytoplasm Ø Control movement of substances between nucleus & cytoplasm Nucleolus: Dense region where ribosomes are made DNA (genetic material) is combined with histones and exists in two forms: Ø Chromatin (Loose, threadlike DNA) Ø Chromosomes (Tightly packaged DNA, found during cell division) Functions Ø House and protect cell’s genetic information (DNA) Ø Ribosome synthesis Endoplasmic Reticulum (ER) An extensive network of flattened membranous sacs/tubules (cisterns) Two types of ER: Ø Rough Endoplasmic Reticulum (RER) o continuous with nucleus membrane & usually unfolds into a series of flattened sacs o Outer surface is studded with ribosomes o Protein synthesized by ribosomes attached to rough ER enter cisterns within ER for processing & sorting o Functions: v Synthesis and modification of proteins v Synthesis of cell and organelle membranes v Packaging, and transport of proteins that are secreted from the cell e.g.: Antibodies Ø Smooth Endoplasmic Reticulum (SER) o Extends from rough ER to form a network of membrane tubules o Without ribosomes on the outer surface of its membrane o Functions: v Lipid Synthesis: Phospholipids, fatty acids, and steroids (sex hormones) v Breakdown harmful substances (alcohol, antibiotics, etc.) v Helps develop tolerance to drugs and alcohol v Regulates sugar release from liver into the blood v Calcium storage for cell and muscle contraction Ribosomes Site of protein synthesis (translation), present in all eucaryotic & procaryotic cells Made up of protein and ribosomal RNA (rRNA) May be found free in the cytoplasm or associated with the rough endoplasmic reticulum (RER) Eucaryotic ribosomes (80S):larger & more dense than procaryotic ribosomes (70S) Eucaryotic ribosomes have two subunits: Ø Large subunit: 60S Ø Small subunit: 40S Free ribosomes unattached to any structure in cytoplasm>>synthesize proteins Membrane bound ribosomes attached to nuclear membrane & endoplasmic reticulum>>> synthesize proteins destined for insertion in plasma membrane/export from the cell Located within mitochondria>>synthesize mitochondrial proteins Golgi complex Consists of 3-20 cisterns that resemble a stack of pita break, cisterns are often curved, giving a cuplike shape Works closely with the ER to secrete proteins Functions: Ø Receiving side receives proteins in transport vesicles from ER Ø Modifies proteins into final shape, sorts, and labels them for proper transport Ø Shipping side packages and sends proteins to cell membrane for export or to other parts of the cell Ø Packages digestive enzymes in lysosomes Lysosomes Formed from Golgi complexes & look like membrane enclosed sphere Have only a single membrane & lack internal structure containing at least 40 different digestive enzymes, which can break down carbohydrates, proteins, lipids, and nucleic acids Optimal pH for lysosomal enzymes is ~5 Found mainly in animal cells Functions: Ø Molecular garbage dump and recycler of macromolecules (e.g.: proteins) Ø Destruction of foreign material, bacteria, viruses, and old/ damaged cell components. Important in immunity Ø Digestion of food particles taken in by cell Vacuoles Membrane bound sac Different types, sizes, shapes, and functions: Central vacuole: In plant cells. Store starch, water, pigments, poisons, & wastes. May occupy up to 90% of plant cell volume Contractile vacuole: Regulate water balance, by removing excess water from cell. Found in many aquatic protists Food or Digestion Vacuole: Engulf nutrients in many protozoa (protists). Fuse with lysosomes to digest food particles Mitochondria (Singular: mitochondrion) Spherical/rod shaped organelles appear throughout the cytoplasm of most eukaryotic cells Structure: Ø Inner/outer membrane Ø Intermembrane space Ø Cristae (inner membrane extensions) Ø Matrix (inner liquid) Number of mitochondria per cell varies among cell types Powerhouses of the cell-Site of cellular respiration: Food (sugar) + O2-----> CO2 + H2O + ATP Change chemical energy of molecules into the useable energy of the ATP molecule Contain their own DNA, 70S ribosomes, & machinery necessary to replicate, transcribe & translate the information encoded by their DNA Can reproduce more/less on their own by growing & dividing in two Chloroplasts A membrane enclosed structure that contains both the pigment chlorophyll & the enzymes required for the light gathering phases of photosynthesis Disc shaped with three membrane systems: Ø Outer membrane: Covers chloroplast surface. Ø Inner membrane: Contains enzymes needed to make glucose during photosynthesis. Encloses stroma (liquid) and thylakoid membranes. Ø Thylakoid membranes: Contain chlorophyll, green pigment that traps solar energy, stacks of thylakoids are called grana (singular: granum) Capable of multiplying on their own within the cell-by increasing in size and then dividing in two Bacterial Growth Growth is defined as an increase in the number of cells , A few bacterial species reproduce by budding not an increase in the size of the individual cells Form a small initial outgrowth (bud) that enlarge until its size approaches Bacteria normally reproduce by binary fission (“binary”- that of the parent cell and then it separate that yields totally new daughter two cells have arisen from one) cell, with the mother cell retaining its original identity cells elongate to ~twice their original length then form a partition that constricts the cell into two daughter cells Some variations in binary fission Ø Bacillus subtilis>>septum forms without cell wall constriction Generation time Time required for a cell to divide and its population to double Under ideal circumstances, a growing bacterial population doubles at regular intervals In bacteria, each new fission cycle/generation increases the population by a factor of 2/double of it Growth is by geometric progression: n=the number of generations Assumption: Individual generation time is the same for all cells in the population Varies among organisms & with environmental conditions (e.g. temperature) Most bacteria generation time: 1-3 hrs Generation time Mean growth rate constant (k) = n/t Generation time (g) or doubing time = t/n Bacterial Growth Curve Growth of cells over time Four basic phases Ø Lag Ø Log Ø Stationary Ø Death Lag phase Immediately after inoculation of the cells into fresh medium, population remains temporarily unchanged (adaptation to new environment) No apparent cell division occurring, cells may be growing in: Ø Volume/mass Ø Synthesizing enzymes Ø Proteins Ø RNA Ø Increasing in metabolic activity Length of the lag phase is dependent on a wide variety of factors: Exponential (log) phase Ø Size of inoculum Best condition for growth Ø Time necessary to recover from physical damage/shock All cells are diving regularly by binary fission & are growing by geometric in the transfer progression Ø Time required for synthesis of essential Cells divide at a constant rate depend on : coenzymes/division factors Ø Composition of the growth medium Ø Time required for synthesis of new (inducible) enzymes Ø Conditions of incubation that are necessary to metabolize the substrates present Rate of exponential growth of a bacterial culture is expressed as generation in the medium time, also the doubling time of the bacterial population Even through population of cells is not increasing , individual Growth is balanced and genetically coordinated cells are metabolically active Stationary phase Direct measurement of Microbial Growth Measure number of viable cells (≥24 hrs to form) Population growth (size) is limited A colony is from short segments of a chain/from a Ø Exhaustion of essential nutrients bacteria clump Ø Accumulation of inhibitory metabolites/end product Reported as colony forming units (CFU) Ø Depletion of oxygen The Food & Drug administration convention count Ø Development of an unfavourable pH only plates with 25-250 colonies/30-300 colonies Ø Exhaustion of space, lack of “biological space” To obtain countable colony counts, serial dilution Number of cells able to divide (viable cells)= number that are unable needed on the original inoculum to divide (non viable cells) Time consuming & tedious to perform, used only Like lag phase, is not necessarily a period of quiescence Plate counts viability/in some statutory tests of food/drinking Bacteria produce secondary metabolites (e.g. antibiotics) during water stationary phase of the growth Secondary metabolites are defined as metabolites produced after active stage of growth During stationary phase, spore forming bacteria have to induce/unmask the activity of dozens of genes that may be involved in sporulation process Death phase Very short phase, Viable cell population declines (turbidimetric measurements/ microscopic counts cannot observe death phase) Number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during log phase Factors contribute to cell death: Ø Cell lysis by autolytic enzymes Ø Effects of toxic metabolites Guidelines for calculating the cfu Guidelines for calculating the colony forming unit per g or mL ü 25 – 250 or 30 – 300 (excluded by spreaders or lab accidents) colonies & average the counts * 1 ml was added in each plate v If ….. Ø Only one plate of a duplicate pair yields 25 – 250 or 30 – 300 colonies 1. count both plates, unless excluded by spreader or lab accidents 2. 3. Ø If count ratio > 2 4. take lowest value 5. Ø Spreaders 6. count area that has well distributed cfus and estimate counts by multiplying 7. total area 8. Ø No colonies 9. check for inhibitory substances, if none, report estimated count as less than 10. the lowest dilution 11. Ø No plate with 25 – 250 or 30 – 300 colonies & ≥ 1 plates have more than 25 – 250 12. or 30 – 300 colonies 13. Select plate(s) having nearest to 250 or 300 colonies & report count as est. CFU TNTC: Too numerous to count per ml/g Ø All plates have fewer than 25 – 250 or 30 – 300 colonies Record the actual number of colonies on the lower dilution & report count as est. CFU per ml/g Filtration Ø Crowded plates (>250 or 300 colonies) Count colonies in portion of the plate that are representative of colony distribution Total area of Petri plates 56 cm2, if colony < 10 colonies/cm2, count 12 boxes (6 vertical and 6 horizontal across the plates= 12cm2) Rapid method to estimate bacterial populations in water useful when evaluating large sample volumes or performing coliform tests Volume of sample depends on types of sample Ø drinking water/bottled water: 100 ml Ø Polluted water: 0.001-10 If sample volume 10 colonies/cm2, count 4 boxes or squares with each 1 cm2 distilled water/deionized water before filtration (ensure an even distribution of bacteria across the entire filter surface) v When > 100 colonies/cm2, report as greater than (>) the plate area 100 x Non-potable water samples must be diluted to a level at which the 56 x highest dilution factor (not as TNTC) bacteria can be measured Sample volume should be adjusted to obtain plates with 20 to 200 Ø Spreader or spreading colony > 50% of plate area CFU/filter label as Spr. If not, count area that has well distributed cfus and estimate Ø Total coliform:~ 20-80 coliform colonies /filter counts by multiplying total area Ø Fecal coliform:~ 20 to 60 coliform colonies/filter. Ø *Analyze 3 different sample volumes when coliform number is uncertain Filtration set preferably autoclavable Membrane (usually modified cellulose e.g. nitrocellulose, nylon or PVDF) must be sterile, pore size 0.45 um or 0.2 um After filtration the filter is place on suitable media either with or without absorbent pad Sample volume by sample type—total coliform test Sample volume by sample type—fecal coliform test Reporting results Direct Microscopic Count Report test results as the number of colonies per 100 mL of sample mL sample: actual sample volume and not the diluted volume Indistinct colonies—If growth covers the entire filtration area of the membrane or a portion of it, and colonies are not discrete, report the test results as “Confluent growth with or without coliforms.” High colony density—If the total number of colonies exceeds 200 per membrane or the colonies are too indistinct for accurate counting, report the results as “Too numerous to count, (TNTC)” When testing non-potable water, if no filter meets the desired minimum colony count Direct counting the cells in a population Viable, dead, viable but nonculturable (VBNC) & viable but difficult to culture (VBDC) organisms Do not distinguish between living & dead cells Ø Direct microscopic observation on specially etched slides (Petroff-Hausser chambers/hemacytometers) o a very small sample (e.g. 10 µL of a cell suspension) of the population is placed into a counting chamber with known volume o The number of bacterial cells visible in the chamber is counted Ø Electronic counters The Most Probable Number Method (Coulter counters, count microorganisms as they flow through a small hole/orifice) o A sample from the population is placed in the machine o Sample is passed between two electrodes, every time a cell passes between the electrodes it causes a disturbance in the electrical field, and the cell is counted Population count is determined immediately, no incubation time is required Epifluorescence microscopy Black polycarbonate membrane filter with 0.2 um pore size is used Sample can be stained in the funnel itself with fluorochrome Ø Acridine orange (AO)-acridine orange direct tube-dilution method (mostly 3 or 5-tube series) where multiple tube counts (AODC) ->green (high amounts of dilution is carried out to extinction RNA)/ orange (high amounts of DNA) Ø 4',6-diamidino-2-phenylindole (DAPI) It uses a medium that is less sensitive to toxicity and supports the Ø Fluorescein isothiocyanate (FITC) growth of stressed organisms Ø LIVE/DEAD® BacLight™ stain from molecular Applicable to examination of total coliforms in chlorinated primary probe-live bacteria appear green (SYTO 9) effluents and under other stressed conditions and dead bacteria appear red (propidium iodide) Suitable to examine of turbid samples, muds, sediments, sludge Cells per ml = Does not provide a direct measure of the bacterial count more variable and tends to yield higher result (Total filterable area on membrane filter / Cells in number of microscope fields counted) Can produce estimates of bacterial concentration that are below Volume of sample filtered detectability of most other methods e.g. plating Three stages III. proper growth medium, temperature and incubation 1. Presumptive conditions have been selected to allow even a single 2. Confirmed viable cell in an inoculum to produce detectable growth 3. Completed IV. population does not contain viable, sub-lethally injured organisms that are incapable of growth in the culture Presumptive test: medium used A series of lauryl tryptose broth containing Durham tubes are inoculated with decimal dilutions of sample Disadvantages: Formation gas at 35˚C, within 48 hr constitutes a ‘+’ tedious and laborious (acid & gas) for members of total coliform group Certain non coliform bacteria may suppress coliforms or act synergistically to ferment Confirmed test: lauryl tryptose broth è false positive results ‘+’ presumptive test are streaked onto Eosine Brilliant green bile broth when chlorinated prima

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