Unit 3 Eukaryotes and Prokaryotes PDF

LL CE TIC YO A R E UK & TI C YO R A Y K O OG R P OL BI OBJECTIVES At the end of the unit, the student w...

LL CE TIC YO A R E UK & TI C YO R A Y K O OG R P OL BI OBJECTIVES At the end of the unit, the student will be able to: 1. Outline the basic difference between prokaryotic and eukaryotic cells; 2. Explain the structure of the prokaryotic cell membrane 3. Explain types of cellular transport mechanisms; 4. Explain the principle of the Gram stain 5. Describe the bacterial endospore as a very heat resistant cell type formed under adverse conditions; 6. Outline methods of investigating microorganisms (e.g. centrifugation, culturing, straining and slide preparation) MAJOR GROUPS  Eukaryote ◦ Algae, Protozoa, Fungi, Slime molds  Prokaryotes ◦ Bacteria, Archaebacteria  Viruses  Prions COMPOSITION OF CELLS Chief distinguishing characteristics of prokaryotes: BOTH CELL TYPE ARE CHEMICALLY  Simple SIMILAR.  DNA not enclosed by a membrane...not associated Comprised of: with chromosomal proteins  Lack membrane bound  NUCLEIC ACID organelles   PROTEINS Cell wall generally contain  LIPIDS peptidoglycan   CARBOHYDRATES Usually divide by binary fission COMPOSITION OF CELLS Chief distinguishing characteristics of eukaryotes:  Membrane bound nucleus  DNA associated with chromosomal proteins  Membrane bound organelles  Cell division usually entails mitosis  Larger...structurally complex. PROKARYOTES VS. EUKARYOTES Prokaryotes  Organelles not bound by a lipid membrane.  No well-defined nucleus (nucleoid)  Usually much smaller than eukaryotic cells.  The cells of the Bacteria and Archaea are prokaryotic.  Bacterial cell walls characteristically contain peptidoglycan, a polysaccharide that is not seen in eukaryotic cell. Eukaryotes  All those cells excepting Bacteria and Archaea.  Contain membrane-bound organelles with well-defined nucleus.  Tend to be larger than prokaryotic cells.  Microbial eukaryotes are called Protist s  Fungal cell walls contain chitin & plants cellulose.  Animal cells do not contain chitin. SIGNIFICANCE OF BEING SMALL The small size of prokaryotic cells affects their physiology, growth rate, and ecology. Why ? SIGNIFICANCE OF BEING SMALL  ELL L C R IA CTE BA THE L TO N A LL TER A EX W S URE UCT S TR CELL WALL  Some eukaryotic cell have cell wall though simpler than those of prokaryotes.  Cellulose  Chitin  Prokaryotes cell wall consists of peptidoglycon PLASMA MEMBRANE  Very similar in eukaryotes and prokaryotes - basic structure and function  There are however differences in the types of proteins found within them  Eukaryotic cell contains carbohydrates as receptor sites.  Semi permeable membrane that facilitates transport of substances CYTOPLASMIC MEMBRANE PLASMA MEMBRANE 1. Endocytosis- enclosure of substances by plasma membrane to bring it into the cell 2. Three types- phagocytosis, pinocytosis and receptor mediated endocytosis CYTOPLASM  Substance in which cellular components are found  Eukaryotic cytoplasm have complex internal structures  Enzymes found in prokaryotic cytoplasm are sequestered in organelles of eukaryotes. PILLI  Are straight hair-like appendages which tend to be short.  They are made of the protein pillin which is arranged helically around a central hollow core.  Pilli function to attach bacterial cells to other cells. VIBRIO VULNIFICUS, ARROWS POINT TO PILLI  The protein called adhesions found in either the tip or side of pilli make the connection possible.  Sex pilli attach one bacterial cell to another during mating.  While others attach them to plant or animal cells or generally anchor bacteria in a favorable environment.  Escherichia coli and Neisseria gonorrhea both have pilli. FLAGELLA  Bacterial flagella are thread-like appendages composed entirely of protein, 12–30 nm in diameter. They are the organs of locomotion for the forms that possess them.  A bacterial flagellum is made up of several thousand moleculesof a protein subunit called flagellin.  highly antigenic (H antigens) SALMONELLA PROTEUS VULGARIS FLAGELLUM STRUCTURE Flagella structure has three distinct parts: 1. An outer helical-shaped filament- Composed of subunits of the protein flagellin arranged in a helical manner around a hollow core. 2. A hook- the filament is attached to a hook which allows the filament to move in different directions. The hook is attached to the basal body. 3. A basal body- Anchors the flagella to the envelope and causes it to rotate.  The part of the basal body that penetrates the envelope has four (in gram negative) or three (in gram positive) rings.  In gram negatives the L ring is embedded in the outer membrane however Gram- positives lack this ring.  The other rings are the P (peptidoglycan), and the inner S(superficial) and M (membrane) rings.  The motor that rotates the flagellum is a bell-shaped structure that sticks into the cytoplasm.  The core of the flagellum (the rod) rotates inside the rings which act as anchors to the envelope.  Different types of bacteria have different numbers of flagella: Monotrichous (genera pseudomonas), amphitrichous, BACTERIAL FLAGELLA (COPIED FROM THE INTERNET) FLAGELLUM (COPIED FROM THE INTERNET) A-MONOTRICHOUS; B-LOPHOTRICHOUS; C-AMPHITRICHOUS; D- PERITRICHOUS;  CAPSULE  Most bacteria secrete a slimy or gummy substance that forms outermost layer of the cell.  Capsules vary in thickness and composition with the organism that produces it.  Most are however made of polysaccharide and a few of protein. Functions of the Capsule:  Principally protect the cell against drying out  Adhere cells to a surface where conditions are favorable for growth  Protect disease causing bacteria against phagocytosis thus play an important role in infection.  Stains of S. pneumoniae that lack a capsule are harmless because they are quickly consumed.  Capsules and slime layers can also provide protection from the loss of nutrients by holding them within the layer.  These extra layers coating the surface of the cell may also potentially mask viral receptors making it more difficult for viruses to attach BACTERIA SURROUNDED BY CAPSULE KLEBSIELLA PNEUMONIAE NUCLEOID  The nucleoid or nuclear region is well defined even though it is not membrane bound.  It is a mass of DNA-carries the cell’s genetic information.  Bacterial DNA (chromosomal) is usually arranged in a single circular molecule.  Usually they also contain smaller circular DNA molecules called plasmids. RIBOSOME  Found in the cytoplasm.  Their great number and small size give the cytoplasm its characteristic grainy appearance.  The ribosome is the site of protein synthesis.  A ribosome is composed of two subunits – both composed of protein and RNA. The large subunit of prokaryotic cells is smaller than that of Eukaryotic cells (80s).  Two complexes of RNA and protein make up the prokaryotic ribosome, the 30S subunit and the 50S subunit.  The 30S subunit is composed of 21 proteins and a single-stranded rRNA molecule of about 1,500 nucleotides, termed the 16S rRNA.  The 50S subunit contains 31 proteins and two RNA species, a 5S rRNA of 150 nucleotides and a 23S rRNA of about 2,900 nucleotides. STORAGE GRANULES  Many bacterial species have several kinds of storage granules.  Granules of carbon containing compounds like glycogen and poly-beta- hydroxyalkanes.  Other granules containing reserves of sulphur and nitrogen, and granules of polyphosphate. OTHER INCLUSIONS  Gas vacuoles: gas-filled regions surrounded by a monolayer of a single protein that allows the bacteria to float at the water level with the conditions best suited for photosynthesis.  Chlorosomes: Seen in photosynthetic bacteria. These structures house pigments necessary for photosynthesis. STAINS  Unstained bacteria are practically transparent when viewed using the light microscope and thus are difficult to see.  Stains serve several purposes: 1. Stains differentiate microorganisms from their surrounding environment 2. They allow detailed observation of microbial structures at high magnification 3. Certain staining protocols can help to differentiate between different types of microorganisms.  Staining protocols can be divided into 3 basic types: simple, differential, and specialized. Simple stains  Single stain reacts uniformly with all microorganisms and only distinguish the organisms from their surroundings.  Differentiation of cell types or structures is not the objective of the simple stain.  However, certain structures which are not stained by this method may be easily seen, for example, endospores and lipid SIMPLE STAIN TECHNIQUE Differential stains  discriminate between various bacteria, depending upon the chemical or physical composition of the microorganism.  Uses a primary followed by a secondary stain Gram stain  The Gram reaction is named after the Danish physician, Christian Gram, who developed this staining technique in 1884  Differentiates bacteria based on the chemical composition of their cell envelope-thick/thin cell wall.  Gram positive organisms stain purple, the colour of the primary stain  Gram negative organisms stain pink, the colour of the secondary stain GRAM REACTION 2.Acid fast Stain  Cells of species of Mycobacterium do not stain readily with ordinary dyes Because of the waxy substance (mycolic acids) present on the cell walls.  Treatment with cold carbol fuchsin for several hours or at high temperatures for five minutes will dye the cells.  Subsequent treatment with a dilute hydrochloric acid solution or ethyl alcohol containing 3% HCl (acid-alcohol) will not decolorize them.  Such cells are thus termed acid-fast in that the cell will hold the stain fast in the presence of the acidic decolorizing agent. Specialized stains  detect specific structures of cells such as flagella and endospores  The nature of the endospore requires a vigorous treatment for staining, but once stained, the endospores are difficult to decolorize.  In the endospore stain, water is the decolorizing agent that removes the primary stain from the vegetative cells.  Endospore stains require heat to drive the stain into the cells.  For an endospore stain to be successful, the temperature of the stain must be near boiling and the stain cannot dry out. BACTERIAL CELL WALL STRUCTURE  Bacterial wall is made mostly of a rigid macromolecule called peptidoglycan.  Peptidoglycan is composed of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) joined by 1,4-glycosidic bonds  Chains of NAM and NAG are cross-linked by peptide chains (differ among bacterial species).  Peptide chains are made of aa of D configuration.  The most common peptides are four amino acid long: L- alanine, D- alanine, D- glutamic acid and lysine or diaminopimelic acid (DAP).  NAM and NAG molecules form a repeating structure. The strength of the bacterial cell wall is proportional to the extent of cross-linkages.  Covalently bound to the thick peptidoglycan are teichoic acid DIRECT VS. INDIRECT CROSS LINKING INDIRECT CROSSLINKING COMPARISON OF GRAM NEGATIVE AND GRAM POSITIVE CELL ENVELOPE COMPARISON OF GRAM NEGATIVE AND GRAM POSITIVE CELL ENVELOPE BATERIAL CELL WALL STEPS IN GRAM STAINING  Bacterial cells are dried onto a glass slide and stained with crystal violet, then washed briefly in water.  Iodine solution (mordant) is added so that the iodine forms a complex with crystal violet in the cells. Alcohol or acetone is added to solubilise the crystal violet - iodine complex.  The cells are counterstained with safranin, then rinsed and dried for microscopy. How does the cell envelope structure influence Gram Stain?  Gram-positive cells retain the crystal violet-iodine complex and thus appear purple, the colour of the primary stain.  Gram-positive bacteria have a relatively thick wall composed of many layers of the polymer peptidoglycan (sometimes termed murein).  The thickness of this wall blocks the escape of the crystal violet- iodine complex when the cells are washed with alcohol or acetone.  Gram-negative bacteria have only a thin layer of peptidoglycan, surrounded by a thin outer membrane composed of lipopolysaccharide (LPS).  The region between the peptidoglycan and LPS layers is termed the periplasmic space.  It is a fluid or gel-like zone containing many enzymes and nutrient-carrier proteins.  The crystal violet-iodine complex is easily lost through the LPS and thin peptidoglycan layer when the cells are treated with a solvent.  Gram-negative cells are decolourised by the alcohol or acetone treatment, but are then stained with safranin (secondary stain) so they appear pink TROUBLE SHOOTING GRAM STAIN 1. The cultures to be stained should be young - incubated in broth or on a solid medium until growth is just visible (no more than 12 to 18 hours old if possible).  Old cultures of some gram-positive bacteria will appear Gram negative.  This is especially true for endospore-forming bacteria, such as species from the genus Bacillus. TROUBLE SHOOTING GRAM STAIN 2. When feasible, the cultures to be stained should be grown on a sugar-free medium.  Many organisms produce substantial amounts of capsular or slime material in the presence of certain carbohydrates.  This may interfere with decolorization, and certain Gram-negative organisms such as Klebsiella may appear as a mixture of pink and purple cells.  ENDOSPORES  Endospores are dormant alternate life forms produced by the genus Bacillus (obligate aerobes found in the soil) and  the genus Clostridium (obligate anaerobes often found as normal flora of gastrointestinal tract of animals)  and several other less common genera  function: An endospore is not a reproductive structure but rather a resistant, dormant survival form of the organism.  Endospores are quite resistant to high temperatures (including boiling), most disinfectants, low energy radiation, drying, etc.  The endospore can survive possibly thousands of years until a variety of environmental stimuli trigger germination, allowing outgrowth of a single vegetative bacterium FORMATION OF ENDOSPORES  Under conditions of starvation, especially the lack of carbon and nitrogen sources, a single Endospores forms within some of the bacteria.  The process is called sporulation : First the DNA replicates and a cytoplasmic membrane septum forms at one end of the cell forming a forespore.  The remainder of the vegetative cell engulfs the forespore.  Then there is synthesis of peptidoglycan in the space between the two membranes surrounding the forespore to form the first protective coat, the cortex.  Calcium dipocolinate is also incorporated into the forming endospore.  A spore coat composed of a keratin-like protein then forms around the cortex. Sometimes an outer membrane composed of lipid and protein and called an exosporium is also seen.  Finally, the remainder of the bacterium is degraded and the endospore is released. Sporulation generally takes around 15 hours. ENDOSPORE STRUCTURE  The completed endospore consists of multiple layers of resistant coats including:  a cortex  a spore coat  and sometimes an exosporium These layers surround a nucleoid, some ribosomes, RNA molecules, and enzymes.  Exosporium- thin covering made of protein and lipids  Spore coat- highly cross-linked keratin and layers of spore-specific proteins  Cortex- loosely cross-linked peptidoglycan  Innermost spore cell- components of the vegetative cell RESISTANCE TO ENVIRONMENTAL CONDITIONS  Bacterial endospores are resistant to antibiotics, most disinfectants, and physical agents such as radiation, boiling, and drying. RESISTANCE OF ENDOSPORES IS DUE TO A VARIETY OF FACTORS: 1. Proteinaceous spore coat- confers resistance to lysozyme and harsh chemicals 2. Calcium-dipicolinate, abundant within the endospore, may stabilize and protect the endospore's DNA. 3. Specialized DNA-binding proteins saturate the endospore's DNA and protect it from heat, drying, chemicals, and radiation. SPORULATING CLOSTRIDIUM BOTULINUM MECHANISMS BY WHICH SUBSTANCES ARE TRANSPORTED ACROSS THE MEMBRANE  Materials move across plasma membrane of both prokaryotic and eukaryotic cell by one of two processes:  Passive processes- substances cross from area of high concentration to area of low conc. along conc. Gradient (no energy)  Active processes- Cell uses energy (ATP) moving substances from areas of low conc. to area ofhigh conc. Against a conc. gradient PASSIVE PROCESS This includes simple diffusion, diffusion, osmosis Simple diffusion:  Net movement of molecules/ions from area of high to area of low conc. to maintain even distribution  Used in the transport of molecules such as O2 and CO2 PASSIVE PROCESS Facilitated diffusion  Membrane proteins act as carriers across plasma membrane  Examples of theses proteins are, transportases, permease  Cell does not expend energy (movement from high to low conc)  Molecules so carried, glucose vitamins etc. PASSIVE PROCESS OSMOSIS Net movement of a solvent molecules across a semi permeable membrane from an area of high conc. of solvent to an area of low conc. of solvent molecules OSMOSIS  Bacterial cells may be subject to any of three kinds of osmotic solutions:  Isotonic solution-cell content in equilibrium  Hypotonic solution- causes cell lysis  Hypertonic solution- plasmolysis (shrink) (describe conc. of solutions outside cell) ACTIVE PROCESS  When bacterial cell is in an environment with low nutrient concentration the cell uses active processes to acquire required substances ACTIVE TRANSPORT  Use of energy in the form of ATP to move substances across plasma membrane ACTIVE PROCESS GROUP TRANSLOCATION  Seen exclusively in prokaryotes  Substances are chemically altered during transportation  Allows cell to accumulate various substances even if they are in low conc. outside the cell  Requires energy IDENTIFICATION OF MICROORGANISMS Methods of microbial identification fall within three groups: 1. Phenotypic- microscopic and macroscopic morphology 2. Genotypic- Genetic technique 3. Immunologic- serological analysis PHENOTYPIC IDENTIFICATION  Initial presumptive identification  Stains e.g. gram, endospore  Morphology – size, shapes, arrangement  Macroscopy  Biochemical characteristics e.g. enzyme (catalase test), sugar fermentation etc. GENOTYPIC TECHNIQUES  Examination of the genetic material of the organisation.  Fast and accurate methods  Examples: PCR, Nucleic acid probes etc IMMUNOLOGICAL METHODS  Involves antigen antibody interactions  Testing of microbial antigen or host antibody production is often easier than direct microbial tests  Examples include ELISA, EIA  LABORATORY SAMPLES PROCESS AND ANALYSIS  Pre analytical phase  Specimen collection  Analytical phase  Specimen receipt, processing, and testing  Post Analytical Phase  Interpretation, Reporting THREE PHASES OF LABORATORY ANALYSIS PREANALYTICAL ANALYTICAL POST ANALYTICL Patient variables Performance of Test reporting selected laboratory variables test Specimen variables Recording Collection Reporting Handling Interpreting Processing PRE ANALYTICAL PROCEDURES: SEE ATTACHED LINK  http://www.mayomedicallaboratories.co m/articles/communique/2008/12.html PREANALYTICAL IMPACT ON LABORATORY OUTCOME The success identification of microbes is dependent on:  Use of aseptic technique  Correct collection of specimen  Correct handling of specimen  Speedy transportation to lab  REFERENCES  http://www.merck.com/media/mmhe2/figures/MMHE_17_190_01_e ps.gif  http://www.microbiologytext.com/index.php?module=Book&func= displayarticle&art_id=70  http://www.arn.org/docs/mm/flag_labels.jpghttp://answers.yahoo.c om/question/index?qid=20090420112848AATic8k  http://botit.botany.wisc.edu/images/130/Bacteria/Growth_Forms/B accili_cocci.jpg  http://www.lima.ohio-state.edu/biology/images/spirl.jpg  www.ehinger.nu/.../files/Sarcina_lutea.html  www.answers.com/topic/leptospirosis  bioinfo.bact.wisc.edu/.../medical.html  www.sportsspecialtychemicals.com/info.php?t=b...  pathmicro.med.sc.edu/.../CNS%20infections.htm  path.upmc.edu/cases/case53/dx.html