Scope of Microbiology Microorganisms are widely distributed Earth air water solid liquid

1. Basic Microbiology and ImmunologyPM 401 Dr Mohammed A. RamadanProfessor of Microbiology and Immunology

2. Scope of Microbiology • Microorganisms are widely distributed • Earth air water solid liquid • Pathogenic: Ability to cause disease • non-pathogenic ????? Useful • - General microbiology • - Medical microbiology • - Veterinary microbiology • - Agricultural microbiology • - Industrial microbiology • - Environmental microbiology

3. Microbiology and Pharmacy • 1- Biology of organisms • 2- Applications of antiseptics and Disinfectants • 3- Production of antibiotics and vitamins • 4- production of vaccines • 5- Production of therapeutic peptides by genetic engineering • 6- Sterile products • 7- Epidemiology of microorganisms • 8- Infectious diseases

4. History of microbiology • Egyptian Ancients • 1-Microbial products mushroom fermented beverages medicine • 2-Microbial symptoms Quarantine for plague • Transmission of diseases Certain properties of diseases bread making using yeast • ِAnton van Leuwenhoek (1676) used his microscope • to describe bacteria & protozoa (animolecules) • Spontaneous generation Theory: : Aristotle (1700) • Living organisms (animolecules) are created do novo • Needham supported this theory • Spallanzani (1776) opposed this theory : • by sealing the container containing meat broth after boiling • The broth does not develop microorganisms • Gram staining of bacteria

5. Louis Pasteur(1822-1895) The Father of Microbiology • 1- Microbes are responsible for fermentation???????? destroyed • 2- Pasteurization development • 3- Chemical changes occurring during fermentation • 4- Development of vaccines from killed or attenuated organisms • Robert Koch (1843-1910) • 1- Developed solid artificial medium for cultivation of mo • 2- Isolated B. anthracis in a pure form • 3- developed staining of organisms • 4- Discovered Tubercle bacilli • 5- few mo are pathogenic while others are non pathogenic • 6- Koch’s postulate • - mo should observed in every stage of the disease • be isolated on artificial medium • When mo infect healthy animal it should gives same symptoms

6. Petri petri dishes • Christian Gram (1884) gram staining • Lister father of antiseptic surgery phenol as a disinfectant • Nomenclature • Staphylococcusaureus • Genusspeciesstrain • Escherichia coli colon • Classification of Biota (living cells)

7. Prokaryotes and Eukaryotes • Pro: primitive Eu : true karyo : nucleus • Prokaryotes bacteria and cyanobacteria • Eukarotes fungi; single cell algae and protozoa • CharacterProkaryotesEukaryotes • Size microscopicmacroscopic • Nucleus absent present • Cell division binary fission mitosis • Cell membrane absence of sterol sterol • Ribosome 70 s 80 s • Mitochondria absent present • Cell wall peptidoglycan absent or • polysaccharide • Mesosome present absent • Endospores present absent • Example bacteria fungi, protozoa

8. Kingdome Woese system Molecular level genetic analysis • 1- Kingdom Archaebaceteriae • 2- Kingdom Eubacteria • 3- Kingdom Eukaryotes • Kingdom Wittaker’s system • 1- Monera (bacteria) • 2- Fungi • 3-Protista ( Protozoa and algae) • 4- Plants • 5- animals

9. Bacteria: Unicellular prokaryotes • Rickettsia: ????? • Viruses: Obligate intracellular parasites

10. Nomenclature • Staphylococcusaureus • Genus species species strain • Archaebacteria • - Prokaryotes • - resist high salt concentration • - grow high temperature • - grow at acidic pH • - thermo-acidophiles • - some are methanogenic • - Cell wall ----- glycoprotein

11. Eukaryotes • Fungi: mold and yeast • - non photosynthetic Heterotrophic • - aerobic or facultative anaerobic • Mold: long filament cells -- hyphae septated non-septated • Yeast : long oval cells multiply by budding pseudo mycelium • Filamentous fungi are multiply by sexual and a sexual spores • Fungi are classified into: • 1- Phycomycetes • Hyphae - ---- non-septated • Spores - ---  inside sac (sporangium) • Examples: Mucor and Rhizopus 2- Ascomycetes - asexual spores born on tips of hyphae -- conidiospores - septated hyphae - Examples: Aspergillus and Penicillium

12. 3-Basidiomycete • Sexual spores --- clup shaped (basidium) ( 4 basidiospores) • Example: Agaricus • 4- Deutromycetes (fungi imperfecti) • Sexual fusion is not observed • Examples: Epidermophyton • Asexual spores include: • 1- Sporangiospores; hyphae - sporangiophore sporangium • 2- Conidiophores conidiophore conidium • 3- Arthrospores Fragmented hyphae • 4- Blastospores from parent cell as a bud some yeast • 5- Chlamidospores formed by enlargement within hyphae Dimorphism : pathogenic fungi could exist in 2 forms a- yeast like at 37 C in tissue b- mould like at 25 C in artificial medium

14. Slime Molds • - have fungal and amoeboid characteristics • - Life cycle--- mold • - Some stage---- creeping amoeboid movement • Algae • - Photosynthetic organisms • - Cellular with cell wall • - Cell wall --- cellulose and pectinate; alginate • - autotrophic • Protozoa • - Eukaryotic unicellular without cell wall • - Motile through pseudopodia cilia or flagella • - Non photosynthetic • - Reproduction by sexual and asexual

15. Other Structures • Prions • 1- smallest infectious agent • 2- infect cattle and cause Bovine spongiform encephalopathy (BSE) • 3- Neurological disease • 4- can infect human also Viroids 1- cause for plant diseases 2- short pieces of RNA with no protein coat 3- does not code for protein Viruses 1- obligate intracellular parasite 2- non living 3- small filtrable 4- specific host 5- phage 6- contain either DNA OR RNA 7- Have a protein coat (capsid) 8- some viruses are enveloped

16. Lipid Envelope Nucleic Acid Protein Capsid Virion Associated Polymerase Spike Projections Virus structure

17. Viral replication • 1- Attachment or adsorption chemical interaction • 2- Penetration • Injection of DNA into bacterium • Phage release lysozyme which lysis of bacterial cell wall • Capsid remains outside the bacterium 3- Biosynthesis - Phage use its template DNA instead of bacterial DNA for synthesis a new DNA - use of bacterial ribosome for synthesis of the capsid (protein) - Eclipse period 4- Maturation Bacteriophage DNA and capsid are assembled into virion 5– Release Phage lysozyme - lysis of cell wall

19. Structure of Bacterial cell

20. Bacterial cell • Eubacteria Archaebacteria Rickettsia Actinomycetes • Size of bacterial cell 1 u • weight 1 g - 5 x 10 11 cells • No of species 200,000

21. Bacterial cell structures • Main structure • - cell wall • - cell membrane • - cytoplasm • - nuclear materials • - ribosomes • ُExtra-cellular • structures • - capsule • - flagella • - pili • - endospore • - plasmid

22. CELL WALL • - maintain the shape of the cell • - Semirigid structure - virulent factor • - Site of action of antibiotics • Structure of cell wall • Peptidoglycan: composed of N-acetyl glucosamin and N-acetyl muramic acid linked with bridge composed of tetrapeptide chain of both D and L • Two these aa are unique for bacteria: diaminopemilic acid & D-alanine • In Gram positive Bacteria • Teichoic acid -- polymer of glycerol or sorbitol linked to phosphate • 1- role in cell growth 2- antigenic structure • In Gram negative Bacteria • It contains outer membrane (OM) - lipoprottein + LPS+ phspholipid • The space between OM and plasma membrane  periplasmic space • 1- evade phagocytosis and the action of complment • 2- barrier to certain antibiotics, lysoszyme, detergent, heavy metals • 3- Part of permeability of OM is due to protein channel (Porins)

23. Upon lysis of cell wall of –ve ---- LPS --- endotoxins or pyrogens  • fever and schock • LPS --- (i) outer polysaccharide (O antigen) • (ii) phospholipid called Lipid A ---- endotoxin • Damage to cell wall • 1- Peptidoglycan except archaebacteria 2- no cell wall PG in human • 3- rupture in hypertonic solutions • N.B. Archaebacteriae : glycoprotein • Mycoplasma : have no cell wall • Cell wall could be removed by lysozyme or by chemical • Protoplast spheroplast L-form • LPS ------ pyrogen or endotoxin LPS ????? Function of cell wall • it is a rigid structure protect the cell from mechanical stress • It maintains the shape of the cell

24. Gram positive Gram negative • - peptidoglycan thick thin • - teichoic acid present absent • - outer membrane absent present • - Lipopolysaccharide absent present • - Gram reaction purple red • - Periplasmic space absent present • - Lysozyme high low • A typical cell wall: • - acid fast bacteria mycolic acid a waxy lipid, and peptidoglycan • - Archaebacteria have peptidoglycan cell wall

25. Cell membrane (plasma membrane) • Composed of : bilayer of phospholipid and protein • Phospholipid molecule: hydrophilic (PO4 and glycerol) • hydrophobic (fatty acids) • Sterol is available in eukaryotic bacteria: EXCEPT: Mycoplasma • Protein molecule • 1- peripheral: easily removed by mild treatment • 2- Integral : can be removed after disruption of bilayer (by detergent) • Function of cell membrane: • 1- selective permeability • Macromolecules are degraded by extracellular enzyme into micromolecules • - passive transport: movement according to concentration gradient • - simple diffusion from high from high to low conc simple molecule ), CO2 • - facilitated diffusion glucose bound to protein as a carrier • - active transport 2 - respiration and energy production 3 – site of synthesis of cell wall subunits 4 – site of enzyme and toxin production

29. Extracellular structures Glycocalyx - mucopolysaccharidemucilagenous - exhibit mucoid structure - attachment to surfaces dental caries - biofilm (growing of bacteria on a surface) - virulence factor - can protect cell against dehydration Capsule - polysaccharide polypeptide - protect the cell from phagocytosis - might help in bacterial adherence

30. Flagella • Whip-like structure; used for motility -Composed of protein (flagellin) • Originating from cell membrane • movement of bacteria by hydrolysis of ATP • The number and distribution of flagella is a characteristic for each organism • Monotrichous amphitrichous lophotrichous peritrichous • Flagella composed of • 1- Filament long contain flagellin 2- Hook slightly wider • 3- Basal body small rod inserted into a series of ring • -movement is resulted from rotation of its basal body • -Some bacteria like Proteus (surrounded by many flagella) - swarming • -Taxis: is the movement of bacteria away from a specific stimuli -- • Chemotaxis Phototaxis • Axial filament: specific for spirochetes for spiral movement at one end • - Fimbria and pili • - gram negative bacteria hair like shorter straight thin than flagella • Fimbria • Function: adhere and colonize the mucous membrane • =Virulent factor

31. Pili : • similar to fimbrai But longer and only 1 or 2 per cell • Functions 1- adherence 2- sexual conjugation • Bacterial endospores • 1- a structure developed under unfavorable conditions • 2- difficult in staining 3- does not multiply ( no cell division) • one gram negative species - Coxiella  forms endospore like structure • 4- containing a. ca dipicolinate b. hard cortex • c. low water content d. presence of disulfide bond • Spore forming bacteria: Bacillus (aerobic) • Clostridium (anaerobic) Spherical or oval central terminal subterminal Bulging or non bulging Germination: transfer of spore into vegetative cell

32. Ribosomes • 1- site for protein synthesis • 2- 2 subunits protein + rRNA • 3- 70 s 80 s s Svedberg units sedimentation • 4- 70 s 30s one mol of rRNA 50 s 2 mol of r RNA

33. Osmosis: movement of solvent across membrane from high to low • In living system - water • Osmotic pressure: • pressure required to prevent movement of water into a solution • Isotonic • Conc of solute are equal on both side • Hypotonic • Conc of solute outside are lower than that inside the cell • Result: swelling---- cell lysis • Hypertonic • Conc of solute outside are ligher than that inside the cell • Result: shrink and collapse

34. Bacterial Metabolism • Metabolism: all chemical reactions that takes place in a living cell • Catabolism: biodegradation of organic molecule into simple molecules • Anabolism : assimilation of simple molecules into structural unit • Requirement for survival and growth: • -Nutritive requirements: C source : structural and functional molecules E source : anabolic (building ) and catabolic ( degradation) N source : synthesis of amino acids, vitamins and nucleic acids P source : for synthesis of nucleic acids • Environmental sources: • pH - temp - oxygen requirement • C SOURCE • Autotrophes: C from CO2 • Heterotrophes : C from organic compounds • Chemoorganotroph • Chemolithotrophes

35. Energy sources • Oxidative phosphorylation coupling • Oxidation : release of electrons reduction ; accept electrons • Oxidation-reduction intermediates: • These are compounds which are easily capture and release of electrons • Examples : NAD; NADP; NADH; FAD Terminal electron acceptor : O • Phosphorylation reactions: formation of phosphate bond ???????? • Carried out at the cell membrane • AMP + PO3 --------------------- ADP + PO3 -------------- ATP energy energy ENERGY GENERATION Glycolysis Glucose ---- pyruvate 2 ATP ATP is synthesized by substrate phosphrylation Pyruvate pathways Pyruvate ----- acetyl co A kreb’s cycle Kreb’s cycle -------- electron transport chain

36. Conclusion • Catabolism of glucose is completed by: • (i) glycolysis 2 ATP • (ii) kreb’s cycle 2 ATP • (iii) oxidative phosphorylation 34 ATP • Oxidative phosphorylation is the main source for ATP • A total of 38 ATP is formed from glucose metabolism • Anaerobic respiration • Inorganic compound act as a terminal electron acceptor, instead of O • For examples, nitrate, sulfate • Fermentation • Organic compound act as a terminal electron acceptor • For examples: glucose, fructose and lactose