MICR20010 Lecture 1-2 Microbiology UCD

MICR20010 Lecture 1 Microbiology Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science [email protected]  Practicals Practicals will be carried out face to face and you should be able to s...

MICR20010 Lecture 1 Microbiology Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science [email protected]  Practicals Practicals will be carried out face to face and you should be able to see your practical slots in your timetable. Please check your allocated lab rotation is correct in brightspace and identify your lab room and seat number. Please view introductory lecture on Brightspace PRINT OUT THE LAB MANUAL AND BRING IT WITH YOU AND MARKER!!!  Assessments Practical accounts for 30% 15% on the two Practical reports to be submitted online after the practicals 15% on the Practical Exam to be held at date TBC 70% on an end of term Final MCQ exam.  Remember Check you practical assignment on brightspace matches your timetable and that you can identify your lab room and seat number The lab manual and pre-practical talk for practical 1 will be on brightspace before the lab. Do not leave the lab without asking all questions about the write up. We will not address lab write up outside of lab. If you have any questions please E-mail the module coordinator at [email protected] Learning Outcomes Define Microbiology Different types of microbes Role and application of microbes Importance of microbiology in: – Agriculture – Food industry – Animal and plant health Role of Microbiologists History of Microbiology  What is Microbiology? The study of microscopic organisms Microbes are tiny single-cell organisms They are the oldest form of life on Earth. Microbe fossils date back >3.5 billion years - when the Earth was covered with oceans that regularly reached the boiling point. Hundreds of millions of years before dinosaurs! → Incredible biodiversity - outpaced higher organisms Without microbes, we couldn’t eat or breathe. Without us, they’d probably be just fine. Understanding microbes is vital to understanding the past and the future of ourselves and our planet. Types of Microbes Types of Microbes Bacteria Often dismissed as “germs” that cause illness, bacteria help us do an amazing array of useful things, like make vitamins, break down some types of garbage, and maintaining our atmosphere. Archaea These bacteria look-alikes are living fossils that are providing clues to the earliest forms of life on Earth. Fungi From a single-celled yeast to a 3.5-mile-wide mushroom, fungi do everything from helping to bake bread to recycling to decomposing waste. Protista Plant-like algae produce much of the oxygen we breathe; animal-like protozoa (including amoeba) help maintain the balance of microbial life. Viruses Unable to do much of anything on their own, viruses go into host cells to reproduce, often wreaking havoc and causing disease. Their ability to move genetic information from one cell to another makes them useful for cloning DNA and could provide a way to deliver gene therapy. Role and Application of Microbes Role of Microbiology in Biotechnology and pharmaceutical industry Production of important pharmaceuticals: – Glucose polymers – Vitamins – Amino acids – Ion chelating agents – Enzymes – Antibiotics Bacteria as producers of human substances. The hormone erythropoietin, which is absolutely necessary for the proper development of red blood cells (erythrocytes), but very, very, difficult to isolate, is now available in high quantity Erythropoietin gene/ human insulin gene Cloned into bacteria EPO: abused by some professional Overproduced and purified athletes. Increases RBC count Administered to patients who cannot make these substances themselves Importance of Microbiology in Agriculture Decomposition and Recycling Waste treatment Soil fertility Food Production Diary industry Spoilage Animal and plant health Benefits Disease Agriculture: Legumes – plants with root nodules containing bacteria that fix nitrogen – reduce dependence on fertilisers Ruminant animals – cattle and sheep have special digestive vessel called the rumen filled with bacteria. Bacteria digest cellulose in grass and hay, without which animals would not thrive Nutrient cycling – carbon, nitrogen and sulphur. Microbial activities in soil and water convert these elements into forms plants can use (Plant nutrition) Microbial diseases – Foot and mouth virus, mad cow disease, potato blight (fungus) Role of Microbiology in the Food Industry Food spoilage – enormous economic losses every year Food borne pathogens – serious health risk Dairy products – cheese, yogurt, buttermilk all produced by microbial activity Baked goods, alcoholic beverages result from yeast activity Animal Feed (Single cell protein – microbial biomass or proteins extracted from large scale cultivations of bacteria, yeast or fungi) Food supplements (Probiotics) Probiotics Only 1% of microbes cause infection Probiotics are live microorganisms administered in adequate amounts which confer a beneficial health effect on the host. 1. Favorably alter the intestinal microflora balance 2. Inhibit the growth of harmful bacteria. Probiotic bacteria also produce substances called bacteriocins, which act as natural antibiotics to kill undesirable microorganisms 3. Promote good digestion 4. Boost immune function and increase resistance to infection. Beneficial bacteria (probiotics) are present in fermented dairy products namely live culture yogurt Yogurt is the original probiotic preparation—used as a folk remedy for hundreds, if not thousands, of years. However, different brands of yogurt can vary greatly in the bacterial cultures used and potency. Supplements in powder, liquid extract, capsule, or tablet form containing beneficial bacteria are other sources of probiotics.  Role of Microbiology In plant and animal Health Antimicrobial Use in Food Animals In the US large quantities of antibiotics are consumed by animals being raised for food, such as cattle, dairy cows, pigs, and poultry Contributes to antibiotic resistance. Most of the antimicrobials given to food-producing animals each year are not used to treat sick animals. Instead, antibiotics are routinely added to feed and water to prevent disease and to promote growth.  Role of Microbiology In plant and animal Health This long-term, low-dose exposure to antibiotics is more likely to result in resistant bacteria than short-term antibiotic use to treat sick animals. Transmission to human pathogens In 1999, the European Union banned the use of four antibiotics as growth promoters.  Microbiology is an important medical discipline Prevention and treatment of infectious disease RTI, diarrhoeal diseases, mycobacteria are principal causes of death worldwide Drug resistance a major problem Emerging infections in immunocompromised patients Hospital-acquired infections Role of Microbiologists Bacteriologists focus specifically on bacteria and how they help or hurt us. Virologists specialize in viruses and how they infect cells. Mycologists study fungi. Protozoologists devote their efforts to protozoa. Epidemiologists investigate infectious disease outbreaks to learn what caused them and if we’re facing a deadly new microbe. Immunologists study how the body defends itself against microbial invaders.  History of Microbiology Discovery of microscopic life Invisible living creatures were thought to exist and were thought to be responsible for disease long before they were observed Mid 1600’s single celled organisms were discovered Considered to be at an early stage of development into complex organism 1684 Antony van Leeuwenhoek Cloth merchant in Holland – used a magnifying glass to inspect quality of cloth Developed into an amateur microscope builder – “wee animalcules” Leeuwenhoek did not invent microscopes. In fact compound microscopes (with two lenses) were invented 40 years before he was born. Only 20-30X at that time. Leeuwenhoek’s skill in grinding and polishing lenses with great curvature achieved 200X. “Father of Microscopy” Antonie van Leeuwenhoek would not tell anyone how he built his microscopes. It was over 100 years after Leeuwenhoek's death before anyone could manufacture a microscope that could match or surpass the magnifying quality of the microscopes he built. Although Leeuwenhoek's scientific discoveries were monumental, his secretive nature delayed the onset of the widespread study of microbiology for over a century..  Spontaneous Generation Living things produced from vital forces in non-living or decomposing matter Abiogenesis versus Biogenesis Spontaneous generation Organisms arose Production of life from from seeds or germs vital forces in non-living that had entered the matter food from the air 200 year debate among scientists  Countered the argument that spontaneous generation could take place if broth was exposed to air, since the neck of the flask freely admits oxygen Countered the arguments that heating would kill the "life force" of the broth, since the heated broth supported growth after it was exposed to dust  Louis Pasteur (1822-1895) Pasteur's explanation for re-growth in previously sterile media was due to contamination - the result of the ubiquitous presence of microbes. 1. Microorganisms present in air resembled those present in decaying material 2. Postulated that these microorganisms were constantly settling on all objects 3. To prove this he demonstrated that food treated to destroy microorganisms would not putrefy 4. Used HEAT to kill microorganisms & prevent putrification Importance of Sterilisation The experiments of Pasteur and other microbiologists in the 1800s also highlighted the importance of killing all the bacteria and other microorganisms in or on objects This process is termed sterilisation Mastitis in cattle is reduced by sterilising milking equipment  Discovery of Penicillin 1929 Alexander Fleming discovers penicillin. Produced by the fungus Penicillium. Fleming noticed that the bacteria seemed to dissolve and cultures were contaminated with the fungus. Not produced in major quantities until 1940s – launches the “Antibiotics Era,”. Fleming is awarded the Nobel Prize in Medicine or Physiology in 1945.  Further Reading Microbiology an Introduction, Tortora, Funke and Case 12th Ed. Chapter 1 “The Microbial World and You”  MICR20010 Lecture 2 Culturing Microbes Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science  Any questions? Email module coordinator Dr. Tadhg O Croinin [email protected] View intro lecture video on brightspace for all module info  Lecture 1 Define Microbiology Different types of microbes Role and application of microbes Importance of microbiology in: – Agriculture – Food industry – Animal and plant health Role of Microbiologists History of Microbiology  Learning Outcomes How to culture microbes Difficulties working with microbes Sterile growth media Handling microorganisms – Inoculation – Incubation – Isolation – Inspection – Identification Disposal of cultures Disinfectants and Antiseptics  How to culture microbes? During 1880s Scientists realised that the study of microorganisms would require ways to visualise and handle them. 1. Preparation of sterile growth media 2. Separating microbes from each other 3. Growing microbes under controlled conditions 4. Preparing specimens for microscopic examination  Difficulties working with microbes Most microbes exist in complex communities e.g. soil or human mouth – Individual bacterial species need to be isolated before they can be studied Microbes need to be grown under artificial conditions Microbes are invisible to the human eye – problems with contamination Aseptic technique to prevent contamination  Sterile Growth Media Before handling and growing microbes sterile growth media is required Bacteria are grown in a medium (latin for middle) containing nutrients Medium can be liquid, water based (broth) or solid (agar Petri plates)  Common methods of sterilising bacterial growth media Boiling – 100OC for 30 mins - Kills cells Autoclave – 121OC for 30 mins - Kills all cells and spores Dry heat – 150OC for 120 mins - Kills all cells and spores Handling Microorganisms: The five I’s ➊ Innoculation ➋ Incubation ➌ Isolation ➍ Inspection ➎ Identification  Innoculation or producing a culture To grow/cultivate/culture microbes, a tiny sample (the inoculum) is introduced or inoculated into nutrient medium, which provides an environment in which the organisms multiply. Growth in nutrient broth can be observed as a cloudy suspension which is termed a culture. Nutrient agar provides a surface for colonies to develop. The inoculum may be a clinical specimen e.g. blood; a soil sample, a water sample, a sewage sample, a food sample etc.  Growth of bacteria on agar nutrient medium AGAR: MELTS AT 100OC, SOLIDIFIES AT 40OC STERILIZED BY AUTOCLAVING BACTERIA GROW AS COLONIES SINGLE COLONY PURIFICATION  Single colony purification Each bacterial colony is derived from a single cell Single colony purification = Single bacterial cell purification Obtaining Pure Cultures from an Isolation Plate Growth of bacteria in liquid nutrient medium Bacterial growth Sterile  Incubation Microorganisms are grown in an incubator, which provides optimal temperature and gas content. An incubator speeds up the process of multiplication and production of a culture  Isolation Concept of separating cell from other cells and providing it with adequate nutrients and space to grow The ability to grow microbes in pure form in essential in the study of their biology. The nutrients required to grow bacteria in medium vary depending on the bacterial species. – Each microorganism has its own nutritional requirements. – Can be exploited in identifying microbes. – > 500 different media for growing bacteria – reflects bacterial diversity COLONIES OF BACTERIA  Inspection Examination of colonies to determine if culture is pure – Each colony forms from a single cell – therefore the colony is an isolated population of an individual bacterial species The appearance of the colony is useful in identifying the bacterial species – Therefore in isolating a bacterial species, it is important that all the colonies appear the same Different colony type means that the culture is not pure or is mixed. – If culture is contaminated or mixed, a single colony of the desired species is subcultured In broth culture, not possible to determine if growth of more than one bacterial species has occurred.  Identification Macroscopic or colony morphology Microscopic morphology Biochemical characteristics Genetic characteristics  Disposal of cultures Sterilisation: Removal and destruction of all microbes in or on an object Physical Methods: Heat - Moist Heat. Boiling water, flowing steam. - Cells & most viruses. Not spores (Tyndallisation –intermittent boiling) – Steam (Autoclaving) 121oC - 15-30min. All spores/viruses/cells. Media & equipment - Dry Heat (Hot air), 1 hour at 171oC, – Incineration (burning) 1 sec or more at 1000oC) How an autoclave works  Disposal of cultures Radiation – Ionising e.g. X rays, gamma rays, secs-hrs. OH- radicals, damage to DNA. – Sterilize pharmaceuticals, medical supplies – Nonionising e.g. UV light. DNA damage. Operating theatres, kitchens  Disinfection Related process Reduction in bioload including the removal of pathogens Methods – Chemical – heat, e.g. pasteurisation – filtration Often easier to achieve than sterilisation and adequate for instruments in contact with mucous membranes, e.g. endoscopes Pasteurisation: Use of heat (e.g. 75°C, 15 seconds) to kill pathogens and reduce the number of spoilage micro-organisms in food and beverages (milk, fruit juice, wine, beer). Balance between removing microbes and affecting taste or quality of product  Antiseptics and disinfectants Antiseptics: microbicidal agents harmless enough to be applied to the skin and mucous membrane – should not be taken internally. Examples: mercurials, silver nitrate, iodine solution, alcohols, detergents.  Antiseptics and disinfectants Disinfectants: Agents that kill microorganisms, but not necessarily their spores, not safe for application to living tissues; they are used on inanimate objects such as tables, floors, utensils, etc. Examples: chlorine, hypochlorites, chlorine compounds, copper sulfate, quaternary ammonium compounds.  Antiseptics and disinfectants Note: disinfectants and antiseptics are distinguished on the basis of whether they are safe for application to mucous membranes. Often, safety depends on the concentration of the compound. For example, sodium hypochlorite (chlorine), as added to water is safe for drinking, but "chlorox" (5% hypochlorite), an excellent disinfectant, is hardly safe to drink.  Appropriate handwashing facilities Antiseptic hand wash  Further Reading Microbiology an Introduction, Tortora, Funke and Case 12th Ed. Chapter 6 “Microbial Growth” Chapter 7 “The control of Microbial Growth”  MICR20010 Lecture 3 Microscopy & Introduction to Microbial diversity Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science  Lecture 2 How to culture microbes Difficulties working with microbes Sterile growth media Handling microorganisms – Inoculation – Incubation – Isolation – Inspection – Identification Disposal of cultures Disinfectants and Antiseptics  Learning Outcomes Light Microscopy Preparing bacterial cells for microscopy Light microscope resolution Electron Microscope – Scanning Electron Microscope – Transmission Electron Microscope Domains of Life Types of microorganism Eukaryotic Cell Structure Eukaryotic Versus Prokaryotic Cells  Light Microscopy Magnification versus resolution Can be increased without limit Cannot Magnification is how much an image is enlarged under a microscope Resolution is the amount of detail you can see in an image. You can enlarge a photograph indefinitely using more powerful lenses, but the image will blur together and be unreadable. Therefore, increasing the magnification will not improve the resolution. This is also known as the resolving power. Light microscope resolution = 0.2mm Electron microscope = 1000x light microscope Light Microscope 100x, 400x, 1000x 10x 10x, 40x, 100x (oil) Preparing bacterial cells for microscopy Resolution Light microscope Cannot distinguish objects that are smaller than half the wavelength of light. White light has an average wavelength of 0.55 micrometers, half of which is 0.275 micrometers Any two lines that are closer together than 0.275 micrometers will be seen as a single line, and any object with a diameter smaller than 0.275 micrometers will be invisible or, at best, show up as a blur  Electron Microscopy I Uses electrons instead of light photons to image cells and cell structures Electrons provide "illumination" with a shorter wavelength than light photons Electromagnets function as lenses Entire system is held in a vacuum  Light Microscopy Electron Microscope Electron Microscopy  Electron Microscopy II Electrons are speeded up in a vacuum until their wavelength is extremely short, only one hundred-thousandth that of white light. Beams of fast-moving electrons are focused on a cell sample and are absorbed or scattered by the cell's parts so as to form an image on an electron-sensitive photographic plate. Most electron microscopes can magnify objects up to 1 million times Resolving power of EM = 0.2 nm versus 0.2 mm for light microscope (1000X) Electron microscope  Electron Microscopy III Scanning electron microscopy – Used to observe external features of cells. Specimen coated with thin film of metal e.g. gold. – Electrons scattered by the metal are collected to produce an image Transmission electron microscopy – Used to observe internal cell structures. – Unlike light photons, electrons do not penetrate the cell – Hence thin sections of the cells are prepared (one bacterial cell cut into many thin sections)  SEM bacterial cells E. Coli Salmonella Staphylococcus TEM bacterial cells E. Coli Streptococcus  Light versus Electron microscopy No living specimen can survive under high vacuum and chemical fixatives used in EM Light microscopes enable the user to see living cells in action. – Primary challenge for light microscopists has been to enhance the contrast between pale cells and their paler surroundings so that cell structures and movement can be seen more easily. – Phase contrast light microscopy  Light versus Electron microscopy New strategies involving: – video cameras, – polarized light – Fluorescent dyes – digitizing computers Yields vast improvements in contrast, fueling a renaissance in light microscopy Domains of Life  Types of Microorganism 1. Prokaryotic microorganisms: – Bacteria, Archaea 2. Eukaryotic microorganisms: – Fungi, Protozoa, Algae 3. Non-cellular microorganisms: – Viruses, Prions Prokaryotic Cell Structure Eukaryotic Cell Structure Eukaryotic Versus Prokaryotic Cells Prokaryotic Cell Eukaryotic Cell No nucleus Nucleus All have cell wall Some have cell wall, many do not No cell organelles Cell organelles e.g. – Mitochondria, chloroplasts, – Endoplasmic reticulum – Golgi Eukaryotic Versus Prokaryotic Cells Nucleic acid Eukaryotic cell 1. Nucleic acid in organelle called a nucleus. Bounded by a nuclear membrane. 2. Contains one or more paired, linear chromosomes composed of DNA associated with histone proteins Prokaryotic cell 3. Nucleic acid not bounded by a nuclear membrane 4. Usually contains one circular chromosome composed of DNA associated with histone-like proteins. Eukaryotic Versus Prokaryotic Cells Cell division Eukaryotic cell 1. By mitosis 2. Sex cells in diploid organisms are produced through meiosis. Prokaryotic cell 3. Usually by binary fission. No mitosis. 4. Organisms are haploid. No meiosis needed. Eukaryotic Versus Prokaryotic Cells Cytoplasmic membrane Eukaryotic cell Cytoplasmic membrane is a fluid phospholipid bilayer containing sterols Prokaryotic cell Cytoplasmic membrane is also a fluid phospholipid bilayer. Eukaryotic Versus Prokaryotic Cells Cytoplasmic structures Eukaryotic cell – Ribosomes composed of a 60S and a 40S subunit forming an 80S ribosome. – Internal membrane-bound organelles e.g. mitochondria, endoplasmic reticulum, Golgi apparatus, are present – Chloroplasts serve as organelles for photosynthesis. – Cytoskeleton responsible for cell shape. Prokaryotic cell – 70S Ribosomes composed of a 50S and a 30S subunits – Internal organelles are absent – No chloroplasts. Photosynthesis usually takes place in infoldings of the cytoplasmic membrane. – Cell wall responsible for cell shape.  Eukaryotic Versus Prokaryotic Cells Respiratory enzymes & Electron Transport chains Eukaryotic cell Located in the mitochondria. Prokaryotic cell Located at the cytoplasmic membrane  Eukaryotic Versus Prokaryotic Cells Cell wall Eukaryotic cell – Plant cells, algae, and fungi have cell walls, usually composed of cellulose or chitin but never containing peptidoglycan – Animal cells and protozoans lack cell walls Prokaryotic cell – Bacteria and Archaea have cell walls composed of peptidoglycan, protein or unique molecules – Obligate intracellular bacteria – mycoplasma, chalmydia, ureaplasma have no cell walls  Eukaryotic Versus Prokaryotic Cells Locomotor organelles Eukaryotic cell – May have flagella or cilia. Flagella and cilia are organelles involved in locomotion and in eukaryotic cells. Prokaryotic cell – Some have flagella.  Further Reading Microbiology an Introduction, Tortora, Funke and Case 12th Ed. Chapter 3 “Observing Microorganisms through a Microscope” Chapter 4 “Functional Anatomy of Prokaryotic and Eukaryotic Cells”  MICR20010 Lecture 4 Basic microbial morphology Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science  Lecture 3 Light Microscopy Preparing bacterial cells for microscopy Light microscope resolution Electron Microscope – Scanning Electron Microscope – Transmission Electron Microscope Domains of Life Types of microorganism Eukaryotic Cell Structure Eukaryotic Versus Prokaryotic Cells  Learning Outcomes Prokaryotic cell morphology Bacterial cell structure The gram stain – Gram stain mechanism Bacterial shapes – Different morphological shapes Bacterial cell structure – G+ve G-ve Archaea – Cell membrane – Cell wall – Outer membrane – Cell appendages  Prokaryotic cells Bacteria Archaea Gram positive Gram negative Bacterial Cell Structure  In the lab! YOU MUST PRINT OUT THE LAB MANUAL BEFOREHAND!!! YOU MUST BRING A NOTEBOOK TO RECORD OBSERVATIONS!!! YOU MUST READ THE INSTRUCTIONS AND VIEW ONLINE MATERIAL BEFORE THE LAB!!!!!!! Bunsen Burner  The Gram Stain Most important differential staining method in Microbiology Gram-positive Gram-negative (staphylococci) (Escherichia coli) 1. Crystal Violet 2. Iodine 3. Alcohol 4. Neutral Red Gram Stain Use tongs to fix smear!!  The microscope Oil must be removed from 100x lens immediately after use using lens tissue.  Gram Stain Mechanism Differential lipid content of G+ and G- cell envelopes Crystal violet-iodine complex forms within the cells (Blue colour) Alcohol treatment G+ cell envelope has low lipid G- cell envelope has content and is dehydrated by high lipid content which alcohol - making it impermeable is extracted by alcohol to permeabilise the membrane Crystal violet-iodine complex diffuses out and neutral red is taken up Streaking out a mixed culture Microbes are everywhere a. Air, outside and inside; b. fingertips, before or after washing or after touching these to your hair; c. Soil; d. Water, a drop from a tap; e. Blade of grass; f. A drop of milk; g. Leaf of a plant Incubate at 27°C  LAB WRITE UP Instructions on page 2 of practical manual!!! Bring sharpie to write on petri dishes. You have one week to write up and submit on Brightspace. Ask all questions to your demonstrator in the lab. Do not leave until all your questions are answered! Some bacteria don’t stain using the Gram method Mycobacteria have a high wax content in their cell envelope and suspected mycobacteria are stained using the Ziehl- Nielson stain Mycoplasmas, the smallest known bacteria, have no cell wall to stain  Bacterial Shapes Cocci (spherical) Bacilli (rod shaped) Curved or spiral shaped Morphological Shapes of Different Bacteria Thiomargarita magnifica  Bacterial Cell Structure Chromosome The bacterial chromosome contains the bacterial genetic information. Plasmids may also be present. Cytoplasmic Membrane The cytoplasmic membrane surrounds the cytoplasm Cell Wall Rigid layer surrounding the cytoplasmic membrane Outer Membrane of Gram-negative bacteria Covers the cell wall and acts as a molecular sieve  Typical Gram-negative and Gram-positive Cell Envelopes  Cytoplasmic Membrane Composed primarily of lipids and phospholipids Osmotic barrier – Only molecules smaller than glycerol diffuse into the cytoplasm Site of energy production oxidative phosphorylation Transport of important molecules via Permeases Facilitated diffusion (passive) and Active transport Synthesis of new cell wall Anchor the chromosome  Cell Wall Domains of Life Cell wall composed 1. Eukaryotes primarily of peptidoglycan 2. Bacteria Prokaryotes 3. Archaea Lack peptidoglycan - wall composed of other polysaccharides or proteins  Function of the cell wall Bacterial cells contain high concentrations of dissolved solutes (salts, sugars etc). Generates a high pressure within the cell caused by the cytoplasm pressing against the cell envelope (similar to pressure in car tyre) Cell wall allows cell to withstand turgor pressure Gives the cell shape and rigidity  Bacterial Cell Wall Peptidoglycan = the principal component of the cell wall, is a unique polysaccharide which gives the cell its characteristic shape and prevents osmotic lysis Gram-positive Gram-negative Many peptidoglycan layers One peptidoglycan layer (90% of cell envelope material) (2-20% of cell envelope material) Penicillin disrupts peptidoglycan synthesis Many antigens are presented on cell wall surface  Peptidoglycan NAG = N-acetylglucosamine NAM = N-acetylmuramic acid  Basic structure of the peptidoglycan disaccharide unit (left) and multiple peptidoglycan units liked to give the cell wall structure (right Amino acids G = N-acetylglucosamine G-M: b 1,4 glycosidic bond M = N-acetylmuramic acid Gram-positive cell envelope  Cell walls of Archaea No peptidoglycan S-layer composed of a ordered layer of protein or glycoprotein – Examples: Many thermophiles, halophiles, methanogens Few Archaea contain pseudopeptidoglycan – (Repeated sugar units, however, ab1,3-linked) – Example: Methanogens Polysaccharides  Gram-negative Cell Envelope: Outer Membrane Phospholipid-Lipopolysaccharide (LPS) Bilayer (extra lipid layer - mechanism of the Gram stain) Bacterial cell adhesion Resistance to phagocytosis Molecular sieve - access of some molecules to cell wall and cytoplasmic membrane LPS vs Phospholipid Gram-negative cell envelope  Gram positive cell surface Note different surface textures Gram negative cell surface  Cell Appendages and other Cell Structures Flagella and Pili extend from the cell surface Flagellae rotate and are required for motility (chemotaxis) Bacteria swim towards chemoattractants and away from chemorepellents Flagella Bacteria use flagella to swim. Changing the direction of the flagellar rotation can cause the cell to tumble and change direction.  Pili (from latin for hair) Common Pili - adherence UTI’s Conjugative Pili - plasmid transfer  Further Reading Microbiology an Introduction, Tortora, Funke and Case 12th Ed. Chapter 4 “Functional Anatomy of Prokaryotic and Eukaryotic Cells”  MICR20010 Lecture 5 Growth and Physiology Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science  Lecture 5 Prokaryo&c cell morphology Bacterial cell structure The gram stain – Gram stain mechanism Bacterial shapes – Di)erent morphological shapes Bacterial cell structure – G+ve G-ve Archaea – Cell membrane – Cell wall – Outer membrane – Cell appendages  Learning Outcomes Microbial Growth and Physiology Growth of Bacteria – Bacteria Divide by Binary Fission – Growth of Bacteria on Solid Medium – Growth of Bacteria in Liquid Medium Growth Phases of liquid Bacterial Culture Measurements of Bacterial Growth Direct Measurements of Bacterial Growth: Indirect Measurements of Bacterial Growth: Growth Requirements Microbial growth and physiology In the laboratory Liquid broths and Nutrient Agar plates  GROWTH OF BACTERIA: ASEPTIC TECHNIQUE STERILE GROWTH MEDIA BOIL – KILL ALL CELLS 100⁰C / 30 MIN AUTOCLAVE – KILL ALL CELLS & SPORES 120 ⁰ C / 30 MIN DRY HEAT – 150 ⁰ C / 120 MIN  Bacteria divide by Binary Fission Binary Fission Chromosome divides to produce two iden&cal copies These copies segregate to opposite ends of the cell Cell wall is laid down the middle of the cell to ul&mately produce two new cells which are iden&cal Binary Fission  Bacterial growth is Exponen'al 1->2->4->8->16->32->64->128->256->512 etc Bacterial growth proceeds exponen&ally Genera&on &mes (&me for bacterial mass to double) can be as fast as 20 minutes Contributes to the remarkable adaptability of bacteria Growth in a hos&le environment can create a selec&ve pressure for mutant cells which can persist. One mutant cell which can survive will rapidly grow and take over. GROWTH OF BACTERIA ON SOLID MEDIUM AGAR: MELTS AT 100⁰C, SOLIDIFIES AT 40⁰C STERILIZED BY AUTOCLAVING BACTERIA GROW AS COLONIES SINGLE COLONY PURIFICATION GROWTH IN LIQUID MEDIUM COTTON WOOL BUNG GAS EXCHANGE KEEP CONTENTS STERILE INCUBATE STANDING OR AGITATED TURBID CULTURE ~ 109 CELLS/ML Growth Phases of a Bacterial Culture 1. Lag Phase – Adapta&on 2. Logarithmic Phase – Cells mul&ply at the maximum rate 3. Sta&onary Phase – Lack of nutrients and build up of toxic metabolic intermediates means mul&plica&on is balanced by cell death 4. Phase of decline  Genera'on Times of Bacteria Bacterium Medium Genera'on Time (minutes) Escherichia coli Glucose-salts 17 Bacillus megaterium Sucrose-salts 25 Streptococcus lac&s Milk 26 Streptococcus lac&s Lactose broth 48 Staphylococcus aureus Heart infusion broth 27-30 Lactobacillus acidophilus Milk 66-87 Rhizobium japonicum Mannitol-salts-yeast extract 344-461 Mycobacterium tuberculosis Synthe&c 792-932 Treponema pallidum Rabbit testes 1980  Measurements of bacterial growth Direct measurements of bacterial growth: I. Total cell count. Using microscope and coun&ng chamber II. Total viable count. Cells in culture are diluted and spread on nutrient agar plates. Only viable cells will reproduce to give rise to a colony. Direct measurements of bacterial growth: Coun&ng chamber Direct measurements of bacterial growth: The area and volume under each square is known. Can determine the number of cells in sample volume. Total Viable Count Serial 10-fold dilu&ons  Total Viable Count: Spread plate method and pour plate method Indirect measurements of bacterial growth Turbidity (Cloudiness) Measures live and dead cells How a spectrophotometer measures turbidity (cloudiness)  Chemostat culture 1. Cell density controlled by nutrient conc. 2. Growth rate controlled by Oow rate of nutrient  Growth requires Energy, The building blocks required for the construc&on of cellular machinery Appropriate environmental condi&ons  Growth Requirements Nutrient Requirements – Water – Carbon (carbohydrate) – Nitrogen (protein) – Inorganic salts Iron - siderophores – Oxida&on of organic compounds – (carbohydrates, lipids, proteins) Temperature pH Atmosphere 20⁰C- 110 ⁰ C !! 4.0 - 9.0 O₂ / No O₂  Energy Derived from the enzyma&c breakdown of organic substrates (carbohydrates, lipids or proteins) in a process called Catabolism Energy generated from catabolism is used to synthesise cellular cons&tuents in a process called Anabolism Catabolism + Anabolism = Metabolism Bacterial growth in diverse environments In addi&on to carbohydrates, lipids and proteins bacteria can also derive energy from plas&c, rubber and toxic compounds like phenol. Important implica&ons for decontamina&on of environmental pollu&on Exxon Valdez Oil Spill in Alaska: Engineered bacteria that “eat” hydrocarbons” were fer&lised onto beaches contaminated with oil. Known as bioremedia&on, this method was successful on several beaches where the oil was not too thick.  Auxotrophs The ability of individual bacterial species to produce their own cellular components will dictate its nutri&onal requirements E.g. some species can synthesis all essen&al amino acids whereas others need amino acids to be added to their growth media (auxotrophs).  Oxygen Obligate aerobes – grow only in presence of O2. Obligate anaerobes – grow only in absence of O2, killed by O2 Faculta&ve anaerobes – grow in presence & absence of O2  Temperature Psychrophile - cold loving bacteria (10-20⁰C) Mesophile (20-40 ⁰ C) – human body temperature – pathogens – opportunists Thermophile - heat loving (>60 ⁰ C)  pH Many bacteria grow best at neutral pH Some can survive/grow – acid – alkali  Extremophiles Antar&ca Hot geyser at Yellowstone Na&onal Park  Further Reading Brock, Biology of Microorganisms, Madigan, Mar&nko and Parker 10th Ed. Chapter 5 “Nutri&on, Laboratory Culture and Metabolism of Microorganisms” Chapter 6 “Microbial Growth”  MICR20010 Lecture 6 Bacterial Physiology and Metabolic Diversity Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science  Lecture 5 Microbial Growth and Physiology Growth of Bacteria – Bacteria Divide by Binary Fission – Growth of Bacteria on Solid Medium – Growth of Bacteria in Liquid Medium Growth Phases of liquid Bacterial Culture Measurements of Bacterial Growth Direct Measurements of Bacterial Growth: Indirect Measurements of Bacterial Growth: Growth Requirements  Learning Outcomes Metabolic diversity Chemical basis of energy produc*on Simpli+ed model of energy produc*on Energy storage and release Chemotrophs Phototrophs Chemotrophs – Chemoorganotrophs – Chemolithotrophs Autotrophs Heterotrophs Photosynthesis  Carbon and Energy All cells need carbon and energy sources for their metabolic ac*vi*es Di/erent microorganisms have evolved every conceivable means of obtaining carbon and energy This results in signi+cant metabolic diversity Hence microbes have been able to colonise environmental habitats which are too extreme for other life forms. h2ps://www.teagasc.ie/news--events/daily/other/the-soil-microbiome-and-soil-health.php  Chemical Basis of Energy Producon 1. Chemical reac*ons used to generate energy 2. Speci+cally chemical reac*ons involving the release of electrons 3. Electrons have stored energy and when an atom or molecule loses that electron (becomes oxidized) that energy is released 4. Oxida*on: atom or molecule loses one or more electrons 5. Reduc*on: atom or molecule gains those electrons. 6. Energy sources are oxidised to release electrons which have stored energy 7. Energy generated is stored in the form of ATP OIL RIG Oxida*on Is Loss of electrons Reduc*on Is Gain of electrons  Simpli!ed Model of Energy Producon Light e- e- ATP oxidised Iron Enzymes e- e- e- e- idised s ox yme Glucose Enz Example of simple oxida*on reac*on: Fe2+ ⇋ Fe3+ + e- Ferrous iron Ferric iron  ENERGY Energy Storage: Used to trap energy released from chemical reac*ons Energy stored as high-energy phosphate bond Inorganic phosphate group a2ached to adenosine diphosphate (ADP) Adenosine triphosphate (ATP) The energy currency of the cell Energy Release: Phosphate enzyma*cally removed from ATP to release energy  How to get Energy? Chemotrophs: Derive energy from chemicals Chemotrophs – Chemoorganotrophs (Use organic chemicals) – Chemolithotrophs (Use inorganic chemicals) Phototrophs – Derive energy from light  Chemoorganotrophs Derive energy from organic chemicals Organic chemicals are compounds containing carbon All cells require carbon as a major nutrient, hence these chemicals are a good source of carbon and energy for chemoorganotrophs 1000’s of di/erent organic chemicals present on Earth ALL can be broken down by microorganisms to derive energy  How do chemoorganotrophs derive energy from organic chemicals? Answer: Oxida*on of the compound releases electrons, which are ul*mately used to generate ATP. Electrons have stored energy and when an atom or molecule loses that electron (becomes oxidized) that energy is released Oxida*on: atom or molecule loses one or more electrons Reduc*on: atom or molecule gains those electrons. Energy sources are oxidised to release electrons which have stored energy Aerobes Some chemoorganotrophs can only produce energy in the presence of oxygen Anaerobes Microbes that can only produce energy in the absence of oxygen Facultave anaerobes Microbes that produce energy in the presence or absence of oxygen  Methanogens Livestock produce signicant amounts of methane as part of their normal digestive processes. Some feed additives can inhibit the microorganisms that produce methane in the rumen and subsequently reduce methane emissions. Ruminant livestock – cattle, sheep, bu alo, goats, deer and camels – have a fore-stomach (or rumen) containing microbes called methanogens, which are capable of digesting coarse plant material and which produce methane as a by-product of digestion (enteric fermentation): this methane is released to the atmosphere by the animal belching. h2ps://www.agric.wa.gov.au/climate-change/carbon-farming-reducing-methane- emissions-ca2le-using-feed-addi*ves#:~:text=Feed%20addi*ves%20or%20supplements%20can  Supplements Methane-reducing feed additives and supplements inhibit methanogens in the rumen, and subsequently reduce enteric methane emissions. Methane-reducing feed additives and supplements are most e ective when grain, hay or silage is added to the diet, especially in beef feedlots and dairies.  Reducing Methane Methane-reducing feed additives and supplements can be: synthetic chemicals natural supplements and compounds, such as tannins and seaweed fats and oils. Feeding one type of seaweed at 3% of the diet has resulted in up to 80% reduction in methane emissions from cattle.  Ac*ve inhibitors trihalomethanes, such as bromoform, which is an active ingredient that decreases methane emissions Tannins  Bene+ts: The reduced volume of methane formation may lead to better e)ciency of feed utilisation, given that methane emissions represent a gross energy loss from feed intake of about 10%.  Chemolithotrophs Derive energy from inorganic chemicals Inorganic chemicals are compounds which do not contain carbon, e.g. H2, H2S, Fe2+ These inorganic compounds are oxidised to release electrons for ATP synthesis However all cells require carbon as a major nutrient  Chemolithotrophs Name Examples Source of energy and electrons Respiraon electron acceptor Iron bacteria Acidithiobacillus ferrooxidans Fe2+ (ferrous) → Fe3+ (ferric) + e- O2 → H2O Nitrosifying bacteria Nitrosomonas NH3 (ammonia) → NO2- (nitrite) + e- O2 → H2O Nitrifying bacteria Nitrobacter NO2- (nitrite) → NO3- (nitrate) + e- O2 → H2O Chemotrophic purple sulfur S2 (sul+de) → S0 (sulfur) + e- O2 → H2O bacteria Halothiobacillaceae Sulfur-oxidizing bacteria Chemotrophic S0 (sulfur) → Sulfate (SO2−4) + e- O2 → H2O Rhodobacteraceae Aerobic hydrogen bacteria Cupriavidus metallidurans H2 (hydrogen) → H2O (water) + e- O2 → H2O Thiobacillus denitri+cans Thiobacillus denitri+cans S0 (sulfur) → Sulfate (SO2−4) + e- NO3- (nitrate) Sulfate-reducing bacteria: H2 (hydrogen) → H2O (water) + e- Sulfate (SO2−4) Hydrogen bacteria Sulfate-reducing bacteria: Desulfo*gnum PO3−3 (phosphite) → PO3−4 Sulfate (SO2−4) Phosphite bacteria phosphitoxidans (phosphate) + e- Methanogens Archaea H2 → H2O + e- CO2 (carbon dioxide) Carboxydothermus carbon monoxide (CO) → carbon Carboxydotrophic bacteria dioxide (CO2) + e- H2O (water) → H2 (hydrogen) hydrogenoformans  Chemolithotrophs Chemolithotrophs obtain carbon from CO2 – autotrophy Ecological niche and compe**on Lithotrophy is advantageous because organisms deriving energy from inorganic compounds do not have to compete with chemoorganotrophs. In addi*on some of their energy sources (H2, H2S) are waste products from the chemoorganotrophs  Heterotrophs and Autotrophs Heterotrophs Microbial cells which use one or more organic compounds as their carbon source are called heterotrophs Autotrophs Microbial cells which use CO2 as their carbon source are called autotrophs – CO2 +xa*on or Calvin cycle  Autotrophs Autotrophs are called primary producers because they produce organic ma2er from CO2 in the air. Chemoorganotrophs and other organisms can ul*mately use this organic ma2er by feeding on the autotrophs or their waste products All organic ma2er on the planet has been synthesised from CO2 by autotrophs  Phototrophs Use light as energy source Phototrophs contain pigments which allow them to use light as an energy source These pigments give the cells colour  Photosynthesis Phototrophs obtain energy from light using photosynthesis Photosynthesis involves reac*ons in which ATP is generated Oxygenic photosynthesis – oxygen is produced as a bi-product Anoxygenic photosynthesis – no oxygen is produced What are the Pigments in Phototrophic Cells? Chlorophylls Carotenoids  Chlorophylls Green colour Similar to the pigments responsible for photosynthesis in plants Phototrophic bacteria contain chlorophylls called bacteriochlorophylls In plant cells photosynthesis takes place in chloroplasts In bacteria photosynthesis takes place in a specially developed cytoplasmic membrane.  Carotenoids Yellow, red, brown and green colours Carotenoids are closely associated with bacteriochlorophyll but play no direct role in photosynthesis Transfer light energy to bacteriochlorophyll Carotenoids have a photoprotec*ve role Photosynthec Bacteria  Further Reading Brock Biology of Microorganisms Chapter 5 “Nutri*on, Laboratory Culture and Metabolism of Microorganisms” MICR20010 Lecture 7 Bacterial Genecs Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science  Lecture 6 Metabolic diversity Chemical basis of energy producon Simpli&ed model of energy producon Energy storage and release Chemotrophs Phototrophs Chemotrophs – Chemoorganotrophs – Chemolithotrophs Autotrophs Heterotrophs Photosynthesis Learning Outcomes DNA DNA replicaon Gene structure – Transcripon Protein synthesis – Translaon Anbiocs The Genec Code Mutaon Genec Exchange  DNA Deoxyribonucleic Acid (DNA) Monomer building blocks called deoxyribonucleodes: – 5-carbon sugar deoxyribose – a nitrogenous base – a phosphate group 5 4 1 3 2  Bases There are four nitrogenous bases found in DNA – Adenine (A) – Guanine (G) – Cytosine (C) – Thymine (T) DNA strand  DNA DNA consists of two complementary strands (double stranded) Complementary base pairing A=T G≡C G≡C  DNA Replica#on The process of generang an idencal set of genes during cell division Very accurate process carried out by DNA polymerases Occasional inaccuracies give rise to a slightly altered nucleode sequence – a mutaon  DNA Replica#on Iniaon Elongaon Proofreading Terminaon  DNA Polymerase DNA polymerase can add free nucleodes to only the 3' end of the newly-forming strand. This results in elongaon of the new strand in a 5'-3' direcon. No known DNA polymerase is able to begin a new chain (de novo). DNA polymerase can add a nucleode onto only a preexisng 3'-OH group DNA strand Gene#c Code DNA contains the genec informaon (genes) required for all cellular processes Genes can occur individually or in groups (operons) Gene Expression: Transcripon Iniated at the promoter region upstream of the gene RNA polymerase copies the DNA and produces an RNA transcript (mRNA) Translaon mRNA is decoded by ribosomes and tRNA molecules to specify the exact sequence of amino acids in a Gene Structure Protein Expression  The Gene#c Code Codon A set of three adjacent nucleodes that encode a parcular amino acid. Specifying the type and sequence of amino acids for protein synthesis.  An#bio#cs and DNA/RNA/Protein Some anbiocs target DNA replicaon, transcripon and translaon Rifampicin a=ects RNA polymerase Macrolides (erythromycin), Kanamycin, Tetracycline a=ect ribosome & protein synthesis Mutaons in the anbioc target can lead to bacterial resistance Ciprofoxacin Ciprofoxacin targets DNA gyrase, the enzyme which unwinds bacterial DNA during replicaon. Ciprofoxacin prevents cell division Quinolone anbioc Plasmids Circular extrachromosomal DNA Replicate independently and can move between cells Phenotypic advantage for the host cell Plasmid genes: – Anbioc resistance genes (oBen mulple) – Virulence genes (e.g. toxins) – Metabolic genes Hospital-acquired Infec#ons Plasmids with mulple anbioc resistance genes predominate within hospital bacteria Infecons caused by such bacteria (nosocomial or hospital-acquired infecons) are therefore parcularly serious and diCcult to treat. Anbioc resistance genes existed before the era of anbioc treatment but have become prevalent due to selecve pressure. Highlights bacterial adaptability. Transmission of AMR genes between species Muta#on Most common source of genec variaon Spontaneous or induced (mutagens) Three types – Substuon – Deleon – Inseron Codons - Muta#on  Gene#c Varia#on Important implicaons for microbial virulence: Resistance to anbiocs New virulence factors (e.g. E. coli 0157)  Mutagenesis Mutagens Physical - Radiaon Chemical mutagenesis – Base analogues – Intercalang agents – Metals - ROS Biological agents – Virus – Transposon Gene#c Exchange Modes of Genec Transfer between Bacterial Cells  Transforma#on DNA fragments can be taken up directly by bacterial cells Normally degraded Somemes integrated into host genome Some bacteria are naturally competent e.g. Streptococcus pneumoniae  Conjuga#on Describes plasmid transfer between bacterial cells Requires cell-to-cell contact & can occur between di=erent bacterial species and even between G+ve and G-ve tra genes encode pilus (channel) between the cells through which the plasmid moves Plasmids replicate in the donor cell prior to transfer into the recipient cell  Transduc#on DNA transfer between bacteria via infecon with a bacteriophage Phage infect the bacterial cell and replicate Involves incorporaon of phage DNA into phage capsids (heads) Occasionally host genomic DNA is also packaged  Transposi#on Transposons are DNA sequences that can ‘jump’ within the bacterial genome and from the genome to plasmids within the same cell Transposons carry the enzymes required for their own transposion (homology not needed). This can result in gene disrupon Transposons oBen contain anbioc resistance genes Transposion into broad host range plasmids has facilitated rapid disseminaon of anbioc resistance genes among di=erent bacterial species  Genec Variaon and Anbioc Resistance Mutaon – (e.g. drug resistance in tuberculosis) Transformaon/transposion – (e.g. Penicillin-resistant gonorrhea) Conjugaon – (e.g. mul-resistant shigella)  Further Reading Brock Biology of Microorganisms Chapter 10 “Bacterial Genecs”  MICR20010 Lecture 8 Microbial responses to unfavourable environments Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science  Learning Outcomes Environmental conditions Temperature pH Osmolarity Oxygen Spore formation Bacterial Biofilms What are ideal growth conditions for a microbe? A temperature where all their enzymes are folded properly and working at the optimum rate Plenty of food The correct atmosphere for their own type of respiration Available water Environmental conditions dictate microbial growth and the distribution and habitats of microbes Temperature pH Osmolarity (water concentration/availability) Oxygen  Effect of temperature on growth Perhaps the most important environmental factor controlling microbial growth Too hot or too cold can prevent growth However different microbes have evolved to growth at very different temperatures Cardinal temperatures Minimum Optimum Maximum Temperature controls chemical and enzymatic reactions Above the maximum temperature, enzymes and proteins are denatured Below the minimum temperature, the cell membrane may no longer function Cell Membrane is required for nutrient transport and for energy production. Cell Membrane composition is altered depending on growth media – Maximum and minimum temperatures supporting growth are different in rich media versus minimal media  Growth at cold temperatures Organisms adapted for growth at cold temperatures do better when the temperature is constant e.g. deep ocean (approx 2oC, arctic/antarctic waters. Environments with high summer temperatures and cold winter temperatures are less suited to growth. Psychrophiles have an optimal growth temperature of 15oC or lower Found in environments that are constantly cold and rapidly die at room temperature – difficult to isolate and grow in laboratory Enzymes in psychrophiles are denatured/inactivated at even moderate temperature Cold-active enzymes are structurally different to normal enzymes Membrane structure is different in psychrophiles – allows normal nutrient transport at cold temps.  Frozen but not dead Liquid water is required for microbial growth Freezing prevents microbial growth but does not always cause cell death Effects of freezing: 1. Dehydration 2. Ice crystal formation Water-miscible liquids (e.g. glycerol, DMSO – liquids which mix well with water) at a low concentration (10%) are protective: These penetrate the cell and reduce the severity of the effects if freezing Routinely used for storing bacterial cultures at -20oC and -80oC. Can occur to varying degrees in natural environments  Growth at high temperatures Microbial life flourishes at high temperatures up to and including boiling point of water Above 65oC only prokaryotic life (bacteria and archaea) exists Thermophiles – optimum growth temperature >45oC Hyperthermophiles – optimum growth temperature >80oC High temperature environments found in nature are associated with volcanic phenomena – hot springs, hydrothermal vents in deep oceans Archaea are more thermophilic than bacteria Protein/enzyme stability at high temperature Critical amino acid substitutions facilitate heat stable folding Membrane stability at high temperature Alternative membrane composition maintains structure and function DNA stability at high temperature Double stranded DNA molecule usually separates at high temperature In hyperthermophiles, an enzyme called reverse DNA gyrase prevents this from happening. This enzyme is absent in organisms that grow below 80oC. Introduces positive supercoils into DNA, resulting in increased stability  Bacterial DNA Gyrase Heat labile Heat stable  Effect of pH on growth pH refers to the concentration of hydrogen ions (H+) in a solution and is commonly expressed in terms of the pH scale, which is a log scale. A log scale is used because the large variations in H+ ion concentration in different solutions. Low pH corresponds to high hydrogen ion concentration High pH corresponds to low hydrogen ion concentration. pH values are calculated as (-) the log of the H+ concentration (- log is used to get positive values for the pH scale). pH = -log [H+]  Most microorganisms grow best at pH 6 – pH 8 (Neutrophiles) Acidity and alkalinity can greatly affect growth Acidophiles – acid loving Alkaliphiles – alkaline loving Tolerance of extreme pH may depend in part on altered membrane stability Important: Internal cell pH must be close to neutral (between pH 5 – pH 9) even if external pH is very acidic or alkaline At pH extremes – the cell macromolecules (enzymes, proteins, nucleic acid) are destroyed How does Helicobacter withstand Stomach acid?  Effect of osmolarity on growth Water is required for growth of all cells – water is the solvent of life Water availability is dictated not only by how moist or dry an environment is but is also dependent on the concentration of solutes (e.g. NaCl or sugar) in the water Why? Because dissolved solutes have an affinity for water and make it unavailable to the microbial cell Osmosis is the diffusion of water from high water concentration (low solute concentration) to low water concentration (high solute concentration. Controlled in cells by the cytoplasmic membrane  Osmosis Diffusion of water Water molecules diffuse easily across the CM Water molecules associated with other molecules in solution do not Dissolved sugar limits that availability of water to the cell In nature, absence of water inhibits life and thus biologically relevant water availability linked to solute (usually NaCl) concentration Halophiles – NaCl loving Osmophiles – can grow high sugar concentrations Food preservatives: Salt and sugar are commonly used as preservatives to inhibit microbial growth How do microbes grow under conditions of low water availability? NaCl NaCl NaCl NaCl H2O NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl H 2O NaCl NaCl NaCl NaCl Compatible solute H2O NaCl H2O NaCl Compatible solute H2O NaCl H2O NaCl NaCl Cell NaCl Cell NaCl NaCl NaCl H2O Compatible solute H2O NaCl H2O NaCl NaCl Compatible solute H 2O H2 O NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl H2O NaCl NaCl NaCl H2O Increase in internal solute concentration High NaCl, low H2O availability - Increased uptake of H2O Compatible Solutes Microbes in high solute, low water environments Obtain water by increasing intracellular solute concentration Synthesising or accumulating an organic solute This solute must be non-toxic to cellular metabolism – hence called compatible solute Compatible solutes are highly water soluble – thus they “attract” water into cell  Effect of oxygen on growth Because most higher animals require oxygen – doesn’t mean the same is true for microbes O2 is only weakly water soluble – hence many aquatic habitats are anoxic Aerobes – grow at full oxygen tensions (air is 21% O2) Microaerophiles – grow at reduced O2 concentrations (may have oxygen sensitive enzymes Anaerobes - cannot use oxygen for respiration – strict and aerotolerant Facultative anaerobes – can grow in presence or absence of oxygen Oxygen killing bacteria in neutrophils ROS  Bacterial Sporulation Some Gram-positive bacteria can form Spores which provide protection from adverse conditions Spores introduced into a wound site can germinate and cause infection Gram-negative bacteria cannot form spores  Spore Formation Adverse environmental conditions trigger spore formation. The spore is surrounded by a peptidoglycan-rich cortex layer and a keratin-like spore coat.  Spores are resistant to: Heat, Drying, Radiation, Freezing, Toxic chemicals Antibiotics & Can be difficult to eradicate with standard disinfectants. The existence of bacterial spores highlights the need for proper sterilisation - 121oC, 15psi Spores can persist for hundreds and possibly thousands of years, before germinating under the right conditions Although harmless themselves until they germinate, they are involved in the transmission of some diseases to humans including: *anthrax, caused by Bacillus anthracis; *tetanus, caused by Clostridium tetani; *botulism, caused by Clostridium botulinum; and *gas gangrene, caused by Clostridium perfringens  B. anthracis - Anthrax Spores persist in soil - animals and animal products are usual source of human infection Woolsorters were often infected - woolsorters’ disease Disease: Treatment: High fever, bacteraemia Penicillin, ciprofloxacin Massive swelling (oedema) Systemic affects Death Anthrax endospores as bioterrorism agents ? Bacterial Biofilms In all natural environments, microbes grow in complex communities called biofilms. Biofilms offer safety in numbers and provide increased resistance to adverse environmental conditions The majority of bacterial infections treated by clinicians involve biofilms Pseudomonas aeruginasa infections in cystic fibrosis patients Staphylococcus epidermidis catheter related infections Coagulase-negative staphylococci form biofilms or slime on implanted biomaterials and catheters Biofilms form when bacteria adhere to surfaces and excrete slimy glue-like substances which anchor the cells  Why are biofilm infections difficult to treat? Antibiotic Doses 1 1000 Planktonic Biofilm cells cells  Further Reading Brock Biology of Microorganisms Chapter 6 “Microbial Growth” MICR20010 Lecture 9 Theory behind Practical 2 Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science To be carried out in Laboratory: 1. Streaking bacteria onto selective Media (Baird Parker and McConkey Agar) using Streak Plate technique 2. Enumeration of bacteria by serial Dilution Method followed by the Spread Plate technique 3. Personal Hygiene experiment. Extra material to be viewed online with demonstrations in lab: Most Probable Number test for Water Sampling Membrane Filtration The Dye Reduction test Streaking bacteria onto selective Media (Baird Parker and McConkey Agar) using Streak Plate technique SELECTIVE AND DIFFERENTIAL MEDIA Many media have been developed in order to select for certain organisms and to differentiate among them. These media are called selective or differential media. A selective medium is one which favours the growth of certain organisms and represses the growth of others. The choice of a selective medium for a given experiment depends on two factors: o Growth characteristics of the desired organism. o Nature of the material from which it is being isolated. A differential medium is one in which certain bacterial species produce characteristic colony morphology which may be easily recognised. The colonies may be characteristic because of their unusual size, colour or their effect on the immediate environment. Many of the selective media are also differential. In lab 2 we examine 2 commonly used selective and differential media: MacConkey agar and Baird-Parker agar. McConkey Agar MacConkey broth/agar is routinely used for the isolation and differentiation of coliforms and other intestinal bacteria in water, dairy products and biological specimens. The basic ingredients are lactose as sole carbon source, peptone as nitrogen source and bile salts as selective agent. Colonies of organisms capable of fermenting lactose produce a localized drop in pH and the inclusion of the pH indicator, neutral red, causes these organisms to appear as dark red colonies. MacConkey agar allows the isolation and differentiation of lactose fermenting, bile salt tolerant bacteria. McConkey Agar Ingredient g/L Peptone 17 g Polypeptone 3g Lactose 10 g Bile salts 1.5 g Sodium chloride 5g (Agar) (13.5 g) Neutral red 0.03 g Crystal violet 0.001 g Distilled water 1L Final pH 7.1 BAIRD-PARKER AGAR Baird-Parker agar is used principally for the detection and enumeration of staphylococci, particularly coagulase positive organisms such as S. aureus. It is widely used in food safety applications. Lithium chloride and tellurite are included to inhibit many organisms and the presence of glycine and pyruvate enhance staphylococcal growth. Reduction of tellurite yields a grey to black colour and coagulase positive organisms form a clear halo around the colonies. BAIRD-PARKER AGAR Ingredient g/L Casein Peptone 10 g Meat Extract 5g Yeast Extract 1g Lithium Chloride 5g Glycine 12 g Sodium pyruvate 10 g Agar 15 g Final pH (at 25 °C) 6.8±0.2 Cool to 50°C and aseptically add 50ml of Egg Yolk Emulsion and 3ml of Potassium Tellurite 3.5% STREAK BACTERIA ONTO SELECTIVE AND DIFFERENTIAL MEDIA 1. Using the streak plate method carried out in Practical 1 streak one plate of MacConkey agar, with the two organisms provided making sure not to cross-contaminate. 2. Streak one plate of Baird Parker agar, with the two organisms provided making sure not to cross-contaminate. 3. Incubate at 37oC. 4. Photos of plates on brightspace after 48 hours. Note and discuss for practical write up.  Streak each of 2 organisms provided on to 2 different types of media Staphylococcus aureus and Escherichia coli MacConkey agar (1x) and Baird-Parker agar (1x) Plates 1 1 2 2 Half plates for each organism 4 4 3 3 Note: Make sure not to cross-contamination E. coli S. aureus  Selective MacConkey Agar Selects enteric bacteria Bile salts inhibit many bacteria Differential Lactose + pH indicator (Neutral red) Lactose fermenters - Pink Enumeration of Bacteria Serial dilution- agar plate method 0.1 ml Plate counts (CFU/ml) Add sample (milk or contaminated water) 1 ml to diluent (water) Mix Add diluted sample to fresh diluent (fresh tip) Repeat n times Sample Sample H2O H 2O H2O H 2O Spread dilutions on agar plates 9ml 9ml 9ml 9ml Incubate 10-1 10-2 10-3 10-4 Count colonies Diluons Calculate original concentration  Spread plate technique Isolate colonies of interest Counting Enumerate bacteria in a sample of milk Create 10-fold dilution series Use spread Nutrient Agar Plate to detect bacteria in 10-2, 10-3, 10-4 dilutions Calculate number per ml in original sample 10 x ( 288.33 x 1000) (28 X 10000) Personal Hygiene- transfer of microorganisms The potential to transfer the microorganisms through tissue paper Use of hand washing 2  Agar 2 day incubation Agar plate plate 3 day incubation results results Plate 1 Malt Extract Agar Touch plate with Touch Touch unwashed unwashed gloved Glovedfingers fingers glovedFingers fingers to to plate plate Plate 2 #1 #2 Wrap gloved fingers in tissue Remove tissue Wrap gloved fingers in tissue Ppaper and touch plate Paper and touch plate paper aper and touch plate #3 Plate 3 Wrap gloved fingers in tissue Touch Wrap Ppapergloved fingers and touch in tissue plate, remove fingers to plate tissue and then wash with soap provided. Touch  Display for students Dye reduction test Membrane filtration Methylene blue: redox indicator Cellulose acetate or cellulose nitrate: Pore size Strepotcoccus lactis: change from blue to of 0.45 – 0.2 μm colourless upon reduction Bacteria become trapped on the filter, which can be removed and placed on appropriate medium. Advantages: Quick (24 h), field testing, greater reproducibility, etc. Poor milk – decolourised inside 2 h; High quality milk – not decolourised after 8 h. Methylene blue Leuco-Methylene blue  Most Probable Number (MPN) Coliform bacteria are indicators of faecal contamination No. of coliforms in a given water sample can be determined by MPN method double-strength MacConkey broth 2 1 1  MICR20010 Agricultural Microbiology Dr. Tadhg Ó Cróinín Assessments Praccal accounts for 30% 15% on the two Praccal reports to be submi#ed online a$er the praccals. Note these include write ups on praccals as well as online material. 15% on the Praccal Exam online to be held Friday Nov 22nd 2-3pm. 70% on an end of term MCQ exam in the RDS. Microbiology The study of microorganisms. Bacteria/Viruses which cause disease Bacteria which help – anbiocs, probiocs Biotech Industry Why is Microbiology Important? Industrial Microbiology Food and Beverage Industry Health Industry Environmental Microbiology Bacteria and their role in the ecosystem Polluon and Bioremediaon Clinical Microbiology Developing vaccines, anbiocs new treatments Diagnoscs MICR20010 - remaining lectures Lecture 10 – Microorganisms and Disease Lecture 11 – The Immune System Lecture 12 - Pathogenic Bacteria Lecture 13 – Pathogenic Fungi and Viruses Lecture 14 – Anbioc Resistant Microorganisms Lecture 15 – Microbiology in the Food Industry – The Fungi Lecture 16 – Microbiology in the Food Industry - Fermentaons Lecture 17 – The Nitrogen Cycle Microorganisms and Disease Microorganisms play a variety of di?erent roles in disease and we have complex relaonships with these organisms. Mutualism Bene@cial associaons – bacteria providing vitamin precursors in gut Commensalism Passive associaons – non pathogenic Staphylococci Parasism Microorganism causes harm – pathogenic bacteria Key is understanding the relationship How do we prove a pathogen Koch’s postulates 1. The m/o must be present in the diseased and not in a healthy animal 2. M/O must be cultivated in pure culture 3. Pure culture inoculated into 2nd animaldisease 4. Pure culture from 2nd animal should be same as 1st. Koch’s Postulates What about exceptions?  Some pathogens difficult to culture.  Some diseases are caused by combinations of  Pathogens  Physical, environmental  Genetic factors  Animal models and ethics: inoculation of healthy susceptible host not always possible (the postulate could never be fully applied to HIV) Virulence Factors A key di?erenal in pathogens is the presence of virulence factors which help the organisms cause disease. Evoluon allows these toxins to o$en be host speci@c Toxins which have very speci@c targets Adhesins which recognise speci@c receptors Other less so – Endotoxins (e.g. LPS) Virulence factors can thus de@ne host speci@city  Extracellular Enzymes Secreon of enzymes allows microorganisms to alter their environment Avoiding the Immune system– Some blood borne pathogens have the ability to secrete coagulase which leads to coagulaon allowing microorganisms to form clots which in turn can provide a physical hiding place from the immune system. Leukocidins can be used to destroy white blood cells. Catalase can be used to protect from reacve oxygen species in the Macrophage  Toxins  Exotoxins 1. Cytotoxins: kills or affects the functions of host cells 2. Neurotoxins: interferes with nerve cells 3. Enterotoxins: affects cells lining gut tract (clostridia, pathogenic strains of S.aureus and E.coli) Cytotoxins Neurotoxins Clostridum botulinum and Clostridium tetanii secrete extremely potent neurotoxins which lead to two very di?erent forms of fatal paralysis Related toxins Botulinum toxin inhibits the release of acetylcholine which smulates contracon therefore leading to relaxaon Tetanus toxin inhibits the release of Glycine which induces relaxaon of a contracted muscle which thus leads to contracon. Tetanus not a food poisoning toxin! Enterotoxins Clostridium perfringens secretes an enterotoxin which can induce gastroenters on its own Inoculaon with the toxin alone has the same e?ect as inoculang with the bacteria Virulence vs Colonization factors A virulence factor is directly involved in causing disease Toxins etc A colonizaon factor may be necessary for disease to progress but is not directly involved Adhesins and Jagella for molity Clostridium perfringens entertoxin (CPE) clearly a virulence factor Endotoxins Primarily found in Gram negave organisms Key is the constuents of the Outer membrane Lipopolysaccharide (LPS) Released on cell death or through membrane blebbing but has dramac e?ect on Immune system Anti-phagocytic factors  Capsules: many are made of chemicals found in body : no immune responses made  Anti-phagocytic compounds: some m/o make compounds that prevent fusion of lysosomes with phagocytic vesicles… (Neisseria gonorrhea)  Previously mentioned enzymes such as catalase Immune Evasion Phagocytosis blocked by capsule Surviving phagocytosis How is disease transmitted Microorganisms do not simply appear They can be already present and taking advantage of a change in environment – Opportunisc pathogens S. aureus commonly found on skin but pathogenic in blood infecons C. dicile takes advantage of anbioc treatments changing microbiome How are organisms transmi#ed - epidemiology Modes of Transmission A. Contact transmission Direct contact-person to person Indirect contact-needles, toothbrushes Droplet transmission- spread via droplet nuclei B. Vehicle transmission Air, drinking water, food C. Vector transmission Biological and mechanical Basic protections from infection Skin: barrier…tight layer of packed cells…entrance through cuts Mucous membranes: that line the body cavities open to the outside world (nose etc..) To infect: adherence of parasite to cells to allow for establishment of colonies Adaptive immunity Acquired immunity Develops from birth, as we encounter various pathogens Antigens trigger specific response Components of bacterial cells Cell walls, capsules Flagella Proteins (internal + external) Toxins Food may have antigens that provoke allergic reactions What are Antigens? Properties of antigens Antigens recognised by antigenic determinants (epitopes) >5-100KDa better than smaller antigens Proteins, glycoproteins etc…