MICR20010 Lecture 3 2023.pptx

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 In...

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 Electron Microscope Light Microscopy 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 • No nucleus • All have cell wall • No cell organelles Eukaryotic Cell • Nucleus • Some have cell wall, many do not • 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”