Lec 3.pdf

PowerPoint® Lecture Presentations CHAPTER 2&3 2.I Microscopy 3.I Laboratory culture of microorganisms © 2018 Pearson Education, Inc. E. coli on the point of a pin Observing microbes • Microscopes magnify an image and increase resolution • Resolution = Ability to distinguish two adjacent obje...

PowerPoint® Lecture Presentations CHAPTER 2&3 2.I Microscopy 3.I Laboratory culture of microorganisms © 2018 Pearson Education, Inc. E. coli on the point of a pin Observing microbes • Microscopes magnify an image and increase resolution • Resolution = Ability to distinguish two adjacent objects as distinct and separate • Better resolution = more detail • Resolution of human eye = ~100 µM Magnification alone Magnification + enhanced resolution Compound light microscope • Uses visible light + two lenses (objective + ocular) • Light source is focused on the specimen by a condenser Magnification & Resolution • Resolution depends on the wavelength of light and the light-gathering ability of the lens • Immersion oil increases light that enters lens • Best resolution with light microscope = ~0.2 µM • Total magnification = objective magnification x ocular magnification • Upper limit of about 2000X Types of light microscopy • Bright-field • Specimens are visualized because of slight differences in contrast between them and the surroundings • Typically difficult to see bacterial cells, unless the organisms are pigmented Staining • Stains (dyes) can be used to increase contrast for brightfield microscopy • Many dyes are positively charged (basic) • Methylene blue, crystal violet, safranin • Bind to negatively charged cell components (nucleic acids, many proteins, polysaccharides, cell surfaces) Differential stains • Stain different kinds of cells different colours • Most common: Gram stain • Divides Bacteria into two major groups: • gram-positive (purple) • gram-negative (pink) • Arises due to difference in cell wall structure Gram positive Gram negative Acid Fast Stain • Used to identify acid-fast organisms • Mycolic acid is attached to cell wall surface, producing wax-like hydrophobic coating • e.g. Genus Mycobacterium (M. tuberculosis and M. leprae cause tuberculosis and leprosy) • Initial stain (red) penetrates and is retained by mycolic acids, other cells will destain and take up counterstain (blue) Types of light microscopy • Phase-contrast • Different materials slow light by different amounts • A phase ring amplifies these subtle differences, to increase contrast between background and specimen • Darker cells on a light background • Dark-field • Central portion of light beam is blocked so light reaches specimen from sides only • Only light scattered by the specimen reaches the lens • Light cells on a dark background Bright-field Phase-contrast Dark-field Fluorescence microscopy • Fluorescence = emission of light of one colour after absorbing light of another colour • Occurs due to excitation of electrons to a high-energy state • Cells may fluoresce due to naturally fluorescent substances • e.g. Chlorophyll emits red fluorescence • Alternatively, cells can be stained with a fluorescent dye • e.g. DAPI binds with DNA and emits blue light Cyanobacteria bright-field Cyanobacteria fluorescence E coli DAPI Differential Interference Contrast (DIC) • Uses a polarizer and prism to generate two distinct beams of light that pass through the specimen then are recombined • Enhances subtle differences in cell structure, giving cells a more 3-dimensional appearance • Endospores, vacuoles, granules and nuclei are more visible Yeast, bright-field Yeast, DIC Paramecium, DIC Confocal scanning laser microscopy • Uses a computerized microscope coupled with a laser source to generate a three-dimensional image • Computer can focus the laser on single layers of the specimen • Different layers can be compiled to construct a three-dimensional image • Allows for observation of cells in different layers in a bacterial biofilm • Resolution is improved to 0.1 μm Confocal image of a microbial biofilm. Cells are stained with fluorescent dyes. Different colour cells are at different depths. Electron microscopy • Uses electrons instead of visible light (photons) to image cells and cell structures • Wavelength of electrons is much shorter than visible light, resulting in greatly improved resolution • Electrons are poor at penetrating • A single cell is too thick • To view internal cell structures thin sections are needed • Two types: • Transmission electron microscope • Scanning electron microscope Transmission electron microscopy • Electron beam passes through ultrathin tissue sections of cells (20 – 60 nm slices), or very small specimens (virus, protein) • Samples are first stained with high-atomic weight stains • Absorb energy from the electron beam • Electrons will more freely pass through areas that bind less stain • After passing through the specimen the electrons strike a screen and produce an image • Resolution of 0.2 nm (1000 fold improvement) Cytoplasmic Cell wall DNA membrane (nucleoid) Scanning electron microscopy • Specimen is coated with a thin film of heavy metal • Electron beam scans back and forth across the specimen • Scattered electrons are collected and projected onto a monitor to produce an image • Used to view the surface of an object • Produces black and white images but false colour may be added Laboratory culture • To culture microbes in a laboratory, it is necessary to supply all nutrients required for growth • Nutrients are compounds of chemical elements that can be used by the cell to support growth and metabolism • Macronutrients are required in large amounts • Micronutrients are required in trace amounts (Typically <0.01% total mass) Culture media • Nutrient solution used to grow microorganisms in the laboratory • Defined media contain precise amounts of pure chemicals in distilled water • Exact composition is known • Complex media are made from digests of microbial, animal, or plant products • e.g. casein (milk protein), beef extract, tryptic soy broth (soybeans), yeast extract, or other highly nutritious substances • Nutritional composition is not precisely known Culture media • Selective media contains compounds that inhibit the growth of some microorganisms but not others • Differential media contains compounds or additives that allow microorganisms to be distinguished by the appearance of the colony or surrounding media E. Coli (left) and P. aeruginosa (right). Eosin-methylene blue agar Selective: Inhibits growth of Gram positive, but supports growth of Gramnegative organisms. Differential: Lactose fermenting organisms (e.g. E. coli) have green metallic sheen. Culture media • Enriched media is used for nutritionally demanding microbes • Complex media plus additives such as serum or blood • e.g. Chocolate agar includes heat-lysed blood, which produces browncoloured plates • e.g. Blood agar includes ~5% blood (unlysed) • Both enriched and differential – distinguishes between hemolytic & nonhemolytic bacteria Francisella tularenis on chocolate agar Staph aureus (hemolytic) on blood agar Staph saprophyticus (nonhemolytic) on blood agar Laboratory Culture • Culture media must be prepared & sterilized before use • Heating in an autoclave • Liquid culture media are solidified by the addition of 1 – 2% agar & poured into sterile petri plates • Solid media immobilizes cells, allowing them to grow and form isolated masses called colonies • Colonies are various shapes and sizes depending on the organism, culture conditions, nutrient supply, etc. • Some colonies are coloured due to production of pigments Aseptic technique • Aseptic transfer technique must be used to prevent contamination of sterile objects or microbial cultures during handling Making a streak plate • Produces isolated colonies on an agar plate • Primarily used to obtain pure cultures from a sample containing several different microorganisms Subsequent streaks are at angles to the first streak. 1. Loop is sterilized and a loopful of inoculum is removed from tube. Isolated colonies at end of streak 1 2 5 3 4 2. Initial streak is worked in well in one corner of the agar plate. Confluent growth at beginning of streak 3. Appearance of well-streaked plate after incubation shows colonies of the bacterium Micrococcus luteus on a blood agar plate. Making a spread plate • Alternative to streak plate • Sample must be appropriately diluted to obtain isolated colonies