Week 2 LO2 Observing Microorganisms PDF

Introduction to Microbiology SCIMA2-B22 Eduvos (Pty) Ltd (formerly Pearson Institute of Higher Education) is registered with the Department of Higher Education and Training as a private higher education institution under th...

Introduction to Microbiology SCIMA2-B22 Eduvos (Pty) Ltd (formerly Pearson Institute of Higher Education) is registered with the Department of Higher Education and Training as a private higher education institution under the Higher Education Act, 101, of 1997. Registration Certificate number: 2001/HE07/008  Recap: What was covered in week 1? Ways in which microbes affect our lives Scientific nomenclature Major characteristics for groups of microbes Spontaneous generation vs biogenesis Fields of microbiology Golden age history of Microbiology - Scientific discoveries and inventions Pasteur’s, Lister’s and Koch’s notable works Microbes in biotechnology Emerging infectious diseases …........ 1. Who proposed the system of scientific nomenclature? a. Edward Jenner b. Carolus Linnaeus 2. Which of the following is not a characteristic of algae? a. Have cell walls composed of chitin b. Are able to photosynthesize c. Exhibit both sexual and asexual reproductive forms d. Inhabit water, soil and plants e. Are protists 3. Which of the following is the best definition of biogenesis? a. Non-living matter gives rise to living organisms b. Living cells can only arise from preexisting cells c. A vital force is necessary for life d. Air is necessary for living organisms e. Microorganisms can be generated from non-living matter  What will be covered Week 2 in today’s lesson? Lesson 1 Units of measurement LO2: Methods of Parts of light compound light observing microscope microorganisms Types of light microscopy Electron microscopy Staining techniques Resources for this lesson: Tortora et al. (Chapter 3) Lecture slides myLMS notes - Including associated videos Q&A Acid-fast staining of a patient’s sputum is a rapid, reliable, and inexpensive method to diagnose tuberculosis. What colour would bacterial cells appear if the patient has tuberculosis? 6 Units of Measurement Learning Objective 3-1 List the metric units of measurement that are used for microorganisms. 7 Units of Measurement 1 µm = 10–6 m = 10–3 mm 1 nm = 10–9 m = 10–6 mm 1000 nm = 1 µm 0.001 µm = 1 nm 8 Figure 3.2 Units of Measurement Refer to table 3.1 in textbook 9 Observing Microorganisms 10  If a microbe measures 10 μm in length, how long is it in nanometers? 3-1 11 Microscopy: The Instruments Learning Objectives 3-2 Diagram the path of light through a compound microscope. 3-3 Define total magnification and resolution. 12 Microscopy: The Instruments A simple microscope has only one lens Pioneered by Antonie van Leeuwenhoek 13 Figure 1.2b Microscopy: The Instruments Compound microscope: Makes use of multiple lenses Robert Hooke https://www.nagwa.com/en/explainers/ 754176579570/ 14 Light Microscopy Use of any kind of microscope that uses visible light to observe specimens Types of light microscopy: 1- 2- 3- 4- 5- 6- 15 The Compound Light Microscope Figure 3.1a 16  The Compound Light Microscope – Objective Lenses 4x: 10x: 40x: 100x: Adapted from: https://microbenotes.com/objective-lenses/ 17  Compound Light Microscopy In a compound microscope, the image from the objective lens is magnified again by the ocular lens Total magnification = ? 20 Figure 3.1b Compound Light Microscopy Resolution is _____________________ A microscope with a resolving power of 0.4 nm can distinguish between two points at least 0.4 nm apart Shorter wavelengths of light provide greater resolution General principle: The shorter the wavelength, the greater the resolution. For compound light microscopes: ~0.2µm resolution. 22 Compound Light Microscopy The refractive index is _______________________________ The light may bend in air (refract) so much after passing through a specimen that it misses the objective lens Immersion oil is used to keep light from refracting 24 Refraction in the Compound Microscope Figure 3.3 25  Through what lenses does light pass in a compound microscope? 3-2 What does it mean when a microscope has a resolution of 0.2 nm? 3-3 26 Microscopy: The Instruments Learning Objectives 3-4 Identify a use for darkfield, phase-contrast, differential interference contrast, fluorescence, confocal, two-photon, and scanning acoustic microscopy, and compare each with brightfield illumination. 3-5 Explain how electron microscopy differs from light microscopy. 3-6 Identify one use for the TEM, SEM, and scanned-probe microscopes. 27 Light microscopy - Brightfield Illumination Dark objects are visible against a bright background Light reflected off the specimen does not enter the objective lens Internal structures and external covering visible Uses? 28 Figure 3.4a Light microscopy – Darkfield Illumination Light objects are visible against a dark background Opaque disk placed in darkfield condenser Only light reflected off the specimen enters the objective lens Due to no background light, specimen appears light against dark background Uses? 29 Figure 3.4b Light microscopy – Phase contrast Allows examination of living organisms and internal cell structures. Brings together two sets of light rays, direct rays, and diffracted rays (from the specimen) to form an image. Two sets of light rays form singular image on ocular lens – light to grey to dark. Uses? 31 Figure 3.4c Light microscopy – Differential interference contrast (DIC) Similar to phase-contrast Uses two light beams and prisms to split light beams, giving more contrast and color to the specimen (higher res) Uses? 32 Figure 3.5 Light microscopy – Fluorescence microscopy Uses UV (short wavelength) light Fluorescent substances absorb UV light and emit longer wavelength (visible) light Cells may be stained with fluorescent dyes (fluorochromes) if they do not naturally fluoresce Appear as bright objects against dark background Immunoflourscence Figure 33 3.6b Light microscopy – Fluorescence microscopy Figure 3.6 Light microscopy – Confocal microscopy Cells stained with fluorochrome dyes. Short wavelength (blue) light used to excite the dyes. The light illuminates each plane in a specimen to ultimately produce a three-dimensional image. Due to use of pinhole aperture, blurring is reduced. Figure 3.7 Uses? Two-Photon Microscopy Cells stained with fluorochrome dyes Two photons of long- wavelength (red) light are used to excite the dyes Can study living cells up to 1 mm deep (comparison to confocal?). Advantages of TPM? 36 Figure 3.8 Scanning Acoustic Microscopy (SAM) Measures activity of sound waves that are reflected back from a specimen Resolution: 1 µm Uses? Figure 3.10 37 Electron Microscopy Uses beam of electrons instead of light. The shorter wavelength of electrons gives greater resolution. Used for images too small to be seen with light microscopes, such as viruses. Produces images in black and white – coloured artificially. Instead of glass lenses – electromagnetic lenses. Two types – transmission (TEM) and scanning (SEM) 38 Transmission Electron Microscopy (TEM) Ultrathin sections of specimens. A beam of electrons passes through ultrathin sections of a specimen, then through an electromagnetic lens, then focused on a projector lens. Specimens may be stained with heavy-metal salts for contrast - why? Name of image? 39 Figure 3.10a Transmission Electron Microscopy (TEM) 10,000–100,000; resolution 2.5 nm 40 Figure 3.11a 41 Scanning Electron Microscopy (SEM) An electron gun produces a beam of electrons that scans the surface of a whole specimen Secondary electrons emitted from the specimen produce a three-dimensional image 42 Figure 3.11b Scanning Electron Microscopy (SEM) 1,000–10,000 Resolution 20 nm 43 Figure 3.11b 44 TEM vs SEM TEM SEM Image created Magnification Resolution Sectioning required? Application 45 Scanned-Probe Microscopy Uses probes to examine specimen’s surface using electric current. Used to map molecular and atomic shapes, characterize magnetic and chemical properties and determine temperature variations. STM and AFM. 46 Scanning Tunneling Microscopy Scanning tunneling microscopy (STM) uses a tungsten probe to scan a specimen and reveal details of its surface. Resolution 1/100 of an atom. No specimen preparation required. 47 Figure 3.12a Atomic Force Microscopy Atomic force microscopy (AFM) uses a metal- and-diamond probe placed onto a specimen. Used to observe biological processes. Produces three-dimensional images Figure 3.12b 48 Other Microscopes (Not for Studying) Super-Resolution Microscope 49 Other Microscopes (Not for Studying) Optical Frequency Domain Imaging System 50 Other Microscopes (Not for Studying) Digital Scanned Laser Light Sheet Fluorescence Microscope 51 Other Images (Not for Studying) 52 Other Images (Not for Studying) 53 Other Images (Not for Studying) 54 Other Images (Not for Studying) 55 Other Images (Not for Studying) 56 Images from: http://www.businessinsider.com/miscroscope-images-nikon-small-world- 2015-10?op=1 57 Check Your Understanding How are brightfield, darkfield, phase-contrast, and fluorescence microscopy similar? 3-4 Why do electron microscopes have greater resolution than light microscopes? 3-5 What is TEM used for? SEM? Scanned-probe microscopy? 3-6 58 Class Break Preparation of Specimens for Light Microscopy Learning Objectives 3-7 Differentiate an acidic dye from a basic dye. 3-8 Explain the purpose of simple staining. 3-9 List the steps in preparing a Gram stain and describe the appearance of gram-positive and gram-negative cells after each step. 3-10 Compare and contrast the Gram stain and the acid-fast stain. 3-11 Explain why each of the following is used: capsule stain, endospore stain, flagella stain. 60 Preparing Smears for Staining Live or unstained cells have little contrast with the surrounding medium. Live specimens are used to study cell behavior. 61 Figures B and C Preparing Smears for Staining Staining: Coloring the microbe with a dye that emphasizes certain structures. Smear: A thin film of a material containing microorganisms spread over a slide. A smear is usually fixed (attached) to the slide, which kills the microorganisms. Preserves various parts of microbes in their natural state with minimal distortion. 62 Preparing Smears for Staining Smears are air-dried before passing through a flame, or by covering slide for methanol for 1 minute. Stain is applied, washed off with water, the slide is then blotted with absorbent paper. Without prior fixing, the stain would wash the microorganisms off of the slide. 63 Preparing Smears for Staining Stains consist of a positive and a negative ion, one of which is coloured (the chromophore). In a basic dye, the chromophore is a cation (+). In an acidic dye, the chromophore is an anion (-). Which dye would be best suited for bacteria? Staining the background instead of the cell is called negative staining. Three types of staining techniques – simple, differential, special. 64 Simple Stains Simple stain: Aqueous or alcoholic solution of a single basic dye. Highlights the entire microorganism to visualize cell shapes and structures. E.g.’s: methylene blue, carbolfuhsin, crystal violet and safranin. Mordant: chemical added to intensify the stain: Increase affinity of stain Coat structures to make it easier to see (e.g.: flagellum) 65 Differential Stains Used to distinguish between bacteria: 1. Gram stain 2. Acid-fast stain 66 Gram Stain Classifies bacteria into Gram-positive or Gram-negative. Gram-positive bacteria have thick peptidoglycan cell walls Gram-negative bacteria have thin peptidoglycan cell walls and a layer of lipopolysaccharides 67 Figure 3.13 a Gram staining. KEY Crystal violet Iodine Alcohol Safranin Gram-positive Gram-negative Application of Application of Alcohol wash Application crystal violet iodine (mordant) (decolorization) of safranin (purple dye) (counterstain) Gram Stain Gram + cell walls: Thick layer of peptidoglycans (disaccharides and amino acids) Gram – cell walls: Thin layer of peptidoglycans, layer of liposaccharides CV and iodine form CV-I complex after entering cells. Cannot be washed out of thick layer of peptidoglycans in Gram + cells. Alcohol wash removes liposaccharides from Gram _ cells: CV-I complex can exit cells. Gram – cells remain colourless until counter-stained with safranin. 69 Gram Stain Color of Color of Gram-positive cells Gram-negative cells Primary stain: Crystal violet Mordant: Iodine Decolorizing agent: Alcohol-acetone Counterstain: Safranin 70 Micrograph of Gram-Stained Bacteria 71 Figure 3.12b  72 https://universe84a.com/hucker-modification-gram-stain/#google_vignette Check Your Understanding Why doesn’t a negative stain colour a cell? 3-7 Why is fixing necessary for most staining procedures? 3-8 Why is the Gram stain so useful? 3-9 73 Acid-Fast Stain Binds only to bacteria that have a waxy material in their cell walls, which is not decolorized by acid-alcohol. Used for the identification of: Mycobacterium tuberculosis, Mycobacterium leprae Nocardia Mycobacterium tuberculosis 74 Acid-Fast Stain Color of Color of Acid-fast Non–Acid-fast Primary stain: Carbolfuchsin Decolorizing agent: Acid-alcohol Counterstain: Methylene blue 76 Acid-Fast Bacteria Figure 3.14 77 Q&A Acid-fast staining of a patient’s sputum is a rapid, reliable, and inexpensive method to diagnose tuberculosis. What color would bacterial cells appear if the patient has tuberculosis? 78 Special Stains Used to distinguish parts of cells 1. Capsule stain 2. Endospore stain 3. Flagella stain Salmonella typhi https://www.researchgate.net/figure/Flagellar-stain-of-a-Salmonella- Typhi-Like-E-coli-Salmonella-are-motile-by-means-of_fig26_290911856 79 Negative Staining for Capsules Capsules are a gelatinous covering that do not accept most dyes. Presence of capsule is a means of determining microorganism’ virulence. Suspension of India ink or nigrosin contrasts the background with the capsule, which appears as a halo around the cell 80 Figure 3.15a Endospore Staining Endospores are resistant, dormant structures inside some cells that cannot be stained by ordinary methods. Primary stain: malachite green, usually with heat. Decolorize cells: water Counterstain: safranin Figure 3.15b Spores appear green within red or pink cells 81 Flagella Staining Flagella are structures of locomotion. Uses a mordant and carbolfuchsin. Tedious and delicate staining procedure to build up diameters until flagella are visible. 82 Figure 3.15c Check Your Understanding Which stain would be used to identify microbes in the genera Mycobacterium and Nocardia? 3-10 How do unstained endospores appear? Stained endospores? 3-11 83  QUIZ TIME!!! Microscopes | 57 plays | Quizizz Lab 8 Endospore Staining | 310 plays | Quizizz 84 Questions 1. Which type of microscope would be best to observe each of the following: a. A stained bacterial smear b. Unstained bacterial cells (cells are small, no detail is needed) c. Unstained live tissue when it is desirable to see intracellular detail d. A sample that emits light when illuminated with UV light e. Intracellular detail of a cell that is 1µm long f. Unstained live cells in which intracellular structures are shown in colour Questions 2. How would skipping the alcohol-washing step in Gram staining affect the identification of Gram-negative bacteria? 3. Why is staining useful in brightfield microscopy but not in darkfield microscopy?  What’s next? 1. Review notes and readings for LO2. 2. Prepare readings for LO3.