BIOL 203 – Chapter 04 - Microscopy, Staining, & Classification PDF

CHAPTER 04 – MICROSCOPY, STAINING, AND CLASSIFICATION CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 1 LEARNING OUTCOMES 4.1 Identify the two primary metric units used to measure the diameter of microbes 4.2 List the metric units of length in order, from meter to nanometer 4.2.1 Convert...

CHAPTER 04 – MICROSCOPY, STAINING, AND CLASSIFICATION CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 1 LEARNING OUTCOMES 4.1 Identify the two primary metric units used to measure the diameter of microbes 4.2 List the metric units of length in order, from meter to nanometer 4.2.1 Convert between different metric units of length 4.3 Define microscopy 4.4 Explain the relevance of electromagnetic radiation to microscopy 4.5 Define empty magnification 4.6 List and explain two factors that determine resolving power 4.7 Discuss the relationship between contrast and staining in microscopy 4.8 Describe the difference between simple and compound microscopes CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 2 LEARNING OUTCOMES 4.9 Compare and contrast bright-field microscopy, dark-field microscopy, and phase microscopy 4.10 Compare and contrast fluorescence and confocal microscopes 4.11 Contrast TEMs with SEMs in terms of how they work, the images they produce, and the advantages of each 4.12 Describe two variations of probe microscopes 4.13 Explain the purpose of a smear, heat fixation, and chemical fixation in the preparation of a specimen for microscopic viewing 4.14 Describe the uses of acidic and basic dyes, mentioning ionic bonding and pH CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 3 LEARNING OUTCOMES 4.15 Describe the simple, Gram, acid-fast, endospore, Gomori methenamine silver, and hematoxylin and eosin and negative capsule stains and procedure if listed 4.16 Explain how stains used for electron microscopy differ from those used for light microscopy 4.17 Describe the purposes of taxonomy 4.18 Discuss the difficulties in defining species of microorganisms 4.19 List the hierarchy of taxa from general to specific 4.20 Define binomial nomenclature 4.21 Describe a few modifications of the Linnaean system of taxonomy CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 4 LEARNING OUTCOMES 4.22 List and describe the three domains proposed by Woese and Fox 4.23 Describe five procedures taxonomists use to identify and classify microorganisms 4.24 Use a dichotomous key to identify an unknown microorganism 4.25 Describe the common shapes and arrangements of bacterial cells CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 5 UNITS OF MEASUREMENT The most useful units of measurement for microbes are micrometers (µm) and nanometer (nm) The prefixes can be applied to other measurements as well. Examples: 1 liter (L) is equivalent to 1,000,000 microliters (µl) CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 6 UNITS OF MEASUREMENT How wide is the edge of this penny? Describe it in: cm Centimeters Millimeters Micrometers Nanometers Actual approximate width! CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 7 UNITS OF MEASUREMENT 1 millimeter 1 centimeter 1 decimeter 1 meter The difference between 1 m and 1 mm is a thousand-fold. Now consider that there is a thousand-fold difference between mm and µm, and between µm and nm… CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 8 MICROSCOPY Microscopy: the use of light or electrons to magnify objects General principles involved: 1. Wavelength of radiation 2. Magnification of the image 3. Resolving power of the instrument 4. Contrast of the specimen CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 9 WAVELENGTH OF RADIATION Ninja Light is electromagnetic radiation that travels in waves Wavelength (λ): distance between two corresponding parts of a wave To be seen, an object must interfere with the waveform of light Lower wavelengths of light increase resolution CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 10 MAGNIFICATION This is a cell Empty magnification Magnification: ratio of the object’s image size to size in real life Empty magnification: magnification where image is larger, but faint and blurry. Magnification requires resolution and contrast to be useful. CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 11 LENSES Lenses refract light because they are optically dense – light passes through it more slowly than air Images are inverted, reversed, and enlarged CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 12 RESOLUTION This is a high resolution image of a cell Same magnification, different resolutions (trust me!). It’s just really, really small so you can’t see it. Resolution: The minimum distance that two points can be separated and still be distinguished as separate points The smaller this distance is, the greater the resolving power CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 13 ABBÉ EQUATION 0.61 𝜆𝜆 𝑟𝑟 = 𝑁𝑁𝑁𝑁 r = resolution 0.61 = A constant which represents the degree to which two image points can overlap and still be recognized as separate λ = wavelength of light Numerical aperture (NA): Measure of the ability of a lens to gather light. Larger values equate to better resolutions Light microscopy limit of resolution ~ 200nm CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 14 IMMERSION OIL Increases the numerical aperture (and thus the resolving power) by preventing the refraction of light between the glass-air interface Allows more light to be gathered Optical density of glass and immersion oil is the same CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 15 CONTRAST Low and high contrast Contrast: the difference in brightness between the light and dark parts of an image Scientist use stains to label specific structures and increase contrast CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 16 LIGHT MICROSCOPY Light X Simple Compound Bright-field microscopes: Background is illuminated, specimen is darker. Why? Simple: Use a single magnifying glass Compound: Use a series of magnifying lenses CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 17 COMPOUND BRIGHT-FIELD MICROSCOPES Total magnification = ocular magnification x objective magnification You use a compound microscope that has a 10x ocular and 40x objective lens in place. What is the total magnification? CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 18 DARK-FIELD MICROSCOPES Dark-field microscopes: Dark background with a lighter specimen Useful for pale specimens Light is prevented from directly entering the objective Only light scattered by specimen enters the objective CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 19 PHASE MICROSCOPES Phase Contrast Phase contrast microscopes: Uses a phase plate to alter the phase of the background light, increasing contrast DIC Differential interference contrast (DIC) microscopes: Increase contrast and give a 3D shadowed appearance. Uses a polarizer and prisms. CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 20 FLUORESCENCE MICROSCOPY Yersinia pestis Fluorescence microscopy: Microscopy using fluorescent dyes that absorb light at one wavelength (excitation) and give off light at a longer wavelength (emission) Immunofluorescence: link fluorescent dyes to antibodies CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 21 CONFOCAL FLUORESCENCE MICROSCOPY Confocal Fluorescence Microscopy Fluorescence Microscopy Light from all planes Only light from one of focus seen focal plane seen Confocal fluorescence microscopy: Prevents blurry images resulting from the presence of fluorescent structures above and below the plane of focus. CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 22 OUT OF FOCUS LIGHT CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 23 ELECTRON MICROSCOPES Capable of magnifying millions of times – limit of resolution ~0.001 nm depending on type of microscope Ultrastructure: cellular structures only visible using electron microscopy Two general types: 1. Transmission electron microscopes (TEM) 2. Scanning electron microscopes (SEM) CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 24 TRANSMISSION ELECTRON MICROSCOPES (TEM) Similar to light microscopy except uses electrons instead of light, electromagnetic lenses instead of glass lenses Dense areas of the specimen block electrons. Brightness corresponds to electrons hitting a screen Requirements: Specimen must be very thin (or sliced!) Must be performed in a vacuum Can’t be used for living organisms* CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 25 SCANNING ELECTRON MICROSCOPES (SEM) Uses electrons in a vacuum tube but electrons scan the surface of the specimen coated with a metal. Scattered electrons picked up by a detector. Whole specimens can be observed, creates a 3D image Requirements: Must be performed in a vacuum Can’t be used for living organisms* Scans the surface CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 26 PROBE MICROSCOPY Use tiny, pointed electronic probes Can magnify more than 100,000,000x Two variations: https://www.youtube.com/watch?v=GuCdsyCWmt8 1. Scanning Tunneling Microscope (STM) 2. Atomic Force Microscope (AFM) CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 27  SCANNING TUNNELING MICROSCOPE (STM) Probe Uses a probe sharpened to a single atom tip that sweeps just above surface of the specimen Electron flow to and from the specimen’s Electrons surface measured (tunneling current). Sample Proportional to distance to surface Measures distances as small as 0.01nm! Specimen must be electrically conductive More current Tunneling current: CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 28  SCANNING TUNNELING MICROSCOPE (STM) Probe Electrons Sample More current Tunneling current: https://www.rhk-tech.com/scanning-tunneling-microscopy- observation-phonon-condensate/ CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 29 ATOMIC FORCE MICROSCOPY (AFM) Uses a probe that traverses the surface of a Laser Detector specimen Vertical probe movements are detected by a laser shining at the probe Probe Specimen does not need to be conductive Sample No electron beam, no vacuum required Stage CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 30 ATOMIC FORCE MICROSCOPY (AFM) Laser Detector Probe Sample Stage https://link.springer.com/article/10.1007/s40094-015-0187-3/figures/4 CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 31 WHAT KIND OF MICROSCOPY? A B C D Virus 1. Which type of microscopy is shown in each of the pictures above? 2. What type of microscopy would you use if you wanted to visualize the location of a particular protein inside of a human epithelial cell? Be as specific as possible. CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 32 PREPARING SPECIMENS FOR STAINING Staining: colouring specimens with stains (dyes). Improves contrast. Smear: a thin film of organisms on a slide Sample in liquid: drop liquid on a slide Sample on solid medium: add a drop of water on a slide. Obtain culture from plate, and mix with the water Allow slide to air dry CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 33 FIXATION Fixation Kills the specimen Attaches specimen to the slide Preserves shape and size Heat fixation: pass slide through a flame (smear side up!) Chemical fixation: a chemical, like methyl alcohol is applied on the slide for 1 minute CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 34 DYES Cation Anion Methylene Blue Most dyes are salts, composed of a cation (positively charged) and an anion (negatively charged). At least one of the ions is coloured. Coloured portion is known as the chromophore CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 35 DYES Basic Dyes Acidic Dyes Stain acidic structures Stain alkaline structures Cationic chromophores Anionic chromophores Work best under basic conditions Work best under acidic conditions What kind of dye would I use to stain: 1. DNA? 2. A positively charged protein? 3. A negatively charged protein? CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 36 EFFECT OF PH Aspartate at pH 7 Aspartate at pH 2 H H Henderson-Hasselbalch Equation: H3N+ C COO- H3N+ C COOH − 𝐴𝐴 𝑝𝑝𝑝𝑝 = 𝑝𝑝𝑝𝑝𝑎𝑎 + 𝑙𝑙𝑙𝑙𝑙𝑙10 ( ) 𝐻𝐻𝐻𝐻 CH2 CH2 COO- COOH Overall charge: -1 +1 pH can have a large impact on the ability of a dye to function Changes in pH alter the protonation status of ionizable groups on molecules (like amino acids) which can affect their charge and thus the ability of a dye to form ionic bonds with it CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 37 SIMPLE VS DIFFERENTIAL STAINS Unstained Simple Stain Differential Stain Simple stains: are composed of a single dye (usually basic) Examples: crystal violet, safranin, methylene blue Differential stains: are composed of more than one dye to distinguish between different cells, chemicals or structures. Examples: Gram stain, acid-fast stain, endospore stain, Gomori methenamine silver stain, hematoxylin & eosin stain CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 38 GRAM STAIN Differentiates between Gram + and - cells General steps: 1. Flood slide with crystal violet (CV) for 1 minute. Rinse with water. Result: CV acts a primary stain; all cells are stained purple Primary stain: initial dye which colours all cells CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 39 GRAM STAIN 2. Flood slide with iodine for 1 minute. Rinse with water. Result: iodine acts as a mordant; all cells remain purple Mordant: substance that binds to a dye to make it less soluble CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 40 GRAM STAIN 3. Slide rinsed with 95% ethanol for 10-30 seconds. Rinse with water. Result: Ethanol acts as a decolourizing agent; Gram + cells remain purple, Gram – cells lose colour. Decolourizing agent : substance that washes away the primary stain CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 41 GRAM STAIN Why are there colour differences between Gram positive and negative bacteria? CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 42 GRAM STAIN 4. Slide is flooded with safranin for 1 min then rinsed with water and blotted dry. Result: Safranin acts as a counterstain. Gram + cells remain purple, Gram – cells are coloured pink. Note: all cells absorb safranin. Counterstain: substance that provides contrasting colour to the primary stain. CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 43 ACID-FAST STAIN An example mycolic acid Special stain for some bacteria that don’t stain well by Gram staining due to a large amount of waxy lipid (mycolic acids) in their cell walls Used for Mycobacterium and Nocardia Cause tuberculosis and leprosy, respectively CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 44 ACID-FAST STAIN Procedures: 1. Cover smear with tissue paper to retain dye 2. Flood slide with carbolfuschsin (red primary stain) for several minutes while warming is over steam. Heat drives stain through waxy wall. 3. Remove tissue paper, cool the slide and decolourize with HCl and alcohol. Acid-fast cells Which cells are acid-fast cells? (Like TB) retain the red dye, while it is removed from others. 4. Counterstain with methylene blue CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 45 ENDOSPORE STAIN Schaeffer-Fulton endospore stain: Like the acid-fast stain, also uses heat to drive primary stain. Difficult for stains to penetrate endospores Decolourizing agent: water What were the following used as? 1. Safranin 2. Malachite green Green: Endospores Red: Vegetative cells CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 46 HISTOLOGICAL STAINS H&E Stain of Human Lung Tissue Histological stains: stains for tissue samples Common histological stains: Gomori Methenamine Silver (GMS) Stain: screens for the presence of fungi and carbohydrates in tissue. Hematoxylin and eosin (H&E): hematoxylin is a basic dye while eosin is an acidic dye. CC BY 2.0, https://commons.wikimedia.org/w/index.php?curid=437645 Hematoxylin: Violet/dark blue Eosin: Red/pink CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 47 NEGATIVE (CAPSULE) STAIN Negative stains bind to the background and leave the cells colourless Often an acidic dye since most cells have many negatively charged molecules within themselves Bacterial capsules are often negatively charged – negative stains are repulsed by them CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 48 STAINING FOR ELECTRON MICROSCOPY Stains have atoms of heavy metals such as lead, osmium, tungsten or uranium which absorb electrons May bind to molecules in specimen or the background Examples: Osmium tetroxide (OsO4) preferentially stains lipids Note: SEM samples are coated, not stained Osmium crystals By Periodictableru www.periodictable.ru - Own work, CC BY 3.0, https://commons.wikimedia.org/w/index.php?curid=9774411 CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 49 CLASSIFICATION AND ID OF MICROORGANISMS Taxonomy: the science of classifying and naming organisms. Organisms are grouped into nonoverlapping groups called taxa Why is taxonomy important? Chondrus crispus. Also known as: Irish moss Carragheen Curly moss CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 50 LINNAEUS’S SYSTEM Domain: * Drunk Kingdom: King Phylum: Phillip Class: Came Order: Over Family: For Genus: Good Species: Spaghetti Similar groups of species are placed into increasingly inclusive categories. * Not a part of the original system CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 51 SCIENTIFIC NAMES Homo sapiens Genus Species Scientific names are in Latin or are Latinized (except viruses) and consist of two parts (binomial nomenclature). May honour people. Notice that both parts are italicized (or underlined if written) Genus is capitalized and is a noun Species is not capitalized and is usually an adjective CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 52 SPECIES? STRAINS? Several methods to define a species Examples: Morphology, genetic sequences, ability to breed Disagreement on the definition Strain: populations of cells that arose from a single cell that share many stable properties, differ from other strains, and evolve as a group. Multiple strains may belong to the same species CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 53 DOMAINS Carl Woese and George Fox sequenced ribosomal RNA (rRNA) which was used to group organisms into three domains: Eukarya Bacteria Archaea CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 54 TAXONOMIC AND IDENTIFYING CHARACTERISTICS Five common types of information to distinguish microorganisms: Are you the Let’s find out! same as me? 1. Physical characteristics 2. Biochemical tests 3. Serological tests 4. Phage typing 5. Analysis of nucleic acids CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 55 1. PHYSICAL CHARACTERISTICS Cocci Streptococci Baccili Staphylococci Streptobaccili Micro and macroscopic examination of physical characteristics: Morphology Staining for features like endospores and flagella CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 56 BACTERIAL SHAPES AND ARRANGEMENTS Common shapes: Coccus: spherical Bacillus: rod-shaped Common arrangements: diplo-, tetra: arranged in pairs, fours Strepto-: in chains Staphylo-: in clusters CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 57 2. BIOCHEMICAL TESTS Ability to utilize or produce certain chemicals Example: ability to ferment carbohydrates, use amino acids, produce waste products like H2S etc. Automated systems exist as well (MicroScan plate) CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 58 3. SEROLOGICAL TESTS Antigen Serology: study of serum – liquid portion of blood after clotting factors removed Usually the study of antigen-antibody reactions Antigen: substance that is recognized and bound by antibodies. Substances that illicit an immune response are known as antigenic. Example: components of a cell wall or flagella An Antibody Antibodies bind to antigens with very high specificity CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 59 3. SEROLOGICAL TESTS Agglutination test: Serum is mixed with a sample that potentially contains its target antigen If the antigenic cell is present, the antibodies agglutinate (clump) the antigen together CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 60 4. PHAGE TYPING Bacteriophages: Viruses that infect bacteria. Bacteriophages often only infect very specific strains of bacteria Phage typing: ID bacteria based on the ability of certain phages to infect them Bacteria are grown as a lawn that covers the surface of a plate. A dilute sample of virus is added. Rings of death (plaque) indicate a virus killing all the bacteria in an area. CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 61 5. ANALYSIS OF NUCLEIC ACIDS G + C Content An microorganism has the following base composition: A: 5,000 bases T: 5,000 bases % G: 10,000 bases C: 10,000 bases What is the G + C content? The DNA or RNA of a cell is sequenced and used to identify microorganisms A cell’s G + C content can also be used to group microorganisms CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 62 TAXONOMIC KEYS A taxonomic key can help to identify microorganisms Dichotomous key: contains paired statements such that only one of the two choices will apply to any particular organism. CAPILANO UNIVERSITY - EUGENE CHU - BIOL 203 63