Lecture 02 Microscopy, Staining, Classification (Ch3)
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1 Lecture 02 Microscopy, Staining, Classification (Ch3) Topics Methods of Culturing Microorganisms Microscope (History, Types, Definitions) Staining (Gram s) Dichotomous keys Micro for Health Sciences 1 Classification and Identification of Microorganisms Taxonomic Keys Dichotomous keys Series of paired statements, usually only one of two choices applies to an organism Key directs you Why? To identify the organism!! How? Morphology, biochemical results, observations, etc. Micro for Health Sciences 2 Example Dichotomous Taxonomic Key Micro for Health Sciences 3 Figure
2 Microorganism Culturing Methods (How to grow ) Five basic techniques of culturing Media Microbial growth Micro for Health Sciences 4 Five Basic Techniques of Culturing Bacteria 1. Inoculate 2. Incubate 3. Isolation 4. Inspection 5. Identification Micro for Health Sciences 5 A single visible colony represents a pure culture or single type of bacterium isolated from a mixed culture. Fig. 3.2 Isolation technique Micro for Health Sciences 6 2
3 Three basic methods of isolating bacteria. Micro for Health Sciences 7 Media Classified according to three properties Physical state Chemical composition Functional types Micro for Health Sciences 8 Media: Physical State Liquid media Semi-solid media Solid media Micro for Health Sciences 9 3
4 Semi-solid media contain a low percentage (<1%) of agar, which can be used for motility testing. Micro for Health Sciences 10 Solid media contain a high percent (1-5%) of agar, this enables the formation of discrete colonies. Micro for Health Sciences 11 Media: Chemical content Synthetic media Nonsynthetic or complex media Micro for Health Sciences 12 4
5 Synthetic media contain chemically defined pure organic and inorganic compounds (known molecular formula). Micro for Health Sciences 13 Complex or enriched media contain ingredients that are not chemically defined or pure ( animal extracts). Micro for Health Sciences 14 Functional types of growth media Enriched media Selective media Differential media Micro for Health Sciences 15 5
6 Enriched media grows fastidious bacteria. Blood hemolysis Micro for Health Sciences 16 Grows all Differential media - shows different reactions, like color. Selective media - enables one type of bacteria to grow. Micro for Health Sciences 17 Mannitol salt agar is a selective media. MacConkey agar is a differential media. Examples of media that are both selective and differential Micro for Health Sciences 18 6
7 Example miscellaneous media such as reducing, fermentation and transportation media. Carbohydrate fermentation broth Micro for Health Sciences 19 Microbial growth Incubation Varied temperatures, atmospheric states Inspection Mixed culture Pure culture Identification Microscopic appearance Maintenance and disposal Stock cultures sterilization Micro for Health Sciences 20 Microscopy and Staining how we see closer up Intro to Microscopy Evolutionary Trends Some Concepts of Definition and Relationship Some Microscopy Techniques Staining Methods Micro for Health Sciences 21 7
8 Microscope evolution Micro for Health Sciences 22 Evolution of Microscopy Characterized by: A constant search for better resolution Constant search for ability to see better detail in smaller objects Always waiting on Technology Micro for Health Sciences 23 General Principles of Microscopy Wavelength of radiation Magnification Resolution Contrast Micro for Health Sciences 24 8
9 Some Concepts to Consider Definitions and Relationships Resolution Magnification Depth of Focus Field of Vision Numerical Aperture Micro for Health Sciences 25 Resolution Resolution is the ability to distinguish between two points; The closer the two points, the higher the resolution Micro for Health Sciences 26 Resolution distinguishes magnified objects clearly. Micro for Health Sciences 27 Effect of wavelength on resolution 9
10 Resolution can be increased using immersion oil. Magnification comparison Micro for Health Sciences 28 Magnification Relative enlargement of the specimen The power of magnification is calculated by multiplying the power of the eye piece lens by the power of the objective lens. Empty Magnification Micro for Health Sciences 29 Specimen magnified when light passes through objective and ocular lenses. Pathway of light and the two stages of magnification in a compound microscope. Micro for Health Sciences 30 10
11 More Concepts Depth of focus - thickness of a specimen that can be seen in focus at one time; as magnification the depth of focus. Field of vision - the surface area of view; the area as magnification. Numerical aperture (N.A.) the amount of light reaching the specimen; As N.A. resolution. Micro for Health Sciences 31 Always Improving Van Leeuenhoek -- size Zeiss Brothers size and resolution! 1930 s Electron Microscope -- SIZE New Scopes: SEM, TEM, TM, etc. etc.- fine details! Micro for Health Sciences 32 Optical microscopes All have a theoretical maximum magnification of 2000X Bright-field Dark-field Phase-contrast Differential interference Fluorescent Confocal Micro for Health Sciences 33 11
12 Compound microscope Micro for Health Sciences 34 Anatomy of a laboratory microscope Bright-field microscopy Most commonly used Can observe live or preserved Stained or unstained specimens Image from: Micro for Health Sciences 35 Dark-field microscopy Observe live unstained specimens View an outline of the specimens Yeast Cells Image from: Micro for Health Sciences 36 12
13 Phase-contrast microscopy Observe live specimens View internal cellular detail Image from: Micro for Health Sciences 37 Principles of phase microscopy Micro for Health Sciences 38 Figure 4.7 Examples of dark-field, bright-field and phase-contrast microscopy. Three views of a cell Micro for Health Sciences 39 13
14 Example of phase-contrast and differential interference. Visualizing internal structuresmicro for Health Sciences 40 Fluorescent Microscopy Direct UV light source at specimen Specimen radiates energy back as a longer, visible wavelength UV light increases resolution and contrast Some cells are naturally fluorescent; others must be stained Used in immunofluorescence to identify pathogens and to make visible a variety of proteins Micro for Health Sciences 41 Fluorescent microscopy Fluorescence stain or dye UV radiation causes emission of visible light from dye Diagnostic tool Image from: Micro for Health Sciences 42 14
15 Immunofluorescence Micro for Health Sciences 43 Figure 4.10 Example fluorescent microscopy- stained cheek scrapings specimen Micro for Health Sciences 44 Confocal Microscopy fluorescent dyes UV lasers to illuminate fluorescent chemicals in a single plane Resolution increased because emitted light passes through pinhole aperture Computer constructs 3-D image from digitized images Micro for Health Sciences 45 15
16 Confocal microscopy Fluorescence or unstained specimen images combined to form a threedimensional image. Micro for Health Sciences 46 Electron Microscopy Very high magnification (100,00X) Transmission electron microscope (TEM) View internal structures of cells Scanning electron microscope (SEM) Three-dimensional images Micro for Health Sciences 47 Electron Microscopy Light microscopes cannot resolve structures closer than 200 nm Electron microscopes greater resolving power and magnification Magnifies objects 10,000X to 100,000+X Detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms Two major types Transmission electron microscopes Scanning electron microscopes Micro for Health Sciences 48 16
17 Transmission Electron Microscope (TEM) Micro for Health Sciences 49 Figure 4.11 Transmission Electron Microscopy (TEM) Transmission electron micrograph Micro for Health Sciences 50 Scanning Electron Microscope (SEM) Micro for Health Sciences 51 Figure
18 Example SEM Photo False-color scanning electron micrograph ha! No colors with electrons! Micro for Health Sciences 52 Ant in S.E.M. Micro for Health Sciences 53 Ant portrait Micro for Health Sciences 54 18
19 Insect eye at 250x Micro for Health Sciences 55 Insect eye at 2500x Micro for Health Sciences 56 Hydrothermal Worm FEI and Philippe Crassous ( Micro for Health Sciences 57 19
20 Probe Microscopy Magnifies more than 100,000,000 times Two scope types Scanning tunneling Atomic force Micro for Health Sciences 58 Manipulating Atoms w/ STM! Carbon dioxide man, individual carbon dioxide atoms on platinum The word atom, in kanji, written with individual iron atoms on copper From: Micro for Health Sciences 59 Staining why? Increases contrast and resolution by coloring specimens with stains/dyes 1 st make smear Microbiological stains contain chromophore Acidic dyes stain alkaline structures; more commonly, basic dyes stain acidic structures Micro for Health Sciences 60 20
21 Simple stain results? Micro for Health Sciences 61 Figure 4.16 Stain Types Positive stains Dye binds to the specimen Negative stains Dye does not bind to the specimen, but rather around the specimen. Micro for Health Sciences 62 Positive stains are basic dyes (+ charge) that bind negatively charged cells. Negative stains are acidic dyes (- charge) that bind the background. Comparison of positive and negative stains Micro for Health Sciences 63 21
22 Positive stains Simple One dye Differential Two-different colored dyes Ex. Gram stain Special Emphasize certain cell parts Ex. Capsule stain Micro for Health Sciences 64 Staining Technique Example Gram Stain Fix 20 secs Crystal Violet, H2O rinse 15 secs Iodine Mordant, H2O Rinse Alcohol rinse 20 secs Safranin Counter stain, H 2 O Rinse Micro for Health Sciences 65 Preparing a specimen for staining Micro for Health Sciences 66 Figure
23 Staining Simple stains Differential stains Gram stain Acid-fast stain Endospore stain Special stains Negative (capsule) stain Flagella stain Micro for Health Sciences 67 Examples of simple, differential and special stains. Micro for Health Sciences 68 Ziehl-Neelsen acid-fast stain for mycolic acid Micro for Health Sciences 69 Figure
24 Schaeffer-Fulton endospore stain Micro for Health Sciences 70 Figure 4.19 Negative (capsule) stain Micro for Health Sciences 71 Figure 4.20 Flagella stain Micro for Health Sciences 72 Figure
25 Staining for EM Chemicals containing heavy metals used for transmission electron microscopy Stains can bind molecules in specimens or background Micro for Health Sciences 73 Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics Physical characteristics Biochemical tests Serological tests Phage typing Analysis of nucleic acids Micro for Health Sciences 74 Two biochemical tests for identifying bacteria Micro for Health Sciences 75 Figure
26 One tool for the rapid identification of bacteria Micro for Health Sciences 76 Figure 4.25 An agglutination test, one type of serological test Micro for Health Sciences 77 Figure 4.26 Phage typing Micro for Health Sciences 78 Figure
Topic 02 Microscopy, Staining, Classification (Ch3)
Topic 02 Microscopy, Staining, Classification (Ch3) Topics Dichotomous keys Methods of Culturing Microorganisms (9.6) Microscope (History, Types, Definitions) Staining (Gram s) (2.4) Micro for Health Sciences
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