Microbiology Textbook Chapter 4 PDF

CHAPTER 4 Microscopy, Staining, and Classification  Microscopy and Staining Overview MICROSCOPY  The use of light or electrons to magnify objects Microscopy   METRIC UNITS  Millimeter mm...

CHAPTER 4 Microscopy, Staining, and Classification  Microscopy and Staining Overview MICROSCOPY  The use of light or electrons to magnify objects Microscopy   METRIC UNITS  Millimeter mm measuring small organisms (tick)  Micrometer μm measuring cells (5-25 μm)  Nanometer nm measuring viruses (25 nm) Microscopy  General Principles of Microscopy  Wavelength of radiation  Magnification of image  Resolution  Contrast Figure 4.1 Electromagnetic Spectrum Microscopy  General Principles of Microscopy  Wavelength of radiation  Magnification of image  Resolution  Contrast Figure 4.2 – Magnification of an image results when light refracts (bends) as it passes through a lens Microscopy  General Principles of Microscopy  Resolution  Ability to distinguish two points that are close together  The better the resolution, the better two nearby objects are distinguished from one another (clarity)  Contrast  Differences in intensity between two objects or between an object and its background  Staining increases contrast Microscopy  Figure 4.3 Microscopy  Figure 4.6 Four kinds of light microscopy Microscopy  Figure 4.7 Fluorescence microscopy – a dye is used: Mycobacterium tuberculosis stained with auramine O viewed under UV light to fluoresce Microscopy  Figure 4.8 Immunofluorescence – dye is linked to an Ig: Yersinia pestis (cause of bubonic plague) Microscopy Figure 4.11 Scanning electron microscope (SEM) images Microscopy Figure 4.12 Probe microscopy Microscopy Microscopy Microscopy  Identifying Microorganisms  How are MO identified?  Stained: * Simple stains * Differential stains * Special stains  Classified: * Physical characteristics * Biochemical tests * Serological tests https://bit.ly/2Cm9y55 * Phage typing * Nucleic acids (G + C content) Staining  Preparing Specimens for Staining Smear: Fixing does three things: 1. kills organism 2. preserves their morphology 3. anchors specimens to the slide Heat Fixation: Figure 4.13 Staining  Principles of Staining  Dyes are usually salts  Chromophore is the colored portion of the dye  Acidic dyes  Basic dyes https://bit.ly/2LQey14 Staining  Principles of Staining  Acidic dyes, work best at low pH  Contains anionic chromophores (-vely charged)  Bind to +vely charged molecules  Basic dyes, work best at high pH  Contains cationic chromophores (+vely charged)  Binds to -vely charged molecules  These dyes are more common b/c most cells are negatively charged Staining  Simple Stains  Composed of single basic dye  Crystal violet, safranin, or methylene blue  Used to determine  Size of cells  Shape of cells  Arrangement of cells Figure 4.14 Simple stains – E.coli and Staphylococcus aureus Simple Stains  Differential Stains  Uses more than one dye  Distinguish between different cells or structures  Common differential stains include:  Gram stain (developed in 1884, Hans Christian Gram)  Acid-fast stain  Endospore, capsule, flagellar staining  Histological stains (GMS and HE) Differential Staining  Differential Stains: Gram Stain  The Gram stain technique is as follows:  Crystal violet (primary stain) is added to a heat-fixed bacterial smear  Iodine (mordant) is added forming an insoluble crystal violet- iodine complex (CV-I complex)  Acetone-alcohol (decolorizing step) is used to rinse the sample  Safranin (counterstain) is added to the sample Differential Staining  Figure 4.15 The Gram staining procedure: Differential Staining  Differential Stains: Gram Stain Results of the acetone-alcohol treatment on…  Gram negative bacteria:  Dissolves the outer membrane  Damages the thin peptidoglycan layer  CV-I washes out  Gram positive bacteria:  Slightly damages the thick peptidoglycan  Dehydration makes it less permeable  CV-I is retained Differential Staining  Differential Stains: Gram Stain Gram-positive Gram-negative Gram-positive Gram-negative Heat-fixed smear Crystal violet Iodine (acts as a mordant) Acetone-alcohol solution (decolorizer) Counterstain with safranin Differential Staining  Differential Stains: Acid–Fast  Acid–fast bacteria  Contain waxy cell walls rich in mycolic acid  Retain red-colored primary dye after exposure to an acid wash  Non–acid–fast cells  Do not have mycolic acid  Red primary stain is washed away after exposure to an acid wash  Important diagnostic tool for detecting:  Mycobacterium and Nocardia species Differential Staining  Differential Stains: Acid–Fast  Ziehl-Neelsen method  Carbol-fuchsin (primary dye) is added to a heat-fixed smear  Sample is steamed for several minutes to drive the red dye into the bacteria  Acid-alcohol (decolorizing agent) is used to rinse the sample  Methylene blue (counterstain) is added to the sample Differential Staining  Structural Stains  Simple stains that are used to identify specific microbial structures include: * Flagellar stains – determine # and location * Negative stains – capsule stains * Endospore stains – endospore detection Structural Stains  Histological Stains  Two common stains used for tissue specimens:  Gomori methenamine silver (GMS) stain * Screens for the presence of fungi  Hematoxylin and eosin (HE) stain * Uses a basic dye (H) and acidic dye (E) Aspergillus sp. Control Histology Slides * Outline tissue features * Biofilm detection in chronic infections histology.med.yale.edu Differential Staining Staining  Taxonomic and Identifying Characteristics 1. Physical characteristics 2. Biochemical tests 3. Serological tests 4. Phage typing 5. Analysis of nucleic acids Classification/Identification  Physical Characteristics  Used to identify some microorganisms:  Protozoa, fungi, algae, and parasitic worms can often be identified based only on their morphology (shape)  Some bacterial colonies have distinct appearance used for identification Classification/Identification  Biochemical Tests  Distinguish among prokaryotes by their ability to utilize or produce certain chemicals  Biochemical tests to identify pathogens, if they can be grown in a lab Figure 4.21 - CHO utilization test: * Use phenol red, CHO, and durham tube (this is the control) * Colour change-yellow, acid produced * Colour change-yellow, with acid and gas Classification/Identification  Figure 4.22 One tool for the rapid identification of bacteria, the automated MicroScan system Classification/Identification  Serological Tests  The study of antigen-antibody reactions in laboratory settings  Many microorganisms trigger an immune response that results in antibody production, allowing their identification Figure 4.23 An agglutination test, one type of serological test Classification/Identification  Serological Tests  This type of test can ID pathogenic strains of bacteria  E.coli O157:H7  O157 is the antigen of the cell wall  H7 is the antigen of the flagella  This strain of bacteria produces ‘Shiga toxin’  Causes severe foodborne disease  The toxin is the expression of the lambda prophage genes in this E.coli’s genome  A virus infected a bacterium; that bacterium infects you Classification/Identification  Phage Typing  Bacteriophages (phages) are viruses that infect bacterial cells  Phages are specific for the hosts they infect Figure 4.24 Phage typing for Salmonella enterica serotype Typhi Classification/Identification  Analysis of Nucleic Acids  Nucleic acid sequence (DNA or RNA) can be used to classify and identify microbes  Prokaryotic taxonomy now includes the G + C content (percentage) of an organism's DNA  Ranges from 20-80% in prokaryotes G+C x 100 A+T+G+C Classification/Identification