Week 2, Tuesday

Chapter 4- Microscopy, Staining and classification ppt

Microscopy: use of light or electrons to magnify objects

  • Leeuwenhoek Used one of the first primitive microscopes and sparked the study of microorganisms

Wavelength of Radiation: the distance between two corresponding parts of a wave

Magnification: an apparent increase in the size of an object

Resolution: also called resolving power, is the ability to distinguish between 2 pints that are closer together

Contrast: refers to the differences in intensity between two objects or an object and its background

Light Microscope:

  • Bright-field microscopy

    • simple

    • Contains a single magnifying lends

    • Similar to magnifying glass

  • Compound

    • Series of lenses for magnification

    • Light passes through specimen into objective lens

    • Oil immersion lens increases resolution

    • Have one or two ocular lenses

  • Dark-field microscope:

    • Best for observing pale objects

    • Only light rays scattered by specimen enter objective lends

    • Specimen appears light against dark background

    • Increases contrast and enable observation of more details

  • Phase microscopes:

    • Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining

    • Contrast is related by bringing two sets of rays together

    • 2 types

      • Phase-contrast

        • Sharply defined images

        • Useful for cilia and flagella

      • Differential interference contrast

        • Increase the contrast significantly

        • Give image a more dramatic 3D shape/shadow appearance

  • Fluorescence microscopes

    • Direct UV light source at specimen

    • Specimen radiates energy back as a longer visible wavelength

    • UV light increases resolution and contrast

    • Salome cells are naturally fluorescent, others must be stained

    • Used in immunofluorescence to identify pathogens and to make visible a variety of proteins

  • Confocal microscopes

    • Use fluorescent dyes

    • Use UV lasers to illuminate fluorescent chemicals i a single plane

    • Resolution increase because emitted light passes through pinhole aperture (eliminates blurring)

    • Computer constructs 3D image from digitized images

    • Used to identify small structures such as dendritic cells

Electron microscopy

  • light microscopes cannot resolve structures closer than 200nm, this is where electron microscopes come in

  • Magnifies objects 10,000X to 100,000X

  • Detailed views of bacteria, viruses, internal cellular structures, molecules and large atoms

  • 2 types

    • TEM

      • Generates a beam of electrons that produce an image on a fluorescent screen

      • From the source, the electrons pass through the specimen/sectioned, then they pass through magnetic fields that manipulate and focus the beam

      • From there, the beam moves onto a fluorescent screen that changes some of the energy into visible light

      • Dense areas of the specimen blocks electrons, resulting in a dark area

      • Contrast and resolution can be enhancing with electron-dense dyes

    • SEM

      • Uses magnetic field within a vacuum tube to manipulate a beam of electrons

      • Rapidly focuses electrons back and forth across the specimen’s surface

      • One advantage of suing this type of electron microscope is that while specimen can be observed

      • Disadvantages= requires a vacuum, and you can only see external surfaces

Probe microscopy:

  • magnifies more than 100,00,00X

  • 2 types

    • STM (scanning tunneling microscopes)

      • Passes metallic probe Back and forth across and slightly above the surface of the specimen

      • Will measure the flow of electron to and from the probe and the specimen’s surface

      • Called the tunneling current

    • AFM (atomic force microscopes)

      • Also uses a pointed probe to scan the specimen

      • The probe will touch the specimen lightly in this case

      • Capable of magnification of specimens that do not conduct electrons

      • These are capable of magnifying living specimens as this does not use an electron beam or vacuum

      • Used to magnify the surface of bacteria, viruses, proteins and amino acids

Staining:

  • most microorganisms are difficult to view by bright field microscopy

  • Coloring specimen with stain increases contrast and resolution

  • Specimens must be prepared for staining

    • 1st- microorganisms are spread across the surface of the slide (smear)

    • The specimen is left to air dry for a few minutes

    • The slide is passed over a heat source it the slide facing up, this will adhere the specimen t the slide (Killing it) - called heat fixation

    • Alternatively, specimens can be chemically fixed t a slide using a fixing agent such as methyl alcohol

  • Principles of staining

    • Dyes used as stains are usually salts

    • Chromosphere i the colored portion of the dye

  • Acidic dyes (stain alkaline structures)

    • An ionic chromosphere’s (negative charge) work best in acidic environment

    • Binds to positive molecules

  • Basic dyes (stain acidic structures)

    • Cationic chromophores (positive charge) work best in basic environment

    • Binds to negative molecules

    • Used more commonly

  • Simple stains:

    • Composed of single basic dye

    • Crystal violet, safranin, methylene blue

    • Simple, requiring soaking the smear in the dye for 30-60 seconds and then rinsing with water

    • Used to enter mine size, shape, and arrangement of cells

  • Differential stains

    • Use more than one dye

    • Distinguishes between different cells, chemicals or structures

    • Common include:

      • Gram stain

      • Acid-fast

      • Endosperm

      • Histological

    • Gram staining procedure:

      • Slide is flooded with crystal violet for 1 min. Then rinsed with water.

        • All cells are stained purple

        • Primarily stained with crystal violet

      • Slide is flooded with iodine for 1 min. Then rinsed with water

        • Iodine acts as a Mordant (substance that binds to a dye and makes it less soluble

        • All cells remain purple

      • Slide is rinsed with solution of ethanol and acetone for 10-30 seconds. The ruined with water

        • Smear is decolonized

        • Gram-positive remains purple

        • Gram negative cells are now colorless

      • Slide is loaded with safranin for 1 min. Then rinsed with after and blotted dry

        • Gram-positive cells remain purple

        • Gram negative cells are pink

    • Acid-fast (Ziehl-Neelson)

      • Used to stain the genera mycobacterium and nocardia

      • Cause disease like TB, leprosy, and other lung skin infections

      • These cells have a waxy lipid in cell walls, and therefore do not absorb water-soluble dyes

      • Steps:

        • Cover smear with paper to retain dye

        • Flood with red primary stain: carbolfuchsin for several minutes while warming over steaming water

        • Remove tissue paper, cool slide and then add decolonized: hydrochloric acid

          • Acid fast cells retain their red color

        • Counterstain: methylene blue is then added, will Stan the bleached on-acid fast cells

        • Results in pink acid-fast cells that can be distinguished from blue non-acid fast cells

        • Acid fast bacilli in sputum are indicative of mycobacteria’s infection

    • Endospore stain

      • Bacteria in the genera Bacillus and clostridium contain species that cause disease, those species produce endospores

      • Anthrax, gangrene, tetanus

      • These cells are highly resistant to heat, desiccation and harmful chemicals

      • Endospores must be stained with a specialized stain called Schaeffer-Fulton endospore stain

      • Uses heat to drive the primary stain: malachite green into the endospore

      • Decolonized is used with water then the counter stain safranin

      • Results in green-stained endospore and red-colored vegetative cells

    • Histological stains

      • 2 common stains used:

        • Gomori methenamine silver (GMS)

          • used to stain of fungi in certain tissue

          • Surrounding tissue will be green, fungi itself is black

        • Hematoxylin and Eosin (HE)

          • used to stain tissue samples

          • often used to screen for cancer cells

          • structures appear pink or purple

    • Special Stains:

      • simple stains used to identify specific microbial structures

      • include:

        • negative stains

          • used to reveal bacterial capsule

            • aka Capsule Stains

          • acid dyes are used

          • eosin and nigrosine

        • Flagellar stains

          • pararosaniline and carbolfuchsin with a mordant are used

        • Fluorescent stains

          • used for fluorescent microscopy

Taxonomy:

  • consists of classification, nomenclature, and identification

  • allows for the organization of large amounts of information about organisms

  • categorizing organisms allows for predictions to be made about microbes based on knowledge of similar organisms

  • allows for better understanding of evolutionary connections

Domain:

  • Carl Woese compared nucleotide sequences of rRNA subunits

    • proposal of 3 domains as determined by ribosomal nucleotide sequences

      • Eukarya, Bacteria, and Archaea

      • cells in the 3 domains also differ with respect to many other characteristics

Classification:

  • taxonomic and identifying characteristics

    • physical characteristics

      • can often be used to identify microorganisms

      • protozoa, fungi, algae, and parasitic worms can often be identified base only on their morphology (size, shape, structures or the cell)

      • some bacterial colonies have distinct appearance used for identification

    • biochemical tests

      • distinguish prokaryotes by their ability to utilize or produce certain chemicals

      • laboratory scientists use biochemical tests to identify pathogens

      • Automated MicroScan System

        • MicroScan panel s used

        • this is a panel containing numerous wells, each the size of a particular biological test

        • instrument obtains the identity of the organism by reading the pattern of colors in the wells after biochemical tests have been reformed

    • serological tests

      • the study of antigen-antibody reactions in laboratory settings

      • many microorganisms trigger an immune response that results in antibody production

      • antibodies can be used to identify the organism that triggered their production

    • phage typing (identifies bacterial species that viruses may infect- Bacteriophage)

      • Bacteriophage (phage) are viruses that infect and usually destroy bacterial cells

        • phages are specific for the hot they infect

        • phage typing is utilized to identify a specific strain of bacteria

      • Phage test:

        • solution containing bacterium to be identified is spread over a solid surface medium-forms a bacterial lawn

        • drops of solution containing different bacteriophages are added

        • cleared areas indicate where the phage was able to infect and kill bacteria - called a plaque

    • Analysis of nucleic acids

      • nucleic acid sequence can be used to classify and identify microbes

      • prokaryotic taxonomy now includes the G+ C content of an organism’s DNA

Exam will be 30 questions