Tools

How are microorganisms cultured? Inoculation Incubation Isolation Inspection Identification

How is media inoculated? Inoculation Introduce a sample of a microorganism into growth media. Samples obtained from: CSF Blood Urine Sputum Feces Dead tissue

How are microorganisms grown? Incubation Allow microoragnism to grow at appropriate temperature for a period of time 37 C in humans

How are microorganisms separated in a mixed culture? Isolation Separate multiple microorganisms in a mixed culture Make a pure culture Differential media Provide different media with specific nutritive requirements Only certain microorganisms will grow Mannitol-Salt agar used to distinguish S. aureus from other Staphylococcus sp.

Isolation Methods Streak plating Loop dilution Spread plate Pour plate

How are microorganisms inspected? Inspection Observed Media Staining Gram stain Acid fast stain Microscopy

How are bacteria identified? Identification Determine the type of microbe Based on results of differential & selective tests

How are microorganisms visualized? Stain Wet mount Negative vs positive Simple vs differential Microscopy Bright field Dark field Phase contrast Differential interference contrast Fluorescent Confocal Transmission electron Scanning electron

How are live microbes observed? Wet mount

Positive vs Negative Stains Positive Stains Heat fix the specimen Dye sticks to specimen Examples: Negative Stains Dye does NOT stick to specimen Forms a silhouette around specimen Pros: Prevents distortion and shrinkage of cells Heat fixing not used Capsule s Gram stain Endospore stain Acid fast stain Nigrosin stain

Simple Stains vs Differential Stains Single dye used Dye sticks to specimen Examples: Differential Stains Two dyes used Primary dye Counterstain Distinguishes cell types or parts Gram stain Endospore stain Acid fast stain

Anatomy of the Compound Light Microscope Eyepiece Tube Arm Coarse adjustment knob Nosepiece Objective lenses Stage clip Fine adjustment knob Stage Coaxial stage controls Light source Base

Anatomy of the Compound Light Microscope Condenser Iris diaphragm

Anatomy of the Compound Light Microscope

How is Total Magnification Calculated? Total Magnification = (Ocular magnification) x (Objective magnification) Type Ocular Magnification Objective Magnification Total Magnification Scanning 10 4 40 Low High Oil Immersion 100

What is the field of view? The diameter of the field of view is measured in millimeters, micrometers, and nanometers.

What happens to the field of view as the total magnification increases? The diameter of the field of view decreases.

What happens to the field of view as magnification increases? Field of view – the diameter of the field at different magnifications. Total Magnification Field of View (mm) Field 40 X 7 mm 100 X 3 mm 400 X 1 mm

What is “depth of field”? Distance between the nearest and farthest objects in a scene that appear sharp in an image. A lens can precisely focus at only one distance at a time.

How does the depth of field affect microscopy? Cells are 3D. You must be aware of the plane in which the focus lies.

Why do we use oil for the 100X objective? The refractive index is a measure of the light-bending ability of a medium The light may bend in air so much that it misses the small high-magnification lens Immersion oil is used to keep light from bending

Resolution Limits Human eye Compound microscope ~ 0.2 mm 10 m 1 m Human height 1 m Length of some nerve and muscle cells 0.1 m Unaided eye Chicken egg Human eye ~ 0.2 mm Compound microscope 1 cm Frog egg 1 mm 100 µm Most plant and animal cells Light microscope 10 µm Nucleus Most bacteria 1 µm Mitochondrion Smallest bacteria Electron microscope 100 nm Viruses Ribosomes 10 nm Proteins Lipids 1 nm Small molecules 0.1 nm Atoms

Visible Light Microscopy Bright field microscopy Visible light transmitted through specimen Use compound light microscope Multipurpose use Dark field microscopy Visible light reflected of sides of specimen Use compound light microscope with “stop” on the condenser View shape, movement of cells

Visible Light Microscopy Differential interference contrast microscopy Two beams of light Two prisms Add contrasting colors Colored 3D Phase-contrast microscopy Light patterns moving through denser regions of cell transformed into a density map Used to visualize: Internal structures Endospores Granules Organelles External structure Cilia

Ultraviolet Microscopy Fluorescence microscopy Fluorescent dyes attached to specific cell structures DAPI Fluorescein Texas-red View internal/external structures of cells & viruses View interactions of cell biomolecules in 2D Confocal microscopy Fluorescent dyes attached to specific cell structures View internal/external structures of cells/viruses View interactions of cell biomolecules in 2D or 3D Plant embryo Cytoskeleton Proteins

Electron Microscopy Transmission electron microscopy Transmits electrons through specimen View internal structures of cells & viruses Resolution = 0.5 nm Scanning electron microscopy Surface of specimen coated with metal Electrons bounce off metal Produces 3D image of surface Resolution = 10 nm