Tools of the Laboratory: The Methods for Studying Microorganisms Chapter 3

The 5 I’s of culturing microbes Inoculation – introduction of a sample into a container of media Incubation – under conditions that allow growth Isolation – separating one species from another Inspection – looking at specimen (microscope) Identification

1. Inoculation 3

2. Incubation 3. Isolation

4.Inspection 5.Identification

Inspection

Identification

Identification

Media Three categories: Physical state Chemical composition Liquid Semisolid Solid (can go back to liquid) Solid (cannot go back to liquid) Chemical composition Synthetic Nonsynthetic, complex Functional type General Enriched Selective Differential Anaerobic Assay

Most commonly used media: nutrient broth – liquid medium containing beef extract & peptone nutrient agar – solid media containing beef extract, peptone & agar agar is a complex polysaccharide isolated from red algae solid at room temp, liquefies at boiling (100oC), does not resolidify until it cools to 42oC provides framework to hold moisture & nutrients not digestible for most microbes

Enriched media

Selective & Differential Media

Differential media

Microscope Magnification – ability to enlarge objects Resolving power – ability to show detail

Compound Light Microscope

Effect of wavelength on resolution

Oil immersion lens

Types of light microscopes Bright-field – most widely used, specimen is darker than surrounding field Dark-field – brightly illuminated specimens surrounded by dark field Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures

3 views of the same cell a. Bright-field b. Dark-field c 3 views of the same cell a. Bright-field b. Dark-field c. Phase-contrast

Fluorescence Microscope Modified compound microscope with an ultraviolet radiation source and a filter that protects the viewer’s eye Uses dyes that emit visible light when bombarded with shorter uv rays. Useful in diagnosing infections

Electron microscopy Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds. Electron waves are 100,000X shorter than the waves of visible light. Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength. Magnification between 5,000X and 1,000,000X+

2 types of electron microscopes Transmission electron microscopes (TEM) – transmits electrons through the specimen; darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts Scanning electron microscopes (SEM)– provides detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it. (1987)

Transmission Electron Microscope

Transmission Electron Micrograph

Scanning Electron Microscope

Scanning Electron Micrograph

“Carbon monoxide man” (Scanning Tunneling Microscope-Made of 28 CO molecules)

Specimen preparation wet mounts & hanging drop mounts – fixed mounts – allow examination of characteristics of live cells: motility, shape, & arrangement fixed mounts – are made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.

Staining cationic dyes – basic dye, with positive charges on the chromophore Chromophore: chemical(s) that absorb light that is responsible for color in a molecule anionic dyes – acidic dye, with negative charges on the chromophore surfaces of microbes are negatively charged and attract basic dyes – positive staining. negative staining – microbe repels dye & it stains the background

Staining simple stains –one dye is used differential stains – use a primary stain and a counterstain to distinguish cell types or parts. examples: Gram stain, acid-fast stain and endospore stain special stains: capsule and flagellar stains

Types of stains