Chapter 3Observing Microorganisms Through a Microscope. I. Units of Measurement. [Table 1students should be familiar with all except US Equivalents].

a. 1 m = 1000 mm b. 1 mm = 10-3 m = 1000 m c. 1 m = 0.000001 m = 10-6 m = 10-3 mm d. 1 nm = 0.000000001 m = 10-9 m = 10-6 mm e. 1000 nm = 1 m f. 0.001 m = 1 nm g. Typical eukaryotic cells = 10 m to 500 m. h. Typical prokaryotic cells = 1 m to 10 m. i. Typical virus = 20 nm to 1000 nm (1 m). Microscopy: The Instruments. a. A simple microscope has only one lens. b. Compound Light Microscopy. Fig. 1. i. [Discuss parts]. ii. Total magnification = magnification of objective lens magnification of ocular lens.

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Total Magnification Setups for a Typical Light Microscope. Ocular Lens Magnification Objective Lens Magnification 10 X 4X 10 X 10 X 10 X 40 X 10 X 100 X (oil immersion)

Total Magnification 40 X 100 X 400 X 1000 X

c. Resolution is the ability of the lenses to distinguish between two points. Fig. 2. i. A microscope with a resolving power of 0.4 nm can distinguish between two points if they are at least 0.4 nm apart. ii. Shorter wavelengths of light provide greater resolution. iii. The wavelength of white light cannot resolve structures smaller than about 0.2 m; therefore, the best compound light microscope can not magnify something more than about 2000 X. d. Refractive index is the light-bending ability of a medium. i. The light may bend in air so much that it misses the small diameter of the highmagnification lens. Fig. 3. 1. Immersion oil is used to keep light from bending. e. Brightfield Illumination. i. Dark objects are visible against a bright background. ii. Light reflected off the specimen does not enter the objective lens. f. Darkfield Illumination. (Do Not Cover) i. Light objects are visible against a dark background. ii. Light reflected off the specimen enters the objective lens. g. Phase-Contrast Microscopy. (Do Not Cover) i. Accentuates diffraction of the light that passes through a specimen. h. Differential Interference Contrast Microscopy. (Do Not Cover) i. Accentuates diffraction of the light that passes through a specimen; uses two beams of light. i. Fluorescence Microscopy. (Do Not Cover) i. Uses UV light. ii. Fluorescent substances absorb UV light and emit visible light. iii. Cells may be stained with fluorescent dyes (fluorochromes).

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j. Confocal Microscopy. (Do Not Cover) i. Uses fluorochromes and a laser light. ii. The laser illuminates each plane in a specimen to produce a 3-D image. k. Electron Microscopy. i. Uses electrons instead of light. ii. The shorter wavelength of electrons gives greater resolution. l. Transmission Electron Microscopy (TEM). (Do Not Cover) i. Ultra thin sections of specimens. ii. Light passes through specimen, then an electromagnetic lens, to a screen or film. iii. Specimens may be stained with heavy metal salts. iv. 10,000X-100,000X; resolution 2.5 nm. m. Scanning Electron Microscopy (SEM). (Do Not Cover) i. An electron gun produces a beam of electrons that scans the surface of a whole specimen. ii. Secondary electrons emitted from the specimen produce the image. iii. 1000X-10,000X; resolution 20 nm. n. Scanned-Probe Microscopy. (Do Not Cover) i. Scanning tunneling microscopy uses a metal probe to scan a specimen. 1. Resolution 1/100 of an atom. ii. Atomic force microscopy uses a metal and diamond probe inserted into the specimen. 1. Produces 3-D images. Preparation of Specimens for Light Microscopy. a. Live or unstained cells have little contrast with the surrounding medium. However, researchers do make discoveries about cell behavior by looking at live specimens. b. A thin film of a solution of microbes on a slide is called a smear. c. A smear is usually fixed to attach the microbes to the slide and to kill the microbes. d. Preparing Smears for Staining. i. Stains are salts that consist of a positive and a negative ion. 1. In a basic dye, the color is due to a cation (+). 2. In an acidic dye, the color is due to an anion (-). ii. Bacteria have a slight negative charge at pH 7, so basic dyes are attracted to and stain bacteria. 1. Crystal violet, methylene blue, malachite green, and safranin are all basic dyes. iii. Acidic dyes typically stain the background, not the bacterium itself. 1. Eosin, acid fuchsin, and nigrosin are all acidic dyes. iv. Staining the background instead of the cell is called negative staining. e. Simple Stains. i. Use of a single basic dye is called a simple stain. 1. This stains the entire microorganism to show basic shape and structure. ii. A mordant may be used to intensify the stain, increase the affinity of the stain for the specimen, or to coat the specimen to enlarge it. f. Differential Stains. i. These stains react differently with different kinds of bacteria, and allow us to tell the difference between them. ii. Gram Stain. 1. The Gram stain classifies bacteria into two large groups: gram-positive and gramnegative. 2. Steps in making a Gram stain: Fig. 12. a. A bacterial smear is made and heat fixed. b. The smear is flooded with crystal violet for 60 seconds and then rinsed with distilled water. i. Crystal violet sticks to peptidoglycan in the cell wall. 1. It is a primary stain since it colors all cells.

c. The smear is then flooded with iodine for 60 seconds then rinsed with distilled water. i. Iodine is a mordant, increasing the affinity of the crystal violet for the specimen. d. The smear is then flooded with an acetone/ethanol wash (decolorizing agent) for 5 seconds then rinsed with distilled water. i. The acetone/ethanol washes the crystal violet out of Gram-negative cell walls. The Gram-positive cell walls retain the crystal violet as long as the acetone/ethanol wash does not last too long. ii. This step is the differential step in the Gram staining process. Gram-positive cells look purple after this step; Gram-negative cells look clear. e. The smear is then flooded with safranin stain for 1.5-2.0 minutes, then rinsed with distilled water. i. Safranin is pink in color and stains all bacteria. However, Grampositive cells have a purple cell wall due to the crystal violet, and the pink cytoplasm doesnt show through. Gram-negative cells, which have no stain because they were cleared in the differential step show up with pink cytoplasm. 1. Safranin is a counterstain, since it has a different color than the primary stain (crystal violet). f. The slide is then dried in bibulous paper. Once dry, the slide is ready to be viewed. g. Gram staining gives consistent results when done on young, growing bacteria. 3. Gram-positive bacteria. a. They have a thick peptidoglycan layer in their cell walls (disaccharides and amino acids) which is the part that stains purple due to the crystal violet and iodine washes during Gram staining. b. They tend to be killed easily by penicillin, cephalosporins, and detergents. 4. Gram-negative bacteria. a. They have a thin layer of peptidoglycan in their cell walls, and arent able to hang on to the little crystal violet that they pick up once they are flooded with the acetone/ethanol wash during Gram staining. b. They contain a layer of lipopolysaccharide (endotoxin) as part of their cell walls. c. They are more resistant to antibiotics, which cant penetrate the lipopolysaccharide layer very well. g. Differential Stains: Acid-Fast Stain. (Do Not Cover) i. Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast. ii. Nonacid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them. h. Special Stains. (Do Not Cover) i. Negative staining is useful for capsules. ii. Heat is required to drive a stain into endospores to make an endospore stain. iii. Flagella staining requires a mordant to make the flagella wide enough to see.