Tools of the Laboratory: The Microscope Tools of the Laboratory: The Methods for Studying Microorganisms Microbiology: A Systems Approach Tools of the Laboratory: The Microscope Chapter 3 Copyright © The McGraw-Hill Companies, Inc) Permission required for reproduction or display. Chapter 3, pages 55 to 79

Learning Goals Name parts of the microscope and describe their function List and describe the three elements of good microscopy: magnification, resolution, and contrast. Identify a use for darkfield, phase-contrast, differential interference contrast (DIC), fluorescence, confocal, transmission and scanning electron microscopy, and compare each with brightfield illumination. Explain the purpose for simple staining. Differentiate between positive and negative staining. List the steps in preparing a Gram stain, and describe the appearance of G+ and G- cells after each step. Compare and contrast Gram stain and acid-fast stain Explain why each of the following is used: capsule stain, endospore stain, flagella stain.

Viewing Microbial Cells Too small for the human eye Need to magnify the image Leeuwenhoek used simple microscopes Hooke used an early compound microscope

Compound Microscope

Microscope provides Magnification: creates an optical image of the object which appears enlarged. Resolution: capacity to separate two adjacent objects. Contrast.

Refraction Refraction: bending or changing an angle of the light ray as it passes through a medium such as lens

Magnification of an object by a compound microscope occurs in two phases

Resolution Resolution is the ability to distinguish two adjacent objects or points from one another Also called resolving power (smaller is better) Resolving power (RP) = Wavelength of light in nm 2 x Numerical aperture Shorter wavelengths provide a better resolution Numerical aperture is how well the lens gathers light

Effect of Wavelength on Resolution

The Oil Immersion Lens Immersion oil reduces refraction of light More light is gathered Numerical aperture increases Resolution improved Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Objective lens Air Oil Slide

Crystal violet stain of Escherichia coli stain of Corynebacterium Increasing Contrast Most microbes are… Colorless Very Small Difficult to see Staining increases contrast by increasing refractive index Increases size sometimes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. (a) Simple Stains Crystal violet stain of Escherichia coli Methylene blue stain of Corynebacterium a1: © Kathy Park Talaro; a2: © Harold J. Benson

Conclusions: The most common microscope used in microbiology is the compound light microscope. It uses visible light. The total magnification of an object is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens The maximum resolution the ability to distinguish between two points) of a compound light microscope is 0.2m, maximum magnification is 2000x. Immersion oil is used with the oil immersion lens to reduce light loss between the slide and the lens Specimens are stained to increase the difference between the refractive indexes of the specimen and the medium

Light Microscopy Bright-field Dark-field

Light Microscopy Phase contrast DIC

Brightfield Darkfield Phase-contrast

Light Microscopy Fluorescence Confocal

Electron Microscopy Focused electron beam Wavelength = 6pm Magnification100,000 X Offers much better resolution

Conclusions Brightfield illumination is used for stained smears. Unstained cells are more productively observed using dark field, phase-contrast, or DIC microscopy. The darkfield microscope shows a light silhouette of an organism against a dark background. It is most useful for examining living organisms that are invisible in brightfield, do not stain easily, or are distorted by staining. A phase contrast microscope brings direct and reflected light rays together to form an image. It allows detailed observation of living organisms. The DIC microscope provides a colored, three-dimensional image of the object. It allows detailed observation of living cells.

Conclusions: In fluorescence microscopy, specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet as a light source. The organisms appear as bright objects against a dark background. In confocal microscopy, a specimen is stained with a fluorescent dye and illuminated one plane at a time. Using computer, two- and three-dimensional images can be produced. A beam of electrons, instead of light, used in an electron microscope. Electromagnets, instead of glass lenses, control focus, illumination, and magnification. Thin sections of microorganisms can be observed using transmission electron microscopy (TEM). Three-dimensional of the surfaces of microorganisms can be obtained with scanning electron microscope (SEM)

Microbial Stains Simple staining: Positive staining Negative staining Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Simple staining: Positive staining Negative staining Differential staining: Gram staining Acid fast staining Spore staining Special staining Capsule staining Flagella staining Differential Stains (a) Simple Stains (b) (c) Special Stains Crystal violet stain of Escherichia coli Gram stain Purple cells are G+ Red cells are G - India ink capsule stain of Cryptococcus neoformans Acid-fast stain Red cells are acid-fast. Blue cells are non-acid-fast. Flagellar stain of Proteus vulgaris A basic stain was used to build up the flagella. Methylene blue stain of Corynebacterium Spore stain, showing spores (red) and vegetative cells (blue)

Purple cells are gram-positive. Red cells are gram-negative. Gram Staining The most universal diagnostic staining technique Differentiation of microbes as gram positive(purple) or gram negative (red) Gram stain Purple cells are gram-positive. Red cells are gram-negative.

Gram Staining Procedure

Over-decolorized Positive control Under-decolorized

Conclusions: Staining means coloring microorganisms with a dye to make some structures more visible. Fixing uses heat or alcohol to kill and attach microorganisms to a slide. Simple stains make cellular shapes and arrangements visible. Bacteria are negatively charged, and the colored positive ion of basic stain will stain bacterial cells. The colored negative ion of an acidic dye will stain the background of a bacterial smear producing a negative stain. Differential stains differentiate bacteria according to their reaction to the stains.

Conclusions The gram stain procedure uses a purple stain (crystal violet), iodine as a mordant, an alcohol decolorizer, and a red counterstain. G+ bacteria retain the purple stain after the decolorization step; G- bacteria do not, and thus appear pink from the counterstain. Acid-fast microbes, such as members of genera Micobacterium and Nocardia, retain carbolfuchsin after acid-alcohol decolorization and appear red; non-acid-fast microbes take up the methylene blue counterstain and appear blue. Negative staining is used to make microbial capsules visible. The endospore stain and flagella stain are special stains that only color certain parts of bacteria.