Observing Microorganisms Through a Microscope Microscopy Observing Microorganisms Through a Microscope

Microorganisms are so small that metric prefixes may be unfamiliar Units of Measurement Microorganisms are so small that metric prefixes may be unfamiliar centi = 1/100 or 10-1 milli = 1/1000 or 10-2 micro = 1/1,000,000 or 10-6 nano = 1/1,000,000,000 or 10-9

The Instruments

Compound Light Microscope Uses visible light Has at least 2 sets of lenses Can achieve maximum 2000X magnification Resolution of objects as small as 0.2 m

Light Microscopy In a light microscope visible light passes through the specimen and then through glass lenses. The lenses refract light such that the image is magnified into the eye or a video screen.

Light Microscopes Microscopes vary in magnification and resolving power. Magnification is the ratio of an object’s image to its real size. Resolving power is a measure of image clarity. It is the minimum distance two points can be separated and still viewed as two separate points. Resolution is limited by the shortest wavelength of the source, in this case light. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Resolution of Light Microscopes The minimum resolution of a light microscope is about 2 microns, the size of a small bacterium Light microscopes can magnify effectively to about 1,000 times the size of the actual specimen. At higher magnifications, the image blurs. Fig. 7.1 Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Brightfield Illumination Usual operations Specimens must be stained for viewing Best magnification and resolution with the oil immersion objective Oil has same refractive index as glass

Darkfield Microscopy Only light reflected from specimen enters objective lens Organism appears light against a dark field Useful for examining Live organisms Microorganisms which cannot be stained by standard methods Treponema pallidum, the causative agent of syphilis

Phase-Contrast and Differential Interference (DIC) Microscopy Uses wave nature of light One set of light rays are direct and one set are reflected Makes detailed images of internal structure of living microorganisms possible Image in greyscale Uses differences in refractive indices Uses 2 beams of light Resolution higher Brightly colored image Image appears nearly three-dimensional

Electron micrographs

Fluorescence Microscopy When illuminated with short  light some dyes emit light with longer  Enables viewing of cells located on an opaque surface such as a soil particle When illuminated with UV or halogen light source preparations glow Bovine pulmonary artery endothelial cells. Photometrics, Ltd.

Fluorescent stain of cell

Confocal Microscopy Preparations also stained with fluorochromes 3-D confocal microscopy of Salmonella-infected macrophage (green) with XY-slice showing bacteria (red) inside the cell Preparations also stained with fluorochromes Exceptionally clear two-dimensional images Three-dimensional images obtained by computer construct

Electron Microscopy Beam of electrons has shorter  so gives better resolution than visible light Electromagnetic lenses rather than glass Done in a vacuum Can resolve to 0.5nm and magnify up to 100,000 times. Specimen must be dry….dead

Transmission Electron Microscopy (TEM) Resolves objects as close as 2.5nm Magnification 10,000 to 100,000X Ultra-thin sections Specimens must be dehydrated Preparation of specimen may generate artifacts Lambda Bacteriophage DNA (TEM x153,000)

Scanning Electron Microscopy (SEM) Resolves objects as close as 20 nm Magnification between 1,000 and 10,000X Whole specimens 3-dimensional view of specimen Specimen dehydrated Slime Mold Fruiting Structure, Lamproderma sp. (SEM x290)

Scanning Tunneling Atomic Force Thin metal probe scans specimen Resolving power much greater than electron microscopes no special specimen preparation detailed views of silicon chips & DNA molecule Metal and diamond probe forced down along surface of specimen 3-dimensional image no special preparation of specimen is required views of detailed structure of biological molecules

Preparation of Microscopy Specimens Microorganisms must be spread over the surface of a slide (smear) Microorganisms must be attached to the slide (fixed) Microorganisms must be colored (stained)

Making the Smear and Fixing It Put a small amount of organism into a drop of water on a clean microscope slide & spread . When dry pass through the flame of a Bunsen burner or flood with methyl alcohol

Stains Dyes Negative Stain Simple Stains Differential Stains stains the background not the organism Simple Stains everything stained a single color Differential Stains distinguish among bacteria based on particular characteristics

Characteristics of Dyes Basic Dyes Chromophore is in the positive ion Used to stain most bacteria Used alone as simple stains in combination for differential stains Acidic Dyes Chromophore is in the negative ion Used in negative staining & for staining nuclear material

Gram Stain The Gram Stain is the single most important test in microbiology. The principal utility of the Gram Stain rests on its speed and simplicity. Most bacteria may be divided in two groups by this procedure developed by the Danish physician Hans Christian Gram to differentiate pneumococci from Klebsiella pneumonia difference between Gram-positive and Gram-negative bacteria is in the structure of the cell wall

Procedure

Results G+ cocci G- rods Websites with more samples of gram stained bacteria GRAM STAINED IMAGES OF MEDICALLY IMPORTANT BACTERIA Loyola University Medical Center http://www.meddean.luc.edu/lumen/DeptWebs/microbio/med/gram/slides.htm GRAM STAIN TUTORIAL http://www.courses.ahc.umn.edu/pharmacy/5825/GSPage05.html

Acid Fast Stain Acid Fast Staining is used primarily for detection of organisms with a thick outer coat composed of true waxes, mycolic acids and phosphatides Mycobacteria are not decolorized and retain the stain, appearing pink under the light microscope (hence are 'fast', like color-fast clothes). Acid Fast Stain can also be used to identify several protozoa, such as Cryptosporidium and Isospora belli. These two coccidia have recently acquired greater clinical significance because of their widespread occurrence in immuno- compromised patients, such as those infected with HIV.

Special Stains Spore Stain Capsule Stain Flagella Stain Bacillus subtilis Streptococcus pneumoniae Pseudomonas aeruginosa