Observing Microorganisms Through Microscopes

Units of measurement Or Nanometer = 0.000000001 m = 10-9 Metric system The standard unit of length in metric system is the meter (m) --the advantage of m.s is that the units are related to each other by factors of 10 1m=100 cm or 1000mm Micrometer = 0.000001 m = 10-6 Or Nanometer = 0.000000001 m = 10-9

Microscopes There are two basic types of microscopes (according to source of illumination) that are commonly used in Microbiology: light microscopes and electron microscopes. 

illuminator = light source Condenser = has lenses that direct the light rays through the specimen Objective lenses = the lenses closest to the specimen Ocular lens = eyepiece = the image is magnified again by ocular lens

Magnification = enlargement Resolution = is the ability of the lenses to distinguish fine detail & structure Refraction index = is a measure of the light bending ability of a medium

Calculate the magnification. = multiplying the obj. L. power by the ocu.l. power – 10X Low power – 10X == 100X High power – 40X == 400X Oil immersion – 100X == 1000X

Greater resolution can be achieved by using oil immersion, by filtering out with-blue light, and by replacing light with electrons.

Blue Light Increases Resolution Blue light has shorter wavelength than other visible regions of the electromagnetic spectrum. Shorter wavelength results in higher resolution. Blue Light Increases Resolution Blue filter is inserted between light source and condenser.

Oil Immersion Increases Resolution

Oil Immersion Increases Resolution Air has a different Index of Refraction from water (so light bends). Air has a different Index of Refraction from glass (so light bends). Oil Immersion Increases Resolution The Mineral Oil has the same Index of Refraction as glass (so light does not bend).

B- Electron Microscope abeam of electrons is used –free electrons travel in waves (100.000)

EM = Increased Resolution Transmission Electron Microscopy (TEM): electrons are transmitted through substance. EM = Increased Resolution Scanning Electron Microscopy (SEM): electrons bounce off the surface of specimen resulting in a more 3-D image.

Types of Microscopes

Dark field Microscope a- Light Microscopy: Types of Microscopes

Fluorescent Microscope The ability of substance to absorb short wave length (ultraviolet) and give off light at a longer wave length (visible) 15

In fluorescence microscopy specimens are first stained with fluorochromes and then viewed through a compound microscope by using an uv light source The m.o. appear bright objects against a dark background Fluorescence microscopy is used primarily in a diagnostic procedure called f.ab. tequnique

Observation of microorganisms

Observation of microorganisms Colorless A smear must be prepared stained Staining = coloring -Increase contrast of microorganisms Fixed = attached fixing – kills , fix & preserves various parts of m.o. in their natural state

Preparation of smear for light field Microscope

Dyes Organic salts compose of a positive and a negative ion, one of which is colored and is known as the chromophore In Basic dye: positive ion is colored Cystal violet, methylen blue, malachite green, safranin. In Acidic dye: negative ion is colored eosin, acid fuchsine, and nigrosin. As bacteria are slightly negatively charge at pH7, it colored with basic dye

Staining Types of stains Simple stain: Differential stain: Structural or special stains

Differential Stains React differently with different kinds of bac. More than one dye Example : as Gram stain, acid fast Primary dye Mordant Decolorizing step Counter stain

Differential Stain: The Gram Stain

Gram Stain 24

Acid fast stain Mycobacterium Red dye=carbol-fuchsin Gentile heating =enhance penetration & retention of the dye Acid alcohol=decolorizing – removes the red color from not AF bac. Methylene blue

Acid-Fast Staining Note that the acid-fast bacteria are found as red clumps of filamentous cells. Mycobacterium avium complex (MAC) with acid fast stain often has the characteristic appearance shown here with numerous mycobacteria filling macrophages. Such macrophages may be distributed diffusely or in clusters.

India ink Capsule stain Steps India ink Capsule stain 27

Special Stain: Capsule Staining Note that the background is stained as well as the bacteria, plus there is a “halo” around the bacteria. The halo represents the capsule. Special Stain: Capsule Staining

Spore stain Malachite green

Flagella stain Mordant----- iodine