Yasmine Kanaan, Ph.D.

Tel. 202 806 9540

Cancer Center, Rm # 410

Microorganisms?

Much too small to be seen with unaided eyes

Units of Measurement

• The standard unit of length is the meter (m)

• Microorganisms are measured in micrometers, µm (10-6m),

& in nanometers, nm (10-9 m)

Microscope

• Is derived from the Latin word:

Micro: small• And the Greek word:

Skopos: to look at

Antoni van LeeuwenhoekLeeuwenhoek is called "the inventor of the microscope,"

1676

The first clear mention microorganisms and their discovery.• Leeuwenhoek uses of

a self-made, hand-held, single lens microscope magnifying to about 100X,

• Many drawings are highly indicative of species still known to be present in the materials he examined.

Kinds of Microscopes

A. Light Microscopy

B. Electron Microscopy

C. Scanned-Probe Microscopy

A. Light Microscopy

• Refers to use any kind of microscope that uses visible light to observe specimens.

• Several types:1. Compound light microscopy2. Darkfield Microscopy3. Phase-Contrast Microscopy4. Differential Interference Contrast Microscopy5. Flourescence Microscopy6. Confocal Microscopy

1. Compound Light Microscope

• The total magnification of an object is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens.

40X,10X,

100x

10x

• Resolution: resolving power: is the ability of the lenses to distinguish fine details and structure.

• Refractive index: is a measure of the light-bending ability of a medium.

2.Darkfield Microscopy• Uses a special condenser

w/ an opaque disc that block light from entering the objective lens directly.

• Shows a light silhouette of an organism against a dark background.

• It is most useful for detecting the presence of extremely small organisms.

• Examine unstained microorganisms suspended in a media.

3. Phase-Contrast Microscopy

• Brings direct & reflected of diffracted light rays together(in phase) to form an image of the specimen on the ocular lens.

• It allows the detailed observation of living organisms (internal structures).

• No staining required

4. Differential interference contrast (DIC) Microscopy

• Uses 2 beams of light separated by a prisms.

• Provides a colored, 3-dimensional image of the object being observed.

• It allows detailed observations of living cells.

• No staining is required

5. Fluorescence Microscopy

• Specimens are stained w/fluorochromes and then viewed through a compound microscope by using ultraviolet light source.

• The microorganism appear as bright objects against a dark background.

5 cont. Fluorescence Microscopy

• FM is used primarily in a diagnostic procedure called fluorescent-antibody (FA) technique,

• or immunofluorescence to detect and identify microbes in tissues or clinical specimens.

6. Confocal Microscopy

• Uses laser light to illuminate one plane of a specimen at a time

• To obtain 2 and 3-dimensional images of cells for biomedical applications.

B. Electron Microscopy

• Uses a beam of electrons, instead of light.

• Electromagnets, instead of glass lenses, control focus, illumination, and magnification.

B cont.Transmission Electron Microscopy

• To examine viruses or the internal ultra structure in thin sections of cells.

• Electrons pass through the specimen.

• Magnification: 10,000-10,000X

• The image produced is 2-dimensional.

B cont.Scanning Electron Microscopy

• The e- beam is directed at the intact specimen from the top, rather than passing through a section,

• e- leaving the surface of the

specimen (2ry e-) are viewed on a television-like screen.

• To study the surface features of cells and viruses.

• Magnification 1000-10,000X

C. Scanned-Probe Microscopy/Scanning tunneling

• Uses a thin metal probe that scans a specimen and produces an image revealing the bumps and depressions of the atoms on the surface of the specimen

• Detailes views of molecules such as DNA

C. Scanned-Probe Microscopy/Atomic force

• Uses a metal-and-diamond probe gently forced down along the surface of the specimen.

• Produces a 3-dimensional image.

Staining Techniques

1. Simple

2. Differential

3. Special

Staining Reaction

• Stains - salts composed of a positive and negative ion, one of which is colored (chromophore)

• Basic Dyes - chromophore is the positive ion– dye+ Cl-

• Acid Dyes - chromophore is the negative ion– Na+ dye-

Bacteria are slightly negative, so are attracted to the positive chromophore of the

BASIC DYE

• Common Basic Dyes– crystal violet– methylene blue– safranin– Malachite green

• Common acidic dyes

EosinAcid fuchsinnigrosin

Mordant - intensifies the stain or coats a structure to make it thicker and easier to see after it is stained

Example:

Flagella - can not normally be seen, but a mordant can be used to increase the diameter of the flagella before it is stained

Salmonella typhosa

Counterstain: a second stain applied to a smear, provides contrast to the primary stain.

Simple Stains

• A simple stain is an aqueous or alcohol solution of a single basic dye (methylene blue, carbolfuchsin, crystal violet, safranin).

• To visualize shapes and arrangements of cells.

• A mordant may be used to improve bonding between the stain and the specimen.

Differential Stains

• React differently with different types of bacteria

• 2 Most Common– Gram Stain– Acid-Fast Stain

Gram Stain

• 1884 Hans Christian Gram

• most important stain used in Bacteriology

• Divides all Bacteria into 2 groups:– Gram (+)– Gram (-)

Differential Stains

• Such as the Gram stain and the acid-fast stain, differentiate bacteria according to their reactions to the stain.

• Gram stain procedure uses a purple stain (crystal violet), iodine as a mordant, an alcohol decolorizer, and a red counterstain.

Results

• Gram (+) Purple

• Gram (-) Red

• Difference - due to structure of cell wall– Gram (+) Thick cell wall– Gram (-) Thin cell wall

Differential Stains/ Acid-Fast

• Acid-fast microbes, such as members of the genera Mycobacterium & Nocardia, retain carbolfuchsin after acid alcohol decolorization & appear red

• non-acid-fast microbes take up the methylene blue counterstain and appear blue.

Special Stains

• Special stains are used to color and isolate specific parts of microorganisms, such as

EndosporesFlagellaTo reveal the presence of capsules.

Negative staining for capsules

• Microbiologist mix the bacteria in a solution containing a fine colloidal suspension of colored particles (India ink or nigrosin) and then stain, w/ simple stain as safranin.

• Capsules do not accept most stains, & appear as unstained halos around bacterial cells & stand out against a dark background.

Special Stains/Endospore staining

• Schaeffer-Fulton endospore stain.

• Uses Malachite green, the 1ry stain, heat, wash, add safranin (counterstain) to stain portions of the cells other than endospores.

Special Stains/ Flagella staining

• Use a mordant & and the stain carbolfuchsin to build up the diameters of the flagella until they become visible under the light microscope.

Any Questions??

If you have any Qs, please don’t hesitate to stop by my office