1. Framework: arm
2. Stage: holds slide and moves it around stage
3. Lens system

-Oculars: eyepiece lenses

-Objectives: lenses attached to rotable nosepiece (4x,10x,45x,100x-oil immertion lens-)

-Condenser: directs light towards objective lens

-Iris Diaphragm

d, which is the smallest distance between two objects which can be seen as separate, can be decreased by lowering λ(wavelength of light) or increasing the NA(numerical aperture)

d=λ/2NA

Mag(total)= Mag(objective) * Mag(ocular)

-Objective: nosepiece lens (4x,10x,45x,100x)

-Ocular: eyepiece lens (usually 10x)

1. Cocci: more or less spherical organisms
2. Bacilli: Cylindrical organisms
3. Helicoidal: Spiral shaped

• Diplococci(pairs): divide in one plane
• Steptococci(chains): divide in one plane
• Tetracocci(tetrads): divide in two planes
• Staphylococci(clusters): divide in three planes irregularly
• Sarcinae(cuboidal packets): divide in three planes regularly

What are the different cylindrical shapes in which Bacilli can appear?

• Coccobacillus: very short, spherical, slightly longer in one direction than other.
• Fusiform Bacilli: tapered at the ends, 'football' shaped
• Filamentous Bacillary: forms grow in long threds

• Cationic: basic dyes, + charged chromophore

Methylene Blue, Crystal Violet

• Anionic:acidic dyes,- charged chromophore

Acid fuschin, Congo Red, Nigrosin

• Fat soluble:no charge

Sudan Black Stain granules of Poly0B-OH-Butyric acid

• Insoluble Dyes:water insoluble

India Ink

1. Negative Staining
2. Simple Staining
3. Differential Staining
4. Structural Staining

• Stains background, not the cell in brightfield microscopy
• Uses 2 dyes:
• Nigrosin-black anionic charged dye, repelled by the - charged surface of bacterial cell
• India Ink-(colloidal suspension of carbon particels) doesn't penetrate cell surface

• One dye used to stain all cells the same color. Can be used to tell morphology and size. Cationic (+) dyes are attracted by ionic forces to the - charged surface of bacterial cell.
• Common dyes are methylene blue and crystal violet.

Explain differential stain procedure

• Causes cells to stain differently based on properties of the cell
• Two examples are:
• Gram Stain
• Acid Fast Stain

• Causes the cell to stain differently based on characteristics of their cell wall.
• Gram+ microorg. have a higher peptidoglycan and lower lipid content than Gram-.
• Cells are stained with crystal violet, then fixed with iodine forming a crystal violet-iodine complex within the cell. Ethanol is then added as a decolorizer.(Gram- cells are easily decolorized because the ethanol dissolves the high lipid cell wall allowing the crystal violet-iodine complex to readily exit the cell. Gram+ cells resist decolorization due to the difference in cell wall consistency retaining the crystal violet-iodine complex). The Gram- cells are then counterstained with saffranin.

Explain Acid Fast Stain

• Acid fast microorganisms have a high wax content in their walls, which requires the use of steam to allow dye to penetrate the cell.
• Cells are steamed in the presence of carbol fuschin and decolorized with acid alcohol.
• Cells which are "acid fast" will not decoloried and remain red, while non acid fast organisms will readily lose their stain and become colorless.
• These cells are then counterstained with Methylene blue.

• Mycobacterium: Do not gram stain well if mature because of high wax content within walls (if youn appear as gram + tapered rods that sometimes fragment)
• Two important species: Tuberculosis and leprae
• Nocardia

• Spore staining: To stain spores, the cells must be steamed to allow for the dye (malachite green)to enter the spores.Once the spores are stained, all other microorganisms can be easily decolorized with water. The other microorganisms and vegetative cells are then counterstained with Safranin.
• Endospores: green center with a pink sporangium
• Free spores:small green oval bodies

1. Bacillus: areobic, gram+ rod
2. Clostridium: anaerobic, gram+ rod
3. Sporsacinae: cocci

-Anaerobic green: endospore/free spores clostridium

-Aerobic green: endospore/free spores of bacillus

-Anaerobic pink:vegetative/sporangia of clostridium

-Aerobic Pink:vegetative/sporangia of bacillus

1. Pour: 15-20 ml of liquid agar used to pour into plate
2. Broth: 5-7 ml of liquid media
3. Deep: 5-7 ml of media which has solidified in an upright position
4. Slant: 5-7ml of media which has solidified at an angled position
5. Fermentation broth: broth with Durham Tube added

1. Selective media: a media which favors the growth of one type of microorganisms over another. Accomplished by either inhibiting unwanted microorganisms or enriching
2. Differential media: a media which differentiates or distinguishes between different types of microorganisms based on differences in appearance of growth or color changes.