Microbiology Module 1

Magnification occurs because light from specimen is bent as it travels thru lens of diff. optical density than air

Lens is curved, giving rise to patterns of light rays w/paths indicated in bottom figure

Image is inverted and magnified when viewed well beyond focal point

Compound light microscopes have 2 magnifying lenses so final image is not inverted

Light microscope has upper limit of 1500X (set by wavelength of visible light and curvature of lenses)

Condenser acts to focus the light source so therefore nonmagnifying

Resolution describes the ability to distinguish between 2 small objects that are close together

Resolution (d) is expressed as distance that 2 objects must be separated by in order for them to appear distinct

d= 0.5L/NA where d is resolution, L is wavelength of light source and NA is numerical aperture ( relates to ability of lens to gather light

As wavelengths used in microscope get shorter, the resolution gets better (Can distinguish smaller objects)

• Wavelengths (Lambda) of electrons is dependent on applied voltage
• (WV at 10kV is 0.01nm, at 1MV is 0.001nm)

Staining procedures use dyes and techniques that increase contrast of specimen relative to surroundings

Positive strains use colored dyes that bind specifically to microbial surface (cytoplasmic membrane)

Most dyes for simple positive stains are chemically basic (+ charged at neutral pH) (Crystal Violet, Methylen blue)

Negative Stains: Use dyes that do not bind to microbe's surface surface, instead fill spaces around microbe

Chemically acidic at neutral pH ( nigrosin and India Ink)

Do not require heat fixing (can use on fragile or live specimens) because there is no rinse step

Often used for capsule visualization

Both + and - stains reveal general shape, size and type of growth cells

Dyes and techniques that stain different microbes diff. colors

Gram stain distingusiehs microbes based on type of cell walls

• Rosy red colored stains from safarin (Gram - cells)
• Gram (+) bacteria remain purple

Dark-field microscopy and phase contrast use visible light sources

DARK-FIELD: uses alternate optics so that only light that is reflected or refracted by the specimen forms an image

PHASE CONTRAST: Optics are used to emphasize slight diff. in refractive index of cells or subcellular structures. Used to observe live, unstained eukaryotic cells

Variation of phase contrast. Used to see small diff. in refractive indeces as diff. colors of light

Used to see structures in live, unstained specimen

Diff. between phase contrast is the light source which is plane polarized visible light

Image is visualized because a cellular structure or dye that binds to structure is fluorescent (Absorbs light at one wavelength and re-emits it at a longer wavelength)

Microscopes have filters that limit range of wavelengths that illuminate specimen and that reach observer, creating vivid sharp images

"Tags" often are fluorescent dyes covalently attached to antibodies that are specific for a unique cell structure or antigen

Several fluorescent species can be used at same time

Use same optics as light microscope, but light source is an electron beam and lenses are electomagnets

Contrast of specimen (dyes) vs. surroundings done w/electron dense stains (+ or -)

Must be done in vacuum, so specimens are dehydrated and chemically fixed

High resolution, depending on voltage and technique

Optics much diff. than TEM or light microscopy. Visualizes 2ndary electrons ejected from surface features that are then captured by imaging system. No magnifying lenses

Source of 2ndary electrons typically an electron-dense dye (gold) used to stain specimen. Done in vacuum

Specimen size range varies from nm to cm. Resolution is at least 10X worse than TEM

Uses computer integration of images from many diff. focal planes to generate 3D pictures

Usually fluorescent dyes attached to antibodies are used in conjunction w/laser light source to produce sharp images