Microscopy & Cell Structure

L2

Image quality (magnification, resolution, contrast)

1. Magnification

• zoom in size, no upper limit

2. Resolution - higher = more detail

• clarity/sharpness, min distance b/w objects to differentiate

• limited by wavelength of energy, quality of lenses, * magnification

3. Contrast - most microbes are transparent so contrast needed

• difference in color or brightness b/w object & background

• special microscopes / stains can increase contrast

Light microscopy

• lenses magnify img generated from light passing thru specimen

• max magnification = 2000x

• for most bacteria & larger

Compound (brightfield) microscope

• light microscope, white bg

• multiple lenses magnify - objective (4x, 10x, 40x, 100x) & ocular (10x)

• light from lamp + condenser

Darkfield microscope

• light microscope, reverse contrast w/ dark bg

• when staining / brightfield ineffective

  • light passed through specimen is seen; light through glass filtered out

Phase-contrast (PC) microscope

• light microscope

• light thru specimen vs empty glass will bend at diff angles (diff refractive indices)

• utilize different refraction patterns to provide natural contrast for specimen

• no cell fixing/staining required; can view live cells w/ good contrast & resolution

Differential interference contrast (DIC) microscope

• light microscope

• similar to PC, but uses 2 diff light sources & utilizes prisms

  • 1 light into specimen, 1 light into surrounding glass

  • like PC, uses diff refractive indices of ea. to create contrast

• great contrast & resolution w/o staining, almost 3D-like

• prisms can provide diff colored structures

Fluorescence

• absorbs UV, emits visible light — “glow in the dark” green, red, blue, yellow

• track, detect, measure microbes

• Tagging microbes

  • some microbes absorb fluorescent dyes

  • tag microbes w immunofluorescence (fluorescent antibodies) — fixed dead cells

  • GFP tagging - live or fixed cells

  • can engineer microbes to express fluorescent proteins

Fluorescence microscopes

• shoot UV light of specific wavelength at sample

• all visible light emitted is detected/seen

• difficult differentiation of parts due to everything glowing

• simple, cheap, effective

Confocal microscope

• uses laser to excite thin section of thick specimen

• only emitted light from that section - high resolution

• 2D “slices” of sample, computer constructs 3D img

Scanning acoustic microscope

• Shoot sound waves at specimen & creates 3d structure of ricochet/penetration

Electron microscopy

• beam of electrons illuminate sample

• e- travel in short wavelengths

• resolution 1000x better, mag ~100-200x better

Transmission EM

• ultrathin specimen section → fixed → dehydrated → heavy metal coating

• shoot w/ e- beam & measure e- transmitted thru specimen

• high-res 2D image

• downside: may produce artifacts

TEM shadow casting

• stain sample with heavy metal from 45 degree angle, 3D effect

TEM freeze fracturing

• super-freeze sample & break it

• less harsh than fixation

Scanning EM

• sample coated w/ heavy metal & shot w e-

• Electron beam forces metal to release e-

• released e- used to produce 3D img of sample surface

Scanning probe microscopy

• small probe on specimen surface, detects textures

• 3D view

2 major varieties:

1. Scanning tunneling microscopy

2. Atomic force microscopy

Staining

1. smear speci on slide

2. air dry, heat fix (killed)

3. flood slide w/ dye, let sit

4. wash off & rinse

Basic vs acidic stains

Basic = cationic, attaches to most bacteria (slightly - charged @ pH 7)

Acidic = anionic, stains slide not bacteria, no fixation

Simple vs negative stains, mordant

Simple = 1 basic dye on all cells; added mordant = color intensifies

Negative = dyes bg, not cell

Differential stains

• Dyes distinguish bacteria groups

2 types: Gram staining & acid-fast stain

Gram staining

1. stain w/ crystal violet & iodine (helps CV clump up)

2. add alcohol to decolor (stain removed from the thin gram - cells)

3. counterstain clear cells w/ safranin

4. purple (gram +) & pink cells (gram -)

Acid-fast staining

• stains & detects genera Mycobacterium & Nocardia (causes TB & leprosy)

• has lots of lipids in cell wall, prevents uptake of normal dyes

1. carbolfuchsin (red) → heat into cell

2. alcohol decolorization (walls hold stain)

3. counterstain non-acid fast cells w/ methylene blue

Capsule stain

• sugar layer (polysaccharide) envelopes cell

• stain bg (- stain) & cell

• clear halo around cell

Endospore stain

• most resistant form of life; dormant cell (endospore) when at risk

1. heat to force malachite green into spores

2. stain regular cells w/ safranin

Flagella stain

• carbolfuchsin & heat repeatedly

• build up diameter

Coccus, bacillus, spiral

• Coccus - spherical

  • sometimes stay attached → diplo (2), strepto (chains), staphylo (grape-like clumps)

• Bacillus - rod-shaped

  • palisade bacilli (vertically bound), coccobacilli (short, plump rods)

• Spiral - at least 1 bend

  • vibrio (commas), spirillum (short spiral/2), spirochete (long helical, no flagella)

Glycocalyx

• Outermost layer of polysaccharides / sugar around some bacteria

• adherence to surface & e/o & protection from environment

• cell secretes lots of sugar → assembles layer

Capsule

• Type of glycocalyx

• organized & attached to cell wall

Slime layer

• Type of glycocalyx

• Irregular & loosely attached to cell wall

Biolfilm matrix

• Type of glycocalyx

• Outer layer is made by biofilms attached to surface (sugar covers biofilm)

Flagella function & features

• long, thin projections help with movement

• Peritrichous = all over surface

3 major parts:

1. filament - long & hair-like, composed of protein flagellin (hollow helix)

2. hook - curved portion, molecular joint, motor rotation, filament flexibility

3. basal body - anchors into cell wall and plasma membrane via protein rings

Fimbrae & Pili

• Fimbrae = short, hair-like spikes in gram (-) bacteria for attachment

• Pili - slightly longer extensions, can grow & shrink

  • motility, attcahment, trasnfer DNA

  • conjugation = make contact w/ other cells & exchange genetic info