Microscopy and Staining

Microscopy and Staining

Metric system: meter, micrometer (micron) and nanometer (millimicron)

Bacteria are usually usually 0.2 to 2 µm


REFRACTIVE INDEX: ratio of speed of light through two media, usually =  light speed through vacuum  (P 56)  (immersion oil and glass: 1.5150 refractive index) light speed through medium
n25 C  = 1.5150             D = D line of Na emission spectrum (specific wavelength of light)


Chester Hall (1730s) used flint glass and crown glass lenses to correct chromatic aberration (blue refracts more than red) = achromatic


Joseph Jackson Lister (1830) father of Joseph, invented multiple lens components for microscopes (corrected spherical aberration)

Ernst Karl Abbe (1878) oil immersion (increase cone of light, higher resolution, brighter) (p 57)  (1886) invented current condenser

Light microscopes resolve down to 0.3 um (2000x limit of resolution)


Dark field microscopy ( dust in ray of sunlight (p 59) syphilis spirochete not seen previously.

Phase contrast microscopy: depends on minute differences in refractive index: see living cells without staining.

wet mount: liquid suspension under cover slip
hanging drop: to view motility: drop on coverslip, Vaseline, invert on depression slidet
smear: spread carefully, dry over flame to fix (coagulates proteins)

Basic dyes: are positive when ionized, stain negatively charged materials such as bacteria
crystal violet
methylene blue

Acid  stains: are negative when ionized, stain positively charged materials (zB: glass)
nigrosin (spirochete)
results in negative staining because background is usually positive, and so is stained
Fluorescence microscopy:  stain with fluorochromes:
auramine O glows yellow in UV, absorbed by Mycobacterium tuberculosis
fluorescein isothiocyanate apple green for Bacillus anthracis

DIFFERENTIAL STAINS:   Usually four steps:  primary stain, mordant, decolorize, secondary stain)
Gram Stain (p 70): by Hans Christian Gram (1884): Hucker=s stain, Iodine, 95% EtOH, safranin O(Fankhauserser’s page)
Acid fast: (p 70)
1) primary stain: steam carbolfuchsin (fuchsin is a red dye) on specimen, several min (Ziehl-Neelsen)
2) decolorize acid‑alcohol, removed color if not acid fast.
3) counter stain methylene blue
If red (p 69), may be either Mycobacterium tuberculosis or leprae. (or Nocardia, a closely related bacterium)
Negative stain (p 71): demonstrate capsules, usually not stainable,  add India ink (or other acid dyes?), capsule shows up as halo (stains background)
Endospore staining: five genera of bacteria make spores.
(P 71) Very difficult to stain, although easily seen due to different refractive index.

Schaeffer‑Fulton endospore stain: (p 71)
malachite green steamed for five minutes wash 30 seconds with water (spores stay green)
safranin counter stain