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MICROSCOPY AND STAINS

©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
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This page has been accessed Counter times since 8 July 2007.
6/23/83, rvsd 7 July 1995, 5 July 2000, 5 July 2001, 8 Jan 02, 3 July 02, 12 July 04, 08July05, 31Mar06
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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)

  D

IMPROVEMENTS IN MICROSCOPES:

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 lense 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.

 

PREPARATION OF SPECIMEN: (p 67)

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)

STAINING:             Salts of colored compounds, the ionized form either is basic (positive) or acidic (negative):

Stains usually dissolved in an alcohol or water solution,

SIMPLE STAINS

Basic dyes:            are positive when ionized, stain negatively charged materials such as bacteria

                  examples:      crystal violet

methylene blue

safranin

Acid  stains:             are negative when ionized, stain positively charged materials (zB: glass)

examples:      eosin

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 (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