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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)
(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
        Acidstains: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 isothiocyanateapple green for Bacillus anthracis
 

DIFFERENTIAL STAINS:
 Gram Stain (1884) Acid fast: (Ziehl-Neelsen)
Endospore staining:
primary stain
 Hucker's stain carbolfuchsin (a red dye)
malachite green
mordant
 Iodine steam on specimen, several min
steamed for five minutes 
decolorize
 95% EtOH acid-alcohol
wash 30 seconds with water 
secondary stain
 safranin O methylene blue
safranin
purpose
Gram positive or Gram neg
Mycobacterium
spores stay green (five genera)
(p 70):  by Hans Christian Gram : , , , (  Fankhauserser's page)
 
If red (p 69), may be either  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)

 of bacteria make spores.

(P 71)Very difficult to stain, although easily seen due to different refractive index.

Schaeffer?Fulton endospore stain: (p 71)