Observing
Microorganisms
Through
Microscopes
Units of measurement
Metric system
The standard unit of length in metric system
is the meter (m)
--the advantage of m.s is that the units are
related to each other by factors of 10
1m=100 cm or 1000mm
Micrometer = 0.000001 m = 10-6
Or
Nanometer = 0.000000001 m = 10-9
Microscopes
• There are two basic types of
microscopes (according to
source of illumination) that are
commonly used in
Microbiology:
light microscopes
and electron microscopes.
illuminator = light source
Condenser = has lenses that direct the
light rays through the specimen
Objective lenses = the lenses closest to
the specimen
Ocular lens = eyepiece = the image is
magnified again by ocular lens
Magnification = enlargement
Resolution = is the ability of the lenses to
distinguish fine detail & structure
Refraction index = is a measure of the light
bending ability of a medium
Calculate the magnification. = multiplying •
power by the ocu.l. power – the obj. L.
10X
Low power – 10X == 100X •
High power – 40X == 400X •
Oil immersion – 100X == 1000X •
Greater resolution can be achieved by 
using oil immersion, 
by filtering out with-blue light, 
and by replacing light with electrons. 
Blue Light Increases
Resolution
Blue light has
shorter
wavelength than
other visible
regions of the
electromagnetic
spectrum.
Shorter
wavelength
results in higher
resolution.
Blue filter is
inserted between
light source and
condenser.
Oil Immersion Increases Resolution
Oil Immersion Increases Resolution
Air has a different Index of
Refraction from water (so
light bends).
Air has a different
Index of Refraction
from glass (so light
bends).
The Mineral Oil has
the same Index of
Refraction as glass
(so light does not
bend).
B- Electron Microscope
abeam of electrons is used –free electrons travel
in waves
(100.000)
EM = Increased
Resolution
Transmission
Electron
Microscopy (TEM):
electrons are
transmitted
through substance.
Scanning Electron
Microscopy (SEM):
electrons bounce
off the surface of
specimen resulting
in a more 3-D
image.
a- Light Microscopy:
Dark field Microscope
Fluorescent Microscope
The ability of substance to •
absorb short wave length
(ultraviolet) and give off light at
a longer wave length (visible)
In fluorescence microscopy specimens are •
first stained with fluorochromes and then
viewed through a compound microscope
by using an uv light source
The m.o. appear bright objects against a •
dark background
Fluorescence microscopy is used primarily •
in a diagnostic procedure called f.ab.
tequnique
Observation of microorganisms
Observation of microorganisms
Colorless •
A smear must be •
prepared •
stained •
Staining = coloring -Increase contrast •
of microorganisms
Fixed = attached •
fixing – kills , fix & preserves •
various parts of m.o. in their natural state
Preparation of smear for light field
Microscope
 Organic salts compose of a positive and a
negative ion, one of which is colored and is
known as the chromophore
 In Basic dye: positive ion is colored
Cystal violet, methylen blue, malachite green,
safranin.
 In Acidic dye: negative ion is colored
eosin, acid fuchsine, and nigrosin.
 As bacteria are slightly negatively charge at
pH7, it colored with basic dye
Types of stains
Simple stain:
Differential stain:
Structural or special stains
• React differently with different kinds of
bac.
• More than one dye
• Example : as Gram stain, acid fast
 Primary dye
 Mordant
 Decolorizing step
 Counter stain
Differential Stain: The Gram Stain
Gram Stain
24
Acid fast stain
Mycobacterium •
Red dye=carbol-fuchsin •
Gentile heating =enhance penetration & •
retention of the dye
Acid alcohol=decolorizing – removes the •
red color from not AF bac.
Methylene blue •
Acid-Fast Staining
Note that the acid-fast bacteria are
found as red clumps of filamentous
cells
.
Mycobacterium avium complex (MAC) with acid
fast stain often has the characteristic appearance
shown here with numerous mycobacteria filling
macrophages. Such macrophages may be distributed
diffusely or in clusters.
Steps
India ink Capsule stain
27
ecial Stain: Capsule Stai
Note that the
background is
stained as well as
the bacteria, plus
there is a “halo”
around the bacteria.
The halo represents
the capsule.
Malachite green
Flagella stain
Mordant----- iodine