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 101m=100 cm or 1000mm

Micrometer = 0.000001 m = 10-6Or

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 the obj. L. power by the ocu.l. power – 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.

Blu

e L

igh

t In

crea

ses

Res

olu

tio

n

Blue filter is inserted between light source and

condenser.

Shorter wavelength

results in higher resolution.

Blue light has shorter

wavelength than other visible

regions of the electromagnetic

spectrum.

Oil

Imm

ers

ion

Incr

ea

ses

Re

solu

tio

n

Oil

Imm

ers

ion

Incr

ea

ses

Re

solu

tion

The Mineral Oil has the same Index of Refraction as glass (so light does not

bend).

Air has a different Index of Refraction from glass (so light

bends).

Air has a different Index of Refraction from water (so

light bends).

B- Electron Microscopeabeam of electrons is used –free electrons travel in waves

(100.000)

EM

= In

crea

sed

R

eso

luti

on

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.

Dark field Microscope a- Light Microscopy:

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 coloredCystal 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 dyeMordant Decolorizing stepCounter stain

Differential Stain: The Gram Stain

24

Gram Stain

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

Aci

d-F

ast

Sta

inin

g

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.

Note that the acid-fast bacteria are found as red clumps of filamentous

cells.

Steps

India ink Capsule stain

27

Sp

ecia

l Sta

in:

Cap

sule

S

tain

ing

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