CHAPTER 4

DEMONSTRATION OF
MICROORGANISMS
 STAINING
❑ Staining simply means coloring the
microorganisms with a dye that emphasizes
certain structures.
❑ Stains are salts composed of a positive and a
negative ion, one of which is colored and is
called as the chromophore.
❑ The color of the so-called basic dyes is in the
positive ion; in acidic dyes, it is in the
negative ion.
STAINING
❑ Bacteria are slightly negatively charged at pH 7. Thus, the
colored positive ion in a basic dye is attracted to the
negatively-charged bacterial cell.
❑ Basic dyes (safranin, methylene blue, crystal violet,
carbolfuchsin, etc.) are more commonly used than acidic
dyes.
❑ Acidic dyes (eosin, nigrosin, India ink) are repelled by the
bacterial surface so the stain colors the background
instead.
❑ Preparing colorless bacteria against a colored background
is called negative staining. It is valuable in observing
cell shape, size and capsules.
STAINING
❑ Before the microorganisms can be stained, they must be
fixed (attached) to the microscope slide.
❑ Fixing kills the microorganism but preserves the various
parts of the cell with only minimal distortion.
❑ When a specimen is to be fixed, a thin film of material
containing the microorganisms is spread over the surface
of the slide.
❑ This film, called a smear, is allowed to air dry.
STAINING
❑ In most staining procedures, the slide is fixed by passing it
through the flame of a Bunsen burner several times,
smear side up, or by covering the slide with methyl alcohol
for one minute.
❑ Stain is then applied and washed off with water, and then
the slide is blotted with absorbent paper.
❑ Without fixing, the stain might wash the microbes off the
slide. The stained microorganisms are now ready for
microscopic examination.
 TYPES OF STAINS
There are 3 types : simple, differential and special stains.

A. Simple Stains
o A simple stain is an aqueous or alcohol solution of
a single basic dye.
o The primary purpose is to highlight the entire
microorganism so that cell shape and basic
structures are visible.
o Occasionally, a chemical is added to the solution
to intensify the stain; such an additive is called a
mordant.
 TYPES OF STAINS
B. Differential Stains
o Differential stains react differently with different kinds
of bacteria and thus can be used to distinguish among
them.
o The differential stains most frequently used are the
Gram stain and the acid-fast stain.
B. Differential Stains
1. The Gram stain
PRINCIPLE BEHIND GRAM STAINING
Structural differences in the cell wall account for the varying
staining reactions of bacteria. Gram + bacteria have a thick
peptidoglycan layer while G- bacteria have a thin peptidoglycan
layer beneath an outer lipopolysaccharide layer in their cell wall.
The crystal violet and iodine readily enter all cells, and they
combine to form the CV-I complex. This complex is large in
size and cannot be washed out of the thick peptidoglycan layer
of G+ cells by alcohol.
Consequently, G+ cells retain the color of the primary stain.
In G- cells, the alcohol disrupts the outer LPS layer,and the
CV-I complex is completely washed out of the thin
peptidoglycan layer.
As a result, G- bacteria are colorless until counterstained
with a red dye, after which they appear pink.
 STAINING
B. Differential Stains
2. Acid-fast stain
 ACID-FAST STAINING PROCEDURE

1. A heat-fixed smear is covered with carbolfuchsin, a basic red

dye (primary stain). The slide is gently heated for five
minutes to enhance penetration and retention of the dye.
Then the slide is cooled and washed with water.

2. The smear is then treated with acid alcohol for 15 seconds, a

decolorizer, which removes the red stain from non-acid-fast
bacteria. The acid fast bacteria retain the red or pink color
because the carbolfuchsin is more soluble in the cell wall
waxes than in the acid alcohol.

3. The smear is then stained with methylene blue

(counterstain) for one minute. Non-acid-fast cells appear
blue after application of the counterstain.
ACID FAST STAIN
C. Special Stains
- 3 types: capsular, flagellar and endospore stains
1. Capsular staining
Many microorganisms contain a gelatinous covering called a
capsule, which will be discussed in chapter 5. Capsule staining
is more difficult than other types of staining procedures because
capsular materials are soluble in water and may be dislodged
and removed during rigorous washing.

a. Negative staining
The bacteria in a solution can be mixed with a negative
stain such as India ink or nigrosin to provide a dark background
and then stain the bacteria with a simple stain, such as safranin.
Because of their chemical composition, capsules do not accept
most biological dyes like safranin, and thus appear as halos
surrounding each stained bacteria.

b. Anthony’s method
C. Special Stains

Capsular staining: Negative staining

C. Special Stains

Capsular staining:
Anthony’s Stain Method/ Hiss Staining Method
C. Special Stains
2. Endospore staining
C. Special Stains
3. Flagella staining
“Science would not be where it is today if it were not for
microscopes.”
C. Phase Contrast Microscopy
D. Difference Interference Contrast Microscope
E. Fluorescence Microscope
Streptococcus Fluorescent Antibody Test
Direct Fluorescent Antibody Test- Rabies
Image of an ant viewed under SEM
•