Microbiological

Preparations For
Examining Microorganisms
Jeem Carlo F. Pula, Instructor

3 Different for Studying the

Morphology of Microorganisms
1. Wet Mount- prepared by placing a drop of
suspension on a clear glass slide and covering with
a cover slip.
2. Hanging Drop Preparation-prepared by placing a
drop of the suspension on a cover slip which is then
inverted over the concave portion of the hollowground slide.
Conditions where these preparation are
preferred:
a. Study of the morphology of spiral bacteria
b. Motility of bacteria
c. Cell inclusion which are easily damaged
d. Reaction to chemicals or specific sera
e. Cytologic changes during cell division.

3. Fixed Stained Smear- a bacterial

suspension is dried and heat fixed so that it
will adhere to the slide. Such smear is then
stained with colored dyes.
Advantages over wet preparations:
a. Cells and cell structures are more clearly
visible
b. Differences between cells id the same or
different species can be demonstrated using
special staining solutions.

Definition of Terminologies
Smear- is a thin layer of material spread across a
slide for microscopic study.
This thin layer of material on the slide is air dried
and fixed by heating.
Staining- is a process of artificially coloring
microorganisms with dyes in order to facilitate
their study under the microscope.
Dye- is an organic compound consisting of
benzene rings with chromophore and auxochrome
group.
Chromophore- any chemical group which gives a
specific color to a compound.
Auxochrome- furnishes salt-forming properties
responsible for transferring the color of a dye to a
substance upon which it acts.

Staining Methods
1. Direct Staining- the organism to be studied absorbs
the stain thus making the cell appear colored.
a. Simple Staining- an ordinary dye is used for the
general study of organisms with no need of
comparisons of physiological characteristics.
b. Differential Staining- used to contrast two or
more organisms of the same or different species
that are being studied. It is used to compare the
response of the organism with different
physiological characteristics to certain staining
techniques.
Types:
1. Gram Staining-differentiates gram (+) from
gram (-)
2. Acid Fast Staining- differentiates the Genus
Mycobacteria which are acid fast form the nonacid fast bacteria.

C. Selective Staining- specific cell structures

are selectively colored by special dyes such
that these are distinguish from the vegetative
cell.
Ex. Fulton-Schaeffers Method for spores
Loefflers Method for flagella
2. Indirect/Negative/Relief- the background is
colored while the organism or structure to be
studied remains unstained for contrast. This is
due to the fact that some stains have lesser
staining affinity with the cell structure.
Ex. India ink- the cell and its background are
colored black while the capsule is unstained.

Reagents Used In
Differential and Selective
1.
Initial Stain- also known as the primary stain, it is the first
Staining
stain that is applied on the specimen wherein the cell will
appear colored.
2. Mordant- any substance which will form a bridge between
the cell and the initial stain. There are two types of mordant:
a. Physical Mordant- such as heat or cold
b. Chemical mordant- such as iodine, ferrous sulfate, tannic
acid
3. Decolorizer- any substance that maybe used to removed
the initial stain. This is especially important to contrast the
staining affinity of some parts of the cell to the initial stain.
The decolorized stain will then be replaced by the secondary
stain.
4. Secondary Stain- also known as the counterstain. It is the
stain that is applied to the decolorized cell or cell parts. This
will help in differentiating the physiological characteristic or
some special structures that are present in the cell.

DIFFERENTIAL STAINING:
Gram Staining
(1884)- Hans Christian Gram- Danish physician devised a
staining procedure that can divide al thee true bacteria into two
physiological groups.
This is the most important staining method done in bacteriology
to facilitate the identification of the bacterial species.
The cells are exposed to more than one dye or staining
reagents.
Research involved in the determining the comparison of
bacterial cell walls indicated that there was a close correlation
between the gram staining results and the presence or absence
of lipid cell wall constituents.
This basic chemical difference between gram(+) and gram (-)
cells provides a reasonable explanation for the differential
mechanism associated with the Gram stain.

Theories Supporting Grams

Staining Procedures
1. Gram negative cell walls have a high lipid content, as much
as 20% by weight, while gram (+) cells contain little or no
lipid in the cell walls.
2. The lipids found in the gram (-) cell walls dissolve readily in
acetone-alcohol. The dissolution of lipid material by acetonealcohol allows the Crystal violet-Iodine complex to be lost
from the gram (-) cells resulting in decolorization.
3. Since gram (+) cells contain little or no lipid in their cell wall,
the cell walls act as a barrier to the loss of the CV-I complex
from these cells.
4. When the gram (+) cells are treated with the enzyme
lysozyme, their cell walls dissolve resulting in the formation
of protoplasts. The protoplast will stain with crystal violet
indicating that other components beside the cell wall are
stained during the gram staining procedure. However, the
addition of acetone-alcohol to these protoplasts results in
decolorization demonstrating a loss of gram (+)
characteristic along with the loss of the bacterial cell wall.

Reaction and Appearance of

Bacteria in Grams Staining
Reagent

Gram (+)

Gram (-)

Crystal Violet

Cells stain violet

Cells stain violet

Iodine

CV-I complex formed

within cells; cell remain
violet

CV-I complex formed

within cells; cell
remain violet

Alcohol/
Acetone

Cell walls dehydrated;

shrinkage of pores occur
with decreased
permeability, CV-I
complex cannot pass out
of cell; cells remain violet

Lipid extracted from

cell walls, increased
porosity, CV-I is
removed from the cell;
cells become colorless

Safranin

Cells are not affected;

remains violet

Cells take up the

strain; becomes red.

Acid Fast Staining

Some bacteria are not readily stained by the gram
staining procedure, therefore, a more rigorous staining
procedure maybe required using more concentrated
biological dyes and longer staining time.
The acid-fast stain is a differential staining procedure
commonly used to stain Mycobacterium and Nocardia
organisms.
This staining procedure divides bacteria into two major
groups, the acid fast organisms; which retain the
primary dye throughout the staining procedure, and
the non-acid fast organisms; which are decolorized
and then counterstained.
The bacterial cell wall of acid fast organisms contains a
substantial amount of lipid material.
The lipid material is not only difficult to stain, but it
appears to resist the penetration of the primary dye to
the underlying cellular cytoplasm.

 Because of this factor, long stain time is required to

stain the acid-fast organisms.
However, once stained, they are very resistant to
decolorization. Acid-fast organisms are alsso very
resistant to drying.
They main remain viable and infectious in a room
environment for as long as weeks, following
contaminations by a patient having a disease like
tuberculosis.
Again, this characteristic appears to be related to the
wax-like lipid composition of the cell wall associated
with acid-fast bacteria.
The two important acid-fast pathogenic organisms
are Mycobacterium tuberculosis, the causative agent
of human tuberculosis and Mycobacterium leprae,
the causative agent of leprosy.
Acid fast staining of sputum from patients suspected
to have tuberculosis is routinely carried out, and the
presence of acid-fast bacteria is diagnostic.

Types of Acid-Fast Staining

Method
1. Ziehl-Neelsen- acid fast organisms are stained red; non-acid
fast are stained blue due to the color of the counterstain used.
2. Kinyouns Cold Staining Method- this method utilizes no
steaming with carbol fuschsin and tergitol is instead applied
for three minutes. The decolorizer is brilliant green. The
procedure is basically the same as that of Ziehl-Neelsen.
3. Truants Auramine Rhodamine Method- two fluorescent
dyes are used: auramine and rhodamine instead of carbol
fuchsin. The tubercle bacilli glows with a bright yellow
fluorescence.
4. Pappenheims Method- this method makes use of rosolic
acid added to alcohol as its decolorizer. It differentiates
Mycobacterium smegmatis (blue in color) from Mycobacterium
tuberculosis (red in color). My.Smegmatis is found in
sebaceous secretions of the sskin of man but is not known to
cause any disease.
5. Baumgartens Method- the decolorizer used is alcohol with
nitric acid. It differentiates m.tuberculosis (blue) from
m.leprae (red).

Reaction and Appearance of

Bacteria in Ziehl-Neelsen Method
Reagents

Acid Fast

Reactions

Non-acid Fast

1. Carbolfuschsin

Cells stain red

Stain is taken up
by the organism
and penetrates
lipid cell wall.

Cells stain red

2. Steam

Cells remain red

Fixes the stain

and aids dyepenetration of
the cell wall

Cells remain red

3. Acid-alcohol

Cells remain red

Acid-fast remains Cells are

while non-acid
colorless
fast fails to
retain color of
initial stain and
is decolorized

4. Methylene
blue

Cells remain red

Stain is taken up
by non-acid fast
organism

Cells are blue in

color