Introduction

In this experiment, another set of staining methods are used to observer different
given microorganisms and to understand the importance of these methods. These methods
are Acid-fast stain, Endospore stain, Capsule stain and Flagella stain.
Result
Discussion
In acid-fast stain, sputum is observed under caution because it may be pathogenic.
The diagnostic value for acid fast are pathogens retain color even in presence of acid and it
can distinguiseh Mycobacterium, which cause of tuberculosis and leprosy, with rod shapes
and Nocardia, which can cause of a pulmonary disease called nocardiosis, with branching,
filamentous shapes.
When decolorizing, acid-fast uses acidic alcohol instead of 95% ethanol because it is
more powerful than ethanol. Acid alcohol destains non-acid fast bacteria but not
Mycobacteria, which are resistant to the procedure due to the presence of mycolic acid. In
the Ziehl Neelsen procedure, Mycobacteria remain red from the carbolfuchsin primary stain
after destaining and non-acid fast bacteria (or tissue) which lose the primary stain during the
destaining procedure are counterstained blue by methylene blue (VanMeter and Hubert,
2010)
Both acid-fast and endospore staining undergone heating of the slides with the
bacteria. Heating is used in acid fast to breakdown the mycolic acid which enhances the
penetration and retention of the dye. For endospore, heat helps the stain to penetrate the
into the relatively impermeable spore coats (Sumbali and Mehrotra, 2009).
In Schaeffer-Fulton method, the primary stain, Malachite Green, is added over the
heat fixed bacterial smear and heated over a steam bath for few minutes which cause
softening of the hard layer of the spore and the primary stain gets stick to the spore. When
taken from the steam bath followed by further cooling hardens the outer layer of the spore.
During this stage both the spore and vegetative cells appear as green in color. But later the
thick outer layer makes the spore resistant to the action of decolorizing agent (water), but
however, water can easily decolorize the vegetative cells. When counterstained with
Safranin, vegetative cells are easily stained with Safranin, and the cells appear in red or pink
color. The spore is still green after the counterstain so it has different color with the
vegetative cells. Endospores gives advantages to member genera Bacillus like being
resistant to harsh chemical and physical conditions. This makes the bacteria able to
withstand disinfectants, radiation, desiccation and heat. Clostridium has given advantage of
producing endospores.
In capsule stain, water is not used as decolorizer because the capsules surround the
cell is made up of highly ordered polymers of sugars and proteins, where heat or water
would dislodge the capsules from the bacterial cell wall. So copper sulphate is used not only
as decolorizer but also as counterstain. The bacterial capsules provide a mechanism for
these pathogens to evade the hosts immune system to go undetected. The capsule also
provides protection from drying out (Lakomia-Grover and Fong, 1999).
The Quellung reaction, comes from a German word meaning swelling, is
a biochemical reaction in which antibodies bind to the bacterial capsule of Streptococcus
pneumoniae, Klebsiella
pneumoniae, Neisseria
meningitidis,
Haemophilus
influenzae, Escherichia coli, and Salmonella. The antibody reaction allows these species to

be visualized under a microscope. If

becomes opaque and appears to enlarge.

the

reaction

is

positive,

the

capsule

Flagella, is a lashlike appendage that protrudes from the cell body of certain
prokaryotic and eukaryotic cells. It comes from the Latin word meaning whip. Flagella is
important to bacterium because is provides motility to the bacteria in order to move and
provide adhesive organelle in order for the bacterium to stick to its host. Unstained flagella
alone is cannot be seen easily due to its very small size. With the use of mordant, it stains
and coat the flagellum until it is thick enough to be seen in the microscope.

References
Grover-Lakomia, L. I, Fong, E. (1999), Microbiology for Health Careers, Delmar Publishers,
Albany, New York.
Sumbali, G., Mehrotra, R. S. (2009),Principles of Microbiology, Tata McGraw-Hill, New Delhi,
India.
VanMeter, K. C., Hubert, R. H. (2010),Microbiology for Healthcare Professional, Elsevier
Inc., St. Louis, Missouri.
Fisher, Bruce; Harvey, Richard P.; Champe, Pamela C. Lippincott's Illustrated Reviews:
Microbiology (Lippincott's Illustrated Reviews Series). Hagerstwon, MD: Lippincott Williams &
Wilkins.