OBSERVATION-

20 cases were collected to different age group patient suffring from

upper respiratry tract infection were collected with the help of strile
cotton swab .
Gram stain smear were made and inclubated an blood agar and
maconkey media .
After 24-28 hours identification of different microbial flora was based on
colony marphplogy,microscopy and biochemical test.

Age wise

SERIAL NUMBER AGE GROUP NUMBER OF PATIENT %

1. 10-15 05
2. 16-20 04
3. 21-25 08
4. 26-30 03
Identification of bactria(age wise) number of patient 20 bactiria pathogenic and non
pathogenic

S.NUM AGE PTAHOGENIC % NON PATHOGENIC %

1 10-15 03 02
2 16-20 02 02
3 21-25 06 02
4 25-30 03 00

10-15 16-20 21-25 26-30

H.infulenzae 1 1 2 1

S.pneumonaie 2 1 3 1

Moraxella catarrhalis 1 - 1 -
Age distributation graph

3
 1
10-15 15-16 16-20 21-25

History of the Gram

Stain and How it Works
The Gram staining method, named after the Danish
bacteriologist who originally devised it in 1882 (published
1884), Hans Christian Gram, is one of the most important
staining techniques in microbiology. It is almost always the first
test performed for the identification of bacteria. The primary
stain of the Gram's method is crystal violet. Crystal violet is
sometimes substituted with methylene blue, which is equally
effective. The microorganisms that retain the crystal violet-
iodine complex appear purple brown under microscopic
examination. These microorganisms that are stained by the
Gram's method are commonly classified as Gram-positive or
Gram non-negative. Others that are not stained by crystal
violet are referred to as Gram negative, and appear red.

Gram staining is based on the ability of bacteria cell wall to

retaining the crystal violet dye during solvent treatment. The
cell walls for Gram-positive microorganisms have a higher
peptidoglycan and lower lipid content than gram-negative
bacteria. Bacteria cell walls are stained by the crystal violet.
Iodine is subsequently added as a mordant to form the crystal
violet-iodine complex so that the dye cannot be removed
easily. This step is commonly referred to as fixing the dye.
However, subsequent treatment with a decolorizer, which is a
mixed solvent of ethanol and acetone, dissolves the lipid layer
from the gram-negative cells. The removal of the lipid layer
enhances the leaching of the primary stain from the cells into
the surrounding solvent. In contrast, the solvent dehydrates
the thicker Gram-positive cell walls, closing the pores as the
cell wall shrinks during dehydration. As a result, the diffusion of
the violet-iodine complex is blocked, and the bacteria remain
stained. The length of the decolorization is critical in
differentiating the gram-positive bacteria from the gram-
negative bacteria. A prolonged exposure to the decolorizing
agent will remove all the stain from both types of bacteria.
Some Gram-positive bacteria may lose the stain easily and
therefore appear as a mixture of Gram-positive and Gram-
negative bacteria (Gram-variable).

Finally, a counterstain of basic fuchsin is applied to the smear

to give decolorized gram-negative bacteria a pink color. Some
laboratories use safranin as a counterstain instead. Basic
fuchsin stains many Gram-negative bacteria more intensely
than does safranin, making them easier to see. Some bacteria
which are poorly stained by safranin, such as Haemophilus
spp., Legionella spp., and some anaerobic bacteria, are readily
stained by basic fuchsin, but not safranin. The polychromatic
nature of the gram stain enables determination of the size and
shape of both Gram-negative and Gram-positive bacteria. If
desired, the slides can be permanently mounted and preserved
for record keeping.

Besides Gram's stain, there are a wide range of other staining

methods available. By using appropriate dyes, different parts
of the bacteria structures such as capsules, flagella, granules,
and spores can be stained. Staining techniques are widely used
to visualize those components that are otherwise too difficult
to see under a light microscope. In addition, special stains can
be used to visualize other microorganisms not readily
visualized by the Gram stain, such as mycobacteria, rickettsia,
spirochetes, and others. In addition, there are modifications of
the Gram stain that allow morphologic analysis of eukaryotic
cells in clinical specimens.
List of Reagents and Instruments

Equipment
Bunsen burner, alcohol-cleaned microscope slide, water

Reagents
Crystal violet, Gram's iodine solution, acetone/ethanol (50:50 v:v),
0.1% basic fuchsin solution

Procedures

1. Prepare a Slide Smear:

A. Transfer a drop of the suspended culture to be examined on a slide
with an inoculation loop. If the culture is to be taken from a Petri dish
or a slant culture tube, first add a drop or a few loopful of water on the
 slide and aseptically transfer a minute amount of a colony from the
Petri dish. Note that only a very small amount of culture is needed; a
visual detection of the culture on an inoculation loop already indicates
that too much is taken.

If staining a clinical specimen, smear a very thin layer onto the slide,
using a wooden stick. Do not use a cotton swab, if at all possible, as
the cotton fibers may appear as artefacts. The smear should be thin
enough to dry completely within a few seconds. Stain does not
penetrate thickly applied specimens, making interpretation very
difficult.

B. Spread the culture with an inoculation loop to an even thin film over
a circle of 1.5 cm in diameter, approximately the size of a dime. Thus,
a typical slide can simultaneously accommodate 3 to 4 small smears if
more than one culture is to be examined.

C. Air-dry the culture and fix it or over a gentle flame, while moving the
slide in a circular fashion to avoid localized overheating. The applied
heat helps the cell adhesion on the glass slide to make possible the
subsequent rinsing of the smear with water without a significant loss of
the culture. Heat can also be applied to facilitate drying the the
smear. However, ring patterns can form if heating is not uniform, e.g.
taking the slide in and out of the flame.

2. Gram Staining:
A. Add crystal violet stain over the fixed culture. Let stand for 10 to 60
seconds; for thinly prepared slides, it is usually acceptable to pour the
stain on and off immediately. Pour off the stain and gently rinse the
excess stain with a stream of water from a faucet or a plastic water
bottle. Note that the objective of this step is to wash off the stain, not
the fixed culture.

B. Add the iodine solution on the smear, enough to cover the fixed
culture. Let stand for 10 to 60 seconds. Pour off the iodine solution and
rinse the slide with running water. Shake off the excess water from the
surface.

C. Add a few drops of decolorizer so the solution trickles down the

slide. Rinse it off with water after 5 seconds. The exact time to stop is
when the solvent is no longer colored as it flows over the slide. Further
delay will cause excess decolorization in the gram-positive cells, and
the purpose of staining will be defeated.

D. Counterstain with basic fuchsin solution for 40 to 60 seconds. Wash

off the solution with water. Blot with bibulous paper to remove the
excess water. Alternatively, the slide may shaken to remove most of
the water and air-dried.
3. Quality control:
It is a simple matter to prepare a control slide by breadking a clean
wooden applicator stick and picking a small amount of material from
the interproximal space of one's teeth. This should be smeared into a
drop of clean tap water on a clean glass slide. The slide may be stained
as above. This material will consistently display a few neutrophils and
a mixture of Gram (+) and (-) organisms. Neutrophil nuclei should be
pink.

3. Examine the finished slide under a microscope.

A caveat in the examination of the Gram smears is the distortion in

morphology that can be caused by antimicrobial therapy. This is
especially likely to occur in urine speciments. Filamentous and
pleomorphic forms may be observed among the Gram (-) rod species.
Gram reaction of the organism may also change after antimicrobial
therapy, Gram (+) bacterial may become gram variable. Look at areas
that are one cell thick only; observation of thick areas will give variable
and often incorrect results. White blood cells and macrophages should
stain Gram-negative, whereas sqamous epithelial cells are Gram-
positive