YOU ARE GIVEN STAINED SLIDE SAMPLES OF GRAM-NEGATIVE

AND GRAM-POSITIVE BACTERIA. CHOOSE A SUITABLE LIGHT

MICROSCOPE TO USE AND DESCRIBE HOW TO OBSERVE THE
SPECIMENS
INTRODUCTION
The complexity of cells and small microbes were not appreciated until the first
microbes were observed through the lens of microscope. Microscope is very important
to human to define the relationship among the diversity of organisms. Generally,
microscopy is used in biology to initial detection of microbes, definitive identification of
microbes and to define structure of living cells and stained cells. The microscopic
examination of clinical specimens is used to detect bacterial cells, fungi elements,
parasites (eggs, larvae, or adult forms) and viral inclusions present in infected cells. The
microscopic detection of organisms stained with antibodies labeled with fluorescent
dyes, simple staining or differential staining like Gram stain has proved to be very useful
for the specific identification of many organisms.
Gram Stain is a staining procedure that contrasts stained cells against a bright
background. Microbiologists commonly stain bacteria before viewing them because the
cytoplasm of bacterial cells lacks color, making it hard to see the cells on a bright
background. The Gram Stain has been developed to provide contrast for bright-field
microscopy. So, we are able to see the bacteria clearly from the bright background. The
Gram stain technique is a differential procedure. All bacteria stain with the crystal violet
and iodine, but only gram-negative bacteria lose the colour when alcohol is applied.
Subsequently, these bacteria stain with the safranin dye. Gram-positive bacteria remain
blue purple.
CHOOSING THE SUITABLE MICROSCOPE.
Gram stain allows us to view stained cells against the bright background. So the
slide stained with Gram negative bacteria and Gram positive bacteria can be seen
under the bright-field microscopy. This is because the Gram staining procedure colour
the cytoplasm of the cells. The stained cells are contrasts against a bright background.
In light microscope, visible light passes directly through the lenses and specimen.
Such an optical configuration is called bright-field microscopy. Visible light in bright-field
microscopy is projected through a substage condenser lens, which focuses the light into
a sharp cone. The light then passes through the opening of the stage, into the slide,
where the light is reflected, refracted, or passes through the specimen. Next, light
passing through the specimen enters the objective lens to form a magnified
intermediate image inverted from that of the specimen. This intermediate image
becomes the object magnified by the ocular lens (eyepiece) and seen by the observer.
Gram positive and Gram negative bacteria have very tiny in size and can only be
easy to distinguish in the higher resolution, by using 100X lens. The limitation of bright-
field microscopy is the resolution of the image. The resolving power of a microscope is
determined by the wavelength of light used to illuminate the subject and the angle of
light entering the objective lens. On focusing the 100X lens, the resolving power is
greatest when oil is placed between the objective lens and the slide because oil reduces
the dispersion of light. When light rays enter the air, they miss the objective lens.
However, the light remains in the straight line in the oil. This pathway leads them
directly into the lens. The oil thus provides a homogeneous pathway for light from the
slide to the objective, and the resolution of the object increases.
HOW TO OBSERVE THE SPECIMENS.
To observe the specimen stained with Gram stain, place the slide within the
stage clips on the fixed stage. Move the slide to center the specimen over the opening
in the stage directly over the light source.

Raise the microscope stage up as far as it will go. Rotate the scanning lens or
low power lens (4X) into position. Lower the body tube with the coarse-adjustment knob
to its lowest position.

While looking through the ocular lens, use the fine-adjustment knob, rotating it
back and forth slightly, to bring the specimen into sharp focus.

Adjust the substage condenser to achieve optimal focus.

Routinely adjust the light source by means of the light-source transformer setting
and the iris diaphragm, for optimum illumination for each change in magnification.
Magnification is change to 10X lens and 40X lens. The fine-adjustment is readjusted for
each change in magnification to bring sharp image

Once we have brought the specimen into sharp focus with a low-powered lens
(4X), 10X lens and 40X lens, preparation may be made for visualizing the specimen
under oil emersion. Place a drop of oil on the slide directly over the area to be viewed.
Rotate the nosepiece until the oil emersion locks into position. Avoid the high power
objective (40X) to touch the drop of oil. The slide is observed from the side as the
objective is rotated slowly into will be properly immersed in the oil. The fine-adjustment
knob is readjusted to bring the image into sharp focus.



CONCLUSION
The Gram positive bacteria will appear blue purple as it remain that color when
alcohol is applied while the Gram negative bacteria appear orange-red as it lose the
color when alcohol is applied and it stained with the Safranin dye. The bacteria can only
be distinguished easily on oil emersion magnification. The bright background has no
problem to observe the bacteria as their cytoplasm has been stained. The bright-field
microscopy is the most suitable observe it.

Gram negative rod (Pseudomonas spp). Gram positive cocci shape (Staphylococcus aureus)
REFERENCES
1. http://en.wikipedia.org/wiki/Gram_negative
2. http://en.wikipedia.org/wiki/Gram_stain
3. Monica Cheesbrough.1999. District Laboratory Practice in Tropical Countries
Part 1. Cambrige Low Price Edition. Cambrige University Press.
4. Fames G. Cappuccino, Natalie Sherman.2005. International Edition,Microbiology,
A Laboratory Manual, 7
th
Edition. Pearson, Benjamin Cummings.
5. Jeffrey C. Pommerville. 2004. Alcamos Fundamentals of Microbiology, 7
th

Edition. Jones And Bartlett Publishers.