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, 7th Edition. Pearson, Benjamin Cummings.

5. Jeffrey C. Pommerville. 2004. Alcamo’s Fundamentals of Microbiology, 7th Edition. Jones And Bartlett Publishers.