Bacteria Classification By Gram Staining

THE AMERICAN UNIVERSITY IN CAIRO
BIOLOGY DEPARTMENT
SCIENCE 453 : BIOLOGY FOR ENGINEERS REPORT No.1
24/2/96

Objective:

To test a sample of laboratory prepared bacteria and categorise it
according to Christian’s gram positive and gram negative classes and also by
viewing it under a high powered microscope and oil immersions; classify its
shape and note any special characteristics.

Introduction:

Bacteria was categorised into two groups in 1884 by the Danish
Bacteriologist Christian, gram positive and gram negative by a staining
technique where the ability to avoid de-coloration of Crystal Violet solution
by alcohol would render the category of gram positive, and gram negative if the
bacteria is de-coloured. This could be noted by the final colour of the
bacteria: a violet colour where Gram positive and a pink colour of the Safranin
added pending the de-colouring process.

Materials:

1. Bacteria Sample 2. Microscope Slide 3. Gram Staining Kit and Wash Bottles
a. Crystal Violet Solution b. Iodine Solution c. 95% Ethyl Alcohol d.
Safranin e. Distilled Water 4. Bibulous Blotting Paper 5. Microscope 6. Oil

Procedure:

A. Preparation :

1. Bacteria is cultivated on agar jelly in an incubator at 25°C for 24 hours. 2.
Obtain a microscope slide and with a toothpick, smear a thin coat of the
bacteria sample onto the slide 3. Cover the smear with a drop Crystal Violet
and leave standing for 20 seconds 4. Wash off the stain with distilled water;
drain and blot off the excess with bibulous paper. 5. Apply Gram’s Iodine on
the smear and leave to stand for 1 minute. 6. Drain the excess iodine and apply
95% Ethyl alcohol for 20 second duration or till the alcohol runs clearly from
the slide. 7. The smear should rinsed for a few seconds with distilled water to
stop the action of the alcohol. 8. Drain and blot off the excess with bibulous
9. Introduce Safranin to the smear and leave standing for 20 seconds. 10. Wash
off the stain with distilled water; drain and blot off the excess with bibulous
paper. 11. Leave the slide to air dry.

B. Examination:

1. Place the slide under microscope on low powered lens. 2. Move the slide
using the apparatus until the sample can be seen as a blur under the microscope.
3. Focus the lens to ensure that there is a sample directly under the lens. 4.
Move to higher powered lens, repeat step 3. 5. Move to higher powered lens,
repeat step 3 6. Move microscope aside and add Oil immersion, leave for a few
seconds and re-examine the slide.

Note Shape and colour and any other observations.

Results and Observations:

It was evident by visual examination that the alcohol was de-colouring or a
least partially de-colouring the bacteria.
The sample appeared a dark pink or close to violet by the naked eye; a
microscope was needed to ensure results.
Under the low powered microscope shades of pink were noted.
Under the medium power, the shades were more clear but no shape could be made
out.
Under the high powered microscope clumps of pink rod (bacilli) shaped bacteria
cells could be observed.
Under Oil Immersion and high powered lens the cells could seen more spaced out
and thus a clearer indication of the pink colour, bacilli shape and spores could
be made out in the individual cells.

Conclusion:

The Shape was noted as Bacilli (Rod-like) shaped cells; a gram variable
shape, distinct in either Gram Negative or Gram positive bacteria.
The final colour of the cells were stained pink by the Safranin showing
the de-coloration of the crystal violet proving the bacteria is of the gram
negative class.
Under oil immersion the cells became more sparse and under the high
powered lens of the microscope spores could be seen, as little bubbles, in the
cells. This tells us that the bacteria was in its terminal state.
The presence of spores in the bacteria at its terminal state tells us
that the bacteria could be an old culture. Old bacteria cultures which are gram
positive tend to de-colour, yet more slowly than gram negative bacteria. The
speed of de-coloration was not inspected very clearly thus no further conclusion
could be reached, yet it is possible that this an old culture of Bacilli shaped
Gram Positive bacteria.

Recommendation:

It is recommended that the same sample be tested again for de-
coloration; focusing on de-coloration speed. If the de-coloration is fast then
the sample is definitely gram negative, slow de-coloration would tell us it is
gram positive. For future samples it would be recommended to keep the bacteria
sample for this specific test for only 16 hours as recommended to avoid