EXPERIMENT 2

AIM: To study the effect of bacterial endotoxin on intact plant cell.

THEORY: Endotoxins are present in the cell wall of the gram-negative bacteria and are
released only upon the disintegration of these cells. They are composed of
lipopolysaccharides (LPS), present in the outer membrane of gram-negative bacterial cell
wall. They were first found to be produced by Vibrio cholerae bacteria and later by
Salmonella and Serratia and E. coli. Bacterial endotoxins are usually liberated due to cell
sonication or damaging of the cell membrane. They normally damage the nuclear membrane
and the chromatin material. There is also evidence of disruption of the nuclear pore complex.
MATERIALS REQUIRED:
1. A small sized onion
2. sterile distilled water
3. 0.9% Saline solution
4. 1% Eosin solution
5. Overnight bacterial culture
6. Centrifuge
7. Microscope

PROCEDURE:
 ENDOTOXIN EXTRACTION:
1. An overnight culture of E. coli was taken and subjected to sonication to lyse the cells.
2. The solution was centrifuged at 5000 RPM for 10 minutes and the supernatant was
collected.
3. The supernatant collected contained the liberated bacterial endotoxin.

 PERCENTAGE CELL DEATH:

1. The inner onion scale was removed from fleshy leaves of the bud.
2. The scales were treated with -
a) sterile distilled water
b) 0.9% Saline solution
c) Bacterial endotoxin
3. The treated scales were incubated for 30 minutes.
4. After incubation, the scales were stained with 1% Eosin solution and were observed for
the number of dead cells under the microscope.
5. The observations were recorded, and the percentage cell death was calculated.
 OBSERVATION:

Fig: Effect of bacterial endotoxin on inner onion scale in different treatments

a) Sterile water b) 0.9% Saline c) Bacterial endotoxin

No cell damage, intact Cell shrinkage, plasmolysis and Disruption of nuclear pore complex
cells observed. damaged cells observed. and nucleus shifted to edge of the
cells.

RESULT:

No. of cells No. of damaged Percentage

Treatment Nature of damage
studied cells cell damage

Sterile water 34 0 No cell damage -

Plasmolysis, cell shrinkage,

0.9% Saline 45 11 24.44 %
cell wall disruption

Bacterial Disruption of nuclear pore

18 7 38.89 %
endotoxin complex

CONCLUSION:
The application of endotoxin to the plant cells damaged the cell’s nucleus, which was
observed using microscopic techniques.
PRECAUTIONS:
1. The treated onion scales should be properly stained so that the number of dead cells can
be easily calculated under the microscope.
2. Incubation time should not be more than 30 minutes.
3. The supernatant which is formed after centrifugation is to be handled carefully.