Differential Staining

Purpose:

The aim of this experiment is to differentiate the physical and chemical properties of bacterial cells by differential staining.

Hypothesis:

I hypothesize that endospores favor the growth of bacteria in hostile conditions and they can form in different areas of a vegetative cell.

Introduction:

An endospore is a cellular structure formed by bacteria and it is dormant and non-reproductive. On the other hand, a vegetative cell is a cell that is responsible for the formation of an endospore and has an active growth rate. However, a vegetative cell does not take place in the production of gametes. Spores are resistant to factors such as excessive heat, radiation, chemicals, and dehydration. An aerobic genus that can exist as a vegetative cell is Bacillus while an anaerobic genus that can exist as a vegetative cell is Clostridium. The formation of reproductive spores begins with the germination of a free spore under favorable conditions. The coat of the free spore raptures to form a vegetative cell. This vegetative cell can either undergo binary fission or sporogenesis. Binary fission will take place only under favorable environmental conditions while sporogenesis will take place under unfavorable conditions so as to form a forespore that will separate to form an endospore and lead to the formation of spore coat by the degenerating vegetative cell. This will eventually form a free spore that will undergo the same process from the beginning.

Sporogenesis primarily enables the production of spores and reproduction to take place by means of spores. During the spore staining process, malachite green is used as the primary stain since it is water-soluble and it rinses easily from spore cells. In this staining, both the vegetative cells and endospores get stained and heat is used so as to act as a mordant to fix the stain in the cells. Water is used as the decolorizing agent to rinse off the malachite dye. The vegetative cells will become colorless since they had been distorted by heat. A counterstain also known as the secondary stain is added so as to stain the vegetative cells that had been decolorized. The stain used for this purpose is usually safranin. Therefore, at the end of the staining process, the vegetative cells retain a pink color while the spores depict a green ellipse shape.

Methods

Materials required

• 5 grams of Malachite green
• 100ml of distilled water
• 5 grams of safranin

Procedure followed

1. Use a clean slide to make a smear that is sterile
2. Fix the smear by first air drying and later heat fixing the sample on the slide

• Add malachite green to the slide and let it stay for 5 minutes

1. Rinse the slide with distilled water for 2-3 seconds
2. Add the secondary stain safranin and let it stay for 30 seconds
3. Rinse the slide with water and dry it by blotting

• Examine the slide under a microscope to distinguish the colors

Results

Spherical and elliptical shapes of green and pink structures are visible under the microscope. The endospores have light green color while the vegetative cells are pink in color.

Conclusion

Restate: This experiment aims at differentiating endospores from vegetative cells using differential staining.

Explain: In this lab, the cells were stained in two procedures. The first one used primary stain malachite green while the second process used safranin counterstain. After rinsing the cells, the slide was placed under a microscope for observation.

Results: Cells of spherical and elliptical shape were seen and some had green color while others had pink color. These results affirm that endospores are green in color while vegetative cells are pink in color.

Uncertainties: I was not able to establish how endospores are resistant and yet they remain dormant until an opportunity to cause infections arises. I could not further establish the concept of endospores being able to remain resistant in hosting defenses after infection. Moreover, I was unable to find out the reason behind the differences in the shapes of vegetative cells and endospores.

New: Further studies can be conducted to find out how endospores and vegetative cells interact in the same environment and whether there is a symbiotic relationship between the two.